0000 Pectinesterases fromth eorang e fruit- thei rpurification ,genera l characteristicsan d juiceclou ddestabilizin g properties

BIBLIOTHEEK

LANDnot". - . • •• HOOL Promotor:dr .W .Pilnik ,hoogleraa r ind e levensmiddelenleer Co-referent:dr .ir .F.M . Rombouts,wetenschappelij k hoofdmedewerker W <92o\ y&$ C. Versteeg

Pectinesterases from the orange fruit - their purification, general characteristics and juice cloud destabilizing properties

Proefschrift ter verkrijging van de graad van doctori nd elandbouwwetenschappen , op gezagva nd erecto rmagnificus , dr.H.C .va n der Plas, hoogleraari nd eorganisch e scheikunde, inhe t openbaart everdedige n opvrijda g 28septembe r197 9 des namiddagst evie r uuri nd eaul a vand eLandbouwhogeschoo l teWageninge n

Centre for Agricultural Publishing and Documentation

Wageningen - 1979 __ BIBLIOTHEEK

! :• '.< LANDBOIJrt.-.iv.;•••< HOOL II C\C/\ VAGEMiiNGEN Abstract

Versteeg,C . (1979)Fectinesterase s from theorangefrui t- theirpurification , general characteristics andjuic e cloud destabilizing properties.Agric.Res.Rep . (Versl.landbouwk. Onderz.)892 ,ISB N 9022 0070 9X ,(x )+ 10 9p. ,5 3figs ,2 6tables ,24 6refs ,Eng . and Dutch summaries. Also: Doctoral thesis Wageningen.

Twelve forms of pectinesterase were detected in citrus fruits. Two forms, representing over 90% of the total pectinesterase activity in Navel oranges, were purified. These pectinesterases, named Pectinesterase I and II have isoelectric points of 10.05 and > 11.0, respectively. Both pectinesterases have the same molecular weight of 36 200 but differ in amino acid composition. A third pectinesterase, with a higher molecular weight (about 54 000) was partly purified. The optimum pH of these three pectinesterases is about 7.5, but their pH-activity profiles differ and depend on the degree of esterifica- tion of the pectin. The high molecular weight pectinesterase is still active at pH 2.5. The Km values of both purified pectinesterases decrease with decreasing degree of esterification of the pectin substrate and increase with decreasing pH. Pectinesterase II has a tenfold higher affinity for pectin than Pectinesterase I and is more strongly inhibited by pectate. The heat stabilities were determined: the D90 C and Z values in orange juice are 0.00037 min and 6.5 °C for Pectinesterase I, 0.0015 min and 11 °C for Pectinesterase II and 0.375 min and 6.5 °C for the high molecular weight pectinesterase. The orange juice cloud destabilizing properties of the pectinesterases at 5 C and 30 C are remarkably different. The activity measurements were done, amongst other methods, by an improved gas chromatographic methanol assay. The literature on pectinesterase was reviewed.

Free descriptors:molecula r weight,multipl e forms, isolectric point, stability, heat stability, amino acid composition, K , K.K.,, VV ,, mode ofaction , cloud stability,pH , literatur... e review. ,methano .. .l assay, citrum. 1, sma fruitx ,

BIBLIOTHEEKLH . 3 0 AUG. 1979 0HTV. TfJDSCrffl . ADM.

This thesis will alsob epublishe d asAgricultura l Research Reports892 .

© Centre forAgricultura l Publishing andDocumentation , Wageningen,197 9

No part of this bookma yb ereproduce d orpublishe d inan yform , byprint , photoprint, microfilm oran yothe r means without written permission from thepublishers . Stellingen ^^ ?^

1. De klaring van ondergepasteuriseerd sinaasappelsap door pectine-esterase wordt ver­ oorzaakt door slechts enkele van de oorspronkelijk daarin aanwezige vormen van pectine- esterase.

Dit proefschrift, hoofdstuk 8.

2. Het uitdrukken van de Michaelis-Menten-constante van pectine-esterase in een methyl D-galactopyranosyluronaat concentratie, zoals aanbevolen door Rexovâ-Benkovâ & Markovic, is af te raden.

L. Rexovâ-Benkovâ & 0. Markovic, 1976. Advances in Carbohydrate Chemistry and Biochemistry 33: 323-385. Dit proefschrift, hoofdstuk 7.

3. De experimenten van Lee &Macmilla n en Miller &Macmilla n rechtvaardigen niet hun conclusie dat de helft van de enzymactiviteit van pectine-esterase van zowel de tomaat als ook van Aspergillus rrlger begint aan het reducerende einde van de hoogveresterde pectine.

M.Lee & J.D. Macmillan, 1970. Biochemistry 9: 1930-1934. L. Miller & J.D. Macmillan, 1971. Biochemistry 10: 570-576.

4. De relatie tussen de Michaelis-Menten-constante van pectine-esterase van de sinaas­ appel en de veresteringsgraad van gedeeltelijk alkalisch voorverzeepte pectine zoals ge­ vonden door Solms & Deuel is onjuist.

J. Solms & H. Deuel, 1955. Helv. Chim. Acta 38: 321-329. Dit proefschrift, hoofdstuk 7.

5. Het glycoproteïne karakter van pectine-esterases van tomaten en pruimen, zoals ge­ suggereerd door Theron, De Villiers & Schmidt, is vooralsnog niet bewezen.

T. Theron, O.T. De Villiers &A.A . Schmidt, 1977. Agrochemophysica 9: 7-12. T. Theron, O.T. De Villiers &A.A . Schmidt, 1977. Agrochemophysica 9: 93-96.

6. Het verdient aanbeveling om bij de bestudering van zowel fysiologische als technolo­ gische processen in relatie tot de activiteit van een enzym niet alleen de totale enzym­ activiteit, maar ook de activiteit van eventueel aanwezige verschillende vormen van het enzym afzonderlijk in het onderzoek te betrekken. BTTILI^TTTF.EK.

LANDIH.; •;•• . ^IHOOL 7. De door Biran, Gilbert &Giaci n gepubliceerde gegevens over de sorptie van vinyl- chloride in levensmiddelen bemoeilijken de toepassing van de methode van Van Lierop als routinebepaling van vinylchloride in levensmiddelen.

D.Biran ,S.G .Gilber t& J.R .Giacin ,1979 .J .Foo dSei .44 : 56-58. J.B.H,va nLierop , 1979.Determinatio no fpotentiall y carciogeniccompound si nFood . ProefschriftLandbouwhogeschool ,Wageningen .

8. De door Van der Stegen gegeven interpretatie van de door Viani & Horman aangegeven mogelijke afbraak van carboxy-5-hydroxytryptamides bij het branden van koffie als ver­ klaring voor het niet verdragen van koffie door sommige mensen is niet gerechtvaardigd.

G.H.D.va nde rStegen ,1979 .Foo dChem .4 :23-29 . R.Vian i& I .Horman , 1975.Proceeding so fth e7t hInternationa lColloquiu mo nth e Chemistryo fCoffee ,p .273-278 .

9. Behalvebi jkant-en-klaar-maaltijden ,i she tvermelde nva nvoedingsstoffe ne nd e energetischewaard eo plevensmiddele nslecht sva nbeperkt ewaarde .

W.Pilni k &P .Folstar , 1979.Dtsch .Lebensm .Rundsch .I ndruk .

10. Onderzoeknaa rd etoepassin gva nenzyme ni nafwasmachine sdien tgestimuleer dt e worden,aangezie nhe tgebrui kva nenzyme ni nafwasmachine s zowelhe tenergieverbrui kvan , alsd efosfaatverontreinigin gdoo rdez emachine szo ukunne nverminderen .

11. Indienhe toppervla kva nhe tNederlands ewegenne t tevensgebruik tzo ukunne nworde n alszonne-energiecollector ,zo ujaarlijk see nhoeveelhei denergi eopgevange nkunne nworde n diehe tNederlands eelektriciteitsverbrui kbenadert .

12. Wittewij ni snie twit .

13. Degebruikelijk eopdrach tvoori nee nproefschrif tme td estrekkin gvan :"Aa nmij n vrouwe nkinderen "zo uvaa kvolledigheidshalv emoete nluiden :"Aa nmij nvrou we nkinderen , ondankswi edi tproefschrif ttoc hto tstan di sgekomen" .

Proefschriftva nC .Verstee g Pectinesterasesfro mth eorang efrui t- thei rpurification ,genera lcharacteristic san d juiceclou ddestabilizin gpropertie s Wageningen,2 8septembe r197 9 Aan mijn ouders, Mieke, Steven en Edwin Acknowledgments

I wish to thank - my promotor Prof. Dr W. Pilnik for giving me the opportunity of undertaking this work, for his stimulating interest and for the valuable discussions during the experiments and the writing of the manuscript; - the co-referent Dr Ir F.M. Rombouts for his frequent and valuable advice and criticism during the experiments and the writing of the manuscript; - Mrs W.H. van Deventer-Schriemer and Miss C.H. Spaansen for their enthousiastic and reliable technical assistance during part of the experiments; - Mrs C.C. Nieuwenhuis and Mr J.F.M. Höcker for the valuable contributions to this thesis during their graduate work; - Dr Ir J. Visser for his interest during the work and for reading and criticizing parts of the manuscript; - Dr Ir A.G.J. Voragen and Mr M.J. Hooydonk for their assistance in the sugar analyses. - Ir L.B.J, van Boxtel, Miss H. Ouwehand, Ir J.M. Blijham, Dr Ir J.P. Roozen and Mr W.J.H. van Berkel for their assistance in the amino acid analyses; - Miss J.R. van Delden, Mr J.L. Cozijnsen and Dr M.A. Posthumus for their assistance in part of the gas chromatographic work; - Dr Ir P. Folstar, Dr F. Müller and Mrs M.J. Versteeg-Vel for their comments on parts of the manuscript; - Miss H.E. Belling, Mrs L.J. Muller-Kobold, Mrs G. Nass-Olijve and Miss R.A.M. Janssen for typing the manuscript; - Drs J.C. Beunder, Mr H. Groeneveld, Mr H.G. Slijkhuis, Mr B. Ellermeyer, Mr J. Sachte­ leben, Mr H. Paardekoper and several others in the Biotechnion for their willing help whenever it was needed; - Mr M. Schinnel for drawing the figures; - Mrs E. Brouns-Murray for correcting the English text; - Mr J. Castelein for editing the manuscript. Curriculum vitae

De auteur werd op 12 mei 1948 te Maarsseveen geboren. Na het behalen van zijn ULO-B diploma in 1964 werd de Rijks Hogere Tuinbouwschool te Utrecht bezocht en aldaar werd in 1968 het einddiploma behaald met als hoofdvak fruitteelt. Daarop aansluitend werd de studie in de levensmiddelentechnologie gevolgd aan de Landbouwhogeschool te Wageningen. In 1975 werd het doctoraal diploma behaald met als hoofdvak de kennis van levensmiddelen en de bijvakken levensmiddelenmicrobiologie en proceskunde. Een half jaar na zijn afstuderen kwam hij als promotie-assistent in dienst van de Landbouw Hogeschool op het laboratorium voor levensmiddelenchemie en -microbiologie. Sinds 1 maart 1979 verricht hij onderzoek voor Gist-Brocades. Contents

1 Introduction 1

2 Literature on pectinesterase 2.1 Pectinesterasean dothe rpecti cenzyme s 2 2.2 Pectinesterase 3 2.2.1 Occurrence 3 2.2.2 Rolei nviv o 6 2.2.3 Purificationan dpropertie s 8 2.2.4 Specifityan dmod eo factio n 13 2.2.5 Inhibitionan d(in)activatio n 14 2.2.6 Importancei nfoo dtechnolog y 16 2.2.7 Assayan ddetectio n 18 2.2.7.1 Changesi nth esubstrat e 18 2.2.7.2 Methanoldeterminatio n 19

S Materials and methods 22

4 Methanol determination 28 4.1 Experimentsan d results 28 4.2 Discussion 39

5 Purification and molecular properties of two citrus pectinesterases 40 5.1 Experimentsan dresult s 40 5.2 Discussion 49

6 Multiple forms of pectinesterase in Navel orange and other citrus fruit 51 6.1 Experimentsan dresult s 51 6.1.1 Multipleform so fpectinesteras ei nNave lorang e 51 6.1.2 Multipleform so fpectinesteras ei ncitru sfrui t 54 6.2 Discussion 57

7 Kinetics and mode of action of Pectinesterase I and II 58 7.1 Experimentsan dresult s 58 7.1.1 Initialreactio nrate s 58 7.1.2 De-esterificationlimit s 66 7.2 Discussion 72 8 Stability of pectinesterases 76 8.1 Experiments andresult s 76 8.1.1 Stability duringstorage ,assa yan dorang eclou d stability tests 76 8.1.2 Heat stability 78 8.2 Discussion 83

9 Cloud loss of orangejuice 86 9.1 Experiments and results 86 9.1.1 Pectinesterases andth eclou d lossa t 30° Can d5 ° C 86 9.1.2 Cloud stabilizationb ypolygalacturonas e 89

9.2 Discussion 91

10 General discussion and conclusions 93

Summary 95

Samenvatting 98

Literature 101 1 Introduction

Pectinesterase (pectinpectylhydrolas eE C3.1.1.11 ,olde rnames :pecti nraethyleste- rase,pecti n demethoxylase,pecti nmethoxylase ,pectase )i spresen ti nlarg e amountsi n variouscitru s fruits (MacMillan& Sheiman , 1974;Rexovâ-Benkov â& Markovic , 1976).Th e enzymei sfirml y associatedwit hth ecel lwal l fractionsan di ssituate dmainl yi nth e peel,th era gan djuic esa ctissu e (MacDonnelle tal. , 1945;Rouse ,1953 ;Janse ne tal. , 1960b;Manabe , 1973a). Mosto fth eenzym epresen ti nth eextracte djuic ei sbourt dt opul p particles (Rouse, 1953). In fresh citrus juices,pectinesteras e de-esterifies thepectin ,producin g methanol and lowmethoxy lpectin .Subsequently ,precipitatio no fth e lowmethoxy lpecti nwit h calciumion s fromth ejuic eresult si nclou d loss,a seriou squalit y defect (Joslyn& Pilnik, 1961;Krop , 1974). Incitru s juice technology pectinesterasei seithe rinacti ­ vatedb ypasteurizatio no rinhibite db yfroze nstorag eo fjuic e concentratesa t-2 0 Co r below (Joslyn& Pilnik , 1961). Recently,severa l reports appearedo nth estabilizatio no forang e juice cloud,eithe r by theadditio no fpolygalacturonas et odegrad eth elo wmethoxy l pectin (Baker& Bruemmer , 1972; Krop& Pilnik , 1974)o rb yaddin g oligogalacturonic acidst oinhibi t pectinesterase (Termotee tal. , 1977).I nthes estudie sn oattentio nwa spai dt oth epossibl e occurrence ofmultipl e formso fpectinesteras e incitru s fruits,notabl yi norange .Multipl e forms ofpectinesteras e havebee n foundi ntobacc opit h (Glasziou& Inglis , 1958), banana (Hultine tal. , 1966;Brady , 1976;Markovi ée tal. , 1975), tomato (Pressey& Avants , 1972; Markovic& Kuniak , 1974;Rexovâ-Benkov âe tal. , 1977)an dvariou s other fruitsan d vegetables (Roeb& Stegeman , 1975).Multipl e formso fpectinesteras ema ydiffe ri nkine ­ ticpropertie san dtemperatur e stability (Hultine tal. , 1966;Hulti n& Levine ,1963 ) and therefore,i fpresen ti norang ejuice ,ma ypla ya differen t rolei nclou d lossphenomena . Thiswor ko norang epectinesteras ewa s intendedt ob ea ninitia lste pi nth estud yo f themod eo factio no fplan tpectinesteras ean dals ot oobtai na mor e detailedpictur eo f cloud lossphenomen ai ncitru s juice.Extractio nan dextensiv epurificatio n resultedi n the isolationo ftw omolecula r formso fpectinesterase .I nadditio nsevera l other forms couldb edetecte d andon eo fthe m(i nth eimpur estate )wa sals o incorporatedi npar to f thisstudy .Som eo fth emolecular ,kineti c andorang e juiceclarifyin g propertieso fth e different formswer e foundt ob eremarkabl y different. 2 Literature on pectinesterase

2.1 PECTINESTERASEAN DOTHE RPECTI CENZYME S

As this thesisi sconcerne dwit hpectinesterase ,th eliteratur eo npectinesteras ei s reviewedi nthi schapter ,whil e otherpecti cenzyme sar edescribe d onlybriefly .Severa l literature reviews dealingwit hpecti cenzyme shav ebee npublishe d recently (Voragen& Pilnik, 1970a;Rombout s& Pilnik , 1972;MacMilla n& Sheiman , 1974;Rexovâ-Benkov â& Markovic, 1976).Th eimportanc eo fthes eenzyme sfo rth efrui tan dvegetabl e juice technologywa s reviewedb yRombout s& Pilni k (1978)an dals oth eliteratur eo nth epro ­ ductiono fthes eenzyme swa sdiscusse db yth esam e authors (Rombouts& Pilnik , 1980,i n press). Pecticenzyme s degradepectin ,whic hi sa majo r constituento fth eplan tmiddl e lamellaan dprimar y cellwall .The y arever y generallyproduce db yhighe rplant san d micro-organisms.Bu tals o insects (Courtoise tal. , 1968),nematode s (Deubert& Rohde , 1971)an dprotozo a (Mah& Hungate , 1965)hav ebee n foundt oproduc e theseenzymes . Pectini sa (1+4)-polyme ro fpartiall ymethy l esterifieda-D-galacturoni cacid .How ­ ever,i ti sno ta homopolyme ran dcontain s various proportionso fL-rhamnos ei nth emai n chainan darabinose ,xylose ,fucose ,galactos e andpossibl y alsoapios ei nsid e chains (Voragen& Pilnik , 1970b;Fogart y& Ward , 1974;McNei le tal. ,i npress) .Th esecondar y hydroxyl groupsa tC 2 andC 3o fth egalacturonid emonome rma ypartiall yb eacetylated , dependingo nth esource .Pectinesteras e de-esterifies themethy lester so fth e carboxyl groups.Polygalacturonase s splitth emai nchai ni na hydrolyti cwa yan dth e lyases (pectate lyasean dpecti n lyase)spli t themai n chainb ytranseliminatio n (Table 1; Fig. 1). Internalan dtermina l splittingo fpolygalacturonase s andpectat e lyasesar eknow n andal lo fthes eenzyme shav ea preferenc e forsubstrate swit ha lo wconten to fmethy l ester (Pilnike tal. , 1973).Endo-pecti n lyasei sth eonl yenzym ewhic h preferentially attackspectin swit ha hig h contento fmethy lester .Man y chain-splitting enzymesre ­ quire freecarboxy l groups foractivity ,an dthes eenzyme s canonl y degradepectin swit h ahig h contento fmethy l esterwhe n theyoperat ei nconjunctio nwit hpectinesterase .

Table 1.Chain-splittin g pecticenzymes .

Name EC number Preferred Mechanism Mode of substrate atback

Endo-polygalacturonase 3.2.1.15 pectate hydrolytic internal Exo-polygalacturonase 3.2.1.67 pectate hydrolytic terminal

Endo-pectate lyase 4.2.2.2 pectate transeliminative internal Exo-pectate lyase 4.2.2.9 pectate transeliminative terminal Endo-pectin lyase 4.2.2.10 pectin transeliminative internal ^.pectinesterase

COOCH3 COOCH3 CO(ÓCH3 CDOH OOOCHj COOÇH3 (OH^OK^H ÓH | ' ÓH( ' ÓH OH OH -pectin lyase

pectin- esterase COOH COOH COOCH3COO H COOH COOH

OH OHl OH polygalacturonase pectate lyase

COOH COOH COOH COOH .^o^^^J^a-

OH OH OH H pectate lyase COOCH3COOCH 3 COOCH3COOCH 3 <^H^OKPH^to'—--•XO H.^j^V^ 0 Fig. 1.Basi cstructur eo fgalacturona n OH OH OHfH OH withpoint so fattac kan dmod eo fattac k pectinlyas e of pectic enzymes.

From theheterogenou s structureo fpecti ni ti seviden t thatals osevera l other enzymesar eneede d forsplittin g allglycosidi c linkagesi nth emolecule .Th ebasi c structureo fpecti ntogethe rwit hplace s andmod eo fattac ko fsom eo fth e pectic enzymesar egive ni nFig .1 . Becausepecti ni sa structura l elemento fth eplan t cellwal lan damajo r constituent ofth emiddl e lamella,pecti c enzymespla ya nimportan t rolei ngrowth ,ripening ,tissu e deteriorationb yparasite sa swel la sb ysaprophiti c micro-organisms andaffec tth e texturean dappearanc eo fprocesse d fruitsan dvegetables .

2.2 PECTINESTERASE

2.2.1 Occurrence

Pectinesterasewa s discoveredi n184 0b yFrém y (1840)i nvegetabl e juice.Sinc e then numeroushighe rplant s andmicro-organism s havebee n shownt ocontai no rt ob eabl et o producepectinesteras e (Tables2 an d3) .I nfac tal lhighe rplant s seemt ocontai n pectinesterasei nal l living tissues.Fo rinstance ,pectinesteras ewa s demonstrated inseeds ,leaves ,stems ,petioles ,flowers ,gree nan drip e fruito fcucumber s (Belle tal. , 1951).Th eleve lo factivit y varies considerablywit h plant species,variety ,par to fth e plantan dstag eo fgrowth .Als oi nfruit s therear ezone swhic har erelativel y richan d poori npectinesteras e activity,lik ei ncitru s (MacDonnelle tal. , 1945;Rouse , 1953; Rothschilde tal. , 1974;Tahi re tal. , 1975)an dpersimmo n (Nakayama& Iwasaki , 1966). Table 2.Occurrenc e ofpectinesteras e inhighe rplants .

Source References alfalfa Lineweaver &Ballo u (1945) apple Pollard &Kiese r (1951); Lee4 Wile y (1970); Miyairi etal . (1975) apricot Polacsek-Racz & Pozsär-Hajnal (1976) artichoke Glasziou& Ingli s (1958) avocado Zauberman& Schiffmann-Nade l (1972) banana Hultin &Levin e (1963); MarkoviÉe tal . (1975); Brady (1976) barley Fuchs (1965) bean (broad, frenchan dsnap ) Fuchs (1965); Burene tal . (1962) cacao Gamble (1973) carrot Markovic (1978) cauliflower Hoogzand &Doesbur g (1961) cherry (sweet andMontmorency ) Schmid (1975); Al-Delaimy (1966) cranberry Arakji SJan g (1969) cucumber Belle tal . (1951) gooseberry Polacsek-Räcz& Pozsär-Hajna l (1976) grape Montedoro (1968); Datunashvili etal .(1976 ) grapefruit Rouse &Atkin s (1953a); Eagerman& Rous e (1976) guave Shastri &Shastr i (1975) lilac McColloch &Kertes z (1947) lime Evans &McHal e (1978) mandarin Nath &Rangann a (1977a) oat Glasziou (1959) onion Vas etal .(1967 ) orange MacDonnelle tal .(1945) ; Evans &McHal e (1978); Versteeg et al. (1978) papaya Aung & Ross (1965) paprika Vas et al. (1967) peach Buescher &Furmansk i (1978) pea Collins (1970) pear Vase tal .(1967) ; Weurman (1954) persimmon Nakayama& Iwasak i (1966) plum Therone tal .(1977a ) potato Vas etal .(1967) ; Bartolome &Hof f (1972a) radish Polacsek-Räcz& Pozsär-Hajna l (1976) raspberry Polacsek-Räcz &Pozsär-Hajna l (1976) rye Fuchs (1965) strawberry Leuprecht & Schaller (1968) tomato Hills& Motter n (1947); Pressey &Avant s (1972) tobacco Glasziou& Ingli s (1958) wheat Fuchs (1965)

The activity changes infruit san dvegetable s during growth andripenin g dono t followa genera l trend.Fo rinstanc eth epectinesteras e level increases during growthi n orange (Rouse& Atkins , 1953b;Tahi re tal. , 1975), tomato (Sawamurae tal. , 1978;Poszâr - Hajnal &Polacsek-Râcz , 1975),banan a (Hultin& Levine , 1965), apple (Lee,1969b) , cacao (Gamble, 1973), guave (Shastri &Shastri ,1975 )bu tdecrease s incucumber s (Belle tal. , 1951),pear s (Weurman, 1954),pea s (Collins,1970 )an davocad o (Zauberman& Schiffmann - Nadel, 1972). Inorange ,tomat oan dguav a there isa sudde ndro pa ta stag e during ripening.Brad y (1976)suggeste d that theapparen t lowactivit y level inimmatur e bananas is causedb yinactivatio n throughphenoli c compounds. Pectinesterase issituate di nth efre e spacebetwee n thecel lwall sa swa sshow nfo r oat coleoptilesan dtobacc opit h (Glasziou, 1959;Brya n &Newcomb , 1954),an dth emai n Table 3.Fectinesterase-producin gmicro-organisms .

Micro-organisms References

Bacteria Closvridium aurantibutyricum Lund& Brocklehurs t (1978) Clostridium multifermentans Miller& MacMilla n (1970); Sheimane t al. (1976) Corynebacterium (several species) Lange (1970) Ewinia (several species) Lange (1970) Pseudomonas solanacearum Lange (1970) Xanthomonasmalvacearum Abo-El-Dahab (1964) Xanthomonas (several species) Dye (1960);Lang e (1970) Fungi Acroay lindrium Kimurae tal .(1973 ) Alternaria humiaola Abdel-Fattah& Mabrou k (1977) Aspergillus aarbonarius Sreekantiahe tal .(1975 ) Aspergillus japonious Ishiie tal .(1979 ) Aspergillus niger Rexovä-Benkovä4 Slezâri k (1966);Baro n (1978) Aspergillus (severalspecies ) Abdel-Fattah &Mabrou k (1977) Botrytis cinerea Verhoeff4 Warre n (1972) Botryosphaeria ribis Wallacee tal .(1962 ) Byssochlamys fulva Reid (1952) Ceraosporella herpotrichoiaes Hännslere tal .(1971 ) Chaetomiumglobosum Abdel-Fattah 4Mabrou k (1977) Colletotrichum trifolii Hancock& Milla r(1965 ) Coniothyrium diplodiella Endo(1964 ) Corticium rolfsii Yoshiharae tal .(1977 ) Curoularia lunata Abdel-Fattah 4Mabrou k (1977) Diplodia gossypina Wang4 Pinckar d (1971) Epicoccum nigrum Abdel-Fattah4 Mabrou k (1977) Fusarium oxysporum Miller4 MacMilla n (1971) Fusarium roseum Perley& Pag e (1971) Fusariumsp . Abdel-Fattah4 Mabrou k (1977) Gibberella sp.11 7 Abdel-Fattah 4Mabrou k (1977) Gilbertella persicaria Mehrotrae tal .(1971 ) Glomerella cingulata Wallacee tal .(1962 ) Macrosporium cladosporioides Abdel-Fattah 4Mabrou k (1977) Monilia fructicola Paynter (1975) Nigrospora sphaerica Agarwale tal .(1977 ) Oosporasp . Abdel-Fattah 4Mabrou k (1977) Ophiobolus graminis Weste (1970) Pellicularia filamentosa Barker4 Walke r (1962) Pénicillium chrysogenum Phaff (1947) Pénicillium (severalspecies ) Abdel-Fattah &Mabrou k (1977) Vleospora infectoria Agarwale tal .(1977 ) Plylasospora sp. Wallacee tal .(1962 ) Phy8alospora obtusa Wallacee tal .(1962 ) Phytophtora infestons Clarke (1966) Pseudoarachniotus roseus Abdel-Fattah4 Mabrou k (1977) Rhizocotonia solani Bateman (1963) Sclerotina libertiane Oi4 Satomur a (1965) Sclerotina sclerotiorum Astapovich(1976 ) Sclerotium rolfsii Yoshiharae tal .(1977) ;Batema n4 Bee r(1965 ) Stemphylium botryosum Hancock4 Milia r (1965) Trichoderma lignorum Abdel-Fattah4 Mabrou k (1977) Trichothecium roseum Abdel-Fattah 4Mabrou k (1977)

Yeasts Kluyveromyces fragilus Call& Emei s (1978) Torulopsis Candida Call4 Emei s (1978) part isboun db y ionic interaction. Itca nb e liberated fromth ecel lwal l fractionb y raising thep H above 7an dincreasin g the ionic strength,a swa s demonstrated, for example,i norang e (MacDonnelle t al., 1945;Janse ne tal. , 1960b), oat coleoptiles (Glasziou, 1959;Janse n etal. , 1960a)an d tomatoes (Pozsâr-Hajnal &Polacsek-Râcz , 1975). Intomat o cellwall s andoa tcoleoptile s itwa smoreove r demonstrated that therewer e binding sitesspecifi c forpectinesterase .Th ebindin go fpectinesteras ewa s onlypartial ­ lyo rno ta tal laffecte db yprio rsaturatio nwit h otherprotein s orvic evers a (Nakagawa etal. ,1971 ;Janse ne tal. , 1960a). However,th esituatio n isprobabl ymuc hmor ecomplicated .Glaszio u & Inglis (1958) demonstrated threedifferen t fractions ofpectinesteras e fromartichok e and two in tobaccopith .Th epectinesterase s appeared todiffe rwit h respect tobindin g properties tocel lwalls .On epectinesteras e could increasingly beboun db y additiono fauxins ,bu t notb ycalciu m ions,th ebindin g ofanothe rpectinesteras ewa sneithe r affectedb y cal­ ciumion sno rb yauxi nan d thebindin g ofth ethir dpectinesteras ewa sonl yaffecte db y calcium ions andno tb y auxin.Hulti n &Levin e (1963)demonstrate d threedifferen tpectin­ esterases inbanana sb y differential extractionprocedures .Thes e enzymes appeared tob e bound differently andpossesse d differentpropertie swit h respect topH-activity , activationo rinhibitio nb y sodium lauryl sulphate andtemperatur e inactivation.Multipl e forms andisoenzyme s ofpectinesteras e arediscusse d further inSection s 2.2.2 and 2.2.3.

2.2.2 Role inviv o

The role ofpectinesteras e ingrowt h ofplant s andi ngrowt han dripenin g offruit s isno tver ywel lunderstood .A spectinesteras e seems tob epresen t inal l living tissues ofhighe rplant s anessentia l function is likely. Auxins like IAA (3-indolylaceticacid )promot e cellenlargement .I ntobacc opit h this isaccompanie db y aris ei npectinesteras e content (Bryan& Newcomb , 1954). IAA,depen ­ dingo nit sconcentration ,stimulate s orinhibit spectinesteras e activity (Bryan& Newcomb,1954 ;Nakagaw ae tal. , 1970b). IAAan d otherauxin shav ebee nreporte d to affect thebindin go fpectinesteras e tocel lwall so ftobacc opit h (Glasziou, 1957;Glaszio u& Inglis, 1958). Itwa s suggested that immobilization ofpectinesteras e favoursmethylatio n ofpectat eo fth ecel lwal l andthu splasticit y ofcel lwal l increases.However ,Janse n etal . (1960a)an dNakagaw ae tal . (1971)reporte dtha tther ewa sn oeffec to fIA Ao n pectinesterase binding tooa t coleoptile and tomatocel lwalls ,respectively ,bu t itma y bepossibl e that theyteste d thewron g concentration rangeso f IAAo r irrelevant forms of pectinesterase.I twa spointe dou tb yJanse ne tal . (1960a)tha t IAAdi dno tchang eth e degreeo festerificatio no fan ypecti cmateria l incel lwall s and that therefore elongationo fcel lwall smediate d through aneffec t onpectinesteras ewa sunlikely .Sal t inth enutrien tsolutio nreduce dth epectinesteras e concentration inth e leaves of Aster tripolium andpromote d cellelongatio n (Binet, 1976);her e aninvers e relationship betweencel lsiz e andpectinesteras e concentrationwa s founda sb yBrya n& Newcom b (1954)i ntobacc opith . As discussed inSectio n 2.2.1,ther e isn ogenera lrelationshi pbetwee nripenin g and pectinesterase level infruit s andvegetables .Als on orelationshi p couldb e demonstrated betweenavailabl epectinesteras e activity and firmnesso ftomat o fruit (Hall &Dennison , 1960). Itha softe nbee nsuggeste d that tissuesoftenin g during ripening orplan tpatho ­ genesis iscause db ypectinesteras e activity followedby -polygalacturonas e action.How ­ ever,pectinesteras e actionalone ,an dsubsequen tbindin g ofpectat ewit hbivalen t cations increases tissue firmness and resistance againstmaceratio n (Brown, 1969).Ther ear e indications that thismechanis m is involved inresistanc e againstpathogens .I nsevera l cases itwa s demonstrated thathos tpectinesteras e activity increasedupo n infection (Bateman,1963 ;Langcak e et al., 1973). Inbea nhypocotyl s infectedb y Rhizoctonia solani calcium ionsaccumulat e inth einfecte d zone,pectinesteras e activity increases,pecti n isdemethylate dan dinsolubl e calcium-pectatei s formed,whic h cannotb e degradedb y the endo-polygalacturonaseo f thepathoge n (Bateman& Millar , 1966).Durin g pathogenesis Sclerotium rolfsii produces oxalicacid ,whic hbind s calcium ionsan dthu senable s the polygalacturonase todegrad epectate .Also ,th ep H is lowered towards theoptimu m for polygalacturonase activity (Bateman &Beer , 1965).Pectinesteras e ofth epathoge n isno t always needed forvirulence .Lang e (1970)conclude d that therewa sn o relationshipbe ­ tweenvirulenc e andpectinesteras e activityo fpathogeni cbacteria .Paqui n& Coulomb e (1962)foun d thata virulen t straino f Fusarium oxysporum produced 2-3 times more pectinesterase thana navirulen t one.Cell s of Xanthomonas malvacearum adapted toproduc e pectinesterasewer emor epathogeni c thanunadapte d cells (Abo-El-Dahab, 1964).N o relation was foundbetwee nvirulenc e of Vellicularia filamentosa andpectinesteras e production (Barker& Walker , 1962). The amounto fpectinesteras e issometime s veryhig h comparedwit h theamoun t of available pectin.Fo rinstance ,i ncitru s fruit there issufficien t pectinesterase (6000 0unit s (pmol/min)/kg)t ode-esterif y thequantit y ofpecti n (30g/kg )fro mhig h to low degreeo festerificatio n in 10mi n atoptima lp Ho r inles s than 4h atth ep Ho fth e juice.Possibl ypectinesteras e inviv oplay s arol ei nth emethylatio no fpectates . Glasziou (1959)suggeste d thatpectinesteras e mighthav e atransmethylas efunctio n to methylatepectat e topecti n invivo .A transferase activity transferring methyl groups fromS-adenosyl-L-methionin et oth ecarboxy l groups ofpolygalacturoni c acidwa s found inbea nshoot s (Kauss &Hassid , 1967).Kaus s etal . (1969)foun d also that intact particles fromth esam esourc e containedpectinesteras ewhic h couldonl y de-esterify the plant'specti n if the lipidmembrane sprotectin g thepecti c substanceswer e destroyed by detergents orphospholipase .Althoug h theauthor s didno tmentio n thispossibility ,on e mightspeculat e that the transferase becomes anesteras e afterth e lipidmembrane s are destroyed andwate r isintroduce d inth e immediate environment ofsubstrat e andenzyme , andpossibl y alsoafte rsom echemica l orphysica l change of theenzyme .Then ,afte r extractionan dunde rth e aqueous assay conditions,onl ypectinesteras e activitywoul db e measured.Thi s theory is inagreemen twit h the continuously increasing pectinesterase concentration during growth (seeSectio n 2.2.1)a softe n foundwithou t simultaneous decrease indegre e ofesterificatio n ofth epecti c substances.Th e degreeo festerifica ­ tiono fth epecti c substances evenincrease s insom e fruitsan dn o decrease isobserve d until thever y ripestag e (Geee t al., 1959). Insevera lplant smultipl e formso fpectinesteras e havebee n demonstrated, e.g. four toeigh t forms intomatoe s (Pressey &Avants ,1972 ;Delincée , 1976), twot osi xi n bananas (Hultin& Levine ,1963 ;Brady , 1976;Markovic "e t al., 1975), two intobacc opit h (Glasziou& Inglis,1958) ,thre e inartichok e (Glasziou& Inglis,1958 )an d twoi norang e (Evans &McHale , 1978;Verstee ge t al., 1978). Iti sstil lno tknow nwhethe r theseen ­ zymesar ederive d fromth e same forman dar emerel y breakdownproducts ,stil lhavin g pectinesterase activity,o rwhethe r theyal lhav ea different originan d function inth e plant.Severa lexplanation s forth eoccurrenc e ofmultipl e formswer e givenb yHulti ne t al. (1966)an dBrad y (1976).Th e facttha tth etw opectinesterase s incitru swer e found indifferen t tissues (Evans &McHale ,1978 )point s toa functional difference. Ifso , thesituatio n inviv omus tb ever ycomplicate d since eight formswer e detected intomat o (Delincée, 1976)an deve n twelve forms incitrus ,o fwhic h sometimes ten forms inth e samefrui t (thiswork ;Nieuwenhuis , 1978).Multipl e formso fpectinesteras e always seem todiffe ri nisoelectri cpoin t andusuall y differ inon eo rmor e ofth e followingas ­ pects:pH-activit y profile,activatio nb ycations ,substrat e affinity,inactivatio n by detergents,inhibitio nb ysucrose ,hea t stability,molecula rweigh t andbindin gproper ­ tiest ocel lwall s (Pressey &Avants ,1972 ;Hulti n et al., 1966;Hulti n &Levine ,1963 ; Brady, 1976;Glaszio u & Inglis,1958 ;Verstee g et al., 1978).

