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NN08201,442 576.341:58:577.15 the PERMEABILITY of DEAD PLANT CELLS for SOME ENZYMES De Permeabiliteit Van Dode Plantecellen Voor Enige Enzymen

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NN08201,442 576.341:58:577.15 the PERMEABILITY of DEAD PLANT CELLS for SOME ENZYMES De Permeabiliteit Van Dode Plantecellen Voor Enige Enzymen Ze/ "i* l/^z~ THE PERMEABILITY OF DEAD PLANT CELLS FOR SOME ENZYMES Depermeabiliteit van dode plantecellenvoor enige enzymen N. GORIN •IBLIOTHEEK 1ANDBOUV. ;!O Gf :SCHOO* WAGFM\GEN . NN08201,442 576.341:58:577.15 THE PERMEABILITY OF DEAD PLANT CELLS FOR SOME ENZYMES De permeabiliteit van dode plantecellen voor enige enzymen (witha Summaryin English, Dutchand Spanish) PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE LANDBOUWWETENSCHAPPEN OP GEZAG VAN DE RECTOR MAGNIFICUS, DR. IR. F. HELLINGA, HOOGLERAAR IN DE CULTUURTECHNIEK, TE VERDEDIGEN TEGEN DE BEDENKINGEN VAN EEN COMMISSIE UIT DE SENAAT VAN DE LANDBOUWHOGESCHOOL TE WAGENINGEN OP DINSDAG 11 MAART 1969 TE 16.00 UUR DOOR N. GORIN H. VEENMAN & ZONEN N.V.-WAGENINGEN-1969 THEOREMS I In the study ofniacin ,mor econsideratio n should begive nt o the implications ofoverdosag eo fniacin . GOLDSMITH, G. A. and O. N. MILLER. 1968. Niacin. In The Vitamins,Chemistry, Physiology,Methods. Vol. VII, second edi­ tion. Ed. by P. Gyorgy and W. N. Pearson. Academic Press, NewYork , London. 137-167. II The influence of calcium ions on the stability of trypsin and chymotrypsin activities inra t pancreaticjuic ei n the presence ofsodiu m taurocholate deserves moreattentio ntha nhithert oha sbee npai dt oit . VAHOUNY, G. V. and A. S. BRECHER. 1968. Effect of bile salt on the digestive enzymes of rat pancreatic juice. Arch. Biochem. Biophys. 123,247-254. Ill It should be made compulsory to have every newborn child tested for phenylketonuria inth esecon dwee ko flife . FLEURY, P. 1968. Organizing screening programme for inborn metabolic errors in The Netherlands. In Brain damage by inborn errorsof metabolism. Symposium organisedby the Interdisciplin­ ary Society of BiologicalPsychiatry. Amsterdam , 6Octobe r1967 . DeErve n F.Boh nN.V. ,Haarlem .60-66 . IV Intensive investigations should be carried out with a view to ascertaining a suitable method that would permit the determination of the karyotype of the human foetus before it isthre emonth s old. Should the karyotype be abnormal, abortion should be permitted. BROWN, W. M. C. 1968. Criminals and chromosomes. New Scientist 40,235-236. V Crates and boxes of vegetables that are brought into the various markets should beara labe lindicatin gth eproducer . N. GORIN Wageningen, 11thMarc h 1969 VI In publications on the structure ofyeas t cell walls, more attention shouldb e givent oth epossibilit yo fth eoccurrenc eo fmeta lbridges . PHAFF, H. J. 1963.Cel lwal lo fyeasts . Ann. Rev. Microbiol. 17, 15-30. LAMPEN,J .O . 1968. External enzymes of yeasts: their nature andformation . Antonievan Leeuwenhoek, J.Microbiol. Serol.34 , 1-18. ROGERS, H. J.an dH .R .PERKINS . 1968. Cell Wallsand Mem­ branes. EandF. N. SponLtd.,London . 135-152. Thisthesis ,p .41 . VII Someo fth eprotein spresen ti nth ecementin gmateria l inplan t tissues mayb e considered as proteinsbelongin gt oth eplasmodesmata . GINZBURG, B.Z . 