Characterization of Purified Extracellular Lipase Fractions

from Pseudomonas fmgi CRDA 037

by

Aliar Abdul Wahrb

A thesis submitted to the Faculty of Gnduate Studies and Rtseareh in partial fulîiiment of the rquirements of the degree of Muter of Science

O~liaaAbdul Wahab

Department of Food Science and Agncultural Chemistry

McGill University

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EXTRACELLULAR LIPASES FROM PSEUDOMONAS FRQGI ABSTRACT M. Sc. Aliar Abdul Wabab

The partially purified extracellular lipase fkom Pseudornoms fiagîCRDA 037, obtained by ammonium sulfate prccipitation, was subjected to iùrther purification by successive ion-exchange and sue-exclusion chromatographies using the Fast Protein Liquid Chromatography system. The purification of the partially purified lipase resulted in two enzymatic fiactions, FIVa' and FIVb', with a purification-fold of 169 and 295, respectively. Native electrophoretic analyses revded the presence of three bands for fiaction FIVa', with estimated molecular weights (MW) of 16.2, 25.8 and 38.5 kDa and two bands for FIVb', with estimated MW of 15.2 and 25.8 kDa. The two purified fiactions, FNa' and FM', showed an optimum pH of 9.5 and 10.0, respectively, and an optimum temperature of 80°C. The Km values for FIVa' and FIVb' were 3.85 and 5.49 rnM and the V,, values were 2.78 and 2.09 Ulmg, respectively, using triacetin as a substrate. The purified lipase fiactions retained more than 90% of their activity when stored at room temperature for 36 h. The lipase activity of the purified lipase fiactions was compietely inhibited by 10 mM of FeC12, FeC13 and Eliman's ragent. However, 10 mM of CaCIz and EDTA activated the two purified lipase fiactions by 20 to Both fiactions exhibited high specificity towards short- and long-chain fatty acid esters of triacylglycerols. Fraction FIVa' showed higher specificity towards triacetin, tristearin and tripalmitin, whereas fraction FIVb' exhibited higher activity with triacetin, trimyristin and triolein. In addition, the two purified lipase fractions were able to catalyze, to aimost the same extent, the hydrolysis of butter and olive, canola and fish oils. The gas-liquid chromatography analysis of fke fatty acids, obtained by the hydrolysis of the four edible oils, revealed that fraction FIVa' was more specific for the hydrolysis of fatty acid esters chah lengths of C 12 to C 18 whereas fiaction FIVb' showed a non-specific hydrolyzing activity towards fatty acid esters. RÉSUMÉ M. Sc. AIUt Abdul Wahib

La lipase partiellement purifiée de Pseudornoltos Fagi CRDA 037, obtenue par précipitation au sulfate d'ammonium, a été davantage purifiée par chromatographies échangeuses d'ions a de tamisage moléculaire a utilisant le système Fast Protein Liquid Chromatography. Deux fhctions purifiées ont été obtenues, FIVa' et FIVb', avec un facteur de purification de 169 a 195, respectivement. Les analyses d'électrophorèse native ont révélé la présence de trois bandes pour k MonFIVéî', avec des poids moléculaires (MW) estimés de 16,2, 25'8 et 38,s kDa et de deux bandes pour FIVb', avec des MW estimés de 15,2 et 25'8 kDa, respectivement. Les deux nanMns purifiées, FIVa' a Fm', ont montré un pH optimum à 9.5 a 10,0, respectivement, et une température de 80°C. Les valeurs de Km pour FNa' et FIVb' ont été de 3,85 et 5'49 mM et les valeurs de V, ont été de 2-78 et 2,09 Ulrng, respectivement, en utilisant la tracétine comme substrat. Après avoir stockée a température ambiante pour une période de 72 h, les fiactions lipasiques purifiées ont conservé plus de 90 ./. de leur activité pendant 36 h. L'activité lipasique des fiactions purifiées a étt5 compléternent inhibée par FeC12, FeCl3 et le réactif dYEllmanà 10 mM. Cependant, CaC12 a EDTA ont activé l'activité des fiactions purifiées de 20 a 50 %. Les deux fractions ont démontré une forte spécificité vis-à-vis des esters de triglycérides a courtes et a longues chaînes. La fiaction FIVa' a montré une forte spécificité envers la tnacétine, la trimyristine et la trioléine. De plus, les deux &actions purifiées ont catalysé l'hydrolyse de beurre et d'huiles d'olive, de canola et de poisson pratiquement au même degré. Les analyses de chromatographie en phase gazeuse d'acides gras libérés par l'hydrolyse des quatre huiles comestibles ont montré que la fraction FIVa' étaiat spécifique pour les esters d'acides gras a longues chaînes de C12:O à Cl8:O alors que la fraction FIVb' etait non-spécifique, hydroplysant aussi bien les esters d'acides gras à longues chaînes saturées et insaturés. I would like to thank my supervisor, Dr. Seh Kennasha, for the guidance, suppofi and patience he offered throughout my study. 1 would iike to extend my gratitude to Dr. André Morin for his numerous advises and his arrangement for the biomass production at the Agriculture Agri-Food Center, CRDA

1 would aiso like to thank Anwer, Marie-Odile and Tami for their assistance. Also special thanks to Pierre-Yves and Wigdan for their valuable advises and Xavier for writing the résumé. And many thanks to al1 my colleagues for their fnendship and good spirit.