2.2.3 Purification andpropertie s

Though inman yplant s andculture s ofmicro-organism s thepresenc e of pectinesterase was established (Tables 2an d 3),th epropertie s of theseenzyme swer e oftenno t determinedan donl y inrelativel y fewcase s theywer epartiall y orcompletel y purified. Especiallypectinesterase s frommicro-organism s received little attentioneve nthoug h they differmor ewidel y inpropertie s thanth eplan tpectinesterase s seemt odo . Thepropertie s ofpectinesterase s whichhav ebee n studied inmor e detailar e listed inTable s 4a,4 ban d 5.Compariso no fmos tpropertie s hasonl y limitedvalu ebecaus e the assay conditions areno t always thesame .Th edat ao f the impurepreparation s usually represent theoveral lpropertie s ofa mixtur e ofdifferen t formso fpectinesterase .Th e useo fa mixtur eo fdifferen t forms issometime s indicatedb y irregularities reported sucha sp H curveswit h shoulders andirregula rhea t inactivation curves.Eve n insom e of thepur epreparation s more thanon e single formo fpectinesteras e mayb epresent .Thin - layer isoelectric focusing seems tob e themos t sensitivemetho d todetermin e multiple formso r isoenzymes (Delincée, 1976). Purificationusuall y involves extraction (forplan tpectinesterases) ,fractiona l ammonium sulphateprecipitatio n or acetoneprecipitatio n andsevera l typeso f column chromatography,suc ha sge l filtration (separationbase do nmolecula r size),anio no r cationexchang e (separationbase do ncharg e and charge distribution).Sometime s other principleswer eemploye da swell , for instance affinity chromatography (Rexovâ-Benkovâ etal. , 1977;Verstee ge t al., 1978), isoelectric focusing (Brady, 1976),calcium-phos ­ phate adsorption chromatography (Dahodwala et al., 1974;Yoshihar a et al., 1977). A ratherdetaile dsummar yabou tpurificatio nmethod s used forpectinesteras e isgive nb y Rexovâ-Benkovâ &Markovi c (1976). Except foron elo wmolecula rweigh t formfro mbanan a andon ehig hmolecula rweigh t pectinesterase (complexedwit hexo-pectat e lyase)fro m Clostridium multifermentons, all

S pectinesterases havemolecula rweight s inth e rangeo f2 400 0t o3 7 000.Th emolecula r weightswer eno t all determined inth e samewa y andDelincé e& Radol a (1970)showe d that theapparen tmolecula rweigh t forth e sametomat opectinesteras e couldrang ebetwee n 2400 0an d 28000 ,dependin g onth emetho demployed . Theisoelectri cpoint so fplan t pectinesterases were always abovep H 7an dth ep H optimumrange dbetwee n6 an d 8.5. It shouldb enote d that thepH-activit yprofil e isbroadene d atth e acid sideb y raising the concentration ofcation s (Lineweaver& Ballou , 1945).Th ep Hoptimu m formicrobia lpectin ­ esterases depends largelyo n thesourc e of theenzyme .Th eMichaelis-Mente n constants (K values)reporte d forth epurifie denzyme s differu p toa factor 500.Betwee n iso­ enzymes fromth esam esourc e already a 20-folddifferenc ewa s found (Versteeg et al., 1978). Rexovâ-Benkovâ& Markovic '(1976 )suggeste d toexpres s the K onth ebasi so fme - thyl-D-galactopyranosyluronate residues.Bu tthi s seems illogical because free carboxyl groups are regarded tob enecessar y foraffinit y (Solms& Deuel , 1955),se eals o Section 2.2.4. Plantpectinesterase s arecompetitivel y inhibited bypectate ,on eo fthei row n reactionproduct s (Termotee t al., 1977).Th eeffec t ofsalt s onalfalf a pectinesterase was thoroughly studied byLineweave r &Ballo u (1945). Itappeare d that cations andno t anions stimulatepectinesteras e at the acid sideo fit sp Hoptimum .Als opectat einhibi ­ tioni s lessa thighe rp H anda thighe r cationconcentrations . Itwa s concluded thatcat ­ ions dono tstimulat e theenzym ebu t liberate it froma n inactive ioniccomple xwit h the product so that fullactivit y canb eobtained .Als oMacDonnel l et al. (1945)an dHill s & Mottern (1947) found fororang ean d tomatopectinesterase ,respectively , thata highe r concentration ofcation swa s required foractivit y ata lowerpH .Th e optimum concentra­ tiono fsodiu mchlorid e forplan tpectinesteras e is inth e rangeo f0.1-0.2 5mol/ 1an dth e optimum concentrationo fcalciu m chloride isabou t fivetime s lower.Thi s optimum salt concentration isusuall y determined nearp H 7.0 and ishighe r ata lowerpH .Thoug h theoptimu m sodium chloride concentration forfunga lpectinesteras e is approximately the same,th e stimulation ismuc h less.Als o fora fungalpreparatio n itwa s found thatmor e saltwa s required foroptima l activity ata lowerp H (Calesnick, 1950). As canb e seenfro mth e tablesi nthi s chapter,th e optimal temperature forplan t pectinesterases isapproximatel y 55° Cwit h aS of 1.3-1.4i nth e range 20-50°C .Th e optimal temperature formicrobia l pectinesterases isabou t 10° C lower,an dusuall y they areals oles shea tstable .Le e (1969a)suggeste d that tomatopectinesteras ewa sa lipo ­ protein.Markovi c (1974)demonstrate d thaton eo fth emultipl e formso f tomatopectin ­ esterasewa sneithe r a lipo-proteinno ra glyco-protein .Thero n etal . (1977a, 1977b) found indications fora glyco-protein charactero fplu man dtomat opectinesterase .Non e of thesi xbanan apectinesterase s examinedb yMarkovi ^e t al. (1975)wer e lipo-proteins. Baron (1978)foun dindication s fora lipo-protein charactero f Aspergillus niger pectin­ esterase. ' es en m -& \£> CT»

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12 2.2.4 Specificity andmod eo f action

Pectinesterase ishighl y specific formethy l esterso f (1-i-4)-a-D-galacturonanchains . Both theD-galacturona nchai nstructur ean dth e methyl estergrou par eneede d forful l activity.Th emethy leste ro falgini c acid isno tde-esterifie d byorang e pectinesterase (MacDonnell etal. , 1950).Th emethy l esters areno thydrolyse d ifth e galacturonan chains areto oshort ,fo rexampl e trimersar eno tattacke dbu t adegre eo f polymerization of 10i ssufficien t foractivit y (McCready& Seegmiller , 1954).Als o ifpar to fth e galacturonate esterunit s inth emai nchai nar ereduce d togalactos eunit so rpar t ofth e secondaryhydroxy l groupsar eacetylate d thereactio n rate ismuc h lower (Solms& Deuel , 1955).Pectinesteras eha s someactivit y onethy l esterso fD-galacturonan , approx.10 1 of therat eo nmethy lester s atoptima lp H (MacDonnell et al., 1950;Manabe , 1973b).Th e reaction rateo nethy l esters ismuc h lessaffecte db y thep H thanth erat eo nmethy l esters anda tp H 4th erate s areapproximatel y equal (Manabe,1973b) .Som eothe r esters (viz.propyl ,allyl ,propargyl )o fa-D-galacturona nar eattacke d ata ver y lowrat ean d theglycy l andglycery l estersno ta tal l (Deuel,1947 ;Manabe , 1973b). Crudeorang epectinesteras e preparations showactivit y onalky lester so f several acids (MacDonnell et al., 1950).A s theseactivitie s diminishwit h increasingpurit y of thepectinesterases ,MacDonnel l etal . (1950)conclude d that theactivit yo fcrud epectin ­ esterase onthes esubstrate s isdu et ocontaminatin g carboxylesterases other thanpectin ­ esterase.Th eacetylesteras e fromcitrus ,whic h hydrolysis several carboxyl esters and theester s ofaceti c acidbest ,doe sno thav epectinesteras e activity (Jansene t al., 1947). Itshoul db epointe dou t that thesubstrat e specificity asdescribe d above pertains toplan tpectinesteras e (mainly citruspectinesterase ) and thatth e substrate specificity ofmicrobia lpectinesterase s mayappea r tob e different. Iti sbelieve d thatth ede-esterificatio n ofpecti nb yplan t pectinesterases proceeds linearly along thepecti n chains otha tblock so f freecarboxy l groups are produced.Thi s conclusionwa s reachedb y comparing partially acido ralkal i de-esterified 'pectin s (whichhav e randomly distributed freecarboxy l groups)wit hpartiall y enzymatical- lyde-esterifie d pectin andpecti c acidwit h respect toviscosit y ofsolutions ,calciu m gelstrengths ,electrophoreti c andchromatographi c patterns andstabilit y constants of calciumpectinate s (Schultze t al., 1945;Hill s et al., 1949;Her i et al., 1961;Koh n et al., 1968). On thebasi s of theenzymati c activity onhig hmethoxy lpectins ,partiall yde - esterifiedb y alkali,Solm s &Deue l (1955)conclude d thatorang epectinesteras e splits estergroup s next to freecarboxy l groups and thatcarboxy l groups areneede d forth e enzyme-substrate complex.Althoug h carboxyl groups arenecessary ,the y found ahighe r Michaelis-Menten constant (K.) wit h anincreasin g numbero f freecarboxy l groups inth e substrate.Ishi ie t al. (1978; 1979)studie d theelutio npattern s onDEA E cellulosean d thege lstrength s ofpectin s de-esterified with Aspergillus Japoniaus pectinesterase and theysuggeste d thispectinesteras e removesmethoxy lgroup s at random. Forth e complexo fpectinesteras e andexo-polygalacturOnat e lyaseo f Clostridium multifermentons, Sheimane tal . (1976)prove d thatbot hen2yme s attackedhighl yesteri - fiedpecti n (witha H label at the reducingend ) fromth e reducing endi na coordinated

13 marmer.Firs tth epectinesteras e de-esterifies estergroup s andwithou tdissociatio no f theenzyme-substrät ecomplex ,th elyas esplit sof funsaturate ddimers .Fro mth eequiva ­ lentreleas eo fth e H labelan dtota lproduct ,an dfro mth e K and V of thelyase , r m max ' itcoul db eshow ntha tn ostead y stateconcentratio no fth elyas esubstrat ewa s available toaccoun t forth eobserve dinitia lreactio nrate .However ,som edoub tca nb eexpresse d aboutth econclusion so fth esam eresearc hgrou pwit h respectt oth emod eo factio no f tomatopectinesteras e and Fusarium oxysporum pectinesterase.Tomat opectinesteras e and theexo-pectat e lyasefro m Clostridium multifermentans (thepectinesteras e ofth epectin - esterase-pectate lyasecomple xha dbee nhea tinactivated )wer ejointl yincubate dwit h highlyesterifie dpecti n (witha naverag edegre eo fpolymerizatio no f 33).Th e activity ofbot henzyme swa s continuouslymonitored .Th epectinesteras e activity appeared tob e rate limitingfo rth elyas eactivit yan dth e lyaseactivit ywa s alwayshal ftha to fth e pectinesterase activity.I twa s concluded thathal fo fth etomat opectinesteras e activity was initiateda tth ereducin gen do fth epecti n chain (Lee& MacMillan , 1970). However,objection s canb emad et osuc ha conclusion .Suppos eth epectinesteras e initiates itsactio nsomewher e inth epecti nchai nan dmove s toan dreache sth ereducin g endo fth epectin .Wit h theturnove rnumbe ro fabou t 500moles/mole. so fth epectin ­ esterase,onl y0.0 3 sar eneede d fora pectinesteras emolecul e toreac h thereducin g endo fth epecti nchain .I nthi sway ,ever ypectinesteras emolecul eca nde-esterif y 33pecti nchain spe rs .The nn o lagperio dfo r lyaseactivit ywoul db eobserve deither . Ifal lpectinesteras e activitywer e initiated somewhere inth epecti nchai nan di fhal f ofth epectinesteras emolecule swer e tomov e towards andreac hth ereducin gend ,th e experimental resultswoul dhav ebee nth esame .Therefor eth econclusio ntha thal fo fth e pectinesterase activity isinitiate d atth ereducin gen do fth epecti nchai nshoul dno t havebee nmade . A furtherpoin to fcriticis mma yb e raisedb ycomparin gth eobserve dinitia lactivit y ofth elyas ewit h theinitia lactivit yexpecte d fromth eknow nMichaelis-Mente nconstan t (K ),maxima lvelocit y (V J ofth e lyase (Sheimane t al., 1976)an dth emaxima lquan ­ tityo fsubstrat emad eavailabl eb y thetomat opectinesterase .Eve ni fal lpectinesteras e activitywer e initiated atth ereducin gen do fth epecti nchain ,a considerabl eperio d (> 10min )wit h lowerlyas eactivit yshoul dhav ebee nobserved . Similarobjection s canb emad e toth econclusion so nth emod eo fattac ko f Fusarium oxysporum pectinesterase (Miller& MacMillan , 1971).

2.2.5 Inhibitionan d (in)activation

Thehea tstabilit ywa s discussed inSectio n 2.2.3.Pectinesteras e isno t inactivated bya dos eo fy-radiatio n thatca neffectivel y reduceth emicrobia l contaminationo fa n industrialenzym epreparatio n (Delincée, 1978). Table 6show s theeffect so fa variet yo fagent so nth eactivit yo fpectinesterase s fromdifferen tsources .No tal lagent s testedar e listed,onl y thosetha tar ethough tt o givea goo dillustratio no fth earra yo feffectors ,e.g . theeffec to fpotassiu m chloride israthe rsimila rt otha to fsodiu mchlorid ewherea smagnesiu m chloridebehave s like calciumchloride .Als omor esugar s andphenol shav ebee nteste dbu t theresult swer eno t

14 Table 6. Pectinesterase (in)activators and inhibitors. (++: strong stimulation; +: stimula tion; o: no effect; -: inhibition/inactivation; =: strong inhibition/inactivation).

(In)activator/ Approx. Highe r plants Micro-organisms Cross inhibitor cone. refer- (mmol/1) 22 •t» ences st». M ö> ,°> — '«C* <3 T~^S g3 co 3r^ ** i-j s,-j j U i s <0 'ci t^i ta u o 01 0 B (M Co« t S r-i a M a. e O:»l. •-I 01 Soa, a. G u ctj aj a a (M a. « eu U h a g0 i-H o u a ,£> O 00 o a *J r^ 01 •=s(, s^O s >a!

NaCl 100 ++ ++ ++ ++ + + + 1,2,3,4,5, 6,7,10 L12SO1, 100 3,5 (NHc)Cl 100 7,6

(NH^)2S01) 100 3,7,5 NaCN 100 8 NaN3 100 o 8

CaCl2 100 ++ ++ + + + 3,A,7,5,6, 16 CuSOi» o o 8,5 ZnSO., + 3 Hg acetate 5 Hg Cl2 10 8,7 Pb acetate 5

NiCl2 9 ZnCl2 9

Fe2(S0i,)3 5 A1C13 3,5 FeCl3 9,3 SnCl„ 3 glucose 1000 1,10 sucrose 1000 1,11,12,10, 13 glycerol 1000 1,10 ethanol 1000 9 tannic acid 3 14,15 (di)gallic acid 3 IA,15,9 chlorogenic acid 3 14,9 caffeic acid 3 14,9 sodium lauryl sulphate 1 8,2,17 pectate 18,19,6,16 iodine 10 20,21,8

Cross references22 11 12 20 9 4 1 14 15 6 8 5 7 13 17 18 15 10 19 8 2 21 16

1.Chan g e t al. (1965) 2.Presse y &Avant s (1972) 3.End o (1964) 4.MacDonnel l et al. (1945) 5. Yoshihara et al. (1977) 6. Lineweaver & Ballou (1945) 7.Kimur a et al. (1973) 8. McColloch &Kertes z (1947) 9. Montedoro (1968) 10.Brad y (1976) 11.Le e (1969b) 12 . Hultin et al. (1966) 13.Le e &Wile y (1970) 14.Hal l (1966) 15.Fuch s (1965) 16 . Lee &MacMilla n (1968) 17.Hulti n &Levin e (1963) 18.Termot e et al. (1977) 19 . Versteeg et al. (1978) 20.Markovic '(1978 ) 21. Markovig &Patock a (1977) 22. The reference s in the last vertical column refer to inhibitors used and the references at the bottom refer to the source of pectinesterase used. There is always at least one reference in common for apectinesterase-inhibito r combination tested. significantly different fronthos e reportedi nth e table.Th eprincipa leffect so fmono ­ valentan dbivalen tcation so npectinesteras eactivit ywer eals odiscusse di nSectio n 2.2.3.Ammoniu mion shav e aninhibitor yeffec to npectinesteras eo f Acrocylindrium but noto nothe rmicrobia lan dplan tpectinesterases .Mercur y ionsappea rt ob e an effectiveinhibito ro fsom emicrobia lpectinesterases .Som etrivalen tion sar einhibitor y forbot hplan t andfunga lpectinesterases .Som eo fth ecation sma ybloc k thesubstrat e insteado faffectin g thepectinesterase . Theinhibitio nb ysugar san dalcohol s isascribe dt oth ereductio no fth ewate r activity andChan ge t al. (1965)foun da nindicatio nfo rth enon-competitiv e character ofth einhibition . Tannicaci di sth emos teffectiv ephenoli cinhibito ran dclou dstabilizatio no f orangejuic eb ygrap e leaveextract s (Kew& veldhuis , 1961)wa s alsoascribe dt othi s compound (Hall,1966) . Theinactivator yeffec to fsodiu mlauiy lsulphat ediffer ssomewha tdependin go nth e molecular formo risoenzym einvolve d (Pressey &Avants ,1972 ;Hulti n& Levine , 1963). Atrathe rlo wconcentration s iti sa neffectiv e inactivatorfo rplan tpectinesterases . A minimumchai nlengt ho f 8i s requiredfo rth ecompetitiv e inhibitiono forang e pectinesteraseb y oligomers frompectate .Pectat eoligomer swer e successfullyuse dt o increaseth eclou dstabilit yo forang ejuic e (Termotee tal. , 1977). The inhibitionb y iodine (irreversible andnon-competitive )increase dwit hth epurit y oftomat oan dcarro tpectinesteras epreparation s (Markovic& Patocka ,1977 ;Markovic' , 1978).O n thebasi s ofthi sinhibitio ni twa s assumedtha ttyrosin ei spresen ti nth e activecentr eo fth eenzym e (Markovic& Patocka , 1977).

2.2.6 Importance infoo dtechnolog y

Nativepectinesteras ei nfruit san dvegetable sma yproduc edesirabl ean dundesirabl e effectsbefore ,durin go rafte rprocessing . Infres ho runderpasteurize dcitru sjuice spectinesteras ede-esterifie sth epectin , producingmethano lan dlo wmethoxy lpectin .Subsequentl yprecipitatio no fth elo wmetho - xylpecti nb y calciumion s results inclou d loss,a seriou squalit ydefec tbecaus eo fth e appearance,reductio no ftast ean darom acomponent san dth e increasedsensitivit y to oxidation (Joslyn& Pilnik , 1961;Pilnik , 1958;Krop , 1974).Concentrate sma ygel .Th e drinksprepare db y reconstitutionhav ege l lumps,a curd y appearancean da rapidl y setting pulp (Rouse& Atkins ,1952 ;Ke w& Veldhuis ,1961) . Gelatingo fpapay apure ei sals o ascribedt opectinesteras eactivit y (Change tal. , 1965).Th epectinesteras ei ncitru s juice ismor ehea tstabl etha nth emicro-organism swhic h areinvolve d inspoilag e (about 20°C ) (Bissete t al., 1953)an dtha nperoxidas ewhic hma y giveflavou rchange s (Nath & Ranganna, 1977b).Hea tinactivatio no fpectinesteras ebring s theris ko fa cooke doff- flavour (Kew& Veldhuis , 1961). Lemonjuic eo nSicil y isclarifie db ystorag e inbi gtank s formonth s (Uhlig, 1978). Themechanis mo fclarificatio ni sno tclearl yunderstoo dsinc e thep Ho fth ejuic ei s ratherlo w (aboutp H 2.5) fornativ epectinesteras eaction .Nowaday s theproces s canb e speededu pb ycommercia l fungalpreparations ,whic h containsevera lpecti cenzyme s

16 (Uhlig,1978 ;Gonzale ze t al., 1977). Incide rproductio nclarificatio ni sdesirabl ean dcalciu mcarbonat ean dsodiu m chloridear eadde ddeliberatel y toai dclarificatio n (Pollard& Kieser , 1951). Intomat ojuic eproductio nhea to raci dmus tb eapplie dquickl y toproduc ea stabl e juicewit h ahig hviscosit y (hotbrea kprocess) ,wherea s thecombine dactio no fpectin - esterasean dpolygalacturonas esimplifie s theproductio no fconcentrate dproduct s (Fogarty& Ward , 1974;Wagne re t al., 1969;Hobso n& Davies ,1971 ;Paul , 1975). Bymanipulatin gwit hblanchin g temperatures,holdin g timesan dsometime swit h theai d ofcalciu mion so ra p H shift,th etextur eo fcanne dtomatoe s (Hsue t al., 1965),appl e slices (Wiley& Lee , 1970), cauliflower (Hoogzand& Doesburg , 1961),carrot s (Leee t al., 1979)an dpotatoe s (Bartolome& Hoff ,1972a )ca nb e improved.Blanchin g conditionsar e chosens oa sno t toinactivat e toomuc hpectinesteras e andt ous eit sincrease dactivit y atth eelevate dtemperatur e toreduc e thedegre eo festerificatio no fth epecti cmateria l inth etissues .Calciu mbridge s canthe nb e formedwhic hresul t ina bette rfirmnes s retentionafte rcooking .Presumabl y thehea tapplie dfo rblanchin g isals oneede d to liberatepectinesteras eo rt orende rth esubstrat e available forth eenzym esinc ether e isn odirec tcorrelatio nbetwee npectinesteras e levelan dfirmnes so ffres h tomatoes (Hall& Dennison , 1960)o rfirmnes sretentio no fnorma lcanne dpeache s (Shewfelt, 1965). Citrusan dtomat opectinesteras ehav ebee nuse d toproduc e lowmethoxy lpectin , suitablet omak egel swit h lowsuga rconten to reve nwithou tsuga r (Mottern& Hills ,1946 ; Hills etal. , 1942;Le o& Taylor ,1958 ;Le o& Taylor , 1962).Th eprincipa ldifference s inlo wmethoxy lpectin sprepare db y acidde-esterificatio nan db yplan tpectinesteras e arediscusse db yHill se tal . (1949). Lowmethoxy lpecti nproduce dwit hpectinesteras e from Aspergillus japonicus givesa hig hge lstrengt hwit h calciumion s (Ishiie t al., 1979). Pectinesterases areusuall y anintegra lpar to fth ecommerciall y availablepectolyti c enzymeso ffunga lorigin ,whic h areuse d toincreas epres syield so rpressability , improveextractio no fpigments ,reduc eviscosit yo fjuices ,liquef y fruitsan dvegetable s andclarif yjuice s (Pilnike t al., 1975;Pilni k &Rombouts ,1978 ;Sega le tal. ,1966 ; Rombouts& Pilnik , 1978;Gonzale ze t al., 1977;Delecourt , 1972). A possible disadvantage isth emethano lproduction ,bot hb y thenativ ean db yth e addedenzyme si nfrui tjuic ean dwin e technology (Segale tal. , 1966;Dahodwal ae t al., 1974).Methano lconcentration sca nris et o 130mg/ 1 (Bock,1966 )o reve n40 0mg/ 1 (Baumann& Gierschner , 1974)an dconcentration so f5 0mg/ 1ar equit ecommon .I nfres h fruitth emethano lconcentratio ni susuall y 10-20mg/ 1bu tca nals ob emuc hhighe ran d riset onearl y 200mg/ 1 (Segale t al., 1966;Al-Delaim ye t al., 1966;Hulti n& Proctor , 1961).Als o thismethano l isascribe dt opectinesteras e action.I nwin emaking ,th ehea t inactivationo fth enativ epectinesteras ebefor e treatmentwit h thefunga lenzym e preparationconsiderabl y reduces thefina lmethano lconcentratio n (Segale tal. , 1966). Themethano l indistille dspirit s fromfruit si scause db y theactivit yo fpectinesteras e fromth e fruitsdurin g fermentation (Tanner, 1970).

17 2.2.7 Assayan ddetectio n

Severalmethod sar eavailabl et odetermin epectinesteras eactivity .Som edetermin e thechange si nth esubstrate ,other smeasur eth emethano lliberated .

2.2.7.1 Changes in the substrate

Titration of the carboxyl groups Theactivit yo fpectinesteras eca nb efollowe db y titrationo fth eliberate dcarboxy lgroup swit hdilute d (0.1-0.002mol/1 )alkali .I fth e pHi skep tconstant ,th ealkal iuse di sa direc tmeasur eo fth epectinesteras eactivity . Theorigina lmanua ltitratio nwit ha p Hindicator ,used ,fo rexample ,b yLineweave r& Ballou (1945),i snowaday susuall ydon eb yautomati ctitratio nequipment .Th ep H iskep tconstan tb yth eapparatu san dth ealkal iconsumptio ni smonitore dversu stime . Thesetitration sca nb edon ei nsystem swit ha relativel y lowo rn obufferin gcapacit y atth ep Hwher eth eactivit yha st ob edetermined .Th euptak eo fcarbo ndioxid e fromth e airmus tb ecorrecte dfo rb ya blan co rprevente db ysaturatin gwit hnitrogen .Abov ep H7 acorrectio nmus tb emad efo rth echemica lde-esterificatio no fth epectin .Als oth ep H shouldb ewel labov eth ep Ko fth epecti n (about4.0) .Correctio nfo rpartia ldissociatio n ofth eliberate dcarboxy lgroup si sdifficul twhil eth ep Kvarie swit hth econcentratio n ofth epectin ,th edegre eo festerificatio nan dth emod eo fde-esterificatio n (Speiser etal. , 1945).I ti ssimple rt orais eth ep Hquickl yt o6 a tth een do fth ereactio nan d tod oth esam efo ra blan cwit hinactivate denzym e (McColloch& Kertesz ,1947) .However , thisprocedur ema yproduc erelativel y largeerror si fth eactivit yo fth epectinesteras e ismuc hhighe ra tp H6 tha na tth ep Ha twhic hth eactivit ywa smeasured .

Recording pH changes Somogyi& Roman i (1964)propose ddirec trecordin go fth ep Hdro p overa shor tp Hinterva li na nunbuffere dsubstrat esolution .Withi ncertai nlimits ,th e dropi np Hpe rmi nappeare dt ob eproportiona lt oth eamoun to fenzyme .A standar dcurv e mustb emad et orelat ep Hchang et ohydrogen-io nequivalent si na certai nreactio nmix ­ ture.Th emetho di squic kan dlittl esubstrat ei sneeded .Again ,thi smetho di sunsuitabl e ata p Hclos et oth epK-valu eo fpectin .Th emetho di ssuitabl eonl yt omeasur einitia l reactionrate san di fth eenzyme suse dd ono tdiffe ri nconten to fbuffe rsalts .

Colour change of pH indicator Aquantitativ emetho di nwhic hth ecolou rchang eo fa p H indicatori smeasure dwit ha spectrophotomete rwa suse db yBrad y (1976).Th emetho dha s muchi ncommo nwit hth erecordin gmetho dfo rp Hchanges .A semi-quantitativ emetho dwa s describedb yZimmerma n (1978).A p Hindicato ri sinclude di nagar-pecti nplates .Pectin ­ esterasesolution sar epipette dint owell san dth eare ao fth ecoloure d zonesafte ra certainincubatio ntim ei srelate dlogarithmicall y toth eamoun to fenzyme .A suitabl e pHindicato r (sucha smethy lre do rbromothymo lblue )wit hpecti ni ntes ttube sca nb e useda sa sensitiv equalitativ escreenin gmetho dfo rpectinesteras eactivit y (Delincée & Radola,1970 ;Verstee ge tal. , 1978).Pecti nan dindicato rimpregnate dpape rca nb e usedi nprintin gtechnique so ngel safte rge lfiltratio no relectrophoresi s (Delincée& Radola,1970) .

18 Gelation and viscosity Theoldes tqualitativ emetho di stha to fFrém y (1840)wh odis ­ coveredpectinesterase .Afte rde-esterificatio npecti nform sa ge lwit hpouvaien t cations.Usuall ycalciu mion sar eused .Pilni k& Rothschil d (1960)develope da sensitiv e semi-quantitativevariatio nt otes tunde rpasteurizatio no fjuices .Th eincreas ei nvis ­ cosityo fpecti nsolution sha sals obee nuse dt omeasur epectinesteras equantitativel y (Weurman,1954) .Th emetho dha sonl yver ylimite dpossibilitie s forapplicatio nan d calciumals oaffect sth erat eo fviscosit yincrease .I nbot hmethod sth eactivit yo f chain-splittingenzyme sma yinterfere .

Hydroxylamine and methylene blue Hydroxylaminereact swit hth ecarbomethoxy lgroup so f thepectin .Subsequentl yadditio no fferri cion slead st oa brow ncomple xo fferric - pectinhydroxami cacid .McCom b& McCread y (19S8)use dthi sa sa semi-quantitativ emetho d toscree npreparation so npectin-aga rplates .Ge ee tal . (1959)use dth ereactio ni n histochemicalstudie st oestimat eth edegre eo festerificatio no fpecti csubstance si n fruits.Als orecentl yDelincé e (1976)use dth eprincipl efo ra prin ttechniqu eafte riso ­ electricfocusin go fpectinesterases .Th ebufferin gcapacit yo fth eampholite sincorpo ­ ratedi nth ege lreduce s thesensitivit yo fa prin ttechniqu ewit ha p Hindicator .Anothe r detectiontechniqu ewa suse db yRoe b& Stegema n (1975)i nPolyacrylamid e gels.Th epecti n wasinclude di nth ege lan dafte relectrophoresi sth ege lwa sstaine dwit hmethylen eblue . Pectini shardl ycoloure dbu tth ezone swit hde-esterifie dpecti nstrongl yabsor bth edye . Inbot hstainin gtechnique sothe rpecti cenzyme sma yinterfer e (Roeb& Stegeman ,1975 ; Delincée,1978) .

Non-pectin substrates Insteado fpecti np-nitrophenylacetat eha sbee nuse da sa substrat e forpectinesteras e (Zimmerman,1978) .Th epectinesteras eactivit ywa s followedb ya spectrophotometer at40 0nm .I twoul dsee ma simpl eprocedure .However ,fro mth ewor ko f MacDonnelle tal . (1950)i tca nb epresume dtha tno tpectinesteras eactivit yi smeasured , butsom eothe resteras eactivity ;se eals oSectio n2.2.4 .

Manometric assay Glasziou& Ingli s (1958)use dWarbur gmanometr yt omeasur epectin ­ esteraseactivity .Sodiu mhydroge ncarbonat ewa sadde dt oth ereactio nmixtur ean dth e carbondioxid edevelopmen twa smeasured .Th esensitivit ywa shighe rtha nwit ha titri - metricprocedure .

2.2.7.2 Methanol determination

Severalcolou rreaction san dga schromatographi cmethod sar eavailabl et odetermin e methanol.Also ,i nsom ecase sradioactiv eisotope sca nb eused .

Colour reactions and titrations Thecolou rreaction s requirea noxidatio nste pi nwhic h methanoli soxidize dt oformaldehyde .Severa lmethod so foxidatio nwer estudie db y Whright (1927)an dth emetho dwit hpotassiu mpermanganat ewa s foundt ob eth eonl ysatis ­ factoryone .Whrigh t (1927)foun da modifie dSchiff s reagentt ob eth emos tsuitabl e reagentt ocondens eformaldehyd et oa compoun dwit ha (violet)colour .Ethano laffect s

19 thecolou rdéveloppen tan d alsoglycero l andpecti n interfere.Lea f& Zatma n (1952)ad ­ justed their testsolution s to 15g/ lethano l foroptima lsensitivit y and determined methanol concentrations downt o 3mg/ 1i nbod y fluids.Wit h chronotropic acidformalde ­ hydeproduce s aviole tcolou rwhic h ismeasure d at 580n man dsevera lothe r aldehydes dono tinterfer e (Boos, 1948).Ther e againth eabsorbanc e is affectedb yethy l alcohol (Beyer, 1951). Interfering substances aredescribe db y Bricker& Johnso n (1945). The methodwa s notuse dunde r 20mg/ 1methanol .Usuall y inorgani cmateria l all thesemethod s require distillation ofth emethano lbefor e analysis canb e carriedout ,whic h is the maindisadvantage . Wood &Siddiqu i (1971)develope d asensitiv emetho d (downt o 2mg/ 1origina l methanol) inwhic hpentane-2,4-dion e wasuse d toproduc e a colourwit h formaldehyde (absorbance peak at 412nm) .Becaus eo f themil dconditions ,th emetho dca ndirectl yb euse d inpec ­ tinsolutions ,bu t someothe rcompound s interfere.Th emetho dwa s automated ina n analyserb yVijayalakshm ie t al. (1976)wh o found thatpolyphenol s interfere.Sega l et al. (1966)use da metho d inwhic h formaldehyde is furtheroxidize d toformi caci db y hydrochloric acidan da nexces so fsilve rnitrate .Th eremainin g silvernitrat e is titrated andth emethano l is calculated. Methanol canals ob edetermine db y converting itt omethy l iodite,afte rwhic h itca n be titrated (Vieböck& Schwappach , 1930;Clark , 1932;Hoffma n &Wolfrom ,1947 )o r determined gravimetrically (Zeisel,1885 ;Zeisel , 1886).Th esample smus t firstb e dis­ tilledbecaus emethoxy lan dothe ralkoxy l groups react also.Th emethod s are labourous, insensitive andno t specific formethy lalcohol .

Gas àhromatograçhy Several gas chromatographic methods foranalysin g loweralcohol s havebee npublished .Man yo f themwer e developed foranalysi s inwine s andspirit s and wereno tuse d forconcentration s lower than 10-20mg/ 1 (Dyer,1971 ;Venturell ae t al., 1974; Klaushofer &Bandion , 1968).Nowaday s usuallyporou spolymer swithou t liquidphas e areuse d ascolum nmateria l (Porapak,Chromosorb ,Tenax) . Insevera l applications gas chromatographywa s used todetermin emethano l inconcentration s ofabou t 1mg/ 1 (Baker et al., 1969;Berge re t al., 1974;Majchrowic z &Mendelson , 1971;Krop , 1974).However , thesensitivit y ofth e detector canb e affectedb y thewate r inth esampl e (Lucero,1972 ) and lowconcentration s (< 10mg/1 )o fmethano l inaqueou s samples appeared togiv e irregular response inroutin e analysis (Krop, 1974). Forbiologica lmaterial susuall y a precolumn orinle t isneede dwhic hmus tregularl y be replaced topreven t rapid column deterioration (Bakere t al., 1969;Berge re t al., 1974).Als osample s canb e distilled orpretreate dbefor e injection (Baumann& Gierschner , 1974;Majchrowic z &Mendelson , 1971). Lee &Wile y (1970)use da dialysis cell toseparat e themethano l from the reaction mixture.Demethylatio no fcarboxy l groups isthu sprevented ,however ,th ediffusio npro ­ cess limits thepossibilit y tomeasur e initial reaction rates.Usuall y headspace techniques areonl yqualitativel y used (Wolforde t al., 1963;Norman , 1970).Davi s & Chase (1969)measure dquantitativel y thealcohol s ofcitru s juicesb y aheadspac e tech­ nique,bu t the coefficient ofvariatio n determining concentrations of lower than4 0mg/ 1 wasno tgiven . Alcohols canals ob e determined gaschromatographicall yafte rderivatizatio n to

20 alkyliodite s (afterdistillatio nbecaus e estersan dether s such aspectin ,hesperidi n andnaringi n also react). Incontras t to thevolumetri c andgravimetri c determinations ofalky l iodites,th emetho di sspecifi c forvariou s alcoholsbecaus e thealky l iodites areseparate do nth ecolum n (Kratzl& Gruber , 1958;Vertalie r &Martin , 1958).Derivati - zation toalky l iodites isadvantageou s ifa nelectro n capture detectori sused ,whic h has asensitivit y ofabou t 50time s thato fa flameionizatio ndetecto r (Dickes& Nicholas, 1978). Another approach isderivatizatio n ofalcohol s tonitrit e esters,whic h aremuc hmor e volatile thanthei ralcohols .Th eheadspac e issample d and injected and concentrations ofabou t 1mg/ 1ca nb e determinedquantitativel y (Gessner, 1970;Litchma n& Upton , 1972). Ifth emetho d isproperl y used,pecti n isno t de-esterifiedunde r thederivatizatio n con­ ditions andpecti n solutions andplan t tissuehomogenate sca nb e assayedwithou t prior distillation (Bartolome &Hoff , 1972b). Recently Barone t al. (1978)describe d aver y sensitivean d reproducable variation of thisprinciple .A t amethano l concentration of about 1mg/ 1 thecoefficien to fvariatio nwa s only 1.2%,bette r thanan yo fth ega s chromatographic orcolorimetri cmethod s described.Generally ,th e gas chromatographic methods areno tver y timeconsumin g (4-15determinations/h) .

Radioisotopes A sensitivemetho do fanothe rnatur e is theus eo fpecti n labelledwit h '"[C]methy l asa substrate .Th esubstrat e canb eprepare dbiosyntheticall y (Kauss et al., 1969)o rb yesterificatio nwit h 1U[C]diazomethan e (Milner &Avigad , 1973).Afte r the enzymatic reaction thesubstrat e isseparate d from the liberatedmethano lb yprecipita ­ tionwit h alcoholo rb y extraction.Th e radioactivity ofth emethano l isthe ncounte d in a liquidscintillatio n counter.Th e sensitivity of themetho d depends onth especifi c activity ofth e label,bu t asensitivit y of0.0 3 mg/1methano l iseasil y achieved (Milner &Avigad , 1973). Themetho d iso flittl evalu e ifnatura l substrates aret ob euse d (e.g.natura l pectic substances and fruit juices), but canb eadvantageou s instudie so fworkin g mechanisms andi nroutin e assayso fenzymes . Generally a sensitivemethano l analysis offersman ymor epossibilitie s than titration e.g. inbuffere d systems,a t lowreactio n rates,a tp Hvalue sbelo w 5an d ifa limited amount ofsubstrat e isavailable .Bu twher e itca nb e applied,automati c titrationi s themos t simplemethod ,als obecaus e theactivit y iscontinuousl y monitored and deviations from linearity intim ear eeasil y recognized.

21 3 Materials and methods

Citrus fruit Citrusfruit swer epurchase da ssingl elot sfro mloca ldealer si nth e Netherlands.Nave lorange s [Citrus sinensis) fromSpain ,Salustian aorange s {Citrus sinensis) fromSpain ,Shamout iorange s {Citrus sinensis) fromIsrael ,Valenci aorange s (Citrus sinensis) fromth eUSA ,Whit eMars hseedles sgrapefruit s (Citrus paradisi) from Israel,Clementin emandarin s (Citrus reticulata) fromSpai nan dlemon s (Citrus limon) fromth eUSA .

5 Substrates and inhibitors Greenribbo nappl epectin(62 0esterified ;anhydrogalacturoni c acidconten t64%,degre eo fpolymerizatio n368)an dbrow nribbo nappl epectin(72° sesterified ; anhydrogalacturonicaci dconten t631 ,degre eo fpolymerizatio n423 )wer eobtaine dfro m Messrs.Obipecti nLtd. ,Bischofszeil ,Switzerland .Pectin swit hdifferen tdegree so f esterificationwer eprepare db yesterificatio no fth esam ebrow nribbo nappl epecti nt o 95.6%an dsubsequen talkal ide-esterificatio na sdescribe db yva nDeventer-Schrieme r& Pilnik (1976).Th echaracteristic so fth epreparation sar egive ni nTabl e7 .Variou s othersubstrate swer eobtaine d fromcommercia lsupplier s andfro mth elaborator y collectiona sindicate di nTabl e 16(pag e46) .Polygalacturoni caci d (anhydrogalactu­ ronicaci dconten t88% )use da spectinesteras einhibito rwa sobtaine d fromIC NPharma ­ ceuticals,Inc. ,Cleveland ,Ohio ,USA .Pasteurize dfrozen ,pectinesteras einactive , Valenciaorang econcentrat eo f6 5°Bri xfro mLetab a (SouthAfrica )wa ssupplie db yth e Coca-ColaCompan yan dha dth echaracteristic sa sliste db yKro p (1974).

Fractionation of pectins Pectinswer efractionate daccordin gt othei rdegre eo f esterificationb ychromatograph yo na DEA Ecellulos e (WhatmanDE22 )column .Fiv em lo f a 10g/ 1pecti nsolutio nwa sapplie dt oa colum no f2.5x12. 5cm ,equilibrate dwit h startingbuffe r (0.05mol/ 1sodiu mphosphate ,p H4.8 )an delute dwit ha linea rgradien t

Table7 .Characteristic so fpecti npreparations .

Degree ofesterificatio n Anhydrogalacturonic Degreeo f (% ) acidconten t polymerization (g/100 g) (monomers/chain)

95.6 75.0 76 88.0 73.4 84 78.2 73.4 81 67.8 74.6 81 52.6 80.7 77 32.3 82.2 79

22 from0.0 5t o1 mo1/ 1sodiu mphosphat ebuffe rp H4. 8i n3 1 .Th eremainin gmateria lwa s washed fromth ecolum nwit h 250m l0. 1mol/ 1sodiu mhydroxide .Fraction so f1 5m lwer e collectedan danalyse dfo ruroni cacids .Thi smetho di sa modificatio no fth emetho d describedb yva nDeventer-Schrieme r& Pilni k (1976).

Degree of esterification of pectins Thedegre eo festerificatio nwa sdetermine dtitri - metrically.Th epecti n (300mg )wa sdissolve di n10 0m lwate ran dpasse dthroug ha (2x18cm )Dowe x5 0W an da n (2x18cm )Amberlit e4 5I Rio nexchang ecolumn ,place di n series.Th ecolumn swer ewashe dwit h 200m ldistille dwate ran dth etota lvolum e collectedwa s titratedwit h0. 1 mol/1sodiu mhydroxid ewit hPhenolphthalei na sa n indicator (= x ml). Fiftym lo f0. 1 mol/1sodiu mhydroxid ewa sadde dan dth emixtur e waslef ti na glas sstoppere dflas kfo ron ehou ra troo mtemperature .The n5 0m l 0.1mol/ 1sulphuri caci dwa sadde dan dth esolutio nwa sslowl ytitrate dunde rnitroge n with0. 1 mol/1sodiu mhydroxid e (= yml) . Formula:

Degreeo festerificatio n =-^- . 100 1 x+y ^ff- . 176 Anhydrogalacturonicaci dconten t= ™r .100 %

Theio nexchang ecolumn swer eregenerate dseparatel yafte rabou t4 g o fpecti n hadbee ntreated .

Degree of polymerization of pectins Thedegre eo fpolymerizatio no fpectin swa sestima ­ tedviscosimetrically .Th eviscosit yo f4 pecti nconcentration s (2.0,1.5 ,1. 0an d 0.5g/1 )i n0. 1mol/ 1tris-succinat ebuffe rp H6 wit h 0.2mol/ 1sodiu mchlorid ewa smeas ­ uredi na nUbbelohd eviscosimete rNo .1 .Th eintrinsi cviscosit y [TJ]wa sdetermine db y plottingth erati o (TJ-1)/ eagains t aan db yextrapolatin g tozer oconcentratio n (e= pectinconcentration ,1) =viscosit yrelativ et osolvent) .Th eformul a [rj]= 1.4x106 . M1*31*wa suse dt ocalculat eth eaverag emolecula rweigh t (Owense tal. , 1946).Dividin g themolecula rweigh to fth epolyme rb yth eaverag emolecula rweigh to fth emonomer s (176-190,dependin go nth edegre eo festerification )gav eth edegre eo fpolymerizatio n ofth epectins .

Cloud stability of orange juice Formeasurin gth eeffec to fpectinesteras eo nth eclou d stabilityo forang ejuice ,reconstitute dValenci aorang econcentrat ewa sused .Th e cloudwa sdetermine db yoptica ldensit yo fth esupernatan ta t66 0n mafte rcentrifugin g for1 0mi na t360xg .Kro p (1974)gav edetaile dinformatio nabou tth eturbidit ymeasure ­ ments.However ,instea do f1 0m lsamples ,onl y3 m lsample swer eremove dfro mth eglas s stopperedbottle san dsample swer eno treturne dt oth ebottle sbu tuse dfo rmethano l analysis.