1961. Evidence for a protein gel structure cross-linked by metal cations inth e intercellular cement of plant tissues./ . Exp.Botany 12, 85-107. VIII The Food Composition Table can be improved by giving coefficients of digestibility of the various foods based on digestion in vitro by appropriate enzymesunde rstandardize d conditions. Nederlandse Voedingsmiddelen Tabel. 1967. 23edruk . Voor- lichtingsbureau voor deVoeding .De n Haag. IX International standardization ofimmunological-fluorescen t methods should be instituted, so as to render it possible for results to be compared from a Quantitative noint nfvi™ Twenty second Symposium held in October 1968i n London byth e Permanent Section for Microbiological standardization. Round Table Conference on Standardization in Immunofluor­ escence.Internationa l Association of Medical Standards. The assertion that theactio n ofth e Waring Blendor on soybean meal pro­ ducescomplet ecel lwal lruptur e ofth ecell si sno tentirel ytrue . VICKERY,H . B., as referred to by S.J . CIRCLE. 1950. Proteins and other nitrogenous constituents. In Soybeans and Soybean Products. Vol. 1.Ed .b yK .S . Markley. Interscience Publishers, Inc.Ne wYork , p.29 2 XI The South American countries Argentina, Brazil and Uruguay should take steps to encourage the greater consumption of the beverage "Mate" (Ilex paraguariensis)i nth evariou sindustria lcountries . XII Thephilosophica l attitude ofth eresearc hworke r shouldno tb edogmatic . CONTENTS 1. INTRODUCTION 1 1.1. Scopeo fth epresen tinvestigatio n 2 1.2. General considerations 3 2. EXPERIMENTS MADE WITH a-CHYMOTRYPSIN 6 2.1. Materials and methods 6 2.1.1. Chemicals 6 2.1.1.1. Buffer solutions 6 2.1.1.2. Thechoic eofa n antimicrobial compound (Preventol 115) 6 2.1.1.3. Ethylenediaminetetraacetate(EDTA )solutio n 7 2.1.2. Enzymes 7 2.1.2.1. Chymotrypsinogenan d trypsinogen 7 2.1.2.2. a-Chymotrypsin 7 2.1.2.3. Determination ofa-chymotrypsi nactivit y 8 2.1.3. Determination ofth econcentratio no fsubstrat esuspension s 8 2.1.4. Nitrogen determinations and calculations based on nitrogen contents of the substrates 9 2.1.4.1. Nitrogencontaine d inth esampl e 9 2.1.4.2. Nitrogen (%) ondr yweigh to fth esubstrat e 10 2.1.4.3. Calculation ofquantit yo fnitroge ni nth ereactio nmixtur e 10 2.1.4.4. Calculation of thenitroge nrelease dfro m thesubstrat e bya-chymotrypsi n . 10 2.1.4.5. Calculation ofth efractio n ofcel lwal lnitroge n inrelatio n totota lcel lnitroge n 10 2.1.4.6. Calculation ofth eamoun t ofcel lwal lnitroge ni nth ereactio nmixtur e . ... 11 2.1.5. Substrates 12 2.1.5.1. Soybean 12 2.1.5.2. Yeast 14 2.1.5.3. Algae 15 2.1.6. Digestion ofth eprotein si nth esubstrate sb ychymotrypsi n 15 2.1.6.1. Digestion ofsoybea ncell s 16 2.1.6.2. Digestion of soybean sections 17 2.1.6.3. Digestion ofsoybea npowde r (subcellular) 18 2.1.6.4. Digestion ofyeas t 18 2.1.6.5. Digestion ofalga e 18 2.2. Results of theexperiment s on thedigestio n of theprotein si n thesubstrate s by chymotrypsin 19 2.2.1. Determination of the optimal ratio of enzyme quantity to amount of soybean suspension 19 2.2.2. Digestion ofsoybea n cellsb ychymotrypsi n 21 2.2.3. Digestion ofsoybea n sectionsb ychymotrypsi n 23 2.2.4. Digestion ofsoybea npowde r (subcellular)b ychymotrypsi n 26 2.2.5. Digestion ofyeas t bychymotrypsi n 28 2.2.6. Determination of the optimal