1 would like to thank my husband, Osama, my sister, Dalia, my brother, Qussay, my linle sister, Noor, and especiaily my parents for their moral qqmn. TABLE OF CONTENTS

LIST OF FIGURES...... VIII...

1, INTRODUCTION ...... 1

2- LITERATURE REVIEW ...... m~~o~m~ooa~~~o~~o~~~~~~~~~-~moa~m~~~m~ma~oa~a~~a~o--~~~~~~a~~~~ao~~~oa~oam~~o4 2.1. Flavor Production Biotechnology ...... 4 2.2. Pseudomonas fiagi ...... -6 2.2. I . Physical Features ...... 6 2.2.2. FhrPra&ct~on by Pseudomomtsfiagrgr ...... 6 2.2.3. Mechism of Flavor Production by P .fiagrgr ...... 7 2.3. Lipases ...... -8 2.3.1. Nomencluture ...... 8 2.3.2. Sources of Lzpes ...... 9 2.3.3. Characteristics of Lipes-...... 10 2.3.4 Production of Microbiai Lipares...... 12 2.3.5. LipeKinetics ...... 13 2.3.6. Lipaw Substrores ...... I5 2.3.7. Assay of Lipaw Activity ...... 16 2.3.7.1. Titrimetric Method ...... 16 2.3 .7.2. Spectrophotometric Method ...... 17 2.3.7.3. Fluorimetric Method ...... 18 2.3.8. LipeSpecr/ciiq ...... 18 2.3 .8.1. Positional Specificity ...... 19 2.3.8.2. Fatty Acid Specificity ...... 19 2.3.8.3. Stereoselectivity ...... -20 2.3.9. Punijication of li'es...... 21 2-3.10. Industrial Application of Lipes...... 23 3. MATERIALS AND METHODS...... 25 3.1. Bacterial Strain ...... 25 3.2. Chemicals ...... 25 3.3. Biomass Production ...... 26 3.4. Extraction and Partial Purification of the Crude ExtraceIIuar Lipase ...... 26

LIST OF FIGURES

Figure no.

Fig. 1. Lipase-catalyzed hydrolysis of triacylglycerols...... -11

Fig. 2. Lipase-catalyzed esterincation reaction...... -11

Fig. 3. Lipase-catalyzed tramesterification reaction...... -11

Fig. 4. Profile of the purification of the partiaiîy purified lipase fiaaion from Pseudomonas~~~giby ion-exchange chromatography on Source 1SQ, HR 1O/ 10 column ...... -38

Fig. 5. Pronle of the purification of the lipase fiaction Fmd by size-exclusion chromatography on Superdex 75, HR 16/50 column...... -39

Fig. 6. Profile of the purification of the Lipase MonFTVa by re-sire-exclusion chromatography on Superdex 75, HR 16/50 column...... 40

Fig. 7. Profile of the purification of the Lipase fiaction FIVb by re-size-exclusion chromatography on Superdex 75, HR 16/50 column...... -41

Fig. 8. Elearopherogram of the lipase fiactions FII, FIIId, FlVa' and FM'on native polyactylamide gel electrophoresis...... -45

Fig. 9. Standard curve for molecular weight estimation of lipase fiactions using native polyacrylamide gel electrophoresis...... -46

Fig. 10. Standard curve for molecufar weight estimation of lipase fractions using size-exclusion chrornatography...... -48

Fig. 1 1. Effect of pH on the activity of the paxtially purified Lipase fraction FII, fiaction FIIId fiom IEC, fiactions FIVa and FIVb fiom SEC and fiactions FIVa' and FIVb' from re-SEC...... -50 Fig. 12. Effect of temperatwe on the activity of the partially purified Lipase fraction FII, fiaction Fmd fiom IEC and fiactions FIVa' and FIVb' fkom re-SEC...... -52

Fig. 13. Lineweaver-Burk double reciproul plots of the partiaiiy purified Lipase &action FII, fiaction Fmd fiom EC, fiactions FIVa and FWb fiom SEC and fiactions ma'and FIVb' fkom re-SEC...... -54

Fig. 14. Substrate specificity of the purifieci lipase fiactions FNa' and RVb' towards triacylglycerol models...... *...... -65

Fig. 15. Specificity of the purifiai lipase fkdons FIVa' and FIVb' towards four edible oils...... -67 LIST OF TABLES

Table no.