23 Sugar analysis Theneutra l sugarcompositio no fprotein s andpectin swa s analysedga s chromatographicallyaccordin gt oth emetho do fJone s &Albershei m (1972).Protein s (about 2mg )wer edialyse dsal t freean d lyophilized ina glas s tube.Th esampl ewa s dissolvedi n 1m lo fdistille dwater .T o thissolutio n0. 5m lo f 2mo1/ 1tr ifluoro - aceticaci dcontainin g 100p go finosito la sinterna lstandar dwa sadded .Th etub ewa s closedb ymeltin g andheate di na noi lbat ha t 121 °Cfo ron ehour .Th e tubewa sopene d andth ehydrolysat ewa s drieda troo mtemperatur eb ya sof tstrea mo fair .Afte radditio n of0.2 5m lo f 1mol/ 1ammoniu mhydroxid ewit h 10g/ 1sodiu mborhydrid e thesample swer e leftfo ron ehou ra tambien ttemperature .Nex t0.1 5m lo fdistille dconcentrate daceti c acidwa sadde di n3 portions .Afte r additiono f0. 5 mlo f 100g/ 1aceti caci di nmethano l thesampl ewa s driedi na nai rstrea man dthi sprocedur ewa s repeated three timesan d thesam ewa s donewit h 4successiv eaddition s of0. 5m lmethanol .Thereupo n0. 1 mlo f aceticanhydrid ewa s addedan dth etub ereseale db ymeltin gan dplace dfo rthre ehour s ina noi lbat ha t 121°C .Th etube swer e openedan ddrie da troo mtemperatur eb ya sof t streamo fair .T oth eresid u0. 1 mlo fchlorofor m and 0.1 mlo fwate rwer eadde dan d afterdissolving ,th esample swere transferred toa 1m l testtube .Th e aqueous layer was removedwit h asyring ean dth eremainin g chloroformsolutio nwa sdrie di na strea m ofair .Afte rdissolvin g theresidu ei n5 0p io fchloroform , 10p iwa s injectedi na ga s Chromatograph (HewlettPackar d 5750G wit hdua l flame ionization detector)equippe dwit h a dualcolum nsyste mo ftw oglas scolumn so f 190cmx 4m m (I.D.),packe dwit h 3g/k g O.V.275+4g/k gX.F.115 0o nGa sChro mQ 100-120mes h (Darville t al., 1975).Colum nove n temperatureprogramme : 3mi na t 140°C ,140-19 0° Cwit h 1°C/min ,5 0na na t 190° C (toelut eamin osugars) . Peakarea swer emeasure db y anInfotronic sCR S 304computin g integrator. Theneutra lsugar s ofpectin s (about 2mg )were determinedb yvirtuall y thesam e procedure,bu turoni cacid swer eremove db y aDowe x 1x8treatmen tafte rhydrolysi s (batchwise 300m gDowe x 1x8,on ehou r stirring,supernatan twit h 200m gDowe x 1x8,on e hourstirring ,collecte dsediment swashe dwit h 3x 2.5m ldistille dwater ,supernatant s andwashin gwate ruse d forfurthe rprocedure) .Th eholdin g ofth ega sChromatograp ha t theuppe rtemperatur e limitwa somitted . Uronicacid swere determinedb y thecarbazol e testi na nautomate d analysera s describedb yva nDeventer-Schrieme r& Pilni k (1976).

Amino acid analysis About0. 1 mgprotei nwa s dialysedsal t freeagains t distilledwate r andlyophilize dtogethe rwit h 3p gnorleucin e internalstandard .Th esample swere hydro - lysedunde rnitroge nfo r 22h at 110° Cwit h 1m l6 mol/ 1hydrochlori cacid .Th ehydro ­ chloricaci dwa sevaporate da t 40° Cunde rvacuu man dsample swer eanalyse db yeithe ra n aminoaci danalyse ro ra ga schromatographi cprocedure . Forth eanalysi so nth eamin oaci danalyser ,th edrie dsample swere redissolve d in 0.3m lo f0. 2mol/ 1lithiu mcitrat ebuffe r (pH 2.20).Th eamin oaci danalyse ruse dwa s aBiotroni kLC-200 0an dth eamin oacid swere separate do na singl ecolum n (DurrumDC-6A ) bystepwis eelutio nwit h lithium citratebuffers .Amin o acidswer edetecte dwit hnin - hydrina t 570n man d44 0n m (forproline) .Pea karea swere integrate dwit h aSpectr a PhysicsAutola bSyste m Icomputin g integrator.

24 Aminoacid swer eals oanalyse dga schromatographicall ywit hth eprocedur ea s describedb yMackintos h et al. (1977)wit h thederivatizatio n ofamin oacid s to N(0)-heptafluorobutyryl-iso-amylesters . Tryptophanwa sdetermine db y twomethods .Th ecolou rreactio no fOpienska-Blaut h et al. (1963)wa suse dwithou tprio renzymati chydrolysi s andth eabsorbanc eo fstandard s andsample swa smeasure da t45 0n minstea do f54 5nm .Als o thefluorescenc emetho d of Pajot (1976)i n6 mol/ 1guanidin ehydrochlori c acidp H 6.8wa s used todetermin etrypto ­ phan.

Methanol analyses Methanolwa s determined afterth egenera lprocedur eo fLitchma n& Upton (1972),whic h includes conversiono falcohol s tothei rvolatil enitrit eester san d subsequentanalysi sb y gaschromatograph y afterheadspac e sampling. Inthi swor kmuc h attentionwa spai d toth ederivatizatio n conditions,whic h aregive ni nChapte r 4.Th e gaschromatograph ywa s carriedou to na Hewlet tPackar d 5750G researc hga s Chromatographwit h flameionizatio ndetector .Column :9 0cmx 4m m (I.D.)glas s column packedwit h 1mol/ 1potassiu mhydroxid ewashe dtexture dglas sbead s 80-100mes h (Chrom- pack,Middelburg ,th eNetherlands )ove ra lengtho f 15c mstartin g fromth e injection porten dan dpacke dwit hPorapa kR 120-150mes h (WatersAssociates ,Milford ,Mass. )ove r the resto fth ecolum nlength .Colum nove ntemperature : 150 C,temperatur e ofinjectio n portan ddetector : 250°C .Carrie rga s (nitrogen)8 0ml/min .Ne wcolumn swere conditioned at 260° Cfo r 20h .Pea k areaso rpea kheight swere measure dwit ha n InfotronicsCR S 304 computing integrator. Themas sspectr ao falcohol s andthei rreactio nproduct swer edetermine dwit h a combined gasChromatograp h (Pye 204)mas s spectrometer (VGMicromas s 7070F).Th ega s chromatographic conditionswer e thesam ea s forth emethano l analysis describedabove , except thatheliu minstea do fnitroge nwa suse da scarrie rgas .Spectr awere recorde d with theai do fa connecte dV GDatasyste m 2040.

Pectio enzymes Polygalacturonasewa s obtained fromth eyeas t Kluyveromyces fragilis (Jörgenson)v.d .Wal tstrai nCB S 397.Endo-polygalacturonas ei sth eonl ypecti cenzym e producedb y thisorganis m (Phaff,1966) .Th eenzym ewa sprepare da sdescribe db yKro p (1974)bu tfurthe rpurifie do na cross-linke dalginat e column (6x1.5 cm).Th ecross - linkedalginat ewa sprepare da sdescribe db yRombout se t al. (1979).Afte r dialysis against 0.1 mol/1sodiu macetat ebuffe rp H 4.2 the culture filtrate (4000ml )wa spasse d through thecross-linke d alginate columnan d thecolum nwa swashe dwit hstartin gbuffe r untilth eeluat ewa s freeo fglucose .Th epolygalacturonas ewa swashe d fromth ecolum n with 0.1 mol/1sodiu macetat ep H 6wit h 1mol/ 1sodiu mchloride .Specifi cactivit y 100units/mg .Endo-pectat e lyaseo f Pseudomonas fluorescens GK-5,purifie da s described byRombout se tal . (1978),wa sobtaine d fromth elaborator ycollection .Specifi cactivit y 956units/mg .Endo-pecti n lyasewa spurifie dfro mUltrazy m 20 (an Aspergillus nigev preparation fromCIBA-Geig yAG ,Basel ,Switzerland )a sdescribe db yva nHoudenhove n (1975). Theenzym epreparatio nuse dher eonl ycontaine disoenzym eType II,specifi cactivit y 10units/mg .Al lenzym eactivit y units arei nymol/mi na toptima l conditions and 30 C.

25 Enzyme assay and detection Oneuni to fpectinesteras ei sdefine da sth eamoun to fenzym e whichliberate son emicromol eo fcarboxy lgroup spe rmi na tstandar dassa yconditions . Instandar dassay sfo rpectinesteras eactivity ,2 0m lo f5 g/ 1gree nribbo npecti ni n 0.15mol/ 1sodiu mchloride ,p H7. 0wa spipette dint oa glas sbeaker .Afte requilibratio n at3 0 C0.0 1 to 1m lo fpectinesteras esolutio nwa sadded .Whil estirrin gwit ha magneti c stirreran dpurgin gwit hnitrogen ,th einitia lreactio nvelocit ywa smeasure db yautomati c titrationo fth eliberate dcarboxy lgroup swit h0.0 2mol/ 1sodiu mhydroxide ,i na Comb i recordingpH-sta t (E30 0B - E 47 3- E 425 )fro mMetroh mLtd. ,Herisau ,Switzerland .Fo r kineticmeasurement san dpectinesteras eactivitie si njuice sothe rtechnique swer euse d aswell .Activitie sbelo wp H5. 0wer edetermine db yincubatin g 10m lo fbuffere dsubstrat e withenzym ei na 1 0m lglas sstoppere dflas kwit h2 5mg/ 1o fa nalcoho l (ethanolo rpropa ­ noic orpropanol-1 )i na wate rbat ho f3 0°C .A ttim einterval ssample so f 1m lwer etake n andth emethano lconcentration swer edetermine dga schromatographically ,fo rdetail sse e Chapter4 .I ntitrimetri cassay sabov ep H7 th eblan cvalue sfo rchemica lde-esterificatio n weresubstracted .I finitia lreactio nrate swer eto oshor tbecaus eo fshortag eo fsub ­ strateo rproduc tinhibition ,reactio nvolume swer eincrease dan dmor edilute d alkali (0.002mol/1 )wa sused .Also ,i nthi scase ,th ep Ho fth ereactio nmixtur ewa s continuouslyrecorde do na recorde rwit hextende dscal e (1p Hunit/ful lscale) .Fo rmor e sensitivemeasuremen tth ep Hdro pmetho do fSomogy i& Roman i (1964)wa sused .Th ep H shift- alkal iequivalent swei edetermine dwit h0.00 2mol/ 1potassiu mhydroxide .Th e dropi np Hpe rminut ewithou tenzym ei nth ereactio nmixtur ewa ssubstracted .Pectin ­ esterasedetectio no nthin-laye rgel safte risoelectri c focusingo rpH-gradien telectro ­ phoresiswa sdon ewit hth ehydroxylamine-ferri cchlorid eprin ttechniqu eo fDelincë e (1976). Non-esteraseactivitie so fpectinesteras epreparation so nvariou ssubstrate swer e measuredi nreactio nmixture s (0.5ml )containin g 4m gpe rm lo fsubstrate ,0.0 4mol/ 1 sodiumsuccinat ebuffer ,p H5. 0an d3 unit so fpectinesterase .Afte rincubatio na t3 0 C for2 0h ,th eincreas ei nreducin ggroup swa sdetermine db yth eNelson-Somogy imetho d (Spiro,1966) .Phosphatas e (Arnold,1972) ,carboxylesteras e andproteas e (Cabib& Ulane , 1973)activitie swer edetermine dusin gp-nitrophenylphosphate ,p-nitrophenylmyristat ean d azocoll,respectively . Polygalacturonaseactivit ywa sdetermine db yth eNelson-Somogy imetho d (Spiro,1966 ) orb ya viscometri cassa y (Krop,1974) .Pecti nan dpectat e lyasewer emeasure db yth e increasei nabsorbanc ea t23 5n m (Voragen,1972 ;Rombout se tal. ,1978 )o rb yth eNelson - Somogyimetho d (Spiro,1966) .

Chromatography of pectinesterase and analysis of fractions Columnchromatograph ywa s donewit hBio-Ge lP-10 0(100-20 0mesh) ,C MBio-Ge lA (100-200mesh )an dBio-Ge lHT P (hydroxylapatite)obtaine dfrom ,an duse daccordin gt oth especification so fBio-Ra d Laboratories,Richmond ,California ,USA .Cross-linke dpectat ewa sa gif tfro mD rL.Rexovä - Benkovâ,Institut eo fChemistry ,Slova kAcadem yo fSciences ,Bratislava ,Czechoslovakia . Fromth ebe dvolum eo fthi spreparatio n (3.7ml/g )i twa sestimate dtha ti tcontaine d 3.5uroni caci dunit spe ron ecross-lin k (Kohn,1975) .Th eprotei nconcentratio ni n elutedfraction swa sdetermine db yusin gFoli nreagen t (Lowrye tal. , 1951),wit hbovin e

26 serumalbumi n (A-4378,Sigm aChemica lCompany ,St.Louis ,Missouri ,USA )a sstandard . Pectinesteraseactivit yi nfraction swa sassaye dwit ha methyl-re dindicato rtest . Aliquotso f 10- 2 5y lo ffraction swer eadde dt osmal ltes ttube scontainin g 1m lo fa solution (pH7.0 )compose do f5 g/ 1gree nribbo npectin ,0.1 5mol/ 1sodiu mchloride , 0.1 g/1sodiu mazid ea spreservin gagen tan da fe wdrop so fa 1 0g/ 1ethanoli cmethyl - redsolutio npe r2 5ml .Fraction sgivin ga positiv emethyl-re dtes t (redcolour )withi n onehou rwer eals oassaye dtitrimetrically .

Electrophoresis and isoelectric focusing Enzymepurit yi nvariou sstage so fth epurifi ­ cationprocedur ewa schecke db yelectrophoresi so nthi nslab so fPolyacrylamid ege li n thepresenc eo fsodiu mdodecy lsulphat e (Ames,1974) ,wit hth ediscontinuou sbuffe r systemo fLaemml i (1970).Th eseparatin ggel scontaine da fina lacrylamid econcentratio n of12 0g/1 .Stainin go fsla bgel swa sdon ewit hCoomassi eBrillian tBlu e (Fairbankse tal. , 1971).Th emolecula rweigh to fth epurifie dpectinesterase swa sdetermine dwit hth esam e electrophoresissystem ,usin gth efollowin gprotei nstandards :bovin eseru malbumi n (Boehringer,Mannheim ,mol.wt .6 7000) ,bee flive rcatalas e (Boehringer,mol.wt .6 0000) , ovalbumin (Boehringer,mol.wt .4 5000) ,chymotrypsinoge nA (Worthington,mol.wt .2 5700 ) andtrypsinoge n (Boehringer,mol.wt .2 3500) . Isoelectricfocusin gwa sdon ei na 11 0m lcolum n (Type8100-1 ,LKB-Produkte rAB , Bromma,Sweden) ,followin gth einstruction so fWinte r& Karlsso n (1976).Whil emixin g thesorbito ldensit ygradient ,enzym esample swer eintroduce da sa ban da tabou ton e fourtho fth eheigh to fth ecolumn .A constantpowe rsuppl y (Type2103 ,LKB )wa suse dfo r electrofocusinga tmax .4 W andmax .140 0V durin gapproximatel y 40h a t6 C.Whil e continuouslyrecordin g theabsorbanc ea t28 0nm ,th ecolum nwa semptie d fromth ebottom , ata rat eo f2 m lpe rmin ,collectin gfraction so f 1,2 o r4 ml .Th ep Ho fth efractions , kepta t6 °C ,wa sdetermine dwithi n5 mi nwit ha naccurat ean dstabl ep Hmete rpreviousl y calibratedwit hfreshl yprepare dstandar dbuffer s (ElectronicInstruments ,Ltd. ,Chertsey , England)o fp H7.0 8an d9.4 1 at6 °C . pHGradien telectrophoresi s (aseparatio nmetho ddevelope di nthi swork )wa sapplie d ifisoelectri cfocusin gwa simpossibl ebecaus eo finavailabilit yo fcommercia lampholite s inth ep Hrang eneeded .Thin-laye rgel s 11x24c mo fUltrode x 100 (adextra nge lfro m LKB)wit hampholine sfo rth ep Hrang e7-1 0wer eprepare daccordin gt oth einstruction s ofWinte re tal . (1975)fo rth eLK B211 7multipho rfla tbe delectrophoresi sapparatus . Enzymeswer eintroduce d 1c mfro mth eanod esid ean dpowe rwa sapplie dfo rabou t4. 5h . Temperature6 C,powe rsettings :maxima l8 W ,2 0m Aan d 1400V ove rth elengt ho fth egel . Pectinesteraseactivit ywa sdetecte dwit hth eprintin gtechnique .The nth ege lwa sseg ­ mentedwit ha fractionatin ggrid .Th ep Ho fth esegment swa smeasure dwit ha pH-surfac e electrodean dfo rquantitativ epectinesteras edeterminations ,segmente d zonesi nwhic h pectinesteraseactivit ywa sdetecte dwer escrape dof fan dtransferre dt osmal lfilter s (LKB2117-50 2PEG Gelutio ncolumns) .Th epectinesteras ewa selute dint otube swit h 0.1 mol/1potassiu mchlorid ean d0. 1 g/1sodiu mazid esolution san dactivit yi nth e eluateswa smeasure dunde rstandar dassa yconditions .

27 4 Methanol determination

Becauseo fth enee dt omeasur epectinesteras e activitiesunde rcondition swher en o titrimetricassa y couldb eused ,muc h attentionwa spai dt oth ega schromatographi c analysiso fmethanol .Thereb y resultswer eobtaine dwhic har eals ousefu l forth ega s chromatographyo fothe rlowe ralcohols .Th emetho d describedbelo wwa sbase do nth e methodo fLitchma n& Upto n (1972).I nthi smetho dalcohol sar e esterifiedt ovolatil e nitritesan ddetermine dga schromatographicall yafte rheadspac esampling .

4.1 EXPERIMENTSAN DRESULT S

Reaction conditions 0.2g o fure awa sweighe dint o2 5m laluminiu mpainte d flasks (Pierce,Rockford ,Illinois ,Reacti-flask s No. 13320).Th eflask swer epurge dwit h nitrogenfo rabou t5 mi nan dstoppere dwit ha rubbe rstopper .The n1 m lo fa saturate d oxalicaci dsolutio nwa s addedan dth eflas kwa sswirled .Nex t1 m lo fsampl ewa s added (t=0)an dth eflas kwa sswirle dagain .Th erubbe rstoppe rwa s replacedb ya nope nscre w cap (Pierce,No . 13218)wit ha silicon e rubberseptu m (Pierce,No .10155 )an dth eflas k wasswirle dunti l£=3 0sec .A t*=4 5se c0. 5m lo fa sodiu mnitrit esolutio nwa s injected throughth eseptu man dth eflas kwa s swirledunti l £=70sec .Nex tth eflas kwa shel da t a certaintemperature-tim ecombinatio nfo rreaction .Thereupon ,1 m lheadspac ewa swith ­ drawnwit ha ga ssyring ean danalyse db ya ga sChromatograp hunde rth e conditiona sdes ­ cribedi nChapte r3 .Severa l combinationso freactio n time,reactio n temperature,typ e ofga ssyring e andsodiu mnitrit econcentratio nwer e tested.Litchma n& Upto n (1972) prepared theester sa troo mtemperatur ean dBartolom e& Hof f (1972b)a t0 C.Usin ga saturatedsodiu mnitrit esolutio nan dinjectio nwit ha 2. 5m lGastigh tHamilto nsyring e ( #1002 ,Hamilto nCo. ,Reno ,Nevada )i tappeare d thatholdin gth eReacti-flas ki nic e from 4=70se con ,wa sbette r thanholdin gi ta troo m temperature.Fig .2 show s thepea k areaso fmethano l againstdifferen treactio ntim ewherea s Fig.3 show sth erati oo fth e areaso fethano lt omethano lagains tth ereactio ntime .Th epeak sar ename d afterthei r respectivealcohol s (methanol,ethanol ,propanol- 1 andpropanol-2 )an dno tafte rthei r nitriteester sfo rreason swhic har e explained lateri nthi sSection .Th e nitriteester s weremor establ ean dth esensitivit yo fth emetho dwa shighe ra t0 ° C(Fig . 2).Bu tfro m Fig.3 i tca nb esee ntha twit h theus eo fethano la sinterna lstandard ,reactio ntemper ­ aturean dtim ebecom e lesscritical . Withth ereactio na troo mtemperatur ea noverpressur ewa sbuil tu p(th ereactio n isexotherm) ,an dsom ega sescape d fromth esyring e afterwithdrawa l fromth evial .Th e prematureescap eo fth ega sfro mth esyring ewa seffectivel yprevente db yth eus eo fa 2m lPressure-Lo ksyring e (PrecisionSamplin gCorp. ,Bato nRouge ,Louisiana )fitte d witha valv et oclos eth eneedle .Thi s doubledth esensitivit yo fth emetho da troo m

28 peak area methanol (int. units) 6OO-1

400

Fig.2 .Pea karea so fmethano la sa functio no f 200 reactiontim ewit hnitrou sacid .A solutio no f2 5 mg/1methano lwa suse d forth ereactions .Reactio n flasksan dreagent swer ea troo mtemperature .Afte r thereagent swer e combinedi nth eflasks ,th eflask s wereincubate da troo mtemperatur e (A)o ri nic e 40 60 (o)til linjectio no f 1m lo fheadspac ewit ha reaction time(min) Gastigh tsyring ei nth ega sChromatograph .

peak area ethanol/areo methanol (•/.) 100-1 '

80

60-

40- Fig.3 .Pea karea so fethano lrelativ et omethano l asa functio no freactio ntim ewit hnitrou sacid . 20- Thesolutio nuse d forth ereactio ncontaine da mixtureo f2 5mg/ lethano lan d2 5mg/ 1methanol . Reactionan dcondition sa si nFig .2 .Th ereactio n 20 40 60 flaskswer eincubate da troo mtemperatur e (A) reaction time (min) ori nic e(o) .

temperaturebu thardl yaffecte dth emetho da t0 C. Inorde rt opreven tth eregula rblockin go fth einjectio nneedl eo fth esyring euse d toinjec tth esodiu mnitrit esolutio nint oth ereactio nvial ,th esaturate dsodiu mnitrit e solutionwa sreplace db ya 25 0g/ 1sodiu mnitrit esolution . Fig.4 show sth epea karea so f4 alcohol swit hdifferen treactio ntime swhe nth e reagentsan dvia lwer eprechille da t0 Can dth ewhol ereactio nwa sdon ei nice .Fig .5 showsth earea srelativ et oeac hothe runde rth esam econditions .Similarl yFig s6 an d7 showth evalue swhe nth ereagent san dreactio nwer ekep ta troo mtemperature .Pea karea s ofdifferen tfigure s (Figs2 ,4 ,6 ,8 an d9 )ar eno tcomparabl ebecaus esometime s differentsetting so fintegrato ran dga sChromatograp hwer eused .Fro mFig s4 an d6 i t canb econclude dtha ta t0 ° Cth ereactio nproduct sar emor estable .However ,a t0 Ca longerreactio ntim ei srequire dfo roptima lsensitivity .I nspit eo fth erapi dreductio n ofth esensitivit ywit hextende dreactio ntime swit hth emetho da troo mtemperature ,th e relativearea sar eles ssusceptibl et ovariation si nreactio ntim e (Figs6 an d7) . Table8 show sth eabsolut ean drelativ esensitivit yo fsevera lmethod sfo ra mixtur e of4 alcohols .Th edat awer epresente dbot habsolutel yan drelativel yt ovalue sobtaine d bydirec tinjectio no f5 il lliqui dunde rth esam ega schromatographi cconditions .Tabl e 9 showsth evariatio ncoefficient so fsom eo fthes emethods .Fro mTable s8 an d9 i tca n beconclude dtha tth emetho da troo mtemperature ,usin ga Pressure-Lo ksyring e (Table8 , methodNo .8 )i sth emos tsensitive .Th emetho di sals over yreproducibl ei faccuratel y

29 peak area (int.units)

80

Fig. 4. Peak areas of four alcohols as a func­ tion of reaction time with nitrous acid at 0 C. The solution used for the reaction con­ 20 tained a mixture of four alcohols, 25 mg/1 of each. Prechilled reagents and reaction flasks were used. One ml of headspace was injected in the gas Chromatograph with a Pressure-Lok syringe. Alcohols: Methanol (•); 0 10 "3) ' SO ' 7& ethanol (A); propanol-2 (o) and reaction time (min) propanol-1 (•).

relative peak areas

Fig.5 .Relativ epea karea so ffou ralcohol sa s afunctio no freactio ntim ewit hnitrou saci d at0 C.Reactio nan dcondition sa si nFig .4 . Relativepea kareas :methanol/ethano l(1) ; methanol/propanol-2 (2);methanol/propanol- 1 (3 0 10 70 ethanol/propanol-2 (4);ethanol/propanol- 1 (5) reaction time (min) andpropanol-2/propanol- l(6) .

timed (Table9 ,metho dNo .8) . Theshor treactio ntim ei sconvenien ttoo .Thi sprocedur e whichwa sfinall yuse di nth eexperiment s formethano ldeterminatio ni ssummarize di n Table10 . Typicalretentio ntime so na 9 0cmx 4m m (I.D.)colum nwit hth ega schromatographi c conditionsa sdescribe di nChapte r3 ar e4 3s fo rmethanol ,8 2s fo rethanol ,13 6s fo r propanol-2an d19 2s fo rpropanol-1 .Dependin go nth einterna lstandar dused ,10-1 5metha ­ noldeterminations ,derivatizatio nincluded ,coul db edon ei na nhour .

Linearity and reproducibility Thecorrelatio nbetwee nmethano lconcentratio nan dpea k areawa steste dfo raqueou smethano lsolutions ,singl estrengt horang ejuice ,pecti n concentrationsu pt o1 0g/ 1an dhuma nbloo dserum .Fig .8 show sth ecorrelatio nfo r

30 peak area (int units)

180

30 40 reaction time (mi n) Fig.6 .Pea karea so ffou ralcohol sa sa functio no f reaction timewit hnitrou saci da troo mtemperature . The solutionuse dfo rth ereactio ncontaine d amix ­ tureo ffou ralcohols ,2 5mg/ 1o feach .On em lo f headspacewa s injected inth ega sChromatograp hwit h aPressure-Lo ksyringe .Alcohols :methano l (•); ethanol (A);propanol- 2 (o)an dpropanol- 1 (•).

30 40 reaction time (min) Fig.7 .Relativ epea karea so ffou ralcohol sa sa functiono freactio n timewit hnitrou saci da t roomtemperature .Reactio nan dcondition sa si n Fig.6 .Relativ epea kareas :methanol/ethano l(1) ; methanol/propanol-2 (2);methanol/propanol- 1(3) ; ethanol/propanol-2 (4);ethanol/propanol- I(5) ; propanol-2/propanol-l(6) . 31 u « o co CO ON r*. 00 ON ON NO a> o ~ —*U" ) CM CO P- GO ON CO ^- CO CM CO 00 ON ê o o

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m -d* \o co co co

CL — N CO CN >N

a — o — CM — —

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H

CO CO (SI -J- — — c

11* h v^< 10 •O en 4J CcO T3 4J O c CA CM CM CM CO CM — J3 • o »4-1 uc 4J ai CD — vo o oo o m o 4-1 >4-l O CT*CU •o 0) o •-I oc £ J2 4J CO CH O ë co >* m *o r-. oo

33 Table 10.Modifie dprocedur efo rth ega schromatographi cmethano ldeterminatio nwit h reagentsan dreactio na troo mtemperature .

- Weigh0. 2g o fure aint oa 2 5m laluminiu mpainte dvial . - Purgea tleas t4 minute swit hnitrogen . - Closevia lwit hrubbe rstoppe ran dstar tpurgin go fth enex tvial . - Add 1m lo fa saturate doxali caci dsolutio nan dswir lvial . - Add 1m lo fth esampl e (starttimin g : t •0) . - Swirlvial ,reca pwit hope nscre wca pwit hsilicon erubbe rseptum . - Swirlvia ltil l t " 30s . - Inject0. 5m lo fa 25 0g/ 1sodiu mnitrit esolutio nthroug hseptu ma t t =4 5s . - Swirlvia ltil l t =7 0s . - Withdrawwit hPressure-Lo ksyring e 1m lo fheadspac ea tt = 150s. , insertneedl ei ninjectio nport ,precompres s thesample,ope nvalv eo fth e syringean dinject . - Gaschromatographi canalysi sa sdescribe d inChapte r3 .

aqueousmethano lsolution san dFig .9 fo rhuma nbloo dserum .Th ehuma nbloo dseru mcon ­ tained1. 1mg/ 1o fmethanol .Ther ewa salway sa goo dlinea rrelationshi pbetwee nth epea k areasan dpea karea srelativ et oa ninterna lstandar dan dth emethano lconcentration .O n acolum no f18 0cmx 4m m (I.D.)th elinea rrang eextende da tleas tfro m2-40 0mg/ 1me ­ thanolan do na 9 0cmx 4m m (I.D.)colum na tleas tfro m1-20 0mg/ 1methano li nth esample . Inhuma nbloo dseru monl yth econcentration sbetwee n 1an d1 5mg/ 1methano lwer etested . Thepea kareas ,th erelativ earea san dth evariatio ncoefficient so fthes earea sa t fixedalcoho lconcentration so f2 5mg/ 1bu ta tdifferen tpecti nconcentration sar egive n inTabl e 11.Th epecti nconcentratio nslightl yaffecte dth epea kare ao fth emethanol . However,wit hth eus eo fare aratios ,wit hon eo fth eothe ralcohol sa sinterna lstandard , thepecti nconcentratio ndi dno taffec tth erespons efactor .Thereby ,ethano lan dpropa ­ noic wereslightl ybette rinterna lstandard stha npropanol-2 .Additio no f0. 2g/ 1o f sodiumazid eo r30 0g/ 1o fsugar s (100g/ 1sucros e+ 10 0g/ 1glucos e+ 10 0g/ 1fructose ) toth ealcoho lsolutio ndi dno taffec tth erespons efactor .However ,blanc smus tb esub- , stracteda specti ncontain ssom efre emethano lwhic hi srelease ddurin gdissolvin gan d neutralization.Pecti nma yals ocontai nconsiderabl eamount so falcoho luse dfo rpreci ­ pitationdurin gpecti nmanufacture .Severa lmg/ 1o fethano lar efrequentl yfoun di nsuga r solutions. Table1 2show sth evariatio ncoefficient sa t3 methano lconcentration swit hethano l asinterna lstandard .A tlowe rmethano lconcentrations ,th evariatio ncoefficien trise s buti sstil lles stha n 1%a t 1mg/1 .Fo rth ecalculation so fth evariatio ncoefficient s themethano lconcentratio ni nth eblanc swer esubstracted .I nthi scas eth emethano lcon ­ centrationi nth eblanc swa s0. 5mg/1 .Th etota lVariatio ncoefficien ta ta concentratio n of 1mg/ 1methano lwa sles stha n 2%.

Applications Theactio no fpectinesterase sca nb efollowe db ysamplin gfro mth ereactio n mixture.Pectinesteras efro m Aspergillus niger (Pectinmethylesterase ,rein ,Röhm , Darmstadt,Fed .Rep .Germ. )wa sincubate dwit hbuffere dsubstrat ei nglas sstoppere d flasksan dsample da ttim eintervals .Th eresult sar eshow ni nFig .10 .I tca nb esee n thatthi sfunga lpectinesteras ei seffectivel ystoppe db yth eoxali caci dan dals otha t

34 area methanol/are a ethanol (*/•)

300

200

»0-

200 300 400 methanol (mg/l)

Fig.8 .Correlatio n between methanol concentration and peak area after reaction ofmethano l ina aqueous solution with nitrous acid.Pea k areao f methanol (•);pea k areao fmethano l relative topea k areao f 100mg/ lethano l inth esam e aqueous solution(x) .

area methanol/ area 25mg/l propanol-2 08-1

Q6-

10 15 methanol (mg/l)

Fig. 9.Correlatio n between methanol concentration andpea k area after reaction ofmethano l inhuma n blood serum with nitrous acid. Peak areao fmethano l relative topea k area of2 5mg/ l propanol-2 added toth esam e blood serum (•).

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36 Table 12.Variatio ncoefficient so fa 10-foldga schromatographi canalysi s ofmethano l indistille dwate rwit hethano la sinterna lstandard .

Methanol (mg/1) Variation coefficient (%)

25 1.2 5 1.7 1 2.9

methanol(mg/i ) 250-1

Fig. 10.Effec to fsaturate doxali caci do n pectinesterase activityan dpecti nstabilit y 150- uponincubatio na t3 0 C.Brow nribbo npecti n (5g/1 )i n0. 1mol/ 1sodiu msuccinat ebuffe r pH4. 5 and0. 1 g/1sodiu mazid ewa sincubate d witha funga lpectinesteras e (A).I nparalle l experiments, 1:1o fa saturate doxali caci d solutionwa sadde d toa pecti nsolutio n withoutpectinesteras e (•) and toa pecti n solutionafte r 15mi npectinesteras eactivit y (o).A ttim e intervals sampleswer e takenfo r thereactio nwit hnitrou saci dan dsubsequen t gaschromatographi cassay .

thede-esterificatio no fth epecti nb yth eaci dwa snegligible .Simila rresult swer e obtainedwit hcrud ecitru spectinesterase . Themetho dma yals ob euse dt odetermin eth edegre eo festerificatio no fsmal l amountso fpecti csubstances .Thi spossibilit ywa schecke db ydissolvin g 2m go fdiffer ­ entpecti npreparation s inwate r (sodiumchlorid ea sspacin gagen tan dheatin gwit h vigorousstirring) .Afte rcooling ,propanol- 1wa sadde dt ogiv ea fina lconcentratio no f 50mg/ 1an dth evolum ewa sadjuste dt o1 0ml .Th esolution swer edivide dint o2 portion s of5 m li n1 0m lglas sstoppere dflasks .T oon eflas ko feac hse t0. 2m lo f1 mol/ 1pot ­ assiumhydroxid ewa sadded ,thes eflask swer eswirle dan dlef tfo r1 hou ra troo mtemper ­ ature.The n0. 2m lo f1 mol/ 1hydrochlori caci dwa sadde dt othes eflasks .Th econtent s ofal lflask swer eanalyse di ntriplicat efo rmethano lan dfro mth euntreate dflask s sampleswer etake nfo ra triplicat euroni caci danalysi swit hth eautomate dcarbazol e test.Dividin gth edifference si npinole smethano lb yth eumole suroni caci dgav eth e degreeo festerification .Th eresult sar ecompare dwit ha duplicat etitrimetri cassa yo f thesam epreparation si nTabl e 13.Th eresult sar ei ngoo dagreement .Th eadvantag eo f themetho di sth esmal lpecti nquantit yneeded :30-10 0time sles stha nfo ra singl e titrimetricassay .Th emetho dprove dit susefulnes si nth eanalysi so fcel lwal lfractions .

Identity of the detected compounds Gessner (1970)an dLitchma n& Upto n (1972)note dtha t alcoholsha dth esam eretentio ntim ei nth ega sChromatograp ha sthei rreactio nproduct s withnitrou sacid .Th esam eobservatio nwa smad ei nthi sga schromatographi cwork .I nth e Table 13.Compariso n of titriroetric assay and methanol + uronic acids assay for the degree of esterification of pectins.

Degree of esterification(% )

Titrimetric method Gas chromatographic method

94.8 93.4 78.2 78.2 47.3 46.7 28.0 27.9 combined gas chromatograph-mass spectrometer analysis the mass spectra of the compounds eluting after injection of methyl nitrite or 2-propyl nitrite,wer e (computor) corrected for the background spectrum due to the Porapak R bleed. The resulting spectra did not correspond with the spectra of methyl nitrite,o r 2-propyl nitrite (Mass Spectrometry Data Centre, 1974)bu t appeared to be identical to the mass spectra of directly injected corresponding alcohols.Apparentl y the alkyl nitrites are labile and are decomposed upon injection in the gas Chromatograph (250 C).I f the reaction was done in orange juice, an extra peak was foundwit h a shorter retention time than methanol in the gas chromatogram (Fig. 11).Thi s peak was not found when the headspace of untreated orange juice was sampled under the same conditions.T o avoid the temperature effect of the exo­ therm reaction, in both cases the vials were held in ice.Th e gas chromatographic-mass spectrometric analysis revealed that this peak was propene.Th e origin of this product is unknown and itwa s not found in other products like apple juice,pectin , saccharose, glucose and fructose solutions and blood serum.

Problems and sources of errors Formaldehyde and acetaldehyde have nearly the same reten­ tion times as methanol on Porapak R. These were confirmed in this work. The acetone peak (103 s) appeared in between that of ethanol (82 s) and propanol-2 (136 s).Lo w concentra­ tions (25 mg/1) of these compounds did not interfere with the methanol determination. The acetone and acetaldehyde peaks did not increase upon treatment with nitrous acid. The

Fig. 11. Gas chromatographic separation of compounds in _vAJ reconstituted Valencia orange concentrate after reaction with nitrous acid. Compounds:Unknow n (1);methano l (2) 1 0 and ethanol (3). retentiontime(min )

38 effect ofnitrou s acido nth e formaldehydepea kwa sno tverifie dbecaus e thé available commercial 350g/ 1formaldehyd e solutionappeare d tocontai nabou t 100g/ 1methano l as a stabilizer.Blan cvalue so fmethano l couldb e reducedb y theus eo fdistille dwater , whichwa sboile d toabou thal f thevolume ,befor ebein guse d inmakin gu p solutions and reagents.I tappeare dt ob enecessar y toinjec t several blancs (upt o 15fo rrelativel y new columnsan d about 4fo rol d columns)t oeliminat e a falsemethano lpea kwhic happear ­ edwhe nth ecolum nha dbee nou to fus e fora certai ntime .Th e sizeo f thispea k couldb e equivalent toon e representing 10mg/ 1o fmethano l inth esampl ebu tcoul deasil yb e reduced toon eequivalen t to0.3-0. 5mg/ 1o fmethano l by thesepreinjections .Th e samples forconditionin g thecolum n couldb e taken from thesam ereactio nflas kwit h theblan c reagents,hel di nice .Preconditionin g a newcolum na t 260° C insteado f 240° C (Litch- man &Upton , 1972)reduce d this falsepea k considerably.Column s couldb euse d forove r a thousand injections before adecreas e inperformanc e becameapparent . The typeo fga s syringe andth ebatc ho fsilicon e rubber septa for thevial s affected theminimu mblan cvalue .Relativel y softsept acoul dgiv e rise toa pea kcomparabl ewit h thatobtaine dwit h about 3mg/ 1methano lwit ha retentiontim eonl ya fewsecond s longer thanmethanol .Th eminimu mblan cpea kwit h theGastigh t syringeswa s abouttwic e ashig h aswit h thePressure-Lo k syringes.Afte r repeateduse ,th eplunge ro f thesyringe s could leakunde r thepressur e ofth ecarrie rga s inth ega sChromatograph .Th e silicone rubber septafo rth evial s couldb euse drepeatedly ,bu t ifa ver y lowconcentratio n ofmethano l was analysedwit h thesam eseptu mafte ra hig hon e therewa s some carryover.Als o special attentionha d thent ob epai d toth e airwashin g of thega ssyringe .A t lowconcentrations , itappeare d tob e advantageous tomeasur epea kheight s insteado fpea kareas .

4.2 DISCUSSION

Ifproperl y used,th e gaschromatographi c alcohol analysis,afte rconversio no falco ­ hols tonitrit eester s andwit hheadspac e sampling,appear s tob ever y sensitive (<1yg/ml )an d reproducible (variationcoeff .abou t 1.5'»).Th emethod ,a sdescribe dhere , gives abette r reproducibility thanth emethod s describedb yLitchma n &Upto n (1972)an d Bartolome& Hof f (1972b). Baron (1978)reporte d aneve nbette r reproducibility butworke d with a five-fold largersampl e anda ninconvenien t reaction timeo f4 5min .Presumabl y the reproducibility canb e further improvedb y thermostatingth e reactionvial s ina wate r bath.Reproducibilit y atconcentration s of lowertha n 1mg/ 1woul db e improved ifth e blancmethano lpea k couldb e eliminated. Under thega schromatographi c conditionsuse d inthi swor k (150-250 C),th ealky l nitrites areunstable .Baro n (1978)use d a lowertemperatur e inth ega s Chromatograph (50 C). The alkylnitrite s arestabl e then (Baron,persona lcommunication) . Themetho d isver y convenient,becaus e itca nb eapplie ddirectl ywit hpecti nsolu ­ tions,suga rsolutions ,frui tjuices ,bloo d seruman dpresumabl ywit hman yothe rsystems . Thedetail so f theprocedur e areno tver y important,a s longa s theyar e carefully standardized.Mos t time and temperature combinations are suitable,bu tprecis e timing and a standardizedprocedur e are requirements.I flo wconcentration s aret ob eassayed ,th e method described inTabl e 10wil l give thebes tresults .