ratio of enzyme quantity to amount of algae suspension 29 2.2.7. Digestion ofalga eb ychymotrypsi n 32 2.3. Discussion on digestion bychymotrypsi n 35 2.3.1. Antimicrobial compound (Preventol 115) 35 2.3.2. Ethylenediaminetetraacetate(EDTA ) 36 2.3.3. Maceration 37 2.3.4. Soybeantrypsi n inhibitor (Kunitzinhibitor ) 37 2.3.5. Digestion ofsoybea n substrate bychymotrypsi n 38 2.3.5.1. Cells 38 2.3.5.2. Sections 39 2.3.5.3. Subcellular powder 39 2.3.6. Yeastdigestio nb ychymotrypsi n 40 2.3.7. Digestiono falga eb ychymotrypsi n 41 3. FLUORESCENT ANTIBODY TECHNIQUE 43 3.1. Materialsan dmethod s 43 3.2. Results 4g 3.3. Discussion 46 4. PENETRATION OF AMYLOPECTIN INTO SOYBEAN MATERIAL .... 49 4.1. Materialsan dmethod s 49 4.1.1. Ureasetes t 51 4.2. Results . 52 4.2.1. Resultso fth eureas etes t 54 4.3. Discussion 54 5' ™Ec^MENTS MADE WITH ""CHYMOTRYPSIN AND/OR PANCREATIC •Ll"AoE gs 5.1. Preliminaries 5g 5.1.1. Materials,method s andresult s ................. 56 5.1.1.1. Determination of proteolytic activity of the different lipase solutions and the percentage ofproteolyti c inhibition by DFP . 57 5.1.1.2. Influence of inhibition of the proteolytic activity of lipase solution in 10-'3M DFP-20hoursat37°C 58 5.1.1.3. Modified caseindigestio nmetho d 58 5.1.1.4. Determination oflipas eactivit y 59 5.1.1.5. Determination of lipase activity when'lipase and chymotrypsin are present simultaneously 60 5.1.2. Discussion ,. 5'2' ^8vfS!!0n by lipaSe °f substrates (soybean and yeast) treated or not'treated withchymotrypsi n fi, 5.2.1. Materialsan dmethod s . 63 5'2'LL wirhchymotrypS86 ^ SUbStoteS (soybean and yeast)'treated or not treated . 64 5.2.1.2. Digestion ofsoybea ncell sb ychymotrypsi n and/or lipase M 5.2.1.3. Digestiono fsoybea nsection s bychymotrypsi nand/o rlipas e 66 5.2.1.4. Digestion ofyeas tb ychymotrypsi n and/or lipase. ... 66 no/heatednf Wiwi „:i ^ ^T, • °f 0l„l Ve 0il emU™«„'Si0n,' iiv^v-heateud uiivolivce uoinl ememulsion^°n,', emulsio™^<™n oft heateheated oilAT?™,emulsio n o°[f unheateunheated dsu subcellulal ' r soybean powder and emulsion o01f heateneate d subcellulasubcellular soybeasoybeanpowde powder fi6 a S0 h fhigherfaigherfattyaddsliberateyacidsliberatedf s'f ResRes^ou£ o°f/ h h " dfromsoybeancellsbyl'ipas^soybeancellsbyl'ipasee .'. ' .'. ' .'. ' .'. ' .'. ' 66 Kesults of the experiment6 Penmens on th the 1e" digestio n of the sub yeasfeasfbvcLit b r if °? ^rates soybean and „ y chymotrypsinand/o rlipas e . 68 • s^:*^^ 70 lfpasUeltS °f the ana'ySiS °f hi8her fatty acids liberaied from'soybean ceils'by 5.2.4. Discussiono ndigestionb ychymotrypsi n and/orlipas e .' .' .' .' .' .' .' ." .' .' 70 6. GENERAL DISCUSSION 75 SUMMARY. 83 SAMENVATTING 84 RESUMEN 85 ACKNOWLEDGMENTS 86 REFERENCES 88 PRINCIPALABBREVIATION S 92 1. INTRODUCTION The digestibility of plant cells used as food for human beings or for animals, depends, among other factors, to a certain degree on the penetration of the digestive enzymes into them. The first barrier that the enzymes must pass in order to enter the plant cells are the cellwalls . MITCHELL (1942) stated that the cell walls of plant tissues may be an obstacle to the digestion by enzymes of ruminants because they prevent the enzymes from reaching the contents ofth e cellsfro m these tissues.
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