39 5 Purification and molecular properties of two citrus pectinesterases

5.1 EXPERIMENTSAN DRESULT S

Extraction of pectinesterase from oranges Oranges( 5kg )wer ecu ti nhalve san dpresse d ona rotar ykitche ncitru spress .Th ejuic ewa sdraine d throughcheeseclot han dth epul p waspresse di na galeni cpres s (H.Fische r& Co. ,Norf ,Wes t Germany).Th e presscake , togetherwit h thepee lsegment swer efroze na t-4 0 Can dstore da t-2 0 C,til l further use. Afterthawin ga t4 C,pectinesteras ewa sextracte d fromth epreparatio no fpee lan d pulpb ya borat ebuffe raccordin gt oMacDonnel le tal . (1945)an dKro p (1974)wit hextr a 15g sodiu mchlorid epe rlite radded ,t oimprov eextraction .Hundre dgra mportion so fth e preparationwer ehomogenize dwit h 200m lbuffe rfo r5mi ni na Brau nMultimi xhomogenizer . Thetota lhomogenize dpreparatio n( 967 0ml )wa sstirre d foron ehou ra troo mtemperature , thenpacke di ncheeseclot han dpressed .Th epres s liquid( 750 0ml )wa srapidl ycoole dt o 5 °C.Th epres scak ewa shomogenize dwit hbuffe ran dpresse don emor etim et oyiel da secondbatc ho fpres s liquid( 650 0ml) .T obot hbatche s cooledt o5 °C ,1 7g pe r 100m l ofsoli dammoniu msulphat ewa s added (to30 1saturation ;Dixo n& Web b (1964)).Al lensuin g operationswer edon ea t2 t o5 C.Afte rtw ohour sth etw obatche so fpress liquidwer e centrifugeda t1 0OOOx gfo r2 0min .Th e pelletan da noi l layerfloatin ga tth eto pwer e discarded.T oth esupernatant s3 1g pe r 100m lo fsoli d ammonium sulphate (to75 %satura ­ tion)wa sadded .Afte rstandin governigh tth eprecipitate swer e collectedb ycentrifuga - tiona t1 0OOOx gfo r2 0min .Th eprecipitate so fth etw obatche swer e redissolvedi n distilledwate rwit h theai do fa nultrasoni cbat h (Sonicor,SC-100-22H ,Farmingdale ,N.Y. , USA).Undissolve dparticle s (whichcontaine dn oenzym eactivity )wer e removedb ycentri - fugationa t1 0OOOx gfo r2 0min .Th eenzym esolution swer epoole dan ddialyse dagains t fourchange so f1 0volume so f0.0 1 mol/1sodiu mmaleat ebuffe rp H7. 0i n0. 1mol/ 1sodiu m chloride.Dialysi sbag swer e alwayspreviousl y cleanedb yboilin gi n10 0g/ 1aceti caci d andrinsin gwit hdistille dwate ran dbuffer ,sinc e appreciable losseso fenzym e activity werenotice d duringdialysi si nuntreate dbags ,especiall ya tlo wioni cstrengths . The dialysedenzym esolutio nwa s freeze-driedan dstore da sa powde r (541 0m go fprotein )a t -2 0°C .Tabl e 14present sa summar yo fth estep suse di nth eextractio no fpectinesterase .

Purification of Pectinesterase I and II Aportio no fth efreeze-drie dpowde r (715m go f protein)wa sdissolve di n2 0m lo fdistille dwater .Afte rcentrifugatio na t2 0OOOx g for2 0min ,th eclea renzym esolutio nwa sapplie dt oa Bio-Ge lP-10 0colum nfo rge l fil­ tration.Th eelutio npatter ni sshow ni nFig.12 .N omor epectinesteras ewa s detected beyondfractio n37 .Fraction s3 0t o3 3wer epoole dan ddialyse d against threechange so f 11 o f0.0 2mol/ 1sodiu msuccinat ebuffer ,p H6.0 ,containin g0. 1g/ 1o fsodiu mazid ea s

40 Table 14.Extractio no fpectinesteras efro mpeel san dpul po f5 k goranges .

Fraction Volume Pectinesterase Yield (ml) (units) (%)

Homogenizedpreparatio n 967 0 31270 0 100 First extraction Pressliqui d 750 0 20625 0 65.9 Ammoniumsulphat eprecipitatio n 365 16551 0 52.9 Second extraction Pressliqui d 650 0 4356 0 13.9 Ammoniumsulphat eprecipitatio n 201 3588 0 11.5 Poolo fdissolve dammoniu m sulphateprecipitate s 566 20139 0 64.4

preserving agent.The nth eenzym epreparatio nwa s appliedt oa colum no fcross-linke dpec - tatean delute dwit ha sodiu m chloride gradient (Fig.13).Fraction s6 t o1 6(designate d asPectinesteras e I)an dfraction s3 9t o4 4(Pectinesteras e II)wer epoole d separately. Itwa s establishedb yrechromatograph yan dals ob yisoelectri c focusingo fsom eo fth e materialo fPectinesteras eI an do fth epectinesteras e peako ffraction s2 8t o3 2tha t the latterpectinesteras e peakwa s identicalt oPectinesteras e I.Bot h PectinesteraseI andI Iwer e dialysed against0.00 5mol/ 1sodiu mphosphat ebuffe rp H7. 5wit h0. 1g/ 1 sodium azidean dapplie dt oa C MBio-Ge lA column .Th eelutio npattern s areshow ni n Fig.14 (Pectinesterase I)an dFig.1 5 (Pectinesterase II).Apparentl y thebindin go f

protein (mg/ml) pectinesterase (U/ml) 7-I 700

500

300

45 fraction number Fig. 12.Bio-Ge lP-I0 0chromatograph y ofcrud epectin ­ esterase.2 1m lconcentrate d crudeenzym ewa sapplie d toa column (85x3.2cm )o fBio-Ge lP-10 0equilibrate dwit ha solutiono f0.0 1 mol/1sodiu mmaleat ebuffe rp H7.0 , 0.5mol/ 1sodiu mchlorid ean d0. 1 g/1sodiu mazide .Elutio n wasdon ewit h thesam esolutio na ta flo wrat eo f8 m lpe rh andfraction so f8 m lwer ecollected .Protei n(o) ; pectinesterase activity(A) .

41 protein (mg/ml) pectinesterase (u/ml) cross-linked pectote

sodium chloride (M) 1.0- -200

100

50 60 fraction number Fig. 13.Cross-linke dpectat e chromatography ofth epectin ­ esterasepea kfro mBio-Ge lP-100 .3 2m lpectinesteras e from Bio-GelF-10 0wer edialyse dagains t0.0 2mol/ 1sodiu m succinatebuffe rp H6 wit h0. 1g/ 1sodiu mazid e andapplie d toa cross-linke dpectat e column (10.0x2.0cm )equilibrate d withth esam ebuffer .Th ecolum nwa selute dwit h equilibrationbuffe ru pt ofractio n2 2an dthe nwit ha linea r gradiento fsodiu mchlorid ei nth esam ebuffer .Th eflo wrat e was2 5m lpe rh an dfraction so f 10m lwer ecollected . Protein (o);pectinesteras e activity (A)an dsodiu mchlorid e concentration ( ).

PectinesteraseI t oC MBio-Ge lA isno tver ystron gsinc eprolonge dwashin go fth ecolum n withequilibratio nbuffe r (withoutsodiu mchloride )cause sth eenzym et oelut ea sa tail ­ ingpea k (resultno tshown) .Th epurificatio nprocedur eo fPectinesteras e Ian dI Ii s summarizedi nTabl e 15.Th etota lyiel do fpurifie dpectinesterase swa s 26.3?,an dPectin ­ esterase Ian dI Iappeare dt ooccu rapproximatel y ina tw ot oon erati oi nterm so funit s inth ecrud epectinesteras epreparation . Thespecifi cactivit yo fth etw oenzyme scoul dno tb eincrease db yrechromatograph y orapplicatio no fothe rchromatograph ystep ssuc ha shydroxylapatit eadsorptio nchromato ­ graphy.I tma yb eo finteres tt orepor ttha ta neight-fol dpurificatio no fth ecrud e enzymepreparatio ncoul db eobtaine db ychromatograph yo na hydroxylapatit ecolumn , equilibratedwit h0.0 1mol/ 1sodiu mphosphate.buffe rp H6. 8 andelutio nwit ha linea r gradientu pt o0. 2mol/ 1o fth esam ebuffer .

Electrophoretic properties, molecular weights and isoelectric points Sampleso fu pt o 5u go fpurifie dPectinesteras e Ian dPectinesteras eI Iwer eapplie dt oth ewell so fa slabge lfo rsodiu mdodecy lsulphat eelectrophoresis .Onl ya singl eban do fprotei nwa s observedwit hbot hpectinesteras epreparations .Th edetectio nlimi to fthi stechniqu ei s 0.2 ugo fprotei ni na singl eband .Bot henzyme sha dth esam emobility ,whic hcorresponde d toa molecula rweigh to f3 620 0(Fig.16) . Theisoelectri cpoin to fth etw opectinesterase swa sdetermine db yisoelectri c focusingi na colum n (Fig.17).Fo rPectinesteras eI a nisoelectri cpoin to f 10.05+0.05a t 6 Cwa sfound .Tha to fPectinesteras eI Iwa sa to rabov e 11.0,a valu eto ohig ht ob e determinedaccuratel ywit hth ecommerciall yavailabl ecarrie rampholytes .Onl y48 ?o fth e

42 pectinesterase (u/ml)

protein (mg/ml) -600 specific activity ( u/mg) •KH-1000 CM Bio-Gel A

05-500 --200

1 -(-=S= ~r-f—?—y—f—y- ?-.? 10 35 fraction number 50 K» »0 200 250 elution volume (ml) Fig. 14.C MBio-Ge lA chromatography ofPectinesteras e I.Fraction s 6 to 16 (110ml )fro m thecross-linke d pectate columnwer e dialysed against 0.005mol/ 1 sodium phosphate bufferp H7. 5wit h 0.1g/ 1 sodium azide andapplie d toa C MBio-Ge l A column (18x1.1 cm)equi ­ librated with thesam e buffer.Th ecolum nwa swashe d with equili­ bration buffer (40ml )an dthe n eluted with a linear sodium chloride gradient inth esam e buffer.Th eactivit y peakwa scollecte d in5 m l fractions.Protei n (o);pectinesteras e activity (A);sodiu m chloride concentration ( );specifi c activity (*).

protein (mg/ml) pectinesterase (u/ml)

300

200

100

35 45 fraction number 100 200 300 elution volume (ml) Fig. 15.C MBio-Ge lA chromatography ofPectinesteras e II.Frac ­ tions 39t o4 4(6 0ml )fro m thecross-linke d pectate column were dialysed against 0.005mol/ 1sodiu m phosphate bufferp H7. 5wit h 0.1 g/1sodiu m azide andapplie d toa C MBio-Ge l A column (18x1.1 cm)equilibrate d with thesam e buffer.Th ecolum nwa s washed with equilibration buffer (90ml )an d then eluted witha linear sodium chloride gradient inth esam e buffer.Th eactivit y peakwa scollecte d in5 m l fractions.Protei n (o);pectinesteras e activity (A);sodiu m chloride concentration ( );specifi c activity (*).

43 Table 15.Purificatio no forang ePectinesteras eI an dII .

Step of purification Volume Activity Protein Specific Purifi­ Yield (ml) (units) (mg) act ivi ty cation (%) (units/ factor mg)

Homogenized preparation 1 278 40 900 6 1001 7 1 100 Ammonium sulphate precipitation 71 26 350 951 28 4 64.4 Dialysis and freeze drying2 21 20 180 715 28 4 49.3 Chromatography on Bio-Gel P-100 33.5 15 360 48 319 48 37.7 Pectinestera.se I Chromatography on cross- linked pectate 110 8 060 13.6 594 89 19.7 Chromatography on CM Bio-Gel A 15 7 160 10.3. 694 104 17.5 Peetinesterase II Chromatography on cross- linked pectate 60 4 300 8.1 527 79 10.5 Chromatography on CM Bio-Gel A 20 3 600 4.7 762 114 8.8

1.Kjeldah lnitroge ndeterminatio nx 6.25 . 2.Thi sfreeze-drie dpreparatio ni sdesignate d in thetex ta scrud epeetinesteras e preparation.

activityo fPeetinesteras eI Iwa srecovere di nthi sfocusin gexperiment .Apparentl yenzym e inactivationoccurre di nth ealkalin eelectrod esolution .I na focusin gexperimen twhic h wasterminate dprematurely ,th egreate rpar to fth eenzym eremaine dbelo wp H 11an dove r 904o factivit ywa srecovered .Bot hpectinesterase smigrate da ssingl epeak si nth e focusingexperiments .Moreover ,n oothe rprotei npeak swer emonitore da t28 0ran (Fig.17) . Comparedwit hpectat e lyase (Romboutse tal. , 1978)th epectinesterase smove dextreme -

log (molecular weight) 50n'

\,

\ \ \ " \ N,.pectinesterase s Ian d I - \7~ Fig. 16.Molecula rweigh tdeterminatio no f \^5 PeetinesteraseI an dII .Sodiu mdodecy l sulphateelectrophoresi so fenzym ean d standardprotein swa sdon e ina sla bge l witha fina lacrylamid econcentratio no f 120g/1 .Standards :bovin e serumalbumi n(1 ) 42 0.2 0.4 Q6 Q8 beeflive rcatalas e (2);ovalbumi n(3) ; Rfvalu e chymotrypsinogen (4)an d tripsinogen(5) .

44 PH A pectinesterase(u / -X )f—X-. 12- -50 \ x « ji1 1 \ 1 [ E 280

10

^-*-x-x,„ 1 1 0.6-

8 •10

PH pectinesterase (u/ml) 12 h20 / \ 0-

E 280 0.6- 10 I I 0.4-

(* 'v. 02- 0

20 40 60 elution volume (ml) Fig. 17.Isoelectri c focusingo fPectinesteras e I andII .Th eenzyme s (300units )wer efocuse d asdescribe d inMaterial s andMethod s(Chap ­ ter 3). A:Pectinesteras e I,85 %o factivit y recovered.B :Pectinesteras eII ,48 %o factiv ­ ityrecovered .p H (x);pectinesteras eactivit y (A);absorbanc e at28 0n m( );absorb - ancea t28 0n mi na nidentica l runwithou t enzyme ( • ).

lyslowl ya tp Hvalue sclos et othei risoelectri cpoints .Apparentl yth ene tcharg eo f thesemolecule snea rthei risoelectri cpoint si sver ylow .

Occurrence of other enzyme activities Inorde rt oasses sth estat eo fpurit yi nterm so f absenceo fothe renzym eactivities ,variou ssubstrate swer eincubate dwit h3 unit so f pectinesterasefo r2 0h a t3 0° C (Table 16).I nadditio nt opurifie dPectinesteras eI an d IIth epreparation ,obtaine db yammoniu msulphat eprecipitatio n (crudeenzyme ,Tabl e15) , wastested .Th ecrud epreparatio ncontaine da variet yo fothe renzymes ,som eo fwhic hma y beinvolve di nclou dlos sphenomena :ß-(1->-3)-glucanase ,ß-(1->6)-glucanase ,amylase , ß-(1->4)-xylanase,g-(1->-3)-galactanase ,arabanase ,a an d ßglucosidase ,a an dß galactosi - dase,fosfatas ean dcarboxylesterase .Non eo fthes eenzym eactivitie scoul db edetecte d inth epurifie denzym epreparations . Proteaseactivity ,no tdetecte da tp H 5.0wit hazocol la ssubstrate ,coul db edemon ­ stratedi nth ecrud epreparatio na tp H7. 5wit hstrip so fexpose dan ddevelope dphoto ­ graphicnegativ efilm ,a techniqu edescribe db yKro p (1974).N opolygalacturonas ewa s detectedi nth ecrud epreparation .Rio v (1975)reporte dth epresenc eo fextremel ylo w

45 Table 16.Enzymati c activityo fcrud ean dpurifie dpectinesteras epreparation so n varioussubstrates .

Supplier1 Substrate Typeso fmonomer s Activityi n andlinkage s preparations 2

crude I II

Dow methyl cellulose ß-1 ,4(glucose ) Difco cellobiose ß-1 ,4(glucose ) ICN laminarin ß-1 ,3;ß-1,6(glucose ) Lab pachyman ß-1 ,3(glucose ) Calbiochem pustulan ß-1 ,6(glucose ) Lab yeastgluca n ß-1 ,3;ß-l,6(glucose ) K& L lichenan ß-1 ,4;ß-1,3(glucose ) F.C.&R . chitosan ß-1 ,4(glucosamine ) F.C.&R . chitin ß-1 ,4(N-acetylglucosamine ) Difco solublestarc h o-l ,4;a-l,6 (glucose) K& L nigeran a-1 ,4;a-l,3 (glucose) ICN dextran a-I ,6;a-l,3;a-l,4 (glucose) Merck inulin ß-1 ,2(fructose;a-glucose ) K& L xylan ß-1 ,4(xylose ) K& L galactan ß-1 ,3(galactose ) K& L araban a-1 ,5;a-l,3 (arabinose) K& L mannan a-1 ,6;a-l,3;a-l,2(mannose ) ICN polygalacturonate a-1 ,4(galacturoni cacid ) K& L p-nitropheny1-ß-D-glucoside K& L p-nitropheny1-a-D - •glucos ide K& L p-nitrophenyl-S-D-galactoside K& L p-nitropheny1-a-D--galactoside K& L p-nitrophenylphosphate K& L p-nitrophenylmyristät e Calbiochem azocoll

1.Suppliers :Dow ,Do wChemica lInternationa lS.A. , Brussels;Difco ,Difc oLaboratories , Detroit;ICN ,IC NPharmaceuticals ,Inc. ,Cleveland ,Ohio ;Lab ,thi slaboratory' s collection;Calbiochem , SanDiego ,Calif. ;K & L ,Koc han dLigh tLaboratories ,Ltd. , Colnbrook,Bucks ,England ;F.C .& R. ,Foo dChemica lan dResearc hLab. ,Inc. ,Seattle , USA;Merck ,E.Merck ,Darmstadt ,Wes tGermany . 2.Activit y detected (+).n oactivit y detected (-).

activitieso fpolygalacturonas e inpee lan dpul pfro morange .Thi senzym eactivity ,how ­ ever,coul donl yb emeasure dwhe nprecaution swer etake nt oinhibi turoni caci doxidase , alsopresen ti norange .

Amino aaid composition About 100u go fth eenzym ewer ehydrolyse d (induplicate )afte r lyophilization.I na paralle lexperiment ,th esam ewa sdon efo rsolution scontainin g 100u gbovin eseru malbumi n (SigmaA 4378 )an da mixtur eo fcommo nL-amin oacids ,abou t 3u geach .Thes epreparation swer eanalyse do na namin oaci danalyse rtogethe rwit h standardmixture so famin oacid swhic hha dno tbee nsubjecte dt ohydrolysi sconditions , todetermin eth erespons efactors .Bot hi nth eamin oaci dstandar dwhic hha dbee nsub ­ jectedt ohydrolysi scondition san di nth ebovin eseru malbumin ,cystin ean dcystein e werecompletel yoxidize dt ocystei cacid .Usin gth erecover yfactor sfro mth eamin oacid s whichha dbee nsubjecte dt ohydrolysi sconditions ,th ecystein econtent so fth ebovin e serumalbumi nan dth eenzyme swer ecalculated .Methionin ewa snearl ycompletel ylos ti n

46 allth ehydrolyse dprotein san di nth eamin oacid swhic hha dbee nsubjecte dt ohydrolysi s conditions.Th eexperiment swer e repeatedwit hbette rprecaution st oexclud eoxyge ndurin g hydrolysisan dth eamin oacid swer e analysedb yga schromatograph y (Chapter 3).No wth e recoveryo fmethionin ewa snearl y completean dthes evalue swer euse di nth edat apresen ­ ted. The tryptophandeterminatio nb yth emetho do fPajo t (1976)yielde d highervalue s than themetho do fOpleftska-Blaut he tal . (1963),presumabl y becauseth eprotein swer eno t enzymaticallyhydrolyse dbefor eth ereactio na sdescribe di nth elatte rmethod .Besides , inthi smetho dth etryptopha nstandard san dth esample s appearedt ohav ehardl yan y absorbancea tth eindicate dwavelengt ho f54 5n mbu tha da nabsorbanc emaximu mnea r45 0nm . Therefore this latterwavelengt hwa sused . Theamin o acidcompositio no fbovin e serum albuminfoun db yth eamin oaci d analyser was verysimila rt oth eliteratur evalue s (Reeck, 1970).Al lvalue swer ewithi n 2%fro m the literaturevalues ,excep tphenylalanin e (51deviation) ,arginin e (104deviation ) and methionine (751deviation) .I nTabl e 17th eamin o acidcompositio no fth eorang epectin - esterasesar elisted ,usin gth evalue so fth eamin oaci d analysertogethe rwit hth egas - chromatographicvalue sfo rmethionin ean dth espectrometri cvalue sfo r tryptophan. The recoveryo fbovin e serumalbumi ni nth eamin oaci d analyserwa s98 4(valu ecorrec ­ tedfo rtryptopha nan dmethionine) .Assumin gth esam e recoveryfo rth epectinesterases , thecorrelatio n factorbetwee n E28 0n man dth eprotei nconcentratio nfo rPectinesteras e

Table 17.Amin o acid composition ofpurifie d orange pectinesterases.

Aminoaci d PectinesteraseI PectinesteraseI I

mol% mol/mol1 mol% mol/mol1

Cys2 3.13 9 1.79 5 Asp3 11.06 32 9.69 28 Thr 8.62 25 7.94 23 Ser 6.37 19 7.28 21 Glu* 6.77 20 7.34 21 Pro 3.69 11 3.57 10 Gly 8.87 26 11.05 32 Ala 11.55 34 10.80 31 Val 8.00 23 7.87 23 Met5 1.55 5 1.56 5 He 5.33 16 5.31 15 Leu 5.31 15 4.64 14 Tyr 2.12 6 1.36 4 Phe 4.91 14 5.60 16 Trp6 2.28 7(4) 7 1.87 6 (4)7 Lys 3.17 9 4.66 14 His 1.00 3 2.35 7 Arg 6.30 18 5.29 15

1.Neares tinteger . 2.Determine d ascystei c acid. 3.Asparagin e +asparti c acid. 4.Glutamin e +glutami c acid. 5.Methionin e gaschrpmatographically . 6.Tryptopha n withmetho do fPajo t (1976). 7.Tryptopha n withmetho do fOpienska - Blauthe t al. (1963).

47 I is 0.569 (0.569 x E 280ran = mgprotein/ml ) and for Pectinesterase II is 0.626. For 1 TL PectinesteraseI E, canb eestimate da s17.6 ,an dfo rPectinesteras eI Ia s 16.0.The ^ lc m availablequantitie so fpur eenzyme swer einsufficien tt odetermin ethes evalue smor e accuratelyb yweighing . Fig.18show sth eabsorbanc espectr ao fth epectinesterase si nneutra lenvironmen tan d in0. 1mol/ 1potassiu mhydroxide .Usin gth eformul ao fBeave n& Holida y (1952):

MTyr 0.592.g294.4 - 0.263.E280.0 MTrp 0.263.E280.0- 0.170.2294. 4 forth espectr ai n0. 1mol/ 1potassiu mhydroxid egav emuc hlowe rtryptopha nvalue stha n listedi nTabl e 17.Bu ta sstate db yLegge «Bail y (1967),th eabsorbanc espectr ao f tyrosinean dtryptopha ni nprotein sma yshif t 1-3ru nt ohighe rwavelengths .

Glycoprotein character About2 m go fbot hpectinesterase san dovalbumi n (Sigma,A-2512 , GradeVI )wer ega schromatographicall yanalyse dfo rsugar sa sdescribe di nmaterial san d methods (Chapter 3).Standard scontaine drhamnose ,arabinose ,xylose ,mannose ,glucose , inositol (internalstandard) ,glucos eamine ,galactos eamin ean dmannos eamine .Afte r correctionfo rth etrac eo fglucose ,contaminate dfro mth edialysi sbags ,i tappeare d thatnon eo fbot hpectinesterase scontaine dan yo fth esugar smentioned .I nth eparalle l experimentwit hovalbumi n1.7 %mannos ean d1.1 %glucos eamin ewa sfound .Th eliteratur e valuesar e2 %an d 1.2%respectivel y (Winzler,1970) .Th emetho dwa ssensitiv eenoug ht o determine 1mo lo fa sugar/ 1mo lo fenzyme .

Fig. 18.Ultraviole tabsorbanc espectr ao f purifiedpectinesterases .Enzym econcentra ­ tions:46 0units/m lfo rPectinesteras eI ,a 320units/m lfo rPectinesteras eII .I n0. 1 mol/1potassiu mhydroxid e theenzym econ ­ centrationswer e 10%lower .I .Pectinester a I inneutra lenvironment .II .Pectinestera s IIi nneutra lenvironment .I- Aan dII- Ath e 270 310 330 samei n0. 1 mol/1potassiu mhydroxide . wave length (nm)

48 5.2 DISCUSSION

Extraction and purification of the two peotinesterases Thefirs tste pi nth epreparatio n oforang epectinesteras ewa sth eseparatio no fjuic e frompee lan dpulp .Th ejuic ewa s discarded sincei tcontaine donl y traceso fpectinesteras e (

Properties of the enzymes Onth ebasi so fthei rmobilit yi nsodiu m dodecyl sulphate electrophoresis,bot henzyme s appearedt ohav e thesam emolecula rweigh to f3 6200 .Thi s molecularweigh twa s also indicatedb yge l filtrationo nBio-Ge lP-10 0i n0. 5mol/ 1 sodium chloride.Apparentl y bothenzyme sar ecompose do fa singl epeptid echain .I ti s thereforeunlikel y thaton eo fth e formsi sderive d from theother ,e.g .b ydissociatio n ofsubunit so rb ylimite dproteolysis .A ssuggeste db ythei rdifferen t isoelectric points (Fig.17),th etw oenzyme swil l havegeneticall y determined differencesi nprimar ystruc ­ ture,an dcoul d thereforeb ecalle d 'isoenzymes'o r'isozymes ' (CBN, 1971).Thi swa s confirmedb yth eamin oaci danalysis . Twopectinesteras e isoenzymeso fmolecula rweigh t3 000 0wer e recently foundi nbanan a (Brady, 1976).Thes e isoenzymeswer e separatedb yisoelectri c focusingan dthei riso ­ electricpoint swer e8. 8an d9.3 ,wel lbelo w thoseo fth eorang e isoenzymes (10.05an d

49 >11,0).Th emultipl e formso ftomat opectinesteras e have differentmolecula rweight s ranging from 2430 0t o3 5 500 (Pressey &Avants , 1972). Isoelectricpoint so f 7-9.3 have beenreporte d fortomat opectinesterase s (Delincée &Radola ,1970 ;Delincée , 1976). The aminoaci dcompositio n ofth eenzyme s differed considerably oncystin e +cysteine , asparagine +asparti cacid ,glycine ,tyrosine ,lysin e andhystidine .Th e isoelectric points cannotb eexplaine dwithou t alsoknowin g asparagine,asparti c acid,glutamin ean d glutamicaci dseparately .Comparin g theamin oaci dcomposition swit h theamin oaci d com­ position oftomat o (Markoviif& Sajgö , 1977;Nakagaw ae t al., 1970b)o r Citrus natsudaidai orangepectinesteras e (Manabe,1973a )reveal s thatno ton eo fth epectinesterase s has the sameo rnearl y thesam eamin oaci d composition. In literature suggestions havebee nmad e about theglycoprotei n charactero f tomato andplu mpectinesterase s (Therone t al., 1977a; 1977b).Thes ewer e contradicted byMarkovi c (1974)fo ron e formo ftomat opectinesteras e andb yMarkovi ce t al. (1975)fo r6 formso fbanan apectinesterase .Th e twopectineste ­ rasespurifie d inthi swor k couldb e demonstrated not tob e glycoproteins.

50 6 Multiple forms of pectinesterase in navel orange and other citrus fruit

InChapte r5 onl ytw oisoenzyme s arepurifie dan ddescribed ,wherea si nsom eothe r fruitsi xt oeigh t formso fpectinesteras ehav ebee ndetecte d (Delincée, 1976;Markovii f et al., 1975).Difference si npropertie so fpectinesterase s fromdifferen tcitru s fruits areknow nt oexist ,suc ha shea tstabilit y (Rouse& Atkins , 1952;Rous e& Atkins ,1953a ) andjuic e clarifyingpropertie s (Krop,1974) .Fo rthes e reasonsa searc h formor emultipl e formso fpectinesteras ei nNave lorange san dothe rcitru s fruitswa sinitiated .

6.1 EXPERIMENTSAN DRESULT S

6.1.1 Multiple formso fpectinesteras ei nNave lorang e

Search for molecular forms of pectinesterase Crudepectinesterase ,obtaine db yammoniu m sulphateprecipitatio no fth eextrac tfro mpee lan dpul pwa s subjectedt oisoelectri c focusing.Th ecolum nwa s loadedwit ha sampl eo f1 0m lo fthi spreparatio n (250 0pectin ­ esteraseunits ,8 5m gprotein) ,an dcarrie rampholyte swer euse dt oobtai na p Hgradien t fromp H4 t o11 .Larg epectinesteras epeak swer e founda tp H1 0(Pectinesteras eI ) and justabov e 11.0 (Pectinesterase II).I nadditio n fouro rmor esmal lactivit ypeak swer e foundi nth ep Hrang eo f6.0-9.6 .However ,th ecombine d activityo fthes esmal lpeak s accountedfo rles s than4* »o fth etota l activityo fth esample .Moreover ,contaminatin g materialprecipitate di nheav yband s throughoutth ecolum ndurin g focusing.I ti slikel y thatsmal lamount so fpectinesteras ewer e thusprevente d frommigratin gt othei riso ­ electricpoint .Further ,th emino rpea ko fpectinesteras ei nfraction s2 0t o2 7fro mBio - GelP-10 0 (Fig. 12)wa sals osubjecte dt oisoelectri c focusingan dgav ea majo r activity peakwit ha nisoelectri cpoin to f10. 2an da trac eo factivit ywit ha nisoelectri cpoin t >11. Whenth ehea t stabilitieso fth epur ean dcrud eenzym epreparation swer e determined, therewa sa smal lhea tstabl e fraction (about6 1o fth etota lactivity )i nth e crude enzymepreparation ,whic hcoul dno tb eascribe dt oPectinesteras eI o rII .I tappeare d thatmos to fth epectinesteras ei nth e fractions2 0t o2 7fro mBio-Ge lP-10 0 (Fig.12 ) wasmor ehea tstabl e thaneithe rPectinesteras eI an dII .Rechromatograph yo fthes e fractionso nBio-Ge lP-10 0 resultedi nth e sameelutio nvolum ea sbefore .Becaus eo fth e highly impure stateo fth eenzym e (specificactivit y9 units/m gprotein )th emolecula r weight couldno tb edetermine dwit h sodiumdodecy l sulphateelectrophoresis .Rechromato ­ graphyo f1 0m lcontainin g 120unit so fpectinesteras ewa sdon eo nBio-Ge lP-10 0 under thesam eexperimenta l conditionsa sdescribe dbefor e (Fig.12)i na mixtur ewit h1 0m g blue dextran2 00 0(Pharmacia ,Uppsala ,Sweden ,mol.wt .2 00 0000) ,1 0m gbovin e serum albumin (Boehringer,Mannheim ,mol.wt .6 7000) ,an d30 0unit s PectinesteraseI + I I

51 Ve/\*> 2-

fractions 20-27 from \ Bio-Gel P-KX)

" vrmd.wt . 54000 Fig. 19. Molecular weight estimation of fractions 20-27 from Bio-Gel P-100.A sample of fractions 20-27 from Bio-Gel P-100(Fig . 12)wa sre-applie d ,I toth ecolum n together with standard proteins under thesam e column conditions asdescribe dbe - 0+*^ ' ^ ' 5 fore (Fig. 12).Standards : Pectinesterase I+ I I log(molecula rweight ) (1);bovin e serum albumin (2).

(mol.wt. 36200 )t oestimat e themolecula r weight ofthi s small pectinesterase peak. Proteins were measuredb yabsorbanc e at28 0n man dfraction s were assayed forpectineste ­ rase activity. Thelo gmolecula r weightwa splotte d against V /V. {V=elutio n volume protein, 7-=elution volume blue dextran)(Fig. 19).Th emolecula r weightwa sroughl y esti­ mated tob e5 400 0an dthi s pectinesterase will becalle d high molecular weight pectin­ esterase. For crude enzyme preparations column isoelectric focusing was unsuitable todetec t with certainty other molecular formso fpectinesteras e because ofth eflocculatio no f contaminating proteins.Als o thin layero rge lisoelectri c focusing couldno tb euse d because thehighes t obtainable pHi nthes e gels isabou t 10(Winte re tal. , 1975)an d several pectinesterases were expected tohav e isoelectric points abovep H 10.I npreli ­ minary gelisoelectri c focusing experiments, Pectinesterase IImigrate d considerably faster towards thecathod e than Pectinesterase I,bu teventuall y both enzymes disappeared inth ecathod e strip. This observation resulted inth edevelopmen t ofa preparativ e flat bedp Hgradien t electrophoresis ina granulate d gela sdescribe d inmaterial s andmethods . Crude Navel orange pectinesterase (50unit s in0. 3ml )wa sintroduce d asa narro w band over theful l width ofth ege lan dth epowe r was switched offbefor e PectinesteraseII , the fastest migrating pectinesterase, reached thecathod e end. Fig.2 0show s thephoto ­ grapho fa stri p used forpectinesteras e detection with theprin t technique andFig .2 1 shows theactivit y recovered from thescrape d offge lzone s with adrawin g fromth e print. Byusin g pure Pectinesterase Ian dI Ian dth ehig h molecular weight pectinesterase as references,3 o fth e5 pectinesteras e active zones couldb eidentifie d as indicated in Fig. 20.I np Hgradien t electrophoresis most ofth ehig h molecular weight pectin­ esterase migrated slightly faster than Pectinesterase I,a trac e asfas t asPectineste ­ rase IIan da trace slightly slower than Pectinesterase I (results notshown) . Because some ofth eband s arerathe r close toeac h other,th equantitativ e analysis ofth e scraped offzone s showed apoo r resolution (Fig. 21). Homogenizationan dpressin g inth eextractio n procedure ofpectinesteras e wasnormal ­ ly done atroo m temperature (Chapter 5).I na ne wpurificatio n procedure, these initial steps were donea t4 C.Afte rge lfiltratio n ofth ecrud e enzyme onBio-Ge l P-100 (as in Fig. 12),th efractio n ofhig h molecular weight pectinesterase represented exactlya s

52 pole

PectinesteraseI I

highmolecula rweigh tpectine steras e

PectinesteraseI

applicationpoin t

+pol e

Fig.20 .p HGradien telectrophoresi so fcrud e Navelorang epectinesterase .Photograp ho fprin t forpectinesteras edetection ,ligh tzone sar e pectinesterase active (formetho dse eChapte r3) . muchenzym ea si nth enorma lprocedur e (61o fth etota lrecovere dactivity) .Therefor ei t seemsunlikel ytha tPectinesteras eI o rI Iar ederive dfro mth ehig hmolecula rweigh t pectinesterasedu et osom ekin do fdegradatio ndurin gextraction .

pectinesterase units/ fraction PH

K> r / y I1 -"-",11 s / Fig.21 .p HGradien t electrophoresiso f JÜMM jpi crudeNave lorang epectinesterase .Afte r 20 3J0 taking theprin t forpectinesteras ede ­ zone number tection,th ege lwa s segmented and thep H of thesegment smeasured .Th esegment s polei Ipole wereextracte d andassaye d forpectin ­ activity print esterase activity.p H( • •)•

53 Composition of the high molecular weight pectinesterase Thehig hmolecula rweigh tpectin ­ esteras ewa s chromatographedo ncross-linke dpectat eunde rth econdition sa suse di n Section5. 1fo rth e separationo fPectinesteras eI fro mPectinesteras e II.Fig .2 2show s theresult so fthi sexperiment .Th ehig hmolecula rweigh tpectinesteras ewa s separated into3 peak s ofwhic hth efirs tpea ki nth esal tgradien t (HMII )wa sdominan t (85$o f thetota l activity).Thi spea k seemedt ob ea mixtur eo fthre eo rperhap smor e forms, buteve n after repeated chromatographyo fthi smateria li twa s notpossibl et oestablis h thenumbe ro fform sinvolved .Th eenzyme si nthre edifferen t fractionso fthi spea k (fractions 36,3 9an d41 )migrate d equally fasti np Hgradien t electrophoresis runs; slightly faster thanPectinesteras eI .Th eactivit yo fPectinesteras eH MIwa slos tafte r pH gradientelectrophoresi s experiments andPectinesteras eH MII Imigrate da sfas ta s Pectinesterase II.I tca nb esee ni nFig .2 2tha tPectinesterase sH MI Ian dH MII Iar e purifiedt oa goo dexten to nth ecross-linke dpectat e column (about2 0an d1 0fold , respectively).

6.1.2 Multiple formso fpectinesteras ei ncitru s fruit

Fivek gportion so fsevera l citrus fruitswer epresse d andmixe dwel lwit h thepress ­ edjuic e filtratea sdescribe di nSectio n5. 1an dfroze ni nportion so f20 0g .Th epectin ­ esterasewa s extracted froma 20 0g portio n andprecipitate dwit h ammoniumsulphat ea s describedi nSectio n 5.1.Th e30-75 $ ammonium sulphateprecipitat ewa s redissolvedi na minimum amounto fdistille dwate ran ddialyse d against1 0g/ 1glycin e sodiumhydroxid e

pectinesterase (u/ml) 1-15

protein (mg/ml) 1.2

fraction number 100 200 •400 elutkm volume (ml) Fig. 22. Cross-linked pectate chromatography of the high mo­ lecular weight pectinesterase. 700 Units of pectinesterase (9 units/mg protein) from fractions 20-27 of Bio-Gel P-100 (Fig. 12) were applied to a cross-linked pectate column as described in Fig. 13. Protein (o); pectinesterase activity (A) and sodium chloride concentration ( ).

54 bufferp H7.0 .Th edialysi sbag swer epreviousl ytreate dwit haceti caci dan drinse di n wateran dbuffer .Sometime sth eammoniu msulphat eprecipitate swer ehar dt oredissolve . Then 1unit/m lo fyeas tendo-polygalacturonas ewa smixe dthroug hth egel-lik eprecipitat e andlef tovernigh tbefor ebein gtreate dfurther .Thi sste pthe nincrease dgreatl yth e pectinesteraserecovery . Approximately 150 0unit so fth ecrud epectinesterase swer euse dfo rcolum nisoelec ­ tricfocusin gi nth ep Hrang e9-1 1an dapproximatel y 75unit swer euse dfo rth ep H gradientelectrophoresis .Th eextractio nwa srepeate dfo reac hcitru sfrui tan dapproxi ­ mately7 5unit swer euse dfo ra secon dp Hgradien telectrophoresis .I nal lcitru sfruit s Pectinesterases Ian dI Iwer eidentifie db ythei risoelectri cpoint s (10.05an d>1 1res ­ pectively)i ncolum nisoelectri cfocusin gexperiments .Pectinesteras e Ian dI Iwer eth e majorpectinesteras eenzyme si nal lcitru sfruit san dn oothe rmajo rpectinesteras epeak s werefound .Frequentl ya shoulde rcoul db eobserve da tth ehig hp Hsid e (pH10.2 )o fth e PectinesteraseI peak .I nthos einstance s (lemon,grapefrui tan dmandarin) ,p Hgradien t electrophoresisdemonstrate dth ehig hmolecula rweigh tpectinesteras e tob epresen ti na quantityo fmor etha n20 $o ftha to fpectinesteras eI .Th eresul to fth egrapefrui t pectinesterasecolum nisoelectri cfocusin gi spresente di nFig .23 .I nal lfruits ,excep t ingrapefruit ,a smal lpea kcoul db eobserve da sa shoulde ro fPectinesteras e Ia tp H9.6 . Therecover yi ncolum nisoelectri cfocusin gexperiment samounte dt oabou t75 %o fth etota l activity. Drawingso fth eprint sfo rpectinesteras eactivit yi nth ep Hgradien telectrophoresi s experimentsar egive ni nFig .24 .Compare dwit hcolum nisoelectri cfocusing ,thin-laye r pHgradien telectrophoresi sgav emuc hbette rseparatio no fPectinesteras e Ifro mth ehig h molecularweigh tpectinesteras ean dals oallowe ddetectio no fmor emolecula rforms .I n

pectinesterase (u/ml) 75

40 50 elution volume (ml ) Fig. 23.Isoelectri c focusingo fcrud egrapefrui tpectine ste - rase Theenzyme s (1000units )wer efocuse da sdescribe di n Chap ter3 ,activit yrecover y 75%.p H (x);pectinesteras e activity(A) .

55 . applicationZon e „ polet 1 2 3 pole

20 25 30 zonenumbe r Q2 04 0608 10 Rfvalu e Fig. 24.p HGradien t electrophoresis patternso f crude pectinesterases from different citrus fruits. Thep Helectrophoresi s experiments were donea s described inChapte r 3.Drawing s were made from the prints forpectinesteras e detection. Light zones arepectinesteras e active. Identified zones: Pectinesterase I (1);hig h molecular weight pectinesterase (2)an dPectinesteras e II (3).

spite ofth edetectio n ofactivitie s asseparate d zones inth eprints ,th ezone sdi dno t always show distinct activity peaks iconcollection . Therefore theactivit y measurements ofth ecollecte d bands arecompile d in4 group s inTabl e 18.Th evalue s areth eaverage s oftw op Hgradien t electrophoresis experiments.Th egrou p with amigratio n rate between Pectinesterase Ian dI Irepresent s mainly thehig h molecular weight pectinesterasei n recovered activity (90-100%), except forth emandarin s (about 50%).Th emigratio n distance of Pectinesterase IIan dth erelativ e migrations (Äf values)o fPectinesteras e I (flf =0.55) and thehig h molecularweigh t pectinesterase (f?f=0.68 )wer e reproducible.Th eactivit y recovery ofal lpectinesterase s togetherwa srathe r low(abou t 60%). Theprin t technique allowed detection ofzone s toabou t 2%o fth etota l activity (=1 uni t pectinesterase/ zone). This sensitivity couldb einfluence d byth econtac t timeo fge lan dprintpaper . Buta to olon g contact (more than 1min ) resulted infadin g ofdar k zones betweentw o close bands with ahighe r activity.

Table 18.Relativ e quantities ofpectinesteras e forms incitru s fruits.

Fruit Migration rates

Äf<0.55 fff=0.55 0.55<Äf

Pectin­ Pectin­ esteraseI esteraseI I

Navelorang e 5.1 62.0 5.7 27.2 Salustianaorang e 3.1 58.2 11.3 27.4 Shamoutiorang e 10.3 58.4 6.8 24.5 Lemon 10.7 45.6 9.1 33.6 Grapefruit 7.3 45.8 33.2 13.7 Mandarin 14.2 44.2 10.2 31.4

56 6.2 DISCUSSION

Thin-layerp Hgradien telectrophoresi s combinedwit h theprin t technique ofDelincé e (1976)gav ebette rresolutio no fth ealread yknow npectinesterase s and allowed detection ofsevera lothers .Th ehig hmolecula rweigh tpectinesteras ewa sno tdetecte d incolum n isoelectric focusingexperiment s because its isoelectricpoin tdiffer sonl yver y little fromPectinesteras e I.Th esmal lpea ko fhig hmolecula rweigh tpectinesteras e disappeared underth ePectinesteras e Ipeak .Flocculatio n inth euppe rpar t ofth e isoelectricfocu ­ singcolum nmad edetectio no fform swit h isoelectricpoint s lowertha nPectinesteras eI difficult. Themolecula rweigh t ofth ehig hmolecula rweigh tpectinesteras ewa s roughlyestimate d tob e 54000 .Thi s ismuc h lesstha nth edoubl eo f themolecula rweigh to fPectinesteras e I and II (2x36 200= 72400) . Ifth ehig hmolecula rweigh tpectinesteras e is relatedt o Pectinesterase Io r II, asimpl edissociatio no fa doubl emolecul e isunlikel y tob e thecause .Th e complexity ofth esituatio n isillustrate db y Fig. 22.Th emajo r formo f thehig hmolecula rweigh tpectinesteras e (peakH M II,Fig . 22)i scontaminate dwit hothe r forms,whic hhav e thesam emolecula rweigh tbu tdiffe rwit h respect toaffinit y tocross - linkedpectat e and chargeproperties .Th emajo r formo fhig hmolecula rweigh tpectin ­ esterasema y consistou to fsevera lmor e forms.I np Hgradien t electrophoresis,th e three pectinesteraseswit h known isoelectricpoint smigrat e inorde ro f theirisoelectri cpoints . This isno tnecessaril y thecas e foral lpectinesterases . Incitru s fruits twelve formso fpectinesteras e couldb erecognize d (Fig.24) . Maximal­ lyte nform so fthes ewer epresen t inon e citrus fruit (grapefruit).Pectinesteras e Ian d II together always representedmor e than 60% ofth e total activity (Table 8)an d in oranges alwaysmor e than 80%. Inoranges ,th e concentrationquotien t ofpectinesteras eI topectinesteras e IIwa s 2.1-2.4.I ngrapefrui t therewa smor ePectinesteras e Ian d in lemonsan dmandarin s therewa smor ePectinesteras e II.Th ehig hmolecula rweigh tpectin ­ esterase isstrongl y represented ingrapefruit .Nave lorange s contained the leasto fthi s form. Whether thedifference s found canb e ascribed toorang e cultivaran dcitru s species or toothe rfactor s like ripeness andgrowt hcondition s remainsunresolved .Als oth e relativequantitie s inth e componentpart s ofeac h fruitma ydiffe rconsiderabl y aswa s demonstrated byEvan s &McHal e (1978)i norang e fortw o formso fpectinesterase .On e of these formswa s found inth ejuic e sacs andmembran e covers (OPEII )an d theothe r form was found inth epee l (OPE I). Itma yb epresume d fromth eelutio norde ro fOP E Ian dOP E IIo nDEA ESephade x atp H 7.0 (Evans &McHale , 1978)tha tPectinesteras e I is identical toOP E IIan d thatPectinesteras e IIi s identical toOP E Io fthes eauthors .

57 7 Kinetics and mode of action of Pectinesteras I and II

Thekineti cstudie so forang epectinesteras e reportedi nliteratur ewer edon ewit h preparationso fundefine dpectinesteras e composition.I ti sver y likelytha tmixture so f different formswer eused .Ofte npectinesterase swer eno to ronl ypartiall ypurified .I t isimportan tt okno w thepropertie so fth esingl e formso fpectinesteras et ob eabl et o explainphenomen awhic har eobserve di nprocessin go ffruit san dvegetable san dt oadap t processingconditions . Further,som eo fth eexperiment sgiv e informationabou t themod eo factio no fth een ­ zymes.Knowledg eabou t themod eo factio nwil lcontribut et oelucidat eth estructur eo f pecticsubstances . Inth eexperiment st ob edescribe dpurifie dPectinesteras eI an dI Iwer eused .I n somecase s thecrud eNave l orangepectinesteras ean dth epartiall ypurifie d highmolecu ­ larweigh tpectinesteras e containing allth e forms,Pectinesteras eHMI ,HMI Ian dWill i were included.

7.1 EXPERIMENTSAN DRESULT S

7.1.1 Initialreactio nrate s

pH Formos tpectinesterase s thepH-activit y curveswer edetermine dbot ho nbrow n ribbonan dgree nribbo npectin .A tp H5 an dabove ,initia lactivitie swer emeasure db y automatictitratio nan da tp H5 an dbelo w initialactivitie swer edetermine db yth ega s chromatographicmethano l assay.Thi s assaywa s describedi nChapte r4 .Fo r thega s chromatographic assay,pecti nsolution swer ebuffere dwit h 0.04mol/ 1citri cacid , adjustedt oth edesire dp Hwit hpotassiu mhydroxide .Th eioni c strengthwa s adjusteda t 0.1b yaddin gpotassiu m chloride.A tlo wp Hvalue swit hsom eenzymes ,reactio nrate s decreased rapidlywit htime .Th ereactio nrat edecrease d sometimesbefor e1 %o fth e available substratewa sde-esterified .I nsuc hcase s tangentswer edraw nt oth ecurve s todetermin eth einitia lreactio nrate .Th ehig hmolecula rweigh tpectinesteras ewa sth e least susceptiblean dPectinesteras eI Ith emos tsusceptibl et othi sphenomenon .Fig .2 5 showsth emethano l formationa tan dbelo wp H5 b yth ehig hmolecula rweigh tpectineste ­ raseo ngree nribbo npectin .Th ereactio nrate ,decreasin gwit hdecreasin gpH ,increase d againbelo wp H3.5 . Figs2 6t o2 9sho wth epH-activit y curvesfo rth edifferen tpectin ­ esteraseso nbot hgree nan dbrow nribbo npectin .Th eactivitie s determineda tp H5 wit h recordedtitratio ndi dno talway scoincid ewit hth eactivitie sa tp H5 determine db y thega schromatographi cmethano lassay .Th epotassiu m citratebuffere d solutions forth e gaschromatographi cmethano l assaywer e adjustedt oa nioni cstrengt ho f0. 1wit hpotas ­ siumchloride .Th epotassiu m ionconcentration swer e then0.07 3mol/ 1a tp H5 ,0.08 6mol/ 1

58 methanol (mg/l)

120 180 incubation time (min) Fig.25 .Activit yo fth ehig hmolecula rweigh t pectinesterase atlo wp Hvalues .Reactio nmix ­ tures (10ml )containe d5 g/ 1gree nribbo npectin , 0.1 g/1sodiu mazide ,0.0 4mol/ 1potassiu mcitrate , potassiumchlorid e toa nioni cstrengt ho f0. 1an d 0.1 unitspe rm lo fpectinesterase .Incubatio na t 30°C .Sample so f 1m lwer e takenfo rga schromato ­ graphicmethano lassay .Th e figures indicateth e pHvalue so f thereactio nmixtures . atp H4.5 ,0.09 5mol/ 1a tp H4. 0an d0. 1mol/ 1a tth elowe rp Hvalues .I nrecorde dtitra ­ tionexperiment s therewer ealway s0. 1 mol/1potassiu mions .Th epotassiu mion sintroduce d duringth eneutralizatio no fth epecti nwer eneglected .Th epoo rfi to fsevera lcur ­ vesa tp H5 ca nprobabl yb eascribe dt othi sdifferenc ei npotassiu mio nconcentrations , asa tp H5 i nth epotassiu mcitrat ebuffere dsolution sther ewa sa lowe rconcentratio n ofpotassiu mion san dals oth eactivit yo fth epectinesterase swa slowe r (forth einflu ­ enceo fcation sse eSectio n2.2.3) . Onbrow nribbo npectin ,Pectinesteras e Iha sit soptimu ma tp H7. 6an dPectinesteras e IIa tp H8.0 .Further ,Pectinesteras eI i srelativel ymor eactiv ea tlo wp Hvalues ,whic h isparticularl yeviden ta tp H5.0 .A santicipated ,th ecurv efo rth ecrud eenzym eprepa ­ rationrun sbetwee nth ecurve sfo rth etw opurifie denzymes .Th etw opurifie denzyme s accountfo r90 $o fth etota lactivit yi ncrud eNave lorang epectinesteras e (seeSectio n 6.1.2).O ngree nribbo npectin ,th ecurve sar ebroadene da tth eaci dside ,approximately , overa ful lp Hunit .Th edifferenc ei nth ecurve sbetwee ngree nribbo nan dbrow nribbo n pectincanno tb ecause db yth edifferen tquantitie so fpotassiu mhydroxid eneede dfo rth e neutralizationo fth epectins .Th epotassiu mio nconcentratio nwa sabou t0.10 6mol/ 1i n thebrow nribbo npecti nan dabou t0.10 8mol/ 1i nth egree nribbo npecti nsolutions . Noneo fth epectinesterases ,excep tth ehig hmolecula rweigh tpectinesteras eha dan y activitya tp H3. 0o rbelow .Th ecrud eenzym emus thav esom eactivit ya tp H2. 5bu tth e concentrationo fth ehig hmolecula rweigh tpectinesteras ei nthi spreparatio nwa sto o lowt omeasur eactivit ywithi na fe whours . Thethre eenzym epeak so fth ehig hmolecula rweigh tpectinesteras eafte rseparatio n

59 relative activity Cr.) relative activity (•/•) 100 100

80-

60-

Fig. 26.p H Activity curve of pectin­ Fig. 27.p H Activity curve of pectin­ est eras e on brown ribbon pectin (degree of esterase on green ribbon pectin (degree esterification 72%). Reaction mixtures of esterification 62%) .Reactio n mixtures (25 ml) contained 5g/ 1 brown ribbon pec­ and conditions as inFig . 26,excep t that tin;0. 1 g/1 sodium azide; 0.1 mol/1 pot­ 5 g/1 green ribbon pectin instead of brown assium chloride and 0.2 units per ml of ribbon pectin was used. Crude pectin­ pectinesterase. Initial activities were esterase (•); Pectinesterase I(o) ; measured at 30 C by recorded titration. Pectinesterase II (A);hig h molecular Crude pectinesterase (o); Pectinesterase weight pectinesterase (•). I (o);Pectinesteras e II(A) . relative activity(7o ) relative activity (•/.) 15

Fig. 28.p H Activity curve of pectinesterase on brown ribbon pectin (degree of esterification 72%). Reaction mixtures (10 ml) contained 5g/l brown ribbon pectin, 0.1 g/1 sodium azide, 0.04 mol/1 potassium citrate, potassium chloride to an ionic strength of Fig. 29.p H Activity curve of pectinesterase on 0.1 and 0.1 units per ml pectin­ green ribbon pectin (degree of esterification . esterase. Incubation at 30 °C. 62%). Reaction mixtures and conditions as in Samples of 1m l were taken for the Fig. 28,excep t that 5 g/1 of green ribbon pectin gas chromatographic methanol assay. instead of brown ribbon pectin was used. Crude Crude pectinesterase (a);Pectin ­ pectinesterase (•); Pectinesterase I (o);Pectin ­ esterase I (o);Pectinesteras e esterase II (A);hig h molecular weight pectin­ II(A) . esterase (•).

60 l/v(|imol/ml min)"' 90T

.2° i l/[S](mg/ml)" Fig. 30.Lineweaver-Bur k plots forpurifie d Pectineste- rase I with and without inhibitor. Reaction mixtures (30 ml,p H 7.0) contained 0.04 to 1m g perm l of green ribbon pectin, 0 or 0.05m g perm l of polygalacturonic acid inhibitor, 0.1 mol/1 of potassium chloride and 0.05 units perm l of pectinesterase. While purging with nitrogen, initial activities were measured at 30 C by recorded titration with 0.01 mol/1 potassium hydroxide. Without inhibitor (o);wit h inhibitor (•).

l/v (iimol/ml min)-' 700 /

500 A

/ 300 /

___,-- ƒ100

-200 -100 100 200 l/KI(mg/ml ) Fig. 31.Lineweaver-Bur k plots forpurifie d Pectineste­ rase IIwit h andwithou t inhibitor. Reaction mixtures (160 ml,p H 7.0) contained 0.005 to0. 1 mg perm lo f green ribbon pectin, 0 or 0.01 mgpe rm l of polygalac­ turonic acid inhibitor, 0.1 mol/1potassiu m chloride and 0.0095 units perm l of Pectinesterase II.Whil e purging with nitrogen, initial activities were measured at 30 C by recorded titration with 0.01 mol/1potassiu m hydroxide. Without inhibitor (A);wit h inhibitor(A) .

61 oncross-linke dpectat e (Section6.1.1. ;Fig .22 )wer eassaye dseparatel ya tp H2. So n greenribbo npectin .Al lthre epeak sha dactivit ya tp H2. 5bu tth erelativ eactivitie s differed.Th erelativ eactivitie s (theactivitie srelativel yt oth eactivit ya tstandar d assayconditions )wer e4.8 4 forPectinesteras eH MI ,3.5 4 forPectinesteras eH MI Ian d 0.44fo rPectinesteras eH MIII .Differen tfraction s (fractions36 ,3 9an d41 )o fth e broadPectinesteras eH MI Ipea kdiffe rno to rver ylittl efro meac hothe ri nrelativ e activitya tp H2.5 .

K. V and K. Figs3 0an d3 1sho winitia lenzym eactivitie smeasure da tdifferen t concentrationso fgree nribbo npecti ni nth eabsenc ean dpresenc eo fpolygalacturoni c acid,a competitiv einhibitor .Variou skineti cparameter scalculate dfro mthes efigure s aregive ni nTabl e 19.Th evalue sfo r V andturnove rnumbe r (molecularactivity )o f bothenzyme sar evirtuall yequal .However ,th e K valuesfo rgree nribbo npecti nan dals o the K. valuesfo rpolygalacturonat einhibito rdiffe rwidely .Pectinesteras e Iha sa rela ­ tivelylo waffinit yfo rit specti nsubstrat ean di srathe rweakl yinhibite db ypolygalac ­ turonate,wherea sPectinesteras e IIha sa hig haffinit yfo rit ssubstrat ean di sstrongl y inhibitedb ypolygalacturonate .Pectinesteras e IIi smuc hmor eactiv ea tlo wsubstrat e concentrations,a sma yb ejudge dfro mth elargel ydifferen tsaturatio ncurve sfo rth e twoenzymes ,show ni nFig .32 .Activitie smeasure dfo rbot hpurifie dpectinesterase si n thepresenc eo fvariou ssubstrat econcentration s (datafro mFig s3 0an d31 )wer euse d inlo g[S ]versu slo g W^^^-V) plots.Straigh tline swer eobtaine dfro mwhic h Kmva ­ lueswer ecalculate dwhic hwer eth esam ea sthos egive ni nTabl e 19.Th eslope so fth e lineswer e 1+ 0.02 ,whic hi stypica lo fenzyme sfollowin gMichaelis-Mente nkinetic s (Whitaker,1972) .

0.4 [S]

Fig.32 .Substrat e saturationcurve sfo rPectinesteras e I andII .Th erati oo finitia lreactio nvelocit yan d v v s maximuminitia lreactio nvelocit y (• / laa^ * plotte d againstsubstrat econcentration .Reactio ncondition s arespecifie d inFig s3 0an d31 .^ valueswer e takenfro mFig s3 0an d31 . PectinesteraseI (o);Pectinesteras eI I(A) .

62 Table 19.Kineti c datao fPectinesteras eI an dI Ia tp H7. 0o ngree n ribbonpectin . For reaction conditionsse eFig s3 0an d31 .

Enzyme V Turnovernumbe r K K. /ma x, ;• .^% (mole/mole.sec/ 1 / i )\ , ",>,, i l (ymole/min.unit) (mg/ml) (mg/ml)2

Pectinesterase I 0.81 340 0.083 0.416 Pectinesterase II 0.74 338 0.0046 0.0016

1.Expresse d asm go fgree nribbo npecti npe rm lo freactio nmixture . 2.Expresse d asm go fpolygalacturoni c acid (ICN)pe rm lo freactio nmixture .

Relationship of K and degree of esterification of substrate Lineweaver-Burkplot s weremad ewit hpectins ,partiall yde-esterifie db yalkali ,wit hdifferen tdegree so f esterificationa sliste di nChapte r 3.Fo rPectinesteras e Iinitia l reactionrate swer e determinedwit hrecorde dtitration .However ,fo rPectinesteras eI Iinitia lreactio nrate s decreased toorapidl y forth etitrimetri cassay .Th ep Hdro pmetho da sdescribe di nChap ­ ter3 appeare dt ob emor e sensitive andwa suse dfo ral lPectinesteras eI Iassays . For thismetho d conical flaskswer eused ,fille dwit hsubstrate ,an dtoppe dwit h1 c mpetro ­ leumethe r (b.p.60-8 0°C )t oexclud e carbondioxide .Fig s3 3an d3 4sho wth eLineweaver - Burkplot sfo rPectinesteras eI an dII ,respectivel yo nal l substrates.Activit ymeasure ­ mentso nsom epectin sar eonl ypartl y showni nthes e figures.However ,th ekineti c data listedi nTabl e2 0wer eobtaine d fromplot s (notshown )i nwhic h thescale swer e chosen inagreemen twit h thepoint so fmeasurement so neac hparticula r substrate.Bot hpectin ­ esteraseI an dI Ihav ea strongl y increasedaffinit yt osubstrate swit ha lowe rdegre eo f esterification.A sfoun dfo r greenribbo npectin ,th eaffinit yfo r the substratei smuc h higher forPectinesteras eI Itha nfo r PectinesteraseI .Th e K values (Table 19)foun d ongree nribbo npecti n (degreeo festerificatio n 62%)fi tfairl ywel li nTabl e 20.How ­ v ever,th eK maxvalue so nth egree nribbo nsubstrat ewer e lower.Th e max (Table20 ) ap­ pearst ob erathe r independantfro mth edegre eo festerificatio no fth esubstrate ,onl y atth ehighes tan dlowes tdegre eo festerificatio n tested,ther ewa s somedeviation . The slopeso fth elo g[S ]versu slo gV/(V -V) plotswer e1.0 0( +0.05) ,excep tfo r Pectin­ esteraseI Ia tth ehighes tan dlowes tdegre eo festerificatio no fth esubstrat e (slopes 1.21 and1.25 ,respectively) .I tappeare d thatlo g(1/ X) wa slinearl y relatedt olo g (%fre ecarboxy l groupsi nth esubstrate ) forbot hPectinesteras e Ian dII ,wit ha slop e of2.0 8fo r Pectinesterase Ian da slop eo f2.0 3fo r Pectinesterase II(Fig .35) . Anotherobservatio nwa smad edurin g themeasurement sfo r theseLineweaver-Bur k plots. Onhighl yesterifie d pectin,initia l reactionrate s increasedfo r PectinesteraseI afte r someo fth esubstrat ewa sde-esterified .Th ereactio n ratewa smaxima l afterabou t2 %o f thesubstrat ewa sde-esterified ,a si sillustrate di nFig .3 6b yLineweaver-Bur kplots . Thusaffinit y increased andth e V wasno t affected.Thi s phenomenonwa sno t observed atdegree so festerificatio no fth esubstrat eo f78 %an dlower .

63 l/v((imol/min.U)" 1

/[S] (mg/mir Fig. 33. Lineweaver-Burk plots for purified Pectinesterase I on pectins with different degrees of esterification. Reaction mixtures (100 ml, pH 7.0) contained various pec­ tins, 0.1 mol/1 potassium chloride, 0.1 g/1 sodium azide and 0.02 units per ml of pectinesterase. Initial activities were measured at 30 C by recorded titration with 0.01 mol/1 potassium hydroxide. Degrees of esterification of pectins: 95.6% (•); 88.0% (A); 78.2% (•); 68.3% (•) ; 52.5% (*) and 32.3% (+).

l/v(|imol/min.U)"' ./

300 400 1/[S](mg/m l)" ' Fig. 34. Lineweaver-Burk plots for purified Pectinesterase II on pectins with different degrees of esterification. Reaction mix­ tures (500 ml) contained various pectins, 0.1 mol/1 potassium chloride, 0.1 g/1 sodium azide and 0.001 units per ml of pectin­ esterase. Initial activities were measured by the pH drop per min between pH 7.02 and 6.97. Degrees of esterification of pec­ tins: 95.6% (•); 88.0% (A); 78.2% (•); 68.3% (T); 52.5% (*) and 32.3% (+).

64 Table 20. Kinetic data ofPectinesteras e Ian dI Ia tp H7. 0o npectin s with different degrees ofesterification . Forreactio n conditions seeFig s 33an d 34.

Degree ofesterificatio n Pectinesterase I PectxnesteraseI I of pectin K V K V m max (~s m max (mg/ml)' (VJmol e/min .un it) (mg/ml)1 (umole/min.unit) 95.6 1.63 0.87 0.238 0.94 88.0 0.724 1.02 0.074 1.00 78.2 0.231 0.98 0.023 1.00 68.3 0.102 0.98 0.0108 1.02 52.5 0.042 1.06 0.0031 1.04 32.3 0.038 1.30 0.0019 0.96

1.Expresse d asm gpecti npe rm lo freactio nmixture . log d/Km) 3-

Fig. 35. Log (1/Km) versus log (% free carboxyl groups in sub­ I strate). Data calculated from Table 20, reaction conditions as 0 1 2 log (Vofree corboxyl groups) in Figs 33 and 34. Pectinesterase I (o); Pectinesterase II (•).

l/v(umol/min U) 9t

Fig. 36.Lineweaver-Bur k plots forpuri ­ fied Pectinesterase Iwit h increasing initial velocities. Reaction conditions and concentrations asi nFig . 33.Initia l velocity onpecti n 95.6% esterified(•) ; velocity after 2%enzymati c de-esterifi- cation onth esam e substrate (o); initial velocity onpecti n 88.0% esterified(•) ; 3 , S" velocity after 2%enzymati c de-esterifi- 1/[S)(mg/m l)• ' cation onth esam e substrate(•) .

65 Table 21.Kineti c datao fPectinesteras eI an dth ehig h molecularweigh tpectinesteras e ongree n ribbonpecti na tdifferen tp Hvalues .Fo rreactio n conditionsse etext . pH PectinesteraseI High molecularweigh tpectinesteras e

V K K. V K K. max m 1 max m 1 2 (ymole/min.unit) (mg/ml)' (mg/ml)2 (ymole/min.unit) (mg/ml)1 (mg/ml) 7.0 0.81 0.083 0.416 0.83 0.041 0.010 4.0 0.47 1.19 0.002 0.071 1.35 0.002 2.5 0.853 503 0.002 1.Expresse da sm go fgree n ribbonpecti npe rm lo freactio nmixture . 2.Expresse da sm go fpolygalacturoni c acid (ICN)pe rm lo freactio nmixture . 3.Approximat e values because substrate concentrations which couldb euse dwer emuc h lower than the K.

K. K. and V Dependence on pH Lineweaver-Burkplot swer emad ewit hgree nribbo n pectina ssubstrate ,i nth epresenc eo rabsenc eo fpolygalacturoni c acida sinhibitor . Thiswa sdon ea tdifferen tp Hvalue s forbot hPectinesteras eI an dth ehig hmolecula r weightpectinesterase .Determination sa tp H7. 0wer edon ewit hrecorde d titrationunde r thecondition sa si nFig .30 .Measurement sa tp H4. 0an d2. 5wer emad ewit hth ega s chromatographicmethano lassay .Th ereactio nmixture s (10ml )containe d greenribbo n pectin,0. 1g/ 1sodiu mazide ,0. 1mol/ 1potassiu m chloride,2 5mg/ 1propanol- 1an d0.0 1 unitspectinesterase/ml .Durin gth einitia lreactio nstage ,no significan tp Hdro p

(v< 0:0 5p rHunits' )wa sobserve di nthes eunbuffere d substrates.Th e and values ' K , K. V m' i max fromthes eplot s (notshown )ar epresente di nTabl e 21.Th epolygalacturoni c acidex ­ hibited competitive inhibition.Tabl e2 1clearl y showsth e reductiono fth eaffinit yto thesubstrat ewit hdecreasin gp Hfo rbot hpectinesterases .Th epectat e inhibitionin ­ creases,bu tth e K. isa paramete rwhic hdepend so nth e K. I tca nb esee ntha twherea s PectinesteraseI i shardl y inhibiteda tp H7 ,i ti sstrongl y inhibiteda tp H4 b ypolyga ­ lacturonic acid.Th eV isreduce da tp H4 fo rbot henzymes ,bu t remarkablyth e V IH3X HlclX ofth ehig hmolecula rweigh tpectinesteras e increasest oabou tth evalu eo ftha ta tp H7 ifth ep Hi sreduce dt o2.5 .

7.1.2 De-esterification limits

Veetinesteva.se on pectins partially de-esterified by alkali Pectinesteraseswer eincu ­ batedwit halkal ide-esterifie dpectin swit hdifferen tdegree so festerificatio n (pec­ tins listedi nChapte r 3).Th ereaction swer efollowe db yrecorde dtitratio nwit h0.0 2 mol/1potassiu mhydroxide .Whe nth ereactio nrat eha ddecrease dto nearl y zero, the amounto fenzym ei nth ereactio nmixtur ewa sdouble dan dwhe nth ereactio nrat ewa svir ­ tually zeroagai nanothe r formo fpectinesteras ewa s added.Fig s3 7an d3 8sho wth ede ­ creasei ndegre eo festerificatio no fth esubstrat ewit htim e forPectinesteras eI an dII . PectinesteraseI wa s ablet ode-esterif y thehighl yesterifie dpectin sbette r thanPec ­ tinesteraseI Ian da lowe rdegre eo festerificatio n couldb ereached .Th elowe rth e initialdegre eo festerificatio nth e lowerth eultimat edegre eo festerification .I twa s

66 degree of esterification (•/.)

90

Fig.37 .Actio no fpurifie dPectinesteras e Io npectin , partiallyde-esterifie d todifferen tdegree so festerificatio n byalkali .Reactio nmixture s (25ml ,p H7.0 )containe d 1m gpe r mlo fpectin ,0. 1 mol/1potassiu m chloride,0. 1 g/1sodiu mazide . Whilepurgin gwit hnitroge nth ereaction swer efollowe db y recorded titrationwit h0.0 2mol/ 1potassiu mhydroxid e at3 0 C. Pectinesterase I 0.2unit spe rm l ( );Pectinesteras e I 0.4unit spe rm l (==:); Pectinesterase I 0.4unit s perm l+ Pectinesteras e II 0.2unit spe rm l ( ~^-

degree of esterification("/• )

90

Fi g. 38. Action of purified Pectinesterase II on pectin, parirtiallt y de-esterified to different degrees of esterifi cation byalkali .Reactio ncondition san dconcentration s asi nFi g37 . Pectinesterase II0. 2 unitspe rm l ( );Pectinesteras eI I 0.4unit spe rm l ( );Pectinesteras e II0. 4 unitspe rm l+ Pectinesterase I0. 2 unitspe rm l ( )

67 impossiblet oreac ha degre eo festerificatio no flowe rtha n 11%.A ttha tpoin t addition ofmor eenzym eha dn oeffect .I ti sremarkabl e thatwhe nth eamoun to fenzym ei sdouble d duringth ereactio n (especiallywit hPectinesteras e II),th ereactio nvelocit yi smor e thandoubled .Th e original enzymewa sno t inactivated sinceadditio no fgree nribbo n pectinan dsodiu mchlorid et oth e levelo fstandar d assaycondition s restored theacti ­ vityt oth eleve lo fth eadde dunit s (forstabilit yo fth eenzyme sse eals oChapte r8) . WhenPectinesteras eI Ii sadde d afterPectinesteras e Ii sno tlonge ractive ,n omor e carboxylgroup sar eliberate d (Fig. 37).Whe nPectinesteras e Ii sadde d afterPectine ­ steraseI Ii sno tlonge ractive ,th edegre eo festerificatio ni srapidl y loweredt oth e valuetha tPectinesteras eI ca n reachalon e (Figs3 7an d38) .Th ecurve s (notshown )fo r crudepectinesteras ean da1: 1mixtur eo fPectinesteras eI an dI Iresembl ever ymuc hth e figurefo r PectinesteraseI (Fig .37) .

Separation of reaction products Twenty-five unitso fdifferen tpectinesterase s were incubatedwit h 100m lo f5 g/ 1highl yesterifie dpecti n (degreeo festerificatio n 95.61) in0. 1ml/ 1potassiu mchloride .Th ep Hwa shel da t7. 0b yrecorde dtitratio nwit h0. 1 mol/1potassiu m hydroxide.Whe nth eamoun to falkal iequivalen tt oth e loweringo fth e degreeo festerificatio n from 95.6%t o60 %wa sused ,th ereactio nwa s stoppedb yth ead ­ ditiono fconcentrate d hydrochloric acidt op H2.0 .Th epectinesterase swer e inactivated byheatin gfo r1 5mi na t10 0° Can dafte rcoolin g thepecti nwa sprecipitate dwit h2 volumeso fethano l 96%.Th eprecipitat ewa swashe d chloride freewit hethano l 70%,the n washedwit h acetonean dai rdried .Par to fth epecti npreparation swer e fractionatedo n aDEAE-cellulos ecolum na sdescribe di nChapte r 3.Fig s39 A-39 Esho wth e elutionpat ­ ternso fsevera lpecti npreparations .Th e elutionpatter no fpecti n treatedwit hPectin ­ esteraseI Ian dcrud epectinesteras ewer ever y similart oth eelutio npatter no fpecti n treatedwit hPectinesteras eI (Fig .39B) .I tca nb esee nfro mth e figuretha thighl yes ­ terifiedpecti nde-esterifie db ypectinesteras ewa s separatedi na larg efractio nappa ­ rentlyno tde-esterifie dan dfraction sde-esterifie dt o several ratherdifferen t degrees ofesterificatio n (Figs39 Ban dC) . Whereas alkalide-esterifie d pectins eluteda srathe r sharppeak s (Figs 39Dan dE )an dwer emuc hmor eunifor mwit h respectt odegre eo festeri ­ fication.

Sugar analysis of separated reaction products Thehighl yesterifie dpecti nan dfrac ­ tions fromth eDEAE-cellulos e columno fpecti nde-esterifie db yPectinesteras e Iwer e analysed forneutra l sugars.Approximatel y2. 5m go fth eorigina l highlyesterifie d pec­ tin,2. 5m go fth efractio nwhic hwa sno t de-esterified (elutedwit h 0.05 mol/1 sodium phosphate,Fig .39B )an d2. 5m go fth ede-esterifie d fraction (peak fractionelute dwit h 0.45 mol/1 sodiumphosphate ,Fig . 39B)wer edialyse d against distilledwater .Sample s were taken foruroni caci danalysis ,th esolution swer e freezedrie dan danalyze dga s chromatographically forneutra l sugarsa sdescribe di nChapte r 3.Ther ewer e considerable differencesi ngalactos ean dglucos e contentsan donl y slightan dno tver y reproducible differencesi nothe rneutra l sugars.Tabl e2 2list sth eresult sfo rgalactos ean dglucose . The fractionno tde-esterifie dwa s richi ngalactos ean dglucose .I ti sdifficul tt o draw conclusions fromth edifference si nglucos e content sinceth epreparatio n contained

68 20-

.1' '2'.3 '4 .5'. 6. 7A Na-phosphate(M ) Fig. 39.Chromatograph y ofpectin so nDEAE-cellulose . Fivem lo f 10g/ 1pecti nsolution swer eapplie d toa DEAE-cellulose columnan delute dwit ha linea rgradien t ofsodiu mphosphat ebuffer ,p H4. 8a sdescribe d inChap ­ ter3 .The nth ecolum nwa swashe dwit h0. 1 mol/1sodiu m hydroxide (A).A .Highl yesterifie d pectin (degreeo f esterification 95.6%).B .Pecti nde-esterifie dwit h PectinesteraseI (degre eo festerificatio n60.4%) . C.Pecti nde-esterifie dwit hhig hmolecula rweigh tpec ­ tinesterase (degreeo festerificatio n60.8%) .D .Alkal i de-esterifiedpecti n(degre eo festerificatio n 52.6%). E.Alkal ide-esterifie d pectin (degreeo festerificatio n 32.3%).

Table 22.Neutra lsuga rcontent so f thehighl yesterifie dpectin ,th eno tde-esterifie d and thede-esterifie d fractionso fth ehighl yesterifie dpecti ntreate dwit h PectinesteraseI .

Neutral Pectinesterasetreate dpecti n Originalpecti n sugar notde-esterifie d de-esterified weighedsu m galactose (mg/g)2 71.6 6.0 27.8 29.0 glucose (mg/g)2 91.4 14.6 40.2 38.8

1.I nthi spreparatio n 1/3wa sno tde-esterified . 2.mg/ g (uronicaci d+ neutra lsugars) .

69 somestarc hwhic hals oelute swit h0.0 raol/15 sodiu mphosphat efro mth eDEAE-cellulos e column.Highl ypurifie dappl epecti n(th epurificatio no fwhic hinclude sa coppe rpreci ­ pitationstep )neve rcontain sles stha n2 5mg/ ggalactos e (Voragen,Departmen to fFoo d Chemistry,Agricultura lUniversity ,Wageningen ;persona lcommunication) .Thi sgalactos e ispar to fth esid echain si npecti npolymer s (Rombouts,1972) .Apparentl yth ehighl y esterifiedpecti ncontain sa fractio nrelativel yric hi nsid echain swhic hi smor edif ­ ficultt ob ede-esterifie db yth epectinesteras etha nth eres to fth epreparatio nwit h lesssid echains . Thefirs tfractio nfro mth eDEAE-cellulos ecolum nan dth eorigina lhighl yesterifie d pectinwer eincubate dwit hcrud epectinesteras eunde rth esam econdition s (0.3m gpectin / ml,0. 1mol/ 1potassiu mchloride ,0. 1g/ 1sodiu mazid ean d0. 2unit spectinesterase/m l at3 0° Can dp H7.0) .Th ereaction swer efollowe db yrecorde dtitration .Bot hth einitia l reactionrat ean dth ede-esterificatio nlimi tusin gth efirs tfractio no fth ecolum na s asubstrat ewer ehal fth evalu eo nth eorigina lhighl yesterifie dpecti n (figuresno t shown).

Effects of other pectic enzymes PurifiedPectinesteras e Ian dI Iwer eincubate dwit hhigh ­ lyesterifie dpecti na tp H6.0 .Th ereaction swer efollowe db yrecorde dtitration .Whe n thereactio nrat eha ddecrease dconsiderably ,othe rpurifie dpecti cenzyme swer eadded . Thereactio np Ho f6. 0wa schose nt oallo wactivit yo fal ladde dpecti cenzymes :pecti n lyaseha sit soptimu ma tabou tp H6. 0 (Houdenhoven,1975) ,polygalacturonas ea tabou t pH4. 0(Phaff ,1966 )an dpectat e lyasea tp H9. 4 (Romboutse tal. , 1978).Th eamount s ofpecti cenzyme sadde dwer echose nt oobtai ncomparabl erate so fchai nsplittin go fth e pectin,whic hwa sdetermine di npreliminar yexperiments .Tabl e2 3list sth eamount so f pecticenzyme sadde dan dth eaverag enumbe ro f splitspe rpecti nchai nafte ron ehou r ofactivit yi nth ereactio nmixtur etogethe rwit hth epectinesterases .Th edegre eo f polymerizationgraduall ydroppe dt ovalue scorrespondin gt othes enumber so fsplit spe r pectinchai n (about8 0monomer seach ,se eChapte r3) .Fig .4 0show sth eactio no fPec ­ tinesteraseI an dI Io nhighl yesterifie dpecti nbefor ean dafte rth eadditio no fothe r pecticenzyme sa tsom estag edurin gth ereaction . Becausecalciu mion sar ea nabsolut erequiremen tfo rpectat elyas eactivity ,0.2 5 mmol/1calciu mchlorid ewa spresen ti nth ereactio nmixture swher epectat elyas ewa s

Table23 .Quantitie so fpecti cenzyme sadde dt oreactio nmixture swit hhighl yesterifie d pectin,partiall yde-esterifie db ypectinesteras ean dth enumbe ro fsplit spe rpecti n chainafte ron ehou ro fsimultaneou saction .

Pecticenzym e WithPectinesteras eI WithPectinesteras eI I

unitsadded 1 splits/cha in2 unitsadded 1 splits/chain polygalacturonase 1.76 7.5 8.8 7.5 pectin lyase 0.076 2.5 0.076 2.5 pectatelyas e 15.0 2.5 50.0 2.5

1.Pe rreactio nmixtur eo fabou t2 5ml . 2.Averag enumbe ro fsplit spe rpecti nchai n with adegre eo fpolymerizatio no f80 .

70 degree of esterification ("/•) 100-1

0 60 120 180 degree of esterification (%) reaction time (min)

120 180 reaction time (min) Fig. 40.Stimulatio no fpectinesteras eb yothe r pecticenzymes .Reactio nmixture s (25ml ,p H6 ) contained 1g/ 1highl yesterifie d pectin(degre e ofesterificatio n95.6%) ,0. 1 g/1sodiu mazid ean d 0.2unit sPectinesteras e I(A )o r0. 2unit sPec ­ tinesterase II(B )pe rml .I nth ereactio nmixture s wherepectat e lyasewa sadde dals o0.2 5mmol/ 1 calciumchlorid ewa spresent .Whil epurgin gwit h nitrogen,th ereaction swer efollowe db yrecorde d titrationa t3 0°C .Pectinesteras e ( ); pectinesterase +pecti nlyas e ( =•); pectin ­ esterase+ polygalacturonas e ( ^r) ;pectin ­ esterase+ pectat e lyase ( ).

used.Thi sadditio nmarkedl y stimulatedbot hPectinesteras eI an dI I(Fig .40) .Calciu m ionswer eassume dt ob eonl y5-1 0 folda seffectiv ei nstimulatin gpectinesteras ea spo ­ tassiumo rsodiu m ions (seeChapte r 2,Tabl e 4). Inth epresenc eo f0. 1mol/ 1potassiu m chlorideonl ya maximu m stimulationi nreactio nrat eo fabou t 2.5$wa sexpected .Pecti n lyaseha dn oeffec tbu tpolygalacturonas e highly stimulated thepectinesterases ,espe ­ cially PectinesteraseI I(Fig .40) . Alsopectat e lyasestimulate d thepectinesterases , butt oa smalle rextent .I tca nb esee nfro mTabl e2 3tha t thepectat e lyasewa s about 3-fold lesseffectiv ei ndegradin g thepolymer .

71 7.2 DISCUSSION

Thetw opectinesteras eisoenzyme sdescribe dher ediffe rmarkedl yi nthei r K value forgree nribbo npecti nan d K. valuefo rpecti caci dinhibito r (Table 19).Pectineste ­ raseI Iwit hit sisoelectri cpoin to f> 1 1i smuc hmor estrongl yinhibite db ypecti c acidtha nPectinesteras eI wit ha nisoelectri cpoin to f10.05 .Lineweave r& Ballo u (1945)develope da theor yabou tth einhibitio no fplan t (alfalfa)pectinesteras eb y pecticaci d (endproduc tinhibition) .Withou tknowin gth eisoelectri cpoin to fthei r enzyme,the yreasone dtha tbecaus eo fit spositiv echarg ebelo wit sp Hoptimu mfo rac ­ tivityi twoul dfor ma ninactiv eioni ccomple xwit hpectate .The ycarefull ystudie dth e stimulatoryeffec to fvariou ssalt so npectinesteras eactivit yan dconclude dtha tca ­ tions,b yliberatin gth eenzym efro mit sinactiv eioni ccomplex ,stimulat eenzym eacti ­ vityb yremova lo finhibitio nrathe rtha nb yspecifi cactivation .Thi stheor yi ssuppor ­ tedb yth edecrease dinhibitio no forang epectinesteras eb ypecti caci dhydrolysate swit h increasingsal tconcentration ,an dals oa tincrease dp Hvalue s (Termotee tal. , 1977). Inth eligh to fthi stheor yi ti squit ecomprehensibl etha tPectinesteras e II,wit hit s relativelyhig hisoelectri cpoint ,ha sles sactivit ya tlo wpH ,i smuc hstronge rinhibi ­ tedb ypolygalacturonat ean dbind st ocross-linke dpectate . Throughalkal ide-esterificatio npectin swit ha statistica ldistributio no f carboxylgroup sar eproduce d (Kohn& Furda ,1967) .O nalkal ipr ede-esterifie dpectin s the K valuesar elargel yreduce dbu tth e7 valuesar ehardl yaffecte d (Table20) . Theaffinit y (1/X) i sincrease dmuc hmor etha nwoul db eexpecte di fonl yon ecarboxy l groupi sinvolve di nth eenzyme-substrat ecomple xformation .Doublin gth enumbe ro ffre e carboxylgroup si nth esubstrat egive sa fou rfol dincreas eo f1/ X ofbot hPectin ­ esteraseI an dII .Fro mFig .3 5th efollowin grelationshi pca nb ededuce dfo ra wid e rangeo fdegree so festerificatio no fth esubstrate :

log(1/ X) = lo ga + 2 lo g {% freecarboxy lgroups )

2 or1/ X =a ( Ifre ecarboxy lgroups ) wherea i sapproximatel y 10time sa slarg efo rPectinesteras e IIa sfo rPectinesteras e I. Forpectin swit hdifferen tdegree so festerificatio nan dth efre ecarboxy lgroup s randomlydistribute dove rth epolyme rchai nsom ecalculation swer emade .Conside ra pectinwit ha DE(degre eo festerification )o f95 1an da nindefinit echai nlength .Thi s pectincontain s (100-ÖB)= 5 1fre ecarboxy lgroups .Th echanc etha ta ton esid eo fa fre e carboxylgrou panothe rfre ecarboxy lgrou poccur sa tsom efixe ddistanc e (e.g.4 monome r units)i s0.05 .Th epecti ncontain s (100-DE)x 0.0 5= 0.25 $unit so f2 fre ecarboxy l groupsa tsom efixe ddistance .Fo r3 carboxy lgroup si nsom efixe darrangemen tthi si s (100-Zffi)x 0.0 5x 0.0 5= 0.00251 .Thes ecalculation swer emad efo rsevera ldegree so f esterificationo fpectins .Th erelationshi pbetwee nlo g {% unitso f n carboxylgroup si n somefixe darrangement )an dlo g [% freecarboxy lgroup si nth epectin )i sshow ni nFig . 41,wit hn » 1 ,2 an d3 .Th eslop efo rlo g (% unitso f2 carboxy lgroups )i nFig .4 1i s

72 (V . units of n free oarboxyl groups)

Fig. 41.Unit so fn carboxy lgroup sa tsom efixe ddis ­ tancei npectin so fdifferen tdegree so festerificatio n andrandoml ydistribute d carboxylgroups .Th eline s wereconstructe da sdescribe d inth etext . 0 1 2 log ('/.free carboxyl groups) 1. n = 1;2 . n= 2 ;3 . n -3 .

2;equa lt oth eslop eo fth eexperimenta l linesi nFig .35 .I tma yb econclude dtha tfo r bothPectinesteras e Ian dII ,2 carboxy lgroup sa ta certai ndistanc ei nth epecti nchai n arerequire dfo ra noptima lenzyme-substrat ecomple xformation .A twha tdistanc ei sun ­ known.Becaus e (crude)orang epectinesteras ei sonl yinhibite db ygalacturoni caci doli ­ gomerswit ha degre eo fpolymerizatio no f8 o rmor e (Termotee tal. ,1977) ,6 monomer si n between2 monomer swit hfre ecarboxy lgroup sseem slikely .However ,the ni ti sdifficul t tounderstan dho wa pectinesteras eca nprocee dlinearl yalon gth epecti nchai nproducin g blockso ffre ecarboxy lgroup sa sindicate db yth eresult so fsevera lworker s (Schultz etal. , 1945;Hill se tal. ,1949 ;Her ie tal. ,1961 ;Koh ne tal. , 1968).Fo rsuc ha me ­ chanismth eattac ko f2 carboxy lgroup snex tt oeac hothe rseem smor elikely . Solms& Deue l (1955)foun da nopposit erelationshi pbetwee nth e K andth edegre e ofesterificatio no fth esubstrat ea spresente di nTabl e20 .The yals oreporte da n increasing V withdecreasin gdegre eo festerificatio no fth esubstrate .Probabl y theirresult swer eaffecte dstrongl yb ysuboptima lan dvariabl econcentration so f cationsi nth ereactio nmixture s (0.05-0.1 mol/1). Thereduce daffinit ya tlo wp Hvalue s (Table21 )ma ypartl yb eexplaine db yth e decreaseddissociatio no fth efre ecarboxy lgroup so fth epectin .Th ep Kvalu efo r pectini sabou t4. 0 (Speisere tal. , 1945).Bu tth ep Hwil lals ohav eit seffec tb y protonationo facidi cgroup so nth epectinesterase sa sma yb econclude dfro mth echang e in V max.A t lowerpH ,thI T» e pectat r einhibitio nals oincreases .Thi sincreas ecanno tb e understoodi fth erelationshi p (1/X) = a (Ifre ecarboxy lgroups ) holdsfo ral lde ­ greeso festerification .Fo rPectinesteras eI i tca nb ededuce dfro mTabl e2 0tha tth e O/K ) doesno tincreas ebelo w50 1esterificatio no fth esubstrate .Thu sth eaffinit y forpolygalacturoni caci da tp H4 (wher eabou t50 1o fth efre ecarboxy lgroup sar edis ­ sociated)ma yb eth esam ea sa tp H7.0 .Th eaffinit yfo rth esubstrat ei sgreatl y reduceda tp H4. 0s otha tth eapparen t K. valuei slowe ran dth epolygalacturoni caci d

73 ismor eeffectiv ea sinhibitor . Thebroadenin g ofth epH-activit yprofil ea t theaci dsid eb ya substratewit h a lowerdegre eo festerificatio nma y alsob eascribe d toth edecreas eo faffinit ya t low pH (Figs 26-29).A tequa lsubstrat e concentrations,th eenzym eremain smor e saturated with thesubstrat e forwhic h itha s thehighes t affinity. Rexovâ-Benkovâ et al. (1976)suggeste d that thebes tmetho d ist oexpres sa K of pectinesterase asa concentrationo fmethyl-D-galactopyranosyluronat eresidues . Myopinio ni stha tthi s isno t true forcitru spectinesteras e andpresumabl y alsono t forothe rplan tpectinesterases .Expressio no fth e K onbasi s ofth epecti nconcentra ­ tiontogethe rwit ha gooddescriptio n ofth epecti n (whichinclude s anhydrogalacturonic acidcontent ,degre eo festerification ,distributio no fcarboxy l groupsan ddegre e of polymerization)i smor emeaningful . Thevelocit y ofPectinesteras e Io nhighl yesterifie d substrates increases aftersom e substratewa s saponifiedbecaus eo f increase inaffinit y forth echange d substrate (Fig. 36).Apparentl yne wpoint s ofattac kar ecreate db yan d forth epectinesterase .Th ein ­ crease inaffinit y cannotb eexplaine db ya trac eo ffre ecarboxy lgroup swhic hma yb e formedb y spontaneous de-esterificationa tp H 7.0.Perhap s the enzyme startsa ta poin t ofattac k andmove s inon edirection .On e ormor eo fth ecarboxy lgroup sproduce d in thebeginnin g canb ea par t ofth epoin to fattac k inth eopposit edirection .Thi s would implyt osom eexten ta multichai nmod eo fattac kmechanism .Otherwis e onlyin ­ creasingproduc t inhibitionwoul dhav ebee nobserved . Thepectinesterase s areunabl et ode-esterif ypecti ncompletel y (Figs3 7an d38) . Thiswa s alsoobserve db ySolm s &Deue l (1955).Als o thealkal ipr ede-esterifie dpecti n with the lowestdegre eo festerificatio ncoul dno tb ede-esterifie d further thana degre e ofesterificatio no f 11%.Possibl ythes emethoxy lgroup sar elocate dclos et osid echain s orothe r irregularities inth epecti nchain .Anothe rpossibilit y istha tbeside s free carboxylgroup sals o amethoxy l group isrequire d forth eenzyme-substrat e complex,a groupoutsid eth eactiv ecentr ewhic h isno t de-esterified. Thehighl yesterifie dpecti n (degreeo festerificatio n of95.6'» )coul d onlyb ede - esterified toa degre eo festerificatio no fabou t 501.Separatio no freactio nproduct s ofpectinesterase swit h thissubstrat e de-esterified toa degree ofesterificatio n of 60%,reveale dtha tabou t 30%o fth e substratewa sunaffecte db y thepectinesteras e (Fig. 39),presumabl ybecaus ethes e substratepolymer s containedmor e sidechain sa s wasreveale db yneutra l sugaranalysis .Bu tals oth edistributio no ffre ecarboxy l groupsove r thepolyme rwil lhav eplaye d arole . Although 2carboxy lgroup sa ta certai n fixeddistanc ear erequire d foroptima l enzyme-substrate complexformation ,anothe renzyme-substrat e complexmus tb epossible . Highlyesterifie dpecti nwit ha degre eo festerificatio n of95 1contain s 0.25%unit s of2 carboxy lgroup s atsom e fixeddistance .Pecti nwit h adegre eo fpolymerizatio n of8 0 contains lesstha n 201pecti nchain ssuitabl e foroptima lenzyme-substrat ecomple xfor ­ mation.Th eothe r 80% ofsubstrat e chainscoul dno tb eattacke d if2 carboxy l groupsa s described abovewoul db erequired .However ,i tca nb esee ni nFig s 37,3 8an d 39Btha t more than20 %o fth epecti nchain sar eattacked .Thu sanothe rtyp eo f enzyme-substrate complexshoul db epossible .

74 Upondoublin g theamoun to fenzym ewhe nth ereactio nrat eha d decreased tonearl y zero,th ereactio nrat ewa smor e thandouble d (Figs37 ,38) . This resultcoul db e causedb y inhibitory sitesproduce d inth epecti nchai nfo rwhic h thepectinesteras e has amuc hhighe raffinit ytha n forothe rsite sstil lpresen t andno tye tuse d fora n attack.Whitake r (1972)mentione d adsorption ofth eenzym et oth e surfaceo fth ere ­ actioncontaine ro rth erelativ e shortageo fa nessentia lcofacto ra s thecaus eo fsuc h aphenomenon . Thestimulatio nb yendo-pectat e lyasean dendo-polygalacturonas e (bothsplittin g between freecarboxy lgroups )ca npresumabl yb eascribe d toth eremova lo fth ereactio n product:block so ffre ecarboxy l groupsactin ga scompetitiv e inhibitor.Pectinesteras e I isals o stimulatedb y theothe rpecti c enzymes (Fig. 40),wherea s thisenzym e ishardl y inhibitedb ypolygalacturoni c acid (Table 19).I tmus tb ekep t inmin d that theinhibi ­ tionb ya competitiv e inhibitor increaseswhe nth eaffinit y forth e substratedecreases . Theenzym eha sa lowaffinit y forth ehighl y esterified substrate.Moreover ,th ere ­ sultsi nTabl e 19wer eproduce d atp H 7.0,wherea s theexperiment s incombinatio nwit h theothe rpecti c enzymeswer edon ea tp H 6.0. Pectinesterase II,whic h ismor e efficiently inhibitedb ypolygalacturoni c acid,i sstimulate dmor eb y theothe rpecti c enzymes. Calcium ionsals ostimulat epectinesterase s (Fig.40) .Presumabl y theblock so f freecarboxy lgroup sbin d calcium (Kohn, 1975)i na wa y thatmake s them lessaccessibl e toth epectinesterases .Thu s calcium ionsals oreduc eth ecompetitiv e inhibitiono fth e reactionproduct .

75 8 Stability of pectinesterases

Stabilitystudie so fpectinesteras eunde rtechnologicall yrelevan tconditions , re­ portedi nth eliterature ,wer ealway sdon ewit hcrud epectinesteras epreparations ,pre ­ sumablycompose do fsevera l formso fpectinesterase .Manab e (1973a)teste dth ehea t stabilityo fa purifie dcitru spectinesteras ea tnearl yoptima lcondition sfo r activity andstability ,whic har edifferen t fromth econdition sprevailin g incitru sjuice . Factorsknow nt oaffec tpectinesteras e stabilityi ncitru sjuice sar eth ep H(Rous e& Atkins, 1953a)an dth epul pconten t (Rouse& Atkins ,1952) .A smentione di nSectio n 6.1.1,discrepancie sbetwee nhea tstabilit yo fpurifie dpectinesterase san dth eori ­ ginalcrud epreparatio nmad em eawar eo fth etechnologica limportanc eo fth ehig hmole ­ cularweigh tpectinesterase .

8.1 EXPERIMENTS ANDRESULT S

8.1.1 Stabilitydurin gstorage ,assa yan dorang eclou dstabilit y tests

Stability duping storage Thelos so factivit yo fpurifie dPectinesteras eI an dI Ii n solutionso f0.00 5mol/ 1sodiu mphosphate ,p H7.5 ,wit h0. 1mol/ 1sodiu mchlorid ewa s lesstha n 15% afterstorag efo r2 year sa t4 °C .

Stability during activity measurements at pH 4.0 and 7.0 Pectinesteraseswer eincubate d inpecti nsolution so fp H4. 0an d7.0 ,unde rcondition s similart oth econdition sdurin g thekineti cexperiment sdescribe di nChapte r7 .A ttim einterval ssample swer etake nan d theresidua lactivit ywa smeasure db yrecorde dtitratio na tstandar dassa yconditions . Fig.4 2show sth eresidua lactivitie supo nincubatio no fPectinesteras e Ian dI I in mediao fbot hp Hvalues .A tp H7. 0bot henzyme swer e stablefo ra tleas t2 4h .Pectin ­ esteraseI wa sals ostabl ea tp H4. 0bu t PectinesteraseI Ilos tal lactivit ywithi n6 h . Forthi sreaso nPectinesteras eI Iwa sno t usedfo rsom eo fth eexperiment sa tlo wp H valuesdescribe di nSectio n7.1 .Th ecurve sfo rth ecrud eenzym e (notshown )ra ni n betweenth ecurve so fPectinesteras eI an dII ,fo rexampl ea tp H4. 0th eresidua lacti ­ vitydroppe dt oabou t 701withi n5 h an dremaine dconstan tfo rth enex t 12h .

Effect of orange juice constituents on pectinesterase stability Theconcentratio no f someorang ejuic econstituent swa svarie dt otes tit seffec to npectinesteras e stability. Thepectinesterase s (initiallyabou t4 0units/ml )wer e incubatedi nth emedia liste di n Table2 4i ntube sseale dwit hParafil mfo r2week sbot ha t3 0° Can d6 °C .A ttim e intervals,th etube swer e stirredan dsample swer etake nt omeasur eth eresidua lactivit y understandar dassa yconditions .Th elo g {% residualactivity )wa splotte dagains tincu -

76 residual activity (*/•) WO-t"***»

20

u 24 6810 12 14 16 hours Fig. 42.Stabilit y of purified Pectinesterase I and II un­ der the reaction conditions as used in the kinetic experi­ ments. Pectinesterases (about 40 units/ml) were incubated at 30 C with green ribbon pectin (1 mg/ml), 0.1 g/1 sodium azide, 0.03 mol/1 potassium phosphate pH 7.0 or 0.03 mol/1 potassium citrate pH 4.0, potassium chloride was added to give an ionic strength of 0.1.A t time intervals samples were taken tomeasur e activity under standard assay con­ ditions. Pectinesterase I at pH 7.0 (o o);Pectin ­ esterase I at pH 4.0( • •); Pectinesterase II at pH 7.0 (A A);Pectinesteras e II at pH 4.0 (A A).

Table 24.Incubatio n media1 for pectinesterase stability tests.

No. Orcat2 Sugars3 Pectin"*

1 1x 20 g/1 each 1g/ 1 2 3 x 20 g/1 each 1g/ 1 3 1x 60 g/1 each 1g/ 1 4 3 x 60 g/1 each 1g/ 1 5 1x 20 g/1 each + 40 g/1 sucrose 1g/ 1 6 single strength reconstituted Valencia orange juice concentrate 7 as medium No.6 , 3 x concentrated.

1.Sodiu m azide 0.1 g/1 and adjusted to pH 4.0 with potassium hydroxide. 2. Orange cations according to Krop (1974): 0.03 mol/1 potassium citrate, 0.08 mol/1 potassium chloride, 0.43 mmol/1 sodium chloride, 2.4 mmol/1 calcium chloride and 5.2 mmol/1 magnesium chloride. 3. Sucrose, glucose and fructose. 4. Green ribbon pectin.

tog (•/. residual activity) Fig. 43. Stability of pectinesterases in an orange juice-like environment. Pectinesterases (about 40 units/ml)wer e incubated at 30 °C in medium No. 5 from Table 21.A t time intervals samples were taken tomeasur e the residual ac­ tivity under standard assay conditions.Pectin ­ esterase I (o);crud e Navel orange pectinesterase 15" days (•);hig h molecular weight pectinesterase (•).

77 bationtim ea sdemonstrate di nFig .4 3fo r3 pectinesterase si non emediu ma t3 0°C .Fro m these destructioncurve sth edecima lreductio nvalue s (Dvalues )wer e calculated asi scommo npractic ei nthermobacteriolog y (Leniger& Beverloo , 1975). The Dvalue s foral lexperiment sar eliste di nTabl e 25.A sca nb esee ni nFig .43 ,th eline so fth e crudepectinesteras ean dth ehig hmolecula rweigh tpectinesteras ed ono tru n throughth e point t =0 ,lo g(_% residual activity)= 2 ,becaus e thesepreparation sar ecompose do f more thana singl e formo fpectinesterase ,wit hdifferen t stabilities.Th eamoun to f relatively stablepectinesteras ei nthes epreparation swa s calculated from theinter ­ ceptwit hth evertica laxi san di sliste dbetwee nbracket si nTabl e 25.I nth ecrud e preparationth e labilefractio nwil lmainl yb ePectinesteras e II.I nth ehig hmolecula r weightpectinesteras e therear e2 smal lhea t labilefraction s (seeSectio n8.1.2 ) and presumably the initialdro pi nactivit y canb eascribe dt obot ho fthem .Th e Dvalue s overabou t5 0day sar einaccurat ebecaus eo fth erelativel y short incubation timeo f 14days . Itca nb esee nfro mTabl e2 5tha tPectinesteras eI Ii sver y labile,especiall ya t 30°C .Pectinesteras eI i s100-20 0 xmor e stablea tmos t incubations conditions,bu t thehig hmolecula rweigh tpectinesteras ei seve nmor e stable.Generally ,th e Dvalue s ofth ecrud eenzym eresembl eth e Dvalue so fPectinesteras e I,becaus eafte rth e loss ofPectinesteras e II,abou t 901o fth eresidua lactivit yi sPectinesteras eI .Th e orange ionsi n3-fol dconcentratio nreduce dth estability ,wherea sth esugar s increased thestabilit yo fth epectinesterases .A 3-fol dio nconcentratio n combinedwit ha 3-fol d sugar concentration reducedth estabilit yo fth epur e enzymes considerably.Th esam e appliest oa 3-fol dconcentrate djuice .Remarkably ,th ecrud eenzym e followedthes e trendsonl ypartially . Inpreliminar y experimentsi tappeare d thatraisin g thepecti nconcentratio ni n mediumNo .2 (Tabl e 24)fro m1 t o3 g/ 1di dno taffec tpectinesteras e stability (Höcker, 1978).

8.1.2 Heat stability

Residual activities after 5 min at different temperatures Pectinesterases(0. 1ml ,5 units)wer eadde dt o5-m ltes ttube swit h1 m lo fa mediu m (1.1 xfina l strength), cooledi nice .Fo r theseexperiment san dals ofo rth eexperiment s described hereafter, thetube swer e selectedt ob eequa li nlength ,diamete r andweight .Afte rmixin gth e contents,th etube swer e incubatedfo r5mi ni na waterbat ha tvariou s temperaturesan d cooled inwate rwit hmeltin g ice. Immediately,th eresidua l activitywa smeasure d understandar d assay conditions.Fig .4 4show sth estabilitie si n0.0 1 mol/1potassiu m phosphatebuffe rp H7.5 ,Fig .4 5i norang e cationsolution ,p H4. 0(mediu ma si nFoot ­ note2 i nTabl e 24,thi si swithou t sugars,pecti nan dsodiu mazide )an dFig .4 6show s thestabilitie si nsingl estrengt h reconstitutedValenci a orange juiceconcentrate ,p H 4.0.I naddition ,Fig .4 6show sth estabilit yo fth epectinesteras epresen ti nfreshl y pressedValenci a orangejuice .Fo rthi spurpos eprechille dValenci a orangeswer ehalved , pressedo na rotar ykitche ncitru spres san dfiltere d througha 1 m msieve .Al lhand ­ lingswer edon ea t4 °C .Th ejuic e (pH3.85 )wa sadjuste dwit h1 mol/ 1potassiu mhy -

78 6*« fr* &•« &•>« in oo — CO oo m O^ vo ^-^ N-^ •^^ *w CM CM O oo o r*. co o CM O O *cr -* o O O — -* -d- -*

«5-S r*&•«. v O &*&C O* co m a\ vO V—' CM ^-^ CM m — O \o m o

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m o in o CO O o co CM co r^ o m

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A

e-« R ÎÎ vO m co m <7\ *D •o CO co dl CU O o\ CN 4J 3 — nl T-i CM O co r^ O O O o 00 co .o co m O o 3 «ï •a- •S s u > > .-H — CO cd •cH 0e J3 > •r-t CO 4J C ai at O co 3 s*s s-e 6-5 co •O m CN fr«CO •* , 3g h 01 0\ m o\ SO CN aj «H O) u s—* U X 4J vO -^ CU at o co r-t o\ r-t eu u 0) ~ Xi eu a. S g vO O m in O -ï O o n) U Cu •H CO 00 in o H eu al +J U co — -a- CN 'rOt Ol S (U > J3 CO n} M •H 4J -O •H r-t H eu nJ O eu o-in S u u et) CU M cd 4J CO H M JS O M H JS O

,'9 residual activity (*/•) residual activity (•/.) WOn m»-

80 60 80 temperature i'C) temperature (*C| Fig. 44. Heat stability of pectinesterases Fig. 45. Heat stability ofpectinesterases | in phosphate buffer ofp H7.5 . Pectin­ in orcat solution pH4. 0Sam e condi­ esterases (about 5 units/ml) were incu­ tions andprocedure s asi nFig . 44,except | bated in0.0 1 mol/1 potassium phosphate that thepectinesterase s were incubated buffer (pH7. 5) fo r5 mi na t various in orcat solution (orange cations) as des­ temperatures andth eresidua l activity cribed inth etext . Pectinesterase I was determined under standard assay con­ (o o); Pectinesteras e II (A A); | ditions. Pectinesterase I (o o);Pec - crude Navel orange pectinesterase tinesterase II( A A ); crude Navel (ü a); hig h molecular weight pectin­ orange pectinesterase (D O); hig h esterase (• •). molecular weight pectinesterase (• •).

residual activity (•/•) 100

Fig. 46. Heat stability ofpectinesterase si n orange juice ofp H4.0 . Same conditionsan d procedures asi nFig .44 , except thatth e pectinesterases were incubated in single strength reconstituted Valencia orange juice concentrate. Inadditio n also fresh Valencia orange juice (adjusted top H4.0 ) wasuse d with itsnatura l occurring pectinesterase. Pectinesterase I (o o);Pectinesteras e Il| (A A);crud e Navel orange pectinesterase (o o); high molecular weight pectin­ esterase (• •); fresh Valencia orange 60 80 temperature ('O juice with itspectinesteras e (* •—•*•).

80 droxidet op H 4.0.Th ejuic e»a s 13.3 Brixan dcontaine d 1.23unit spectinesteras e per ml.Th ejuic ewa shel da t0 ° Can duse dwithi na fewhour sfo rhea t inactivationstudies . Because ofth e low initialactivity , 10m lportion swer e incubated and assayed. Figs4 4- 4 6sho w that forinactivatio n ofPectinesteras e Ia 10 - 20° Chighe r temperature isrequire d than forPectinesteras e II.Th e crude pectinesterase containsa fraction,onl yabou t 5%o fth etota lactivity ,whic hrequire s a 20 - 30 C higher temperature for inactivationtha nbot hPectinesteras e Ian d II.Thi shea t stable fraction isth ehig hmolecula rweigh tpectinesterase .Th efigure sclearl y showth erela ­ tively greathea tresistanc e ofth ehig hmolecula rweigh tpectinesterase .Thi spectin­ esteraseshowe d 2smal lpeak s (HMI an dH M III)an da majo rpea k (HMII )upo nchromato ­ graphyo ncross-linke d pectate (Chapter 6,Fig .22) .Incubatio no fthes e 3peak ssepa ­ rately injuic ea s inFig .4 6f.o r5 mi na t 80° Cresulte d incomplet e losso fth eacti ­ vityo fPectinesterase sH M Ian dH M III.Th emajo r form,Pectinesteras e HM II,retaine d 734o fit sactivit y andn odifference swer eobserve dbetwee n different fractionso fthi s broadpeak .Th e initialsmal l loss inactivit y atrathe r lowtemperature s ofth ehig h molecularweigh tpectinesteras e inFig s4 5an d4 6ca nb eascribe d toth epectinesterase s inpeak sH M Ian dH MIII . Itca nb e seeni nFig .4 6tha t thenativ epectinesteras e inValenci a orangejuic e ismor ehea t stabletha tth ecrud eNave lorang epectinesteras e but considerably less heat stabletha nth ehig hmolecula rweigh tpectinesterase .Th ewarmin gu ptim e forth e 10m lValenci a juiceportion s islonge rtha nth ewarmin gu p time inth e 1.1 mlportion s with theothe rpectinesterases .Presumabl yunde r identical heating conditions thecurve s forcrud epectinesteras e andValenci ajuic ewoul db esomewha t closer toeac hother . Moreover,th eValenci a orangejuic eappeare d tocontai na pectinesteras e fractionwit h a heat stability lesstha ntha to fth ehig hmolecula rweigh tpectinesteras e but higher thantha to fPectinesteras e I,a s found inth eexperiment s hereafter. Iti sestimate d fromFig .4 6tha tth epectinesteras e inth e freshValenci ajuic e consistso fabou t10 % ofth ehig hmolecula rweigh tpectinesteras e ora nenzym ewit h asimila rhea t stability. Thepectinesterase s aremor e stable inth ereconstitute dValenci a juice (Fig.46 ) thani na norca t (orangecation )solutio n (Fig. 45),probabl ybecaus eo fth enatura l sugarconten t ofth eorang ejuice .

D and Z values Pectinesterases (0.1ml ,1 0units )wer eadde dt otes t tubeswit h 1m l juicereconstitute d fromValenci a concentrate to 1.1 times singlestrengt han d treated as inth ehea t inactivationexperiment s described above,bu t incubation timesa tth e different temperatureswer evaried .Th e log [\ residualactivity )wa s calculated and plotted against incubation time indestructio n curves asshow nfo rth ehig h molecularweigh tpectinesteras e inFig .4 7an d forcrud eNave lorang e pectinesterase inFig . 48.Fo r theexperiment swit ha single formo fpectinesteras e thedecima l reductionvalue s (Dvalues )a t thedifferen t temperatureswer e read fromth e figures (notshow n forPectinesteras e Ian d II)an duse d fortherma ldestructio n curves (Fig. 49). The Zvalue s (therais e intemperatur e (C )necessar y toobserv e a tentime s fasterhea t inactivation)wer e taken froman dar e indicated inthi s figure.Th ewarmin g up timewa s eliminated fromth e Dvalue sb yusin g only thestraigh tpar t ofth e lines in

81 log (°/. residual activity) 2.0

Fig.47 .Hea tstabilit yo fhig hmolecula r weightpectinesteras e inorang ejuic eo f pH4.0 .Hig hmolecula rweigh tpectin ­ esterase (10units/ml )wa sincubate di n singlestrengt h reconstitutedValenci a orangejuic econcentrate ,adjuste d top H 4.0,fo rsevera ltim einterval sa tsevera l temperaturesa sindicate d inth efigure . 2 3 4 Residualactivitie swer emeasure da tp H inactivation time (min) 7.0unde rstandar dassa yconditions .

log (*/o residual activity ) 20-

Fig.48 .Hea tstabilit yo fcrud eNave l orangepectinesteras e inorang ejuic eo f pH4.0 .Crud eNave lorang epectinesteras e (10units/ml )wa sincubate dan dassaye d 2 3 4 inactivation time (min) asi nFig .47 .

82 90 temperature («O Fig.49 .Therma ldestructio n curvesfo rpectin - esterasesi norang ejuic eo fp H4.0 .Method san d conditionso fth edetermination sa si nFig.47 .Pec - tinesteraseI ,I Ian dth ehig hmolecula rweigh tpec - tinesterase (HM)ar eindicate d inth efigure .

thedestructio ncurve sa sshow ni nFig .47 . Theinactivatio no fth ecrud epectinesteras e followsa complicate dpatter n (Fig.48 ) andn orea l Dvalue sca nb eobtained .A t6 0 Cmainl yPectinesteras e IIbu tals oa con ­ siderablepar to fPectinesteras e Iwil lb erapidl y inactivated.A t7 0 C,afte rth ein ­ activationo fPectinesteras e Ian dII ,onl yth ehig hmolecula rweigh tpectinesteras e remainsafte rheatin gfo r 1\ min.Thi spectinesteras ei sver yslowl yinactivate d (from thisfigur eestimate dt ob eabou t 5%o fth etota lactivity) . Thehea tinactivatio no fth epectinesterase si nfres hValenci aorang ejuic eals o followeda complicate dpatter n (resultsno tshown) .I tappeare dt ocontai nabou t10 1o f thehig hmolecula rweigh tpectinesteras e (ora pectinesteras ewit ha simila rhea tstabi ­ lity)an d 20%o fa pectinesteras ewit ha stabilit yles stha nth ehig hmolecula rweigh t pectinesterasebu thighe rtha nPectinesteras eI . Fig.4 9give sa quantitativ eillustratio no fth edifference si nhea tstabilit y betweendifferen tform so fpectinesterase .

8.2 DISCUSSION

Thehea tstabilitie so fth epectinesterase si npotassiu mphosphat ebuffe r (pH7.5 ) mayb ecompare dwit hth eresult so fManab e (1973a)fo rpurifie dan dcrud e Citrus natsu- daidai pectinesterase.Manab edetermine dth ehea tstabilitie sunde ridentica lconditions . Bothth epurifie dan dth ecrud eNatsudaida ipectinesteras ewer ecompletel yinactivate d after5 mi na t8 0 C.Apparentl yneithe rth ecrud eno rth epurifie denzym econtaine da

83 detectablequantit yo fhig hmolecula rweigh tpectinesterase .Th e crudeNatsudaida ipec - tinesterase seemedt ocontai n about 15%o f(a )pectinesterase(s )wit ha stabilit y betweenPectinesteras e Ian dth ehig hmolecula rweigh tpectinesterase ,wherea si nth e purifiedNatsudaida ipectinesteras e this fractionwa sreduce dt oabou t 7%o fth etota l activity. Inolde r literature irregularhea t inactivationpattern sar eals oshow n (Bissette t al., 1953;Rous e& Atkins ,1952 ;Rous e& Atkins , 1953). From the thirdpublicatio ni tca n be estimated thatabou t 7%hig hmolecula rweigh tpectinesteras e (ora pectinesteras e witha simila rhea t stability)wa spresen ti nbot h theHamli nan dPineappl e orange pectinesterasean dabou t 20%i nth eMars hSeedles s grapefruitpectinesterase .Th e relativelyhig hamoun to fhig hmolecula rweigh tpectinesteras ei ngrapefrui twa s also foundi nSectio n6.1.2 ,Tabl e 18.Remarkably ,i nsom erecen twork n oirregularitie s inhea t stabilityo fpectinesterase so fcitru sjuic ear ereporte do rshow n (Eagerman& Rouse, 1976;Nat h& Ranganna , 1977a). Fromth eresult spresente di tca nb ededuce d that allpectinesteras e was ratherstable .Th evalue sobtaine dwit hcitru s juiceb ythes e authorsan dwit hpurifie dpectinesteras epreparation si nthi swork wer e recalculated with the following formulat ofacilitat e comparison:

log {D/D ref.)= (Tref. - T)/Z (Leniger& Beverloo , 1975)

The recalculated values togetherwit hth eincubatio nenvironment sar eliste di nTabl e26 . Itca nb esee ni nTabl e2 6tha tbot hauthor s foundrathe rhig hhea tstabilities ,mor eo r lessth estabilit yo fth ehig hmolecula rweigh tpectinesterase .Th e pectinesterasei nth e mandarin juiceha dals oremarkabl y highZ values .I tma yb epossibl e that themandarin s containconsiderabl e amountso fa pectinesteras eno tencountere di nm ywork .I nthi s work somepectinesteras e formswer e foundi nmandarin swhic hwer eno teviden ti n

Table 26.Compariso no fth e heat stabilityo fpectinesterases .

Sourceo fenzym e Juicefo rincubatio n Stability

origin pH pulp Brix D90° C Z c content t(min • \ ) (° ) (ml/100ml ) PectinesteraseI INave l Valencia 4.0 12.6 0.0015 11.0 Pectinesterase I Navel Valencia 4.0 12.6 0.00037 6.5 Highmolecula rweigh t pectinesterase Navel Valencia 4.0 12.6 0.375 6.5 Hamlin (orange)1 Hamlin 4.1 12 12.8 0.151 4.8 Pineapple (orange)' Pineapple 4.1 12 12.8 0.177 5.1 Valencia (orange)1 Valencia 4.1 12 12.8 0.500 6.8 Duncan (grapefruit)1 Duncan 3.6 10 10.5 0.069 5.2 Mandarin2 Mandarin 3.6 16 12.0 0.793 11.4 Mandarin2 Mandarin 4.0 16 12.0 1.11 10.1

1.Calculate d fromEagerma n& Rous e (1976). 2.Calculate d fromNat h &Rangann a (1977a).

84 grapefruit andorang e (Section6.1.2 ,Fig .24) .Furthe r «relativel yhig hamoun t of pectinesterasewa s foundwhic hmigrate d slowly (i?f< 0.55) inp H gradientelectrophore ­ sis (Section6.1.2 ,Tabl e 18).Nothin g isknow na sye tabou tthes eform so fpectinestera ­ se. Pectinesterase extracted fromth ewhol e orangema yb ever ydifferen tfro mth epectin ­ esterasewhic h canb e found inth ejuice .Bu tth eexperiment swit h thefreshl yprepare d Valencia juicereveale d thatn omor etha n 10%o fth epectinesteras e hada hea t stability comparablewit h thehig hmolecula rweigh tpectinesterase . Iti sdifficul t toexplai nwh y theauthor scite d inTabl e 26di dno tobserv e irregularities inth ehea tstabilit yde ­ terminations.Bu t itseem s likelytha tprocesse sbase d onth e Dan d Zvalue s recommended by theseauthor san dth etota lpectinesteras e activitypresen t inth ejuic ea sa base , result inoverpasteurize d juice.Th eproble mo fhea t inactivation ofpectinesteras e in mostcitru sjuice sma yb e reducedt o inactivation ofth ehig hmolecula rweigh tpectin ­ esterase,whic hrepresent sonl ya par to fth etota lactivity .Th eothe rpectinesterase s areeasil y inactivatedbelo w 80 C. At increased sugarconcentration s thestabilit y ofpurifie d Pectinesterase Ian d II at 30° C isincrease d anda t increased ionicstrength ,th estabilit y isdecrease d (Table 25). Thedestabilizatio nb y the ionicstrengt hwa s strongertha nth estabiliza ­ tionb yth e sugars.Thi seffec tcoul d alsob eobserve d in3-fol dconcentrate d orange juice.Th estabilit yo fth ehig hmolecula rweigh tpectinesteras e at 30 Cwa s toohig h toobserv esuc ha neffec ti fa tal lpresen t (Table 25).Th epositiv e effecto f sugars onth ehea t stabilityma yb epresume db y comparing Figs4 5an d 46.I tma yb emuc h easiert oinactivat e thehig hmolecula rweigh tpectinesteras e inconcentrate d juice thani nsingl estrengt hjuice .Bisset t et al. (1953)indee dobserve d differences in heatstabilit yo fpectinesterase s andclou d stabilityo fValenci ajuic e concentrates depending onth econcentratio n factoro fth ejuice .Th erelation swer e complicated. Thepectinesteras ewa s lesshea tstabl e in2- 4 foldconcentrate d juice thani nsingl e strength juice,bu tmor ehea tstabl e in6-fol dconcentrate d juice.Nevertheles s cloud stabilitywa smos teasil y obtained in6-fol dconcentrate d juice (heattreatmen t less than 1mi na t 71 °C).Inhibitio no fth epectinesteras e by the increased sugar concentration (seeals oSectio n2.2.5 )wil l alsohel p toincreas e cloud stability.

85 9 Cloud loss of orange juice

Destabilizationo fth eclou do fcitru sjuic eha sbee nknow nfo ra lon gtim ea sa n effecto fpectinesteras eactivity .Kro p (1974)reviewe dth eliteratur eo nthi ssubjec t andstudie dth emechanis mo fclou dlos sphenomen ai norang ejuice .U ptil lno w unpasteurizedjuice so rpasteurize djuice swit hadde dcrud epectinesteras epreparation s wereuse di npectinesteras e- clou dlos sstudies .I nadditio nt opectinesterase ,som e otherenzyme sma ypla ya role ,fo rexampl eb ymakin gth esubstrat emor eaccessibl efo r pectinesterase.Therefor ei nth eexperiment sdescribe dbelow ,purifie dPectinesteras eI andI Iwer etested ,bu tals oth ecrud eNave lorang epectinesteras ean dth e(impure ) highmolecula rweigh tpectinesterase .

9.1 EXPERIMENTSAN DRESULT S

9.1.1 Pectinesterasesan dth eclou dlos sa t3 0° Can d5 ° C

Pectinesteraseinactiv eValenci aorang ejuic econcentrat ewa sdilute dwit h 2volume s ofdistille dwater ,potassiu mmetabisulphit ewa sadde dt ogiv ea fina lconcentratio no f 1000mg/ 1sulphu rdioxid ean dth ep Hwa sadjuste dt o4. 0wit h 1mol/ 1potassiu m hydroxideunde rvigorou sstirring .Th evolum ewa sadjuste dt ogiv e5/ 4x singl estrength . To4 0m lo fthi ssolution ,propanol- 2wa sadde dt ogiv ea fina lstrengt ho f2 5mg/1 , 50unit so fpectinesteras ewer eadde dan dth evolum ewa sadjuste dt o5 0ml .Th ejuice s wereincubate di nglas sstoppere dbottle sa t3 0 Co r5 C.Th ejuice sfo rth e5 C incubationwer eprepare da t0 °C .I nthes eexperiment spreservatio nwit hsulphu rdioxid e waspreferre dove rsodiu mazide .Brownin go fth ejuic eupo nincubatio nwit hth elatte r preservativewa sno tprevented . Inpreliminar yexperiment swit hcrud epectinesterase ,i t wasestablishe dtha t2 5mg/ 1propanol- 2affecte dneithe rclou dstabilit yno r pectinesteraseactivity .Als other ewa sn odifferenc ebetwee nth e2 preservative si nthi s respect.A ttim eintervals ,3 m lsample swer eremove dafte rinvertin gth ebottle s1 0 times.Immediatel yth eturbidit ywa smeasure da sdescribe si nChapte r3 .Th esedimen t andth esupernatan t ofth esampl ewer eremixe dan dkep ti nic etil luse dfo rga s chromatographicmethano lassa y (seeChapte r 4),whic hwa sdon ewithi n2 h afte rremova l ofth esampl efro mth eincubatio nbottle .Fig .5 0A show sth eclou dstabilit yo forang e juiceupo nincubatio nwit hdifferen tpectinesterase sa t3 0 Can dFig .5 0B show sth e methanoldevelopmen ti nth esam esamples .Fig s5 1A an d5 1B sho wth esam efo rsample s incubateda t5 °C . Allpectinesteras epreparations ,excep tth epurifie dPectinesteras e II,destabilize d theclou do forang ejuice .A t3 0 CPectinesteras e IIstoppe dbefor ea concentratio no f 20mg/ 1methano lwa sreached .A t5 ° Ca concentratio no f3 5mg/ 1methano lwa sreached ,

86 E 660 nm 15- •>„ a

days

methanol (mg/l) B 60

days Fig.50 .Clou dstabilit yo f (A)an dmethano lformatio ni n(B ) orangejuic eincubate dwit hpectinesterase sa t3 0 C.Singl e strengthreconstitute dValenci aorang ejuic econcentrate , pH4. 0wit h 1000mg/ lsulphu rdioxid ewa s incubatedwit hseve ­ ralform so fpectinesteras e (1uni tpe r ml).Pectinesterases : crudeNave lorang epectinesteras e( • D); purifie dPectin ­ esteraseI ( o o); purifie dPectinesteras eI I (A^—:A ); highmolecula rweigh tpectinesteras e( • •); nopectin ­ esterase (+— — +).

butth eclou dwa sno tdestabilized .Wit hth eothe rpectinesterases ,clou ddestabilizatio n becameapparen twhe nabou t3 0mg/ lmethano lwa sattained .A t5 °C ,th einitial^activit y ofal lpectinesteras epreparations ,excep ttha to fPectinesteras eI ,wa sstil lrathe r high.Th eactivit yo fPectinesteras e Ia t5 ° Cwa sabou ton ehundredt ho ftha ta t3 0 C. At3 0° Ca concentratio no f3 0mg/ lmethano lwa sreached !withi n6 h ,wherea sa t5 Ci t lastedabou t2 5day sbefor ethi sconcentratio nwa sbuil tup .Consequentl yi tlaste d

87 methanol (mg/l) 60n B

Fig.51 .Clou dstabilit yo f (A)an dmethano lformatio n in(B )orang ejuic e incubatedwit hpectinesterase sa t 5 C.Condition s (except temperature)a si nFig .50 . Fectinesterases:crud eNave lorang epectinesteras e (ü D); purifie dPectine steras eI ( o o); puri ­ fiedPectinesteras e II( A A);hig hmolecula r weightpectinesteras e( • ••); nopectinesteras e (+ +).

nearly4 week sbefor eclou ddestabilizatio nbecam eapparen twit hPectinesteras e I.I f after3 day sincubatio na t5 ° Ca bottl ewa sfurthe rincubate da t3 0°C ,th eclou dwa s completely lostwithi n 24h (resultno tshown) . 9.1.2 Cloudstabilizatio nb ypolygalacturonas e

CrudeNave lorang epectinesteras ean dpurifie dPectinesteras e Iwer ecombine dwit h partiallypurifie dyeas tendo-polygalacturonas ei nth eclou dstabilit ytests .Severa l concentrationso fpolygalacturonas ewer etested .Th esample swer eincubate donl ya t 30 C.Th econdition san danalyse swer eth esam ea sdescribe di nSectio n9.1.1 .Fig .5 2 showsth eresult sfo rth ecrud epectinesteras ean dFig .5 3show sth eresult sfo r PectinesteraseI .

E66 0n m

B

Fig.52 .Clou d stabilityo f (A)an dmethano l formation in(B ) orangejuic eincubate dwit hcrud epectinesteras ean ddifferen t concentrations ofpolygalacturonase .Singl estrengt hreconsti ­ tutedValenci aorang ejuic e concentrate,p H4. 0wit h 1000mg/ 1 sulphurdioxid ewa s incubatedwit hcrud eNave lorang epectin ­ esterase (1uni tpe rml )an dsevera l concentrations ofpoly ­ galacturonasea t 30 C.Polygalacturonas e concentrations:n o polygalacturonase (o); 10 —•*unit_pe r ml (•); 5x1 0* uni tpe r ml (A);10~ 3uni t perm l (+); 1 0* uni tpe rm l(*•) .

89 E66 0n m

B

Fig.53 .Clou dstabilit yo f (A)an dmethano l formationi n(B ) orangejuic eincubate dwit hpurifie dPectinesteras eI an d differentconcentration so fpolygalacturonase .Condition sa s inFig .52 ,excep t thatPectinesteras e I( 1uni tpe rml )wa s used.Polygalacturonas e concentrations:n opolygalacturonas e (o); lO-1*uni tpe rm l (•); 5x1 0* uni tpe rm l (A);10~ 3uni t perm l (+);10~ 2uni tpe rm l(*) .

Itca nb esee ni nth efigure stha tth epolygalacturonas estimulate dth emethano l productionfo rbot hpectinesterases .I nspit eo fthi sincrease dpectinesteras eactivity , theorang ejuic eclou dstabilit ywa sincreased .A tth etw ohighes tlevel so f polygalacturonaseactivity ,th eclou dwa sno tdestabilize da tall . Todetermin eth etota lfre ean dboun dmethano li nth ejuice ,3 0m lreconstitute d juice (5/3x singl estrength )wa sadjuste dt op H 11.0wit h2 mol/ 1sodiu mhydroxid ean d incubatedfo r1 h i na glas sstoppere dflas ka troo mtemperature .Th esolutio nwa s neutralizedwit h2 mol/ 1hydrochlori cacid ,5 m lo fa solutio no f25 0mg/ 1propanol- 2 wasadde dan dth evolum ewa sadjuste dt o5 0ml .Th emethano lconcentratio nwa sdetermine d

90 gas chromatographically (seeChapte r 4)an dwa s 62mg/1 .Th e freemethano l concentration was 9mg/ 1 and 53mg/ 1methano l couldb e releasedb y thealkalin e de-esterification. With ananhydrogalacturoni c acid content of 574mg/ 1 (Krop, 1974), theaverag e degree ofesterificatio n ofth epecti n inth ejuic e iscalculate d tob e 5H.

9.2 DISCUSSION

Itwa sno tpossibl e toclarif y theorang e juicewit h Pectinesterase IIa t 30 C.A t 30 C theenzym ewa s inactivated (seeTabl e 25)befor e itcoul dde-esterif y thepecti n toa nexten t thatclarificatio n occurred. Itcoul dno t raise thefre emethano l concentration toove r 20mg/ml ,wherea swit h theothe renzyme s clarification startedwhe n a concentration ofabou t 30mg/ 1methano lwa s reached. The Dvalu e forPectinesteras e II inactivation at 30° Ci njuic e isabou t 5h (seeSectio n 8.1.1,Tabl e 25). However,a t 5 °C,th eenzym e issufficientl y stable (ovalu e forinactivatio n about4 jdays )t o buildu pa concentratio n of 35mg/ 1methanol .I nspit eo f this,th ejuic ewa s not clarified.Thi sma y indicate amod eo factio no nth esubstrat e inth ejuic ewhic h is different oftha to fPectinesteras e Ian d thato fth ehig hmolecula rweigh tpectin ­ esterase.Possibl y shorterblock s offre ecarboxy l groups areforme d thanwit h theothe r pectinesterases.Fo rjuic eclarificatio n thepectat echain smus thav eblock s of lôormore monomerswit h freecarboxy l groups (Termotee t al., 1977). Thisdoe sno t necessarily mean thatPectinesteras e IIremove smethoxy lgroup s atrando m frompecti na s suggested for thepectinesteras e from Aspergillus japonious (Ishiie t al., 1979). Krop (1974) observed differences inth eclarificatio n offres h juices fromdifferen t orange varieties,whic h couldno tb e related tooveral lpectinesteras e activity.Kro p (1974) suggesteddifference s inth emod eo factio no fth epectinesterase s ason eo fth epossibl e explanations. Krop (1974)observe d only asligh t increase inth einitia lpectinesteras e activity when itwa s incubated inth ejuic e togetherwit hpolygalacturonase . Itnow appear s that both the initial activity ofth epectinesterase s and theultimat emethano l concentration areincrease dwit h increasingpolygalacturonas e concentration (Figs 52B and 53B) .Th e stimulationo fpectinesteras e activitymus tb eascribe d tohydrolyti csplittin g of longer sequences ofgalacturoni c acidmonomer s (removal ofen dproduc t inhibition).A t thesam e timeclarificatio n ofth ejuic e isprevente db y thisdegradatio n ofth e longer sequences ofgalacturoni c acidmonomers .Th e remaining galacturonic acid oligomers do notprecipitat ewit h calcium ions. Thecrud epectinesteras e used inthi sstud yappeare dt ob e slightlymor e effective inorang ejuic eclou ddestabilizatio n thanth epectinesteras e preparationuse db y Krop (1974). Also inthi swor kmor epolygalacturonas ewa sneede d tostabiliz e theclou d thani nth eexperiment s ofKro p (1974) (compare'Fig.5 2A from thiswor kwit hFig . 39B from Krop). Because thesam ejuic ean dpolygalacturonas e were used inbot h experiments, thedifference smus tb e ascribed toth epectinesteras e used.Kro p (1974)use d another orange cultivar forth epectinesteras e preparation andals oth eextractio n procedure was somewhatdifferen t (Kropuse d less saltan ddi dno t reextract thepres s cake). Botha t 30 C anda t 5 C,th ehig hmolecula rweigh tpectinesteras ewa s themos t

91 effective clouddestabilize! -.A t 30° Cth edifferenc ewit h thepurifie dPectinesteras e I wasver y small.Th e facttha tth epurifie d Pectinesterase Icoul dclarif y orangejuice , proves thatn oothe renzyme s areneede d inth eproces s ofclarification . Iti spossibl e thati nth eclarificatio nwit h thehig hmolecula rweigh tpectinesteras e oneo rmor e otherenzyme swer e involved,a s thisenzym ewa sno tpur e atall .I nChapte r 5,Tabl e 16 severalenzym e activities are listedwhic hwer e found tob epresen t inth ecrud e enzymepreparation . By alkali de-esterification ofth ejuice ,a methano l concentration of6 2mg/ 1wa s measured.Whe ncombine dwit hpolygalacturonase ,pectinesteras e couldnearl yreac h this samemethano l concentration (Figs 52B and 53 B). Apparently allmethy leste r groups were eventually de-esterifiedb y thepectinesterases .Thi swa s notexpecte dbecaus e it was impossible tode-esterif y thelas t 10%o fth emethoxy l groups,a sdescribe d in Section 7.1.2.Bu t inthos eexperiment s nopolygalacturonas e hadbee nused .

92 10 General discussion and conclusions

Thega schromatographi c methanol assayprove d itsusefulnes si nman y experiments withpectinesterase sa tlo wp Hvalues . Thepresenc eo fsevera l (12)molecula r formso fpectinesteras ei ncitru swa s established.Th edifferen t forms canb edistinguishe db ythei rmobilit yi np Hgradien t electrophoresis.Onl y three forms,representin g 85-954o fth etota l activityi nal l citrus fruits,wer e further investigated.Thes e formsdiffe ri non eo rmor eo fth e followingproperties :isoelectri cpoint ,amin o acid composition,molecula rweight , pH-activityprofile , K forpectin , K. forpolygalacturoni c acid,stabilit yi norang e juice,hea tstability ,orang ejuic eclarificatio npropertie san dtemperature-activit y relationship. Two forms,Pectinesteras eI an dI Ica nb ecalle d isoenzymes.Thes etw oform s represented 604o rmor eo fth e totalactivit yi nvariou s citrus fruits.Th ehig h molecularweigh tpectinesteras ewa sals odetecte di nal lcitru s fruits examined.No tal l 12molecula r formswer edetecte di nal lo fth ecitru s fruits,bu tu pt o1 0form swer e foundi na singl e fruit:th egrapefruit .Th erelativ e amountso fth e formso fpectin ­ esterasevarie dwit h orangecultiva ran dcitru s species.I ti spossibl e thatal lform s ofpectinesteras ewer epresen ti nal lcitru s fruitsexamined ,bu ttha tsometime s their quantitiywa sbelo w thedetectio n limito fth etechniqu eused .Form s representing less than2 4o fth etota lactivit ycoul dusuall yno tb edetected . ThoughPectinesteras eI an dI Itogethe rrepresen t themajo rpar to fth eactivit ya t standard assay conditions,thi si sno tth ecas ea tlowe rp Hvalues .Th eactivit yo f PectinesteraseI Ii srapidl y reduceda tlowe rp Hvalues .I ncitru sjuic ea t3 0C , PectinesteraseI Ii sno ttechnologicall y importantbecaus eo fit s lowactivit y andpoo r stability.Thoug hPectinesteras eI i smor e activea tp H4. 0tha nth ehig h molecular weightpectinesterase ,a tp H2. 5th eonl yactiv epectinesteras ei sth ehig hmolecula r weightpectinesterase .I tma yb epresume d that thenatura l clarificationo flemo njuic e (Uhllg, 1978)i sdu et oth eactivit yo fthi s formo fpectinesterase ,whic h represents onlya smal l fractiono fth eactivit yunde rstandar dassa yconditions . Amongth ethre e forms investigated,th ehig hmolecula rweigh tpectinesteras ei s the onlyon ewhic h canpla ya rol ei nclou d lossphenomen ao f(under )pasteurize d citrus juices.Pectinesteras eI an dI Ica nrapidl yb einactivate da ttemperature sbelo w8 0C . About thispasteurizatio n temperaturei srequire dt oavoi dmicrobia l spoilage (Bissette t al., 1953). Thedifference si nhea t stabilityo fpectinesteras ei ncitru s juicesa tth esam epH , °Brix,io n concentrationsan dpul pconten t (Rouse& Atkins ,1952 ;Rous e& Atkins ,195 3a ; Eagerman& Rouse ,1976 )no wca nb eascribe dt odifference si nth epectinesteras e composition,eve n though thehea t stabilityo fa numbe ro fform so fpectinesteras ear ea s

93 yetunknown .Fro m thewor k ofEvan s &McHal e (1978)i tca nb e seentha tth epectin - esterase composition differswit h thepar to f thefruit .Therefor e a formo fpectin - esterasebein g onlya smal lpar to fth etota l activity inth e fruitma yb epresen ti n a relatively largequantit y inth ejuice .Th epressin gmetho duse d toproduc e citrus juicewil lno tonl yaffec tth epectinesteras e concentrationbu tals oit scomposition . Atpresen tnothin g isknow nabou tth eorigi no fth evariou s forms ofpectinesterase . Ifthe ywer e tob egeneticall y determined itwoul d seempromisin g toeliminat e thehea t stablehig hmolecula rweigh tpectinesteras e throughbreeding . IfPectinesteras e Io rI I were tob e derived franth ehig hmolecula rweigh tpectinesterase ,fo r instanceb y a process invivo ,i twoul db e attractive tostud y thisproces s and tryt ostee r iti na technologically desirable direction. Some lightwa s throwno nth emod eo factio no f thepectinesterases .Th erapidl y increasing affinity forsubstrate swit h alowe rdegre e ofesterificatio n led toth e theory that forth eoptima l enzyme-substrate complex two freedissociate d carboxyl groups ata fixeddistanc e inth esubstrat e are required.Th edecreasin g affinity at a lowerp H is inagreemen twit h this theory.Th emean sb ywhic hMacMilla nan d coworkers (Lee& MacMillan , 1970;Mille r &MacMillan , 1971)reache d theconclusio n thathal fo f thepectinesteras e activitywa s initiated atth ereducin gen dwa scriticize d inSectio n 2.2.4.A n importantquestio nwhic h remains tob e answered ist owha t extent the.variou s pectinesterases attackthei rsubstrat eb y asingl echai nmechanism . Parto fth e conclusions ofSolm s &Deue l (1955)wer equestione d inSectio n7.2 . The specificactivit y and turnovernumber s ofPectinesteras e Ian d IIwer e calculated 1$ with theLowr yprotei n determination.Bu t ifth e estimated E. at 280n mar euse d for theprotei n concentrations,bot hvalue swil lb e approximately twicea shig h forbot h pectinesterases. Ihop e that theknowledg e ofth eexistenc e ofmultipl emolecula r formso fpectin ­ esterasewit hwidel y differentpropertie s incitru s fruitsa spresente d inthi swor kwil l promote research towards theorigi nan dth e functiono fthes eenzyme s andwil lprovid e thefrui tjuic etechnologis twit husefu lbasi c information.

94 Summary

Thepurpos eo fthi sstud ywa s toobtai na bette r insight inth epropertie s of pectinesterase andi nth e clarificationo forang e juice,i nwhic hpectinesteras eplay s arole . Afterth eintroductio n inChapte r 1,th e literatureo npectinesteras e issurveye d anddiscusse d inChapte r 2,coverin g thesubjects :occurence ,rol e invivo ,purificatio n andproperties ,specifit yan dmod eo faction ,inhibitio nan d (in)activation,importanc e forfoo dtechnolog yan dassa yan ddetectio nmethods . Chapter 3give s thematerial suse dan d thestandar dmethod s applied inthi sstudy . Chapters 4 to9 describ eth eexperiment s andresults .Th eresult s arediscusse d ineac hchapter . Detailed informationabou t aga s chromatographic alcoholassa y isgive ni nChapte r4 . Becauseo fth enee d tomeasur epectinesteras e activitya tcondition sunde rwhic h a titrimetricassa y isdifficul t orimpossible ,a ga s chromatographicmethano l assaywa s improved. Inthi smethod ,alcohol s areconverte d tothei r (volatile)nitrit eester s and theheadspac eo fth esolutio ni ssample dan dinjected .Th emetho d israpi d (aboutS min/sample),sensitiv e (<1 mg/1 )an dreproducibl e (variationcoefficien tabou t 1.51). Forsevera lbiologica l fluids themetho d isdirectl y applicablewithou tpretreatmen t of thematerial . Chapter5 describe s thepurificatio no ftw opectinesteras e isoenzymes alongwit h theirmolecula rproperties .Th epectinesteras e fromNave l oranges (6250 0unit spe rk g oranges)wa sextracte dan dpurifie db yfractionate dammoniu msulphat eprecipitatio nan d byge l filtrationo nBio-Ge lP-100 .Th e enzymepreparatio n couldb e separatedb y chromatography oncross-linke dpectat e intoa Pectinesteras e I,whic h didno tbin d to thismatri xan da cross-linke dpectat ebindin gPectinesteras e II.Bot henzyme swer e furtherpurifie db ychromatograph y onC MBio-Ge lA .Thei rspecifi c activitieswer e 694 and 762unit spe rm gprotein ,respectively .Bot hisoenzyme smov ea ssingl eband s of molecularweigh t 36 200durin gsodiu mdodecy l sulphate electrophoresis,an d areals o singleband swit h isoelectricpoint s of 10.05an d >11.0,respectively ,upo n isoelectric focusing.A numbero fcel lwal ldegradin g enzymeswhic hwer epresen t inth ecrud eenzym e preparation,wer e absent from thepurifie denzym epreparations .Th eenzyme s differ in aminoaci dcompositio nan d theyar en oglycoproteins . Chapter6 concerns a survey ofth emultipl emolecula rform so fpectinesteras e in citrus fruits.A special thin-layerp Hgradien t electrophoresis methodwa s developed withwhic h several additional formso fpectinesteras e couldb e detected.Severa l orange cultivarsan dcitru sspecie swer eassaye dfo rthei rform sô fpectinesterase .A totalo f 12form so fpectinesteras ewer e found inal lcitru s fruits together.Pectinesteras e Ian d II togetherrepresente d 60$ -90 $o f thetota lpectinesteras e activity inal lcitru s

95 fruitsexamined .Anothe rpectinesterase ,wit ha highe rmolecula rweight ,whic hwa s also present inal lcitru s fruitsexamined ,wa s also incorporated inth efurthe rstudies .Thi s highmolecula rweigh tpectinesteras ewa s inhomogeneous andinpure . InChapte r 7th ekineti cpropertie s arestudied .O nbrow n ribbonpecti n (degreeo f esterification 724)Pectinesteras e Ian d IIhav eoptimu mpH-value so f 7.6 and 8.0, respectively.O ngree nribbo npecti n (degree ofesterificatio n621 )th e pH-activity curves are about 1p Huni tbroade ra t theaci dsid eo f thecurve .Th ehig h molecular weightpectinesteras e hasa minimu mactivit y atp H 3.5 anda t lowerpH-value s the activity increases again.A tp H 7.0 Pectinesterase Iha s a K valueo f0.08 3m go fgree n ribbonpecti npe rm lan da K. value forpolygalacturoni c acido f0.4 2m gpe rml .Fo r Pectinesterase II thesevalue s are0.004 6an d0.001 6m gpe rml ,respectively .Fo rbot h Pectinesterase Ian dI I the K value is related toth edegre eo festerificatio no fth e substrate inth e followingway : 1/K = a (4fre e carboxyl groups)2 and ai sabou t ten timeshighe r forPectinesteras e IItha nfo rPectinesteras e I.Fro mthi srelationshi p it was reasoned thattw o freecarboxy l groups ata certai n fixeddistanc e inth e substrate are required forth eoptima lenzyme-substrat e complex.Th e V isnearl y independent ofth e degree ofesterificatio no fth esubstrate .Th e K isincrease da t lowerp Hvalues . m The lowerth e initialdegre eo festerificatio n ofth esubstrate ,th e lower thedegre e of esterificationwhic hcoul dultimatel yb ereache db y theenzymes .Pectinesteras e Iwa s moreeffectiv e inthi srespec t thanPectinesteras e II,bu t inn ocas ea degree of esterificationo flowe rtha n 114coul db eachieved .Th eenzymi cde-esterificatio n ofhigh ­ lyesterifie dpecti n (degreeo festerificatio n 95.64)wa s very incomplete.Som e ofth e substratemolecule swer e notde-esterifie d atal lan d these appeared tob e richo f galactose. Itwa s therefore concludedtha tsid echain s inth epecti nmolecul e forma n obstruction forpectinesteras e action.Endo-polygalacturonas ean dendo-pectat e lyase increasedth erat ean d limito fde-esterificatio npresumabl yb yalleviatin g endproduc t inhibition. InChapte r 8th e stability of thepectinesterase s isexamined .A t 30° Ci norang e juice ofp H 4.0,Pectinesteras e Ian d thehig hmolecula rweigh tpectinesteras e are stable,bu tPectinesteras e IIi squickl y inactivated (Dvalu e about 5 h). Sugars increase andion s reduce thestabilit y ofpectinesterases .Th epecti n concentration hasn oeffec t onth epectinesteras e stability.Th eenzyme s differgreatl y inhea t stability inorang ejuic ea tp H 4.0.Th e Dvalue sa t9 0 °Can dZ value sar e0.0003 7mi n and6. 5 °Cfo rPectinesteras e I,0.001 5mi nan d 11° Cfo rPectinesteras e IIan d0.37 5mi n and6. 5 °C forth ehig hmolecula rweigh tpectinesterase .Th ehea t stablehig hmolecula r weightpectinesteras ewa s only asmal l fraction ofth ecrud eNave lorang e pectinesterase preparation (54).Th epectinesteras e infreshl ypresse dValenci aorang e juice contained about 104o fth ehig hmolecula rweigh tpectinesteras e ora pectinesteras ewit h a similarhea t stability. Chapter9 concern s theorang ejuic eclou ddestabilizin gpropertie s ofth epectin ­ esterase at 5° Can d 30°C .Pectinesteras e II isunabl e toclarif yorang e juice,thoug h itsstabilit y at5 C allowsconsiderabl e de-esterificationo f thejuic epectin .A t 30 C Pectinesterase Ian dth ehig hmolecula rweigh tpectinesteras e quickly'destabilize theclou do forang e juice.A t 5 C thehig hmolecula rweigh tpectinesteras e is still

96 effectivebu t theactivit y ofPectinesteras e Ii shundre d timesles s thantha ta t 30 C. Endo-polygalacturonasestimulate s thepectinesteras e activity inorang ejuice .I nspit e of this,th eorang e cloudi sno t destabilized. InChapte r 10th eoveral lresult s arebriefl ydiscusse dan dsom epossibilitie s for furtherresearc h arementioned .

97 Samenvatting

Het doelva n dezestudi ewa smee r inzichtt ekrijge ni nd eeigenschappe nva npectine - esterasee ni nd edestabilisati eva ntroebe lsinaasappelsap ,waarbi jpectine-esteras e een rolspeelt . De literatuurove rpectine-esteras ei ssamengeva te nbediscussieer di nhoofdstu k2 . Hierbijworde nverschillend easpecte nva npectine-esteras ebehandeld , teweten :voor ­ komen,ro li nd enatuur ,zuiverin ge neigenschappen ,specifitei te nwerkingsmechanisme , remminge n(in)activering ,belan gvoo rd elevensmiddelentechnologi ee nanalysemethoden . Inhoofdstu k3 worde nd ebi jhe tonderzoe kgebruikt emateriale ne ngevolgd emethode n beschreven. Dehoofdstukke n4 to te nme t9 omvatte nhe texperimentel egedeelte ,waarbi ji nel k hoofdstukd eresultate nworde nbesproken . Inhoofdstu k4 word tee ngaschromatografisch e methodevoo rd ebepalin gva nalcohole n beschreven.Bi jdez emethod eworde nalcohole nomgeze t totd eovereenkomstig evluchtig e nitrietesters .D edam pbove nd evloeisto fword tbemonster de ngeïnjecteerd .D emethod e isgeschik tvoo rd ebepalin gva nd epectine-esteraseactivitei t onderconditie swaarbi j een titratiemethodemoeilij kuitvoerbaa ris .D egaschromatografisch emethod evraag tvoo r eenmethanolanalys eongevee r5 minute npe rmonster ,heef tee ngevoelighei dva n< 1 mg/ l methanole nee nreproduceerbaarhei dme tee nvariatiecoëfficien tva n1,5% .D emethod e isgeschik tvoo rgebrui ki nverschillend enatuurlijk evloeistoffe n zonderda td emon ­ stersvoorbehandel dbehoeve nt eworden . Hoofdstuk5 beschrijf td eisolati ee nzuiverin gva ntwe epectine-esterase-iso - enzymenui tNavelsinaasappele nalsmed eenig emoleculair eeigenschappe nva ndez eenzymen . DeNave lsinaasappele nbevatte n ruim6 000 0eenhede n (umol/min)enzy mpe rk gvrucht .D e enzymenwerde ngeëxtraheer dme tee n buffere ndaarn agezuiver ddoo rgefractioneerd e amnoniumsulfaatprecipitatiee ngelfiltrati eo pBio-Ge l P-100.He tenzympreparaa tko n dooraffiniteitschromatografi eo pverknoop tpectaa tgescheide nworde ni ntwe eenzymen , tewete npectine-esteras eI ,di e nietaa ndez ematri xgebonde nwer de npectine-esteras e II,di ewe l gebondenwerd .Beid eenzyme nwerde nverde rgezuiver dove ree nC MBio-Ge lA ionenwisselaar.N azuiverin gbedroe gd especifiek e activiteitva npectine-esteras eI e n IIrespectievelij k 694e n76 2eenhede npe rm geiwit .Beid eenzyme nwerde ngetes tm.b.v . natriumdodecylsulfaatelectroforese;hierbi jgave nbeid eenzyme néé nenkel eban dme tee n molecuulgewichtva n3 620 0t ezien .Teven sgave nexperimente nm.b.v . isoelectric focusingvoo rbeid eenzyme nslecht séé nban dt ezien :D eisoelectrisch epunte nva n pectine-esteraseI e npectine-esteras eI Izij nrespectievelij k 10,05e n> 11,0 .Teven s bleekda td eactivitei tva nee naanta lcelwandafbrekend eenzymen ,welk ei nhe t ruwe enzymmengsel aangetoondko nworde nafwezi gwa si nd egezuiverd epreparaten .D eenzyme n verschilleni naminozuursamenstellin ge nbevatte ngee nsuikers .

98 Hoofdstuk 6 betreft een onderzoek naar de verschillende vormen van pectine-esterase in citrusvruchten. Daartoe werd een speciale electroforesemethode ontwikkeld waarbij over een dunne laag gel een pH gradiënt aangelegd werd. Met deze methode werden naast pectine-esterase I en II verschillende andere vormen van pectine-esterase ontdekt. Verscheidene citrusvruchten werden onderzocht op hun pectine-esterasesamenstelling. In alle onderzochte citrusvruchten samen werden totaal 12 vormen van pectine-esterase (in­ clusief pectines-esterase I en II) gevonden. Tevens bleek dat pectine-esterase I en II samen 60-90% van de pectine-esterase hoeveelheid vormen in alle (onderzochte) citrus­ vruchten. Een andere pectine-esterase, met een hoger molecuulgewicht dan pectine- esterase I en II, werd vanwege zijn grote hittestabiliteit betrokken in het verdere onderzoek. Deze pectine-esterase, die aanwezig was in alle onderzochte citrusvruchten, werd in een niet homogene en een onzuivere staat gebruikt. In hoofdstuk 7 worden de kinetische eigenschappen van de pectine-esterases bestu­ deerd. Op bruinbandpectine met een veresteringsgraad van 12% hebben pectine-esterase I en II een pH optimum van respectievelijk pH 7,6 en 8,0. Op groenbandpectine met een veresteringsgraad van 621 zijn de pH-activiteitsprofielen met ongeveer één pH eenheid verbreed aan de zure zijde van de curve. De pectine-esterase met het hoge molecuul- gewicht heeft de laagste activiteit bij pH 3,5 en bij nog lagere pH waarden neemt de acti­ viteit weer toe. Op groenbandpectine heeft pectine-esterase I bij pH 7,0 een K van 0,083 mg pectine per ml en een K. waarde van 0,42 mg polygalacturonzuur per ml. Voor pectine-esterase II bedragen deze waarden respectievelijk 0,0046 en 0,0016 mg per ml. De Km van pectine-esterase I en II is afhankelijk van de veresteringsgraad van de pec­ tine; de volgende afhankelijkheid werd opgesteld: 1/K = a x (t vrije carboxyl groepen)2. De constante a is voor pectine-esterase II ongeveer 10 maal groter als voor pectine-esterase I. Op grond van dit verband werd beredeneerd dat 2 vrije carboxylzuurgroepen op een bepaalde afstand van elkaar in het substraat nodig zijn voor een optimaal enzym-substraatcomplex. De V is nagenoeg on­ afhankelijk van de veresteringsgraad van de pectine. Tevens bleek dat de K hoger wordt bij lagere pH. Hoe lager de initiële veresteringsgraad van het substraat, hoe lager de veresteringsgraad die door de enzymen uiteindelijk bereikt kan worden. Pectine-esterase I bereikte uiteindelijk een lagere veresteringsgraad dan pectine-esterase II; echter een lagere veresteringsgraad dan 111 kon nooit bereikt worden. De enzymatische verze- * ping van hoogveresterde pectine met een veresteringsgraad van 95,6'» is heel onvolledig. Een aantal van de substraatmoleculen wordt in het geheel niet verzeept. Deze moleculen bleken rijk aan galactose te zijn. Daaruit kon geconcludeerd worden dat zijketens in het pectinemolecuul een belemmering vormen voor de pectine-esteraseactiviteit. Endo- polygalacturonase en endo-pectaatlyase verhoogden de verzepingssnelheid en verzepingsli- miet, waarschijnlijk door produktremming op te heffen. In hoofdstuk 8 wordt het onderzoek naar de stabiliteit van de pectine-esterases be­ schreven. In sinaasappelsap van pH 4,0 en 30 C zijn pectine-esterase I en de pectine- esterase met het hoge molecuulgewicht stabiel; pectine-esterase II wordt echter snel ge­ ïnactiveerd; hierbij is een O-waarde van ongeveer 5 uur gevonden. De stabiliteit van de pectine-esterases wordt verhoogd door een mengsel van suikers en verlaagd door een mengsel van ionen. De pectineconcentratie heeft geen invloed op de pectine-esterase

99 stabiliteit. De enzymen verschillen sterk in hittestabiliteit in sinaasappelsap van pH

4,0: De D90 °C-waarden en Z-waarden zijn 0,00037 min en 6,5 °C voor pectine-esterase I, 0,0015 min en 11 °C voor pectine-esterase II en 0,375 min en 6,5 °C voor de pectine- esterase met het hoge molecuulgewicht. De pectine-esterase met het hoge molecuulgewicht vormde slechts 5% van de ruwe pectine-esterase van de Navel sinaasappel. In verse geperst Valencia sinaasappelsap had ongeveer 10% van de pectine-esterase een hittestabiliteit vergelijkbaar met de hittestabiliteit van de pectine-esterase met het hoge molecuul­ gewicht. Hoofdstuk 9 handelt over de sinaasappelsap klarende eigenschappen van de pectine- esterases bij 5 C en 30 C. Het bleek dat pectine-esterase II niet in staat is sinaas­ appelsap te klaren, alhoewel de stabiliteit en de activiteit van het enzym bij 5 C voldoende zijn om een aanzienlijk deel van de pectine in het sap te verzepen. Pectine- esterase I en de pectine-esterase met het hoge molecuulgewicht geven daarentegen een snelle, klaring van sinaasappelsap bij 30 °C. Bij 5 °C is de pectine-esterase met het hoge molecuulgewicht nog goed werkzaam; pectine-esterase I werkt bij 5 C echter honderdmaal zo langzaam als bij 30 °C. Endo-polygalacturonase stimuleert de pectine- esteraseactiviteit in sinaasappelsap. Desondanks bleek de troebelingsstabiliteit van het sap verhoogd te worden. In hoofdstuk 10 wordt het totale resultaat kort besproken en worden enige mogelijk­ heden voor verder onderzoek genoemd.

100 Literature

Abdel-Fattah,A.F .& S.S .Mabrouk , 1977.Productio no fpolygalacturonas epectin-methyl - esteraseb y fungi.Chem .Mikrobiol .Technol .Lebensm .5 :38-41 . Abo-El-Dahab,M.K. , 1964.Productio no fpecti can dcellulolyti c enzymesb y Xanthomonas malvacearum. Phytopathology 54:597-601 . Agarwal,G.P. ,D .Kulhar a& P.S .Bisen , 1977.I nvitr oproductio no fpectolyti cenzyme s by Pleospora infeatoria (Group)an d Nigrospora sphaerica (Sacc.)causin gpo dspot s ofpe a (Pisum sativum) andbea n (Dolichos lablab). Biochem.Physiol .Pflanz .171 : 7-16. Al-Delaimy,K.A. , G.Borgstro m& CL . Bedford, 1966.Pecti csubstance s andpecti cenzyme s offres han dprocesse dmontmorenc y cherries.Q .Bull .Mich .Stat eUniv .49 :164-171 . Ames,G.F.-L. , 1974.Resolutio no fbacteria lprotein sb yPolyacrylamid e gelelectro ­ phoresis onslabs .J .Biol .Chem .249 :634-644 . Arakji,O.A .& H.Y .Yang , 1969.Identificatio nan dcharacterizatio no fth epecti c enzymes ofth eMcFarli ncranberry .J .Foo dSei .34 :340-342 . Arnold,W.N. , 1972.Th estructur eo f theyeas t cellwall .Solubilizatio no fa marke r enzyme,ß-Fructofuranosidase ,b y theautolyti cenzym esystem .J .Biol .Chem .247 : 1161-1169. Astapovich,N.I. ,A.G .Lobano k& M.V .Gre l ,1976 .Kinetic so fgrowt han dsynthesi so f pectolytic enzymes of Sclerotinia sclerotiorum onmedi acontainin g lactose.Appl . Biochem.Microbiol . (USSR)12 :680-682 . Aung,T .& E .Ross , 1965.Hea tsensitivit y ofpectinesteras eactivit y inpapay apuré ean d ofcatalase-lik eactivit y inpassio nfrui t juice.J .Foo dSei .30 :144-147 . Baker,R.A .& J.H .Bruemmer , 1972.Pectinas e stabilizationo forang ejuic ecloud . J.Agric .Foo dChem .20 :1169-1173 . Baker,R.N. ,A.L .Alent y SJ.F .Zac k jr., 1969.Simultaneou s determinationo flowe ral ­ cohols,aceton ean dacetaldehyd e inbloo db yga schromatography .J .Chromatogr .Sei . 7:312-314 . Barker,K.R .& J.C .Walker , 1962.Relationshi p ofpectolyti c andcellulyti cenzym e productionb ystrain so f Pellicularia filamentoaa tothei rpathogenicity .Phyto ­ pathology 52:1119-1125 . Baron,A. , 1978.Contributio nà l'étud e dumécanism ed'actio nd el apectinestéras e A'Aspergillus niger (vanTiegh.) .Doctora lThesi sC—511-190 ,Rennes . Baron,A. ,J .Calve zS J.F .Drilleau , 1978.Dosag edirec te tindirec t dumethano lpa r Chromatographie enphas egazeus eapplicatio npratique .Ann .Fais .Exp .Chim .71 : 29-34. Bartolome,L.G . &J.E .Hoff , 1972a.Firmin go fpotatoes :biochemica leffect so fpreheating . J.Agric .Foo dChem .20 :266-270 . Bartolome,L.G .& J.E .Hoff , 1972b.Ga schromatographi cmethod s forth eassa yo fpecti n methylesterase ,fre emethanol ,an dmethoxy lgroup s inplan t tissues.J .Agric .Foo d Chem.20 :262-266 . Bateman,D.F. , 1963.Pectolyti cactivitie so fcultur e filtrateso f Rhizoctonia solani and extractso f Rhizoctonia-infected tissueso fbean .Phytopatholog y 53:197-204 . Bateman,D.F .& S.V .Beer , 1965.Simultaneou sproductio nan dsynergisti c actiono f oxalic acidan dpolygalacturonas e duringpathogenesi sb y Sclerotium rolfsii. Phytopathology 55:204-211 . Bateman,D.F .& R.L .Millar , 1966.Pecti cenzyme s intissu edegradation .Annu .Rev . Phytopathol.4 :119-146 . Baumann,G .& K .Gierschner , 1974.Untersuchunge n zurTechnologi ede rFruchtsäft eau s schwarzenJohannisbeere n unterEinbeziehun gde sLagerverhalten s undde rgaschromato ­ graphischenBestimmun g desMethyl -un dÄthylalkoholgehalte s derhergestellte nProdukte . Ind.Obs tGemüseverwert .59 :161-171 . Beaven,G.H .& E.R .Holiday , 1952.Ultraviole t absorption spectrao fprotein san damin o acids.In :Advance s inprotei nchemistry .M.L .Anson ,K .Baile y& J.T .Edsal l (Eds), AcademicPres sInc .Ne wYork ,p .375 .

101 Bell,T.A. ,J.L .Etchell s& I.D .Jones ,1951 .Pectinesteras ei nth ecucumber .Arch . Biochem.Biophys .31 :431-441 . Berger,G. ,J.P .Agne l &L .Saint-Lèbe , 1974.Quelque scomposé svolatil s formésa ucour s del'irradiatio n gammad el'amido n demais .Staerk e26 :185-189 . Beyer,G.F. , 1951.Spectrophotometri cquantitativ edeterminatio no fmethano li ndistille d spirits.J .Assoc .Off .Agric .Chem .34 :745-748 . Binet,P. , 1976.Succulenc ee tactivit épectinesteras eche z Aster tripoliwn L.Physiol . Vég. 14:283-295 . Bissett,O.W. ,M.K .Veldhui s& N.B .Rushing , 1953.Effec to fhea t treatment temperature onth estorag elif eo fValenci aorang econcentrates .Foo dTechnol .7 :258-260 . Bock,W. , 1966.Pektin e imFruchtsaft .Lebensm .Ind . 13:298-302 . Boos,R.N. , 1948.Quantitativ e colorimetricmicrodeterminatio no fmethano lwit hchromo - tropicaci dreagent .Anal .Chem .20 :964-965 . Brady,C.J. , 1976.Th epectinesteras eo fth epul po fbanan afruit .Aust .J .Plan tPhysiol . 3:163-172 . Bricker,CE . &H.R .Johnson , 1945.Spectrophotometri cmetho d fordeterminin gformaldehyde . Ind.Eng .Chem. ,Anal .Ed . 17:400-402 . Brown,M.M. , 1969.Pecti nester san d themechanica lpropertie so fplan tcel lwalls .Ph.D . Thesis,Univ .Delaware . Bryan,W.H .& E.H .Newcomb ,1954 .Stimulatio no fpecti nmethylesteras eactivit yo rcul ­ turedtobacc opit hb yindoleaceti cacid .Physiol .Plant .7 :290-297 . Buescher,R.W .& R.J .Furmanski ,1978 .Rol eo fpectinesteras ean dpolygalacturonas ei n theformatio no fwoollines so fpeaches .J .Foo dSei .43 :264-266 . Buren,J.P .van ,J.C .Moye r& W.B .Robinson ,1962 .Pecti nmethylesteras e insna pbeans . J.Foo dSei .27 :291-294 . Cabib,E .& R .Ulane , 1973.Chiti nsynthetas eactivatin g factorfro myeast ,a protease . Biochem.Biophys .Res .Commun .50 :186-191 . Calesnick,E.J. ,C.H .Hill s &J.J .Willaman , 1950.Propertie so fa commercia lfunga l pectasepreparation .Arch .Biochem .29 :432-440 . Call,H.P .& C.C .Emeis ,1978 .Pektinolytisch eAktivitäte nvo nLaktos eun dMilchsäur e assimilierendenHefen .I .Screenin gau fpektinolytisch e undmazerierend eAktivitäten . Chem.Mikrobiol .Technol .Lebensm .5 :137-142 . CBN,1971 .Th enomenclatur eo fmultipl e formso fenzymes .IUPAC-IU BCommissio no nBio ­ chemicalNomenclatur e (CBN),Recommendation s (1971).Eur .J .Biochem .24 :1-3 . Chang,L.W.S. ,L.L .Morit a& H.Y .Yamamoto , 1965.Papay apectinesteras e inhibitionb y sucrose.J .Foo dSei .30 :218-222 . Clark,E.P. , 1932.Th eVieböc kan dSchwappac hmetho d forth edeterminatio no fmethoxy l andethoxy lgroups .J .Assoc .Off .Agric .Chem . 15:136-140 . Clarke,D.D. , 1966.Productio no fpecti cenzyme sb y Phytophtora infestans. Nature (London) 211:649 . Collins,J.L. , 1970.Pecti nmethylesteras e activity insouther npea s (Vigna sinensis). J.Foo dSei .35 :1-4 . Courtois,J.E. ,F .Perchero n &M.J .Foglietti ,1968 .Isolemen te tétud ed umod ed'actio n d'unepolygalacturonas e d'insecte (Pyrrhoaoris apterus) . CR. Acad.Sei. ,Ser .D : 266:164-166 . Dahodwala,S. ,A .Humphre y& M .Weibel ,1974 .Pecti cenzymes :individua lan dconcerte d kineticbehavio ro fpectinesteras e andpectinase .J .Foo dSei .39 :920-926 . Darvill,A.G. ,D.P .Robert s& M.A .Hall ,1975 .Improve d gas-liquid chromatographic separationo fmethylate d acetylated alditols.J .Chromatogr .115 :319-324 . Datunashvili,E.N. ,S.S .Tyurin a& N.K .Kardash , 1976.Som epropertie so fgrap epectin - methylesterase.Prikl .Biokhim .Mikrobiol . 12:49-53 . Davis,P.L .& W.G .Chac ejr. ,1969 .Determinatio no falcoho li ncitru sjuic eb yga s chromatographicanalysi so fheadspace .Hortic .Sei .4 :117-119 . Delecourt,R. , 1972.Applicatio nde senzyme sà l'industri e desju sd efruits .B.I.F. , Bull,d el'Associatio n desancien sElève sd el'Institu t desIndustrie sd e Fermentationd eBruxelle s 15:82-91 . Delincée,H. , 1976.Thin-laye risoelectri cfocusin go fmultipl e formso f tomatopectin ­ esterase.Phytochemistr y 15:903-906 . Delincée,H. , 1978.Irradiatio no findustria lenzym epreparations .II .Characterizatio n offunga lpectinas eb y thin-layerisoelectri c focusingan dge lfiltration .J .Foo d Biochem.2 :71-85 . Delincée,H .& B.J .Radola ,1970 .Som esiz ean dcharg epropertie so ftomat opecti nmethyl ­ esterase.Biochim .Biophys .Act a214 :178-189 .

102 Deubert,K.H .& R.A .Rohdë , 1971.Nematod eenzymes .In :fian tparasiti cnematode sII , B.M.Zuckerman ,W.F .Ma i& R.A .Rohd e (Eds),Academi cPress ,Ne wYor kan dLondon , p. 82-90. Deuel,H. , 1947.Obe rGlykoleste rde rPektinsäure .Helv .Chim .Act a30 :1523-1534 . Deventer-Schriemer,W.H .va n& W .Pilnik ,1976 .Fractionatio no fpectin s inrelatio nt o theirdegre eo festerification .Lebensm .Wiss .Technol .9 :42-44 . Dickes,G.J .& P.V .Nicholas ,1978 .Ga schromatograph y infoo danalysis .Butterworth , London. Dixon,M .& E.C .Webb , 1964.Enzymes .Longmans ,Gree n& Co. ,Ltd. ,London ,2n ded. ,p.40 . Dye, D.W., 1960.Pectolyti cactivit y in Xanthomonas. N.Z.J.Sei .3 :61-69 . Dyer,R.H. , 1971.Compariso no fGL Can dcolorimetri cmethod s fordeterminatio no f methanoli nalcoholi cbeverages .J .Assoc .Off .Agric .Chem .54 :785-786 . Eagerman,B.A .& A.H .Rouse ,1976 .Hea tinactivatio ntemperature-tim erelationship sfo r pectinesteraseinactivatio ni ncitru sjuices .J .Foo dSei .41 :1396-1397 . Endo,A. , 1964.Studie so npectolyti cenzyme so fmolds .Par tXII .Purificatio nan d propertieso fpectinesterase .Agric .Biol .Chem .28 :757-764 . Evans,R . &D .McHale ,1978 .Multipl e formso fpectinesteras e inlime san doranges . Phytochemistry 17:1073-1075 . Fairbanks,G. ,T.L .Stec k& D.F.H .Wallace ,1971 .Electrophoreti c analysiso fth emajo r polypeptideso fhuma nerthrocyt emembrane.Biochemistr y 10:2606-2617 . Fogarty,W.M .& O.P .Ward , 1974.Pectinase san dpecti cpolysaccharides .In :Progres si n industrialmicrobiology ,D.J.D .Hockenhul l (Ed.),Churchil lLivingstone ,Edinburgh , p.59-119 . Frémy,E. ,1840 .Recherche s surl apectin ee tl'acid epectique .J .Pharm .Chim .26 : 368-393. Fuchs,A. , 1965.O nth egelatio no fpecti nb yplan textract san dit sinhibition .Act a Bot. Neerl. 14:315-322 . Gamble,W. , 1973.Th epresenc eo fpecti nmethylesteras ei ncaca opulp .Experienti a29 : 421. Gee, M.,R.M .Reev e& R.M .McCready , 1959.Reactio no fhydroxylamin ewit hpectini cacids . Chemicalstudie san dhistochemica lestimatio no fth edegre eo festerificatio no f pecticsubstance s infruit .J .Agric .Foo dChem .7 :34-38 . Gessner,P.K. , 1970.Metho d forth eassa yo fethano lan dothe raliphati calcohol s applicable totissu ehomogenate san dpossessin ga sensitivit y of Iyg/ml .Anal . Biochem.38 :499-505 . Glasziou,K.T. ,1957 .Th eeffec to f3-indolylaceti caci do nth ebindin go fpecti nmethyl - esterase toth ecel lwall so ftobacc opith .Aust .J .Biol .Sei . 10:337-341 . Glasziou,K.T. ,1959 .Th elocalisatio nan dpropertie so fpecti nmethylesteras eo faven a coleoptiles.Physiol .Plant . 12:670-680 . Glasziou,K.T .& S.D .Inglis ,1958 .Th eeffec to fauxin so nth ebindin go fpecti nmethyl - esterase tocel lwalls .Aust .J .Biol .Sei . 11:127-141 . Gonzalez,A.A. ,F.G .Camar a& L.M .Maguna , 1977.Clarificatio nenzymatiqu ed uju sd e citron.Ind .Aliment .Agric .94 :283-288 . Hall,C.B. , 1966.Inhibitio no f tomatopectinesteras eb y tannicacid .Natur e(London ) 212:717-718 . Hall,C.B .& R.A .Dennison ,1960 .Th erelationshi po ffirmnes san dpectinesteras eactiv ­ ityo ftomat ofruits .Proc .Am .Soc .Hortic .Sei .75 :629-631 . Hancock,J.G .& R.L .Millar , 1965.Relativ e importanceo fpolygalacturonat etrans - eliminase andothe rpectolyti cenzyme s insouther nanthracnose ,sprin gblac kstem , and Stemphylium leafspo to falfalfa .Phytopatholog y 55:346-355 . Hänssler,G. ,G .Menk e& F .Grossmann ,1971 .Productio no fpectolyti can dcellulolyti c enzymesb y Ceroospopella heppotriohoides Fron.Experienti a27 :1022 . Heri,W. ,H .Neukor a& H .Deuel ,1961 .Chromatographi evo nPektine nmi tverschiedene r Verteilungde rMethylestergruppe nau fde nFadenmolekeln . Helv.Chim .Act a44 : 1945-1949. Hills,C.H .& H.H .Mottern , 1947.Propertie so ftomat opeetase .J .Biol .Chem .168 : 651-663. Hills,C.H. ,H.H .Mottern ,G.C .Nuttin g &R .Speiser ,1949 .Enzyme-demethylate d pectinatesan d theirgelation .Foo dTechnol .3 :90-94 . Hills,C.H. ,J.W .Whit e &G.L .Baker ,1942 .Low-suga rjellyin gpectinates .Proc .Inst . FoodTechn .2 :47-58 . Hobson,G.E . &J.N .Davies ,1971 .Th e tomato.In :Th ebiochemistr y offruit san dthei r products,A.C .Hulm e (Ed.),Academi cPress ,London ,2 :p .437-482 . Höcker,J. , 1978.Sapklaringseigenschappe nva nmultipl epectin eesterase si nsinaasappel ­ sap. M.Sei.Thesis ,Dept .Foo dSei. ,Agric .Univ. ,Wageningen .

103 Hoffman,D.O .& M.L .Wolfrom , 1947.Determinatio no fmethoxy lan dethoxy lgroup s inace - talsan deasil yvolatil e alcohols.Anal .Chem .19 :225-228 . Hoogzand,C .& J.J .Doesburg , 1961.Effec to fblanchin go ntextur ean dpecti no fcanne d cauliflower.Foo dTechnol .15 :160-163 . Houdenhoven,F.E.A .van , 1975.Studie so npecti nlyase .Ph.D .Thesis .Meded .Landbouw - hogesch.Wageningen ,75-13 . Hsu,C.P. ,S.N .Deshpand e& N.W .Desrosier , 1965.Rol eo fpecti nmethylesteras ei nfirm ­ nesso fcanne d tomatoes.J .Foo dSei .30 :583-588 . Hultin,H.O .& A.S .Levine , 1963.O n theoccurrenc eo fmultipl emolecula r formso fpectin - esterase.Arch .Biochem .Biophys . 101:396-402 . Hultin,H.O .& A.S .Levine , 1965.Pecti nmethy lesteras e inth eripenin gbanana .J .Foo d Sei.30 :917-921 . Hultin,H.O .& B.E .Proctor , 1961.Change s insom evolatil econstituent so f thebanan a duringripening ,storage ,an dprocessing .Foo dTechnol .15 :440-444 . Hultin,H.O. ,B .Su n& J .Bulger , 1966.Pecti nmethy lesterase s ofth ebanana .Purifica ­ tionan dproperties .J .Foo dSei .31 :320-326 . Ishii,S. ,K .Kiho ,S .Sugiyam a& H .Sugimoto , 1978. Low-methoxylpecti nprepare db y pectinesterase from Aspergillus japonious. Abstr.5t hInt .Congr .Foo dSei .& Technol.,Kyoto ,6a-14 . Ishii,S. ,K .Kiho ,S .Sugiyam a& H .Sugimoto , 1979.Low-methoxy lpecti nprepare db y pectinesterase from Aspergillus japonious. J.Foo dSei .44 :611-614 . Jansen,E.F. ,R .Jan g& J .Bonner , 1960a.Bindin go fenzyme s toaven acoleoptil ecel l walls.Plan tPhysiol .35 :567-574 . Jansen,E.F. ,R .Jan g& J .Bonner , 1960b.Orang epectinesteras ebindin gan dactivity . FoodRes .25 :64-72 . Jansen,E.F. ,R .Jan g& L.R .MacDonnell , 1947.Citru sacetylesterase .Arch .Biochem .15 : 415-431. Jones,T.M .& P .Albersheim , 1972.A ga schromatographi cmetho d forth edeterminatio no f aldosean duroni caci dconstituent so fplan tcel lwal lpolysaccharides .Plan t Physiol.49 :926-936 . Joslyn,M.A .& W .Pilnik , 1961.Enzyme san denzym eactivity .In :Th eorange ,it sbiochem ­ istryan dphysiology ,W.B .Sinclai r (Ed.).Univ .Californi aPrintin gDepartment , p.373-435 . Kauss,H .& W.Z .Hassid , 1967.Enzymi cintroductio no f themethy leste rgroup so fpectin . J.Biol .Chem .242 :3449-3453 . Kauss,H. ,A.L .Swanson ,R .Arnol d& W .Odzuck , 1969.Biosynthesi s ofpecti csubstances . Localisationo fenzyme s andproduct s ina lipid-membran e complex.Biochim .Biophys . Acta 192:55-61 . Kew,T.J .& M.K .Veldhuis , 1961.Clou dstabilisatio ni ncitru sjuice .U.S .Paten t 2,995,448. Kimura,H. ,F .Uchin o& S .Mizushima , 1973.Purificatio nan dpropertie s ofpectinesteras e from Aorooylindrium. Agric.Biol .Chem .37 :1209-1210 . Klaushofer,H .& F .Bandion , 1968.Gaschromatographisch eMethod e zurBestimmun g des Aethylacetat-,Methanol- ,n-Propanol- ,i-Butano lun d i-Amylalkoholgehaltes inSpiri ­ tuosen.Mitteilunge n6 :443-448 . Kohn,R. , 1975.Io nbindin g onpolyuronate s- alginat ean dpectin .Pur eAppl .Chem .42 : 371-397. Kohn,R .& I .Furda , 1967.Calciu mio nactivit y insolution s ofcalciu mpectinate . Collect.Czech .Chem .Commun .32 :1925-1937 . Kohn,R. ,I .Furd a& Z .Kopec , 1968.Distributio no ffre ecarboxy lgroup s inth epecti n molecule aftertreatmen twit hpecti nesterase .Collect .Czech .Chem .Commun .33 : 264-269. Kratzl,K .& K .Gruber , 1958.Zu rquantitative nTrennun g undBestimmun g verschiedener Alkoxylgruppenmittel sGas-Flüssig-Chromatographie .Monatsh .Chem .89 :618-624 . Krop,J.J.P. , 1974.Th emechanis mo fclou d lossphenomen a inorang ejuice .Agric .Res . Rep.830 .Pudoc ,Wageningen . Krop,J.J.P .& W .Pilnik , 1974.Clou d lossstudie s incitru sjuices :clou d stabilization bya yeast-polygalacturonase .Lebensm .Wiss .Technol .7 :121-124 . Langcake,P. ,P.M .Brat t& R.B .Drysdale , 1973.Pectinmethylesteras e in Fusarium-infect- edsusceptibl e tomatoplants .Physiol .Plan tPathol .3 :101-106 . Lange,E. , 1970.Untersuchunge nübe rdi eBedeutun gpektolytischer , cellulolytischerun d proteolytischerEnzym e fürdi eVirulen zphytopathogene rBakterien .Diss. ,Hohenheim . Leaf,G .& L.J .Zatman , 1952.A stud yo fth econdition sunde rwhic hmethano lma yexer t atoxi chazar d inindustry .Br .J .Ind .Med .9 :19-31 .

104 Leammli,U.K. , 1970.Cleavag eo fstructura lprotein sdurin g theassembl yo fth ehea do f bacteriophageT4 .Natur e (London)227 :680-685 . Lee,M.J. , 1969a.Purification ,properties ,an dmode so factio no fpectinesteras efro m tomatoan d Clostridium multifermentons. Ph.D.Thesis ,Rutger sUniv . Lee,Y.S. , 1969b.Measurement ,characterizatio nan devaluatio no fpectinesteras ei nappl e fruits.Ph .D .Thesis ,Univ .Maryland . Lee,C.Y. ,M.C .Bourn e& J.P .Va nBuren , 1979.Effec to fblanchin g treatmentso nth e firmnesso fcarrots .J .Foo dSei .44 :615-616 . Lee,M .& J.D .Macmillan , 1968.Mod eo factio no fpecti cenzymes .I .Purificatio nan d certainpropertie so f tomatopectinesterase .Biochemistr y 7:4005-4010 . Lee,M .& J.D .Macmillan , 1970.Mod eo factio no fpecti cenzymes .III .Sit eo finitia l actiono ftomat opectinesteras eo nhighl yesterifie dpectin .Biochemistr y 9:1930 - 1934. Lee,Y.S .& R.C .Wiley , 1970.Measuremen tan dpartia lcharacterizatio no fpectinesteras e inappl efruits .J .Am .Soc .Hortic .Sei .95 :465-468 . LeggettBaily ,J. , 1967.Technique s inprotei nchemistry .2n ded .Elsevie rPubl .Company , Amsterdam,p .344 . Leniger,H.A .& W.A .Beverloo , 1975.Foo dproces sengineering .D .Reide lPubl .Company , Dordrecht-Holland,Boston ,U.S.A . Leo,H.T .& C.C .Taylor , 1958.Metho do fmakin g lowsuga rpectini caci dgels .U.S .Pat . 2,824,007. Leo,H.T .& C.C .Taylor , 1962.Metho do fmakin g lowsuga rpectini caci dgels .U.S .Pat . 3,034,901. Leuprecht,H .& A .Schaller , 1968.Ergebniss emethodologische rUntersuchunge nhinsicht ­ lichde rMessun gde rPektinmethylesterase-Aktivitä ti naufgetaute mTiefgefrier - Erdbeerpüree.Fruchtsaf tInd . 13:50-74 . Lineweaver,H .& G.A .Ballou ,1945 .Th eeffec to fcation so nth eactivit yo falfalf a pectinesterase (Pectase).Arch .Biochem .6 :373-387 . Litchman,M.A .& R.P .Upton ,1972 .Ga schromatographi cdeterminatio no fresidua lmethano l infoo dadditives .Anal .Chem .44 :1495-1497 . Lowry,O.H. ,N.J .Rosebrough ,A.L .Far r& R.J .Randall ,1951 .Protei nmeasuremen twit h theFoli npheno lreagent .J .Biol .Chem . 193:265-275 . Lucero,D.P. , 1972.Wate rvapo rsensitivit y responseo fhydroge nflam eionizatio n detectors.J .Chromatogr .Sei . 10:463-467 . Lund,B.M .& T.F .Brocklehurst , 1978.Pecti cenzyme so fpigmente d strainso f Clostridium. J.Gen .Microbiol .104 :59-66 . McColloch,R.J .& Z.I .Kertesz ,1947 .Pecti cenzymes .VIII .A comparisono ffunga lpectin - methylesterasewit htha to fhighe rplants ,especiall y tomatoes.Arch .Biochem .13 : 217-229. McComb,E.A .& R.M .McCready , 1958.Us eo fth ehydroxami caci dreactio nfo rdeterminin g pectinesterase activity. StainTechnol .33 :129-131 . McCready,R.M .& C.G .Seegmiller , 1954.Actio no fpecti cenzyme so noligogalacturoni c acidsan dsom eo fthei rderivatives .Arch .Biochem .Biophys .50 :440-450 . MacDonnell,L.R. ,R .Jang ,E.F .Janse n& H .Lineweaver ,1950 .Th especificit yo fpectin - esterases fromsevera lsource swit hsom enote so nth epurificatio no forang epectin ­ esterase.Arch .Biochem .28 :260-273 . MacDonnell,L.R. ,E.F .Janse n& H .Lineweaver , 1945.Th epropertie so forang epectin ­ esterase.Arch .Biochem .6 :389-401 . Mackintosh,R.D. ,J.P .Rooze n& W .Pilnik ,1977 .Analys eva naminozure ndoo rmidde lva n gaschromatografie.Voedingsmiddelentechnol . 10(36) : 27-29. MacMillan,J.D .& M.I .Sheiman ,1974 .Pecti cenzymes .In :Foo drelate denzymes , J.R.Whitake r (Ed.).Advance si nChemistr ySeries ,Am .Chem .Soc ,Washington ,D.C. , 136:101-130 . McNeil,M. ,A.G .Darvil l& P .Albersheim ,i npress .Th estructura lpolymer so fth e primary cellwall so fdicots .In :Progres s inth echemistr yo forgani cnatura l products,Springer-Verlag . Mah,R.A .& R.E .Hungate ,1965 .Physiologica lstudie so nth erume nciliate , Ophryosoolex purkynei Stein.J .Protozool.12 :131-136 . Majchrowicz,E .& J.H .Mendelson ,1971 .Bloo dmethano lconcentration s duringexperimental ­ lyinduce dethano lintoxicatio ni nalcoholics .J .Pharmacol .Exp .Ther .179 :293-300 . Manabe,M. , 1973a.Purificatio nan dpropertie so f Citrus natsudaidai pectinesterase. Agric.Biol .Chem .37 :1487-1491 . Manabe,M. , 1973b.Saponificatio no feste rderivative so fpecti caci db y Natsudaidai pectinesterase (Studieso nth ederivative so fpecti csubstance spar t V). J.Agric . Chem.Soc .Japan ,47 :385-390 . Markovic',0. , 1974.Tomat opectinesterase-characterizatio no fon eo fit smultipl eforms . Collect.Czech.Chem .Commun .39 :908-913 . 105 Markovic,0. , 1978.Pectinesteras efro mcarro t (Dauaus aarrota L.). Experientia34 :561 - 562. MarkoviÄ,0. ,K .Heinrichov â& B .Lenkey ,1975 .Pectolyti cenzyme sfro mbanana .Collect . Czech.Chem .Commun .40 :769-774 . Markovi£,0 .& I .Kuniak ,1974 .Us eo fcross-linke dhydrolyse d starchi nge lelectro ­ phoresis.J .Chromatogr .91 :873-875 . Markovid,0 .& J .Patocka ,1977 .Actio no fiodin eo nth etomat opectinesterase .Expe ­ rientia33 :711-713 . Markovii,0 .& M .Sajgó , 1977.Termina lamin oacids ,peptid ema pan damin oaci d compositiono fon efor mo ftomat opectinesterase .Act aBiochim .Biophys .Acad .Sei . Hung. 12:45-48 . Markovic",0 . &A .Slezärik , 1969.Isolatio nan dpartia lcharacterizatio no fpecti n esterasefro mtomatoes .Collect .Czech .Chem .Commun .34 :3820-3825 . MassSpectrometr yDat aCentre ,1974 .Eigh tpea kinde xo fmas s spectra,2n ded . MassSpectrometr yDat aCentre ,Reading ,UK . Mehrotra,M.D. ,P .Kaise r& C .Reynaud , 1971.Recherch ed el'activit épectinolytiqu ei n vivoe ti nvitro ,d'u nchampigno nphytopathogèn e Gilbertella persioaria (Eddy), Hesseltine.Ann .Inst .Pasteu r (Paris)120 :81-97 . Miller,L .& J.D .Macmillan ,1970 .Mod eo factio no fpecti cenzymes .II .Furthe rpurifi ­ cationo fexopolygalacturonat e lyasean dpectinesteras e from Clostridium multifermen­ tons. J.Bacteriol .102 :72-78 . Miller,L .& J.D .Macmillan , 1971.Purificatio nan dpatter no factio no fpectinesteras e from Fusarium oxysporum f.sp. vasinfectum. Biochemistry 10:570-576 . Milner,Y .& G .Avigad , 1973.A sensitiv eradioisotop eassa y forpecti nmethy lesteras e activity.Anal .Biochem .51 :116-120 . Miyairi,K. ,T .Okun o& K .Sawai , 1975.Purificatio nan dphysicochemica lpropertie so f pectinesteraseo fappl efruit s (Ralls).Bull .Fac .Agric .Hirosak iUniv .24 :22-30 . Montedoro,G. , 1968.L'attivit àpectolitic ad iuv ed idivers ecultivar sdistribuzion e dell'enzimae su acaratterizzazione .Estratt odagl iAnnal idéli aFacolt àd iAgrari a dell'Universitàd ePerugi a23 :1-30 . Mottern,H.H .& C.H .Hills ,1946 .Lo weste rpecti nfro mappl epomace .Ind .Eng .Chem . 38:1153-1156 . Nakagawa,H. ,K .Sekiguchi ,N .Ogur a& H .Takehana , 1971.Bindin go ftomat opectinesteras e andß-fruetofuranosidas et otomat ocel lwall .Agric .Biol .Chem .35 :301-307 . Nakagawa,H. ,Y .Yanagaw a& H .Takehana ,1970a .Studie so nth epectolyti cenzyme . PartIV .Purificatio no ftomat ofrui tpectinesterase .Agric .Biol .Chem .34 :991-997 . Nakagawa,H. ,Y .Yanagaw a& H .Takehana , 1970b.Studie so nth epectolyti cenzyme .Par tV . Somepropertie so fth epurifie d tomatopectinesterase .Agric .Biol .Chem .34 : 998-1003. Nakayama,Y .& Y .Iwasaki ,1966 .Som epropertie s ofpectin-methylesteras e (PM)i n persimmonfruits .J .Jpn .Soc .Foo dNutr .18 :440-443 . Nath,N .& S .Ranganna , 1977a.Time/temperatur e relationship fortherma linactivatio no f pectinesterase inmandari norang e (.Citrus reticulata Blanco)juice .J .Foo dTechnol . 12:411-419 . Nath,N .& S .Ranganna , 1977b.Determinatio no ftherma lproces s forcanne dmandari n orangesegments .J .Foo dSei .Technol .14 :113-119 . Nieuwenhuis,C , 1978.Pectin eesteras eisoenzyme ni ncitrus .M.Sc.Thesis ,Dept .Foo d Sei.,Agric .Univ. ,Wageningen . Norman,S. , 1970.Compariso no ftw oporou spolyme rcolumn s forgaschromatographi canaly ­ siso facetaldehyde ,methanol ,ethano lan dothe rvolatile semanatin g fromintac t "Valencia"oranges .J .Am .Soc .Hortic .Sei .95 :777-780 . Oi,S .& Y .Satomura ,1965 .Increas e inviscosit yan dge lformatio no ffrui tjuic eb y purifiedpectinesterase .Agric .Biol .Chem .29 :936-942 . Opienska-Blauth,J. ,M .Charezinsk i &H .Berbec ,1963 .A new ,rapi dmetho do fdeterminin g tryptophan.Anal .Biochem .6 :69-76 . Owens,H.S. ,H .Lotzkar ,T.H .Schult z& W.D .Maclay , 1946.Shap ean dsiz eo fpectini c acidmolecule sdeduce d fromviscometri cmeasurements .J .Am .Chem .Soc .68 :1628-1632 . Pajot,P. , 1976.Fluorescenc eo fprotein s in6- M guanidinehydrochloride .A metho dfo r thequantitativ edeterminatio no ftryptophan .Eur .J .Biochem .63 :263-269 . Paquin,R .4 L.J .Coulombe ,1962 .Pecti cenzym esynthesi si nrelatio nt ovirulenc ei n Fusarium oxysporum f. lyoopersioi (Sacc.)Snyde ran dHansen .Can .J .Bot .40 : 533-541. Paul,J.K. , 1975.In :Frui tan dvegetabl ejuic eprocessing .Noye sDat aCorp .London . Paynter,V.A. , 1975.I nviv oan di nvitr ostudie so nth epecti cenzyme so f Monilia fruo- tiaola. Ph.D.Thesis ,Clemso nUniv..Xero xUniv .Microfilm s75-22,098 .

106 Perley,A.F .& O.T .Page , 1971.Differentia linductio no fpectolyti cenzyme so f Fusarium roseum (LK.)emend .Snyde ran dHansen .Can .J .Microbiol . 17:415-420 . Phaff,H.J. , 1947.Th eproductio no fexocellula rpecti cenzyme sb y Veniaillium ahryaoge- num. I.O nth eformatio nan dadaptiv enatur eo fpolygalacturonas e andpectinesterase . Arch.Biochem . 13:67-81 . Phaff,H.J. ,1966 .a-1,4-Polygalacturonid eglycanohydrolas e (endo-polygalacturonase) from Saacharomyees fragilis. MethodsEnzymol .8 :636-641 . Pilnik,W. , 1958.Di eTrubstabilitä tvo nCitrus-Konzentraten .In :Ber .Wiss.-Tech.Komm. , Int.Fruchtsaft-Union.Juris-Verlag ,Zurich , 1:p .203-228 . Pilnik,W . &F.M .Rombouts ,1978 .Pecti cenzymes .In :Encyclopedi ao fFoo dScience , M.S.Peterso n& A.H .Johnso n (Eds),Av iPubl .Company ,Westport ,Connecticut,p.604-610 . Pilnik,W. ,F.M .Rombout s& A.G.J .Voragen , 1973.O nth eclassificatio no fpecti ndepoly - merases:Activit yo fpecti ndepolymerase s onglyco lester so fpectat ea sa ne w classificationcriterion .Chem .Mikrobiol .Technol .Lebensm .2 :122-128 . Pilnik,W .& G .Rothschild , 1960.Trubstabilitä tun dPektinesteras e- Restaktivitä ti n pasteurisiertemOrangenkonzentrat .Fruchtsaf tInd .5 :131-138 . Pilnik,W. ,A.G.J .Vorage n& L .d eVos ,1975 .Enzymatisch eVerflüssigun gvo nObs tun d Gemüse.Fluess.Obs t42 :448-451 . Polacsek-Räcz,M . &K .Pozsär-Hajnal ,1976 .Determinatio no fpectinmethylesterase ,poly ­ galacturonasean dpecti csubstance s insom efruit san dvegetables .Act aAliment .5 : 189-204. Pollard,A .& M.E .Kieser , 1951.Th epectas eactivit yo fapples .J .Sei .Foo dAgric .2 : 30-36. Pozsär-Hajnal,K .& M .Polacsek-Räcz ,1975 .Determinatio no fpectinmethylesterase ,poly ­ galacturonasean dpecti csubstance s insom efruit san dvegetables .Act aAliment .4 : 271-289. Pressey,R .& J.K .Avants ,1972 .Multipl e formso fpectinesteras ei ntomatoes .Phyto - chemistry 11:3139-3142 . Reeck,G. , 1970.Amin oaci dcomposition so fselecte dproteins .In :Handboo ko fbiochem ­ istry,H.A .Sobe r (Ed.).Th eChem .Rubbe rCo. ,Cleveland ,2n ded. ,C-286 . Reid,W.W. , 1952.Th epecti cenzyme so fth efungu s Bysaoohlamys fulva. Biochem.J .50 : 289-292. Rexovâ-Benkovâ,L .& 0 .MarkoviC ,1976 .Pecti cenzymes .In :Advance si ncarbohydrat e chemistry andbiochemistry ,R.S .Tipso n& D .Horto n (Eds).Acad .Press ,Ne wYork , 33:323-385 . Rexovä-Benkovä,L. ,0 .Markovic '& M.J .Foglietti ,1977 .Separatio no fpecti cenzyme sfro m tomatoesb yaffinit y chromatography oncross-linke dpecti cacid .Collect .Czech .Chem . Commun.42 :1736-1741 . Rexovä-Benkovä,L .4 A .Slezärik , 1966.Isolatio no fextracellula rpectolyti cenzyme s producedb y Aspergillus niger. Collect.Czech .Chem .Commun .31 :122-129 . Riov,J. , 1975.Polygalacturonas eactivit y incitru sfruit .J .Foo dSei .40 :201-202 . Roeb,L .& H .Stegemann , 1975.Pectin-methylesterases :Isozymes ,propertie san ddetectio n inPolyacrylamid e gels.Biochem .Physiol .Pflanz .168 :607-615 . Rombouts,F.M. , 1972.Occurrenc e andpropertie s ofbacteria lpectat e lyases.Agric .Res . Rep.779 ,Pudoc ,Wageningen . Rombouts,F.M . &W .Pilnik , 1972.Researc ho npecti ndepolymerase s inth esixtie s- a literaturereview .CR CCrit .Rev .Foo dTechnol .3 :1-26 . Rombouts,F.M .& W .Pilnik , 1978.Enzyme s infrui tan dvegetabl ejuic etechnology . ProcessBiochem . 13(8) :9-13 . Rombouts,F.M . &W .Pilnik , 1980.Pecti cenzymes .In :Economi cmicrobiology ,A.H .Ros e (Ed.),vol .5 .Acad .Press ,Londo n (inpress) . Rombouts,F.M. , C.H.Spaansen ,J .Visse r& W .Pilnik ,1978 .Purificatio nan dsom e characteristics ofpectat e lyasefro m Pseudomonas fluoresoens GK-5.J .Foo dBiochem . 2:1-22 . Rombouts,F.M. ,A.K .Wissenbur g &W .Pilnik , 1979.Chromatographi cseparatio no forang e pectinesterase isoenzymeso npectate swit hdifferen tdegree so fcross-linking .J . Chromatogr. 168:151-161 . Rothschild,G. ,Z .Moya l& A .Karsenty , 1974.Pectinesteras e activity inth ecomponen t partso fdifferen tIsrael icitru sfrui tvarieties .J .Foo dTechnol .9 :471-475 . Rouse,A.H. ,1953 .Distributio no fpectinesteras e and totalpecti ni ncomponen tpart so f citrus fruits.Foo dTechnol .7 :360-362 . Rouse,A.H .& CD .Atkins ,1952 .Hea tinactivatio no fpectinesteras e incitru sjuices . FoodTechnol .6 :291-294 . Rouse,A.H .& CD . Atkins,1953a .Furthe rresult sfro ma stud yo nhea tinactivatio no f pectinesterase incitru sjuices .Foo dTechnol .7 :221-223 .

107 Rouse,A.H .& CD . Atkins, 1953b.Maturit y changesi npineappl eorange san d theireffec t onprocesse d frozenconcentrate .Proc .Fl .Stat eHortic .Soc .66 :268-273 . Sawamura,M. ,E .Kneg t& J .Bruinsma , 1978.Level so fendogenou s ethylene,carbo ndioxide , andsolubl epectin ,an dactivitie so fpecti nmethylesteras ean dpolygalacturonas ei n ripening tomatofruits .Plan t CellPhysiol .19 :1061-1069 . Schmid,P. , 1975.pH-statisch ePektinesterase-Bestimmun g inSüsskirschen .Z .Lebensm . Unters.Forsch . 157:23-27 . Schultz,T.H. ,H .Lotzkar ,H.S .Owen s& W.D .Maclay , 1945.Influenc eo fmetho do fde ­ esterificatio no nth eviscosit y andaci dbehavio ro fpectini c acidsolutions .J .Phys . Chem.49 :554-563 . Segal,B. ,R .Sega l& W .Grager , 1966.Studiu mde rFaktoren ,di edi eBildun gde sMethyl ­ alkoholsbe ide rBehandlun gmi tpektolytische nEnzyme nbeeinflussen .Fluess .Obs t 33:151-154 . Shastri,P.N .& N.V .Shastri , 1975.Studie s ofpecti nmethy lesteras eactivit ydurin g development andripenin go fguav afruit .J .Foo d Sei.Technol .12 :42-43 . Sheiman,M.I. ,J.D .Macmillan ,L .Mille r& T .Chas e Jr., 1976.Coordinate d actiono f pectinesterasean dpolygalacturonat e lyasecomple xo f Clostridium multifermentons. Eur.J .Biochem .64 :565-572 . Shewfelt,A.L. ,1965 .Change san dvariation s inth epecti c constitutiono fripenin g peachesa srelate d toproduc t firmness.J .Foo d Sei.30 :573-576 . Solms,J .& H .Deuel , 1955.Obe rde nMechanismu s derenzymatische nVerseifun gvo nPektin ­ stoffen.Helv .Chim .Act a38 :321-329 . Somogyi,L.P .& R.J .Romani , 1964.A simplifie d technique forth edeterminatio no fpecti n methylesterase activity.Anal .Biochem . 8:498-501 . Speiser,R. ,C.H .Hill s& CR . Eddy, 1945.Th eaci dbehavio ro fpectini cacids .J .Phys . Chem.49 :328-343 . Spiro,R.G. , 1966.Analysi so fsugar s foundi nglycoproteins .Method sEnzymol .8 :3-25 . Sreekantiah,K.R. ,S.A .Jaleel ,D .Narayan aRa o& M.R .Ragavendr aRao , 1975.Pectinolyti c enzymesproduce db y Aspergillus oarbonarius (Bainier)Thorn .Eur .J .Appl .Microbiol . 2:173-181 . Tahir,M.A. ,M.S .Chaudhar y& A.A .Malik , 1975.Pectinesteras e (PE)activit y incomponen t partso fJaff aorange s duringripening .Pak .J .Sei .Res .27 :59-60 . Tanner,H. , 1970.liebe rdi eHerstellun g vonmethanolarme nFruchtbranntweinen .Schweiz .Z . ObstWeinba u 106:625-629 . Termote,F. ,F.M .Rombout s &W .Pilnik , 1977.Stabilizatio n ofclou d inpectinesteras e activeorang ejuic eb ypecti caci dhydrolysates .J .Foo dBiochem . 1:15-34 . Theron,T. ,O.T .D eVillier s &A.A .Schmidt , 1977a.Purificatio n ofpecti nmethylesteras e fromSant aRos aplums .Agrochemophysic a9 :7-12 . Theron,T. ,O.T .D eVillier s &A.A .Schmidt , 1977b.Isolatio nan dpurificatio n ofpecti n methylesterase fromMarve l tomatoes.Agrochemophysic a9 :93-96 . Uhlig,H. , 1978.Kenns t duda sLand ,w odi eZitrone nblühen ? RöhmSpektru m 20:44-45 . Vas,K. ,M .Nedbalek ,H .Scheffe r& G .Kovâcs-Proszt , 1967.Methodologica l investigations onth edeterminatio no fsom epecti cenzymes .Fruchtsaf tInd . 12:164-184 . Venturella,V.S. ,D .Grave s& R.E .Lang , 1974.Automate dproo fdeterminatio no fliquor s bygas-soli d chromatography.J .Assoc .Off .Agric .Chem .57 :118-123 . Verhoeff,K .& J.M .Warren , 1972.I nvitr oan d inviv oproductio no fcel lwal ldegradin g enzymesb y Botrytis cinerea fromtomato . Neth.J .Plan tPathol .78 :179-185 . Versteeg,C , F.M.Rombout s &W .Pilnik , 1978.Purificatio nan dsom echaracteristic so f twopectinesteras e isoenzymes fromorange .Lebensm .Wiss .Technol . II:267-274 . Vertalier &Martin , 1958.Microdéterminatio n sélectivede sgroupe s alcoxypa r Chromatographie gaz-liquide.Chim .Anal .(Paris )40 :80-86 . Vieböck,F .& A .Schwappach , 1930.Ein eneu eMethod e zurmassanalytische nBestimmun g der Methoxyl-un dAethoxylgruppe .Ber .Dtsch .Chem .Ges .B 63 :2818-2832 . Vijayalakshmi,M.A. ,J .Sarri s& P .Varoquaux , 1976.Déterminatio n automatiqued e l'activitépectine-estérasiqu edan sun epréparatio npectinolytiqu ecommerciale . Lebensm.Wiss.Technol.9 :21-23 . Voragen,A.G.J. , 1972.Characterizatio n ofpecti n lyaseso npectin san dmethy loligo - galacturonates.Agric .Res .Rep .780 ,Pudoc ,Wageningen . Voragen,A.G.J .& W .Pilnik , 1970a.Pektindepolymerasen .Z .Lebensm.Unters.Forsch .142 : 346-359. Voragen,A.G.J .4 W .Pilnik , 1970b.Pecti nresearc h inth esixties .Dtsch .Lebensm . Rundsch.66 :325-329 .

108 Wagner,J.R. ,J.C .Miers ,D.W .Sanshuc k& R .Becker , 1969.Consistenc y of tomatoproducts . 5.Differentiatio no fextractiv e andenzym e inhibitoryaspect so f theacidifie dho t breakprocess .Foo dTechnol .23 :247-250 . Wallace,J. , J.Ku 2& E.B .Williams , 1962.Productio no fextracellula renzyme sb yfou r pathogens ofappl e fruit.Phytopatholog y 52:1004-1009 . Wang,S.C .& J.A .Pinckard , 1971.Pecti cenzyme sproduce d by Diplodia gossypina invitr o andi ninfecte d cottonbolls .Phytopatholog y 61:1118-1124 . Weste,G. , 1970.Ex tra-c eHu la renzym eproductio nb yvariou s isolateso f Ophiobolus graminis and 0. graminis var. avenae. Phytopathol.Z .67 :327-336 . Weurman,C , 1954.Pectas e inDoyenn eBoussoc hpear s andchange s inth equantit y ofth e enzyme duringdevelopment .Act aBot .Neerl .3 :100-107 . Whitaker,J.R. , 1972.Principle so fenzymolog y forth efoo dsciences .Marce lDekker ,Inc. , NewYork . Whright,L.O. , 1927.Compariso n ofsensitivit yo fvariou s testsfo rmethanol .Ind .Eng . Chem.,Ind .Ed . 19:750-752 . Wiley,R.C .& Y.S .Lee ,1970 .Modifyin g textureo fprocesse d appleslices .Foo dTechnol . 24:1168-1170 . Winter,A .& C .Karlsson , 1976.Preparativ e electrofocusing indensit ygradients . LKBApplicatio nNot e 219,LKB-Produkte rAB ,S-16125 ,Bromma ,Sweden . Winter,A. ,H .Perlmutte r& H .Davies , 1975.Preparativ e flat-bed electrofocusing ina granulated gelwit h theLK B211 7Multiphor .LK BApplicatio nNot e 198,LKB-Produkte r AB,S-16125 ,Bromma ,Sweden . Winzler,R.J. , 1970.Glycoproteins ,eggs ,milk .In :Handboo ko fbiochemistry ,H.A .Sobe r (Ed.).Th eChem .Rubbe rCo. ,Cleveland , 2nded. ,C-47 . Wolford,R.W. ,J.A .Attaway ,G.E .Alberdin g &CD .Atkins , 1963.Analysi so fth eflavo r andarom aconstituent s ofFlorid aorang ejuice sb yga schromatography .J .Foo dSei . 28:320-328 . Wood,P.J .& I.R .Siddiqui , 1971.Determinatio no fmethano l and itsapplicatio nt o measurement ofpecti neste rconten tan dpecti nmethy lesteras eactivity .Anal . Biochem.39 :418-428 . Yoshihara,0. ,T .Matsu o4 A .Kaji , 1977.Purificatio nan dpropertie s ofaci dpectin - esterase from Cortiaium volƒ'sii. Agric.Biol .Chem .41 :2335-2341 . Zauberman,G .& M .Schiffmann-Nadel , 1972.Pecti nmethylesteras ean dpolygalacturonas e inavocad ofrui ta tvariou s stageso fdevelopment .Plan tPhysiol .49 :864-865 . Zeisel,S. , 1885.liebe rei nVerfahre n zumquantitative nNachweis evo nMethoxyl . Monatsh.Chemi e 6:989-997 . Zeisel,S. , 1886.Zu mquantitative nNachweis evo nMethoxyl .Monatsh .Chemi e 7:406-409 . Zimmerman,R.E. , 1978.A rapi dassa yfo rpectinesteras e activitywhic h canb euse d asa prescreen forpectinesteras e inhibitors.Anal .Biochem .85 :219-223 .

109