Table 1. Purification scheme of the Lipase fractions f?om Pseudorno~tasfia~ CRDA 037...... -42

Table 2. Kinetic parameters of the purilied extraceUular lipase fiactions from P. fiagî CRDA 037...... -35

Table 3. Effect of various metai sahs and chernical reagmts on the activity of the p&ed lipase fiactions fiom P. fia@ CRDA 037...... S9

Table 4. GLC analysis of fiee fatty acids produced by the hydrolysis of edible oils using the purihi Lipase fiactions FIVa7 and FIVb7...... 69 1. INTRODUCTION

Lipases are widely distnbuted among animals, plants and rnicroorganisms

(Brokerhoff and Jensen, 1974). These enzymes catalyze the hydrolysis of esters of triacylglycerols, which are the major constituents of fats and oils, to produce fiee fatty acids, giycerol and partial acylglycerols (Macrae, 1983). Lipases form a very distinct group of enzymes that are activated by lipid-water interfaces. Under water-restricted conditions, lipases can also catalyze the reverse reactions of synthesis and group exchange of esters (Le., esterification and transesterification) and the resolution of racemic mixtures into optically active compounds (Godtfiedsen, 1990).

Interest in microbial lipases has markedly increased over the past years. Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial applications (Ghandi, 1997). They are used in the oil and fat industry for the synthesis of structural triacylglycerols (Macrae, 1983) and in the pharmaceutical and agrochemical industries for the production of optically pure products (Godtfiedsen, 1990). They are also employed in the food industry as additives for the development and cnhancement of food flavors (Ghandi, 1997). Recently, increasing consumer demand for products that are natural has led the food industry to produce natural flavors by using the available biotechnological tools, Le., microorganisms and/or microbial enzymes. There are several rasons for using microorganisms and microbial enzymes for the production of flavors. Firstly, the resulting product is considered natural since it is derived by enzymatic action or fermentation. Secondly, complex flavors can be producecl that would otherwise be difficult to produce chemically. Thidly, these flavon can be generated in large quantitics unda controlled conditions.

Lastly, inexpensive waste materials can be used as fermentation media.

Pseudomoms fiagi; a psychrotrophic bacterium, was first associated with the spoilage of milk through the development of hity off-flavor (Stead, 1986). Howwer, under controlled conditions, P. fia* can producc fiavor-active metabolites like fiee fatty acids and faîty acid esters that contribute to the fhity aroma (Morin, 1991). Lipases produced by P. fiagi have been found to be responsible for the aroma production in which milk fat is hydrolyzed into fiee fatty acids that act as flavor precursors (Morgan, 1976).

This knowledge has initiated the need to extract and to purie the lipase enzyme(s) to better understand its (their) properties and to provide a foundation for future work into the structure, mechanism of action and biosynthesis. This study was part of an ongoing research in our laboratory aimed at the purification and characterization of lipases fiom

Pseud~rnonasfia~.The extraction and partial purification of the lipase@) from P. fiagi

CRDA 037 was already accomplished in our laboratory. Therefore. the main objective of this present study was to cany out the chromatognphic purification and characterization of the extracellular lipases fiom P. fiagi.

The specific objectives of the resûuch were:

1. To produce the biomass of Pseudomonas fiagi, to extract and to partially

pune the crude extracellular lipase by ammonium wüate precipitation.

2. To dwelop the methodologies for the purification of the partially purifid

lipase extract using ion-exchange uid size-exclusion chromatographies. 3. To charactaize the purifjeci lipase Mons. in terms of their elcctrophoraic

pronle, optimal pH and temperature, kinetic parameters, stability, the effect of

a number of inhibitors and activaton and substnte specificity ushg modd

substrates and edible oils. 2. LITERATURE REVIEW

2.1. Flavor Production Biotechnology

Biotechnology has been defined as a collection of technologies that employ

biologid or living systems (rnicroorganisrns, plants, or anirnals) or specific compounds

derived fiom these systems, for the production of industrial goods and seNices.

Biotechnology has recently been applied to many different areas including the food

industry for the production of flavors. Flavor may be defined as a wmplex sensation of

taste, aroma and physiological influences. Food flavors in particular depend on odorous

compounds derived from more complex precursors including proteins, fats and sugars.

Initiaily, conventional extraction or chernical synthesis has bcen widcly used for the

production of artificial food flavon. However, due to an increased demand by conamers,

the food industry is moving toward the production of natural flavors. The three

biotechnological techniques pnmarily used in flavor production are: plant ce11 and tissue

culture (PTC), rnicrobial fermentation, and enzyrnatic bioconversion (Vulfwn, 1994).

PTC is a technique that allows plant cells to be grown in solid or liquid media

containing nutrients and plant growth regulators (phytohormones such as auxins and cytokinins). The industrial application of PTC in the production of plant-denved flavor

compounds has been lirnited due to the high cost of production, the low yield of products, and the insufficient knowledge about the biosynthetic pathways and the regulatory mechanisms that govem plant metabolism (Hariander, 1994). On the other hand, the exploitation of microorganisms in the biotechnologid production of femented foads stems fiom the fact that microorganisms are srnall in size, diversified in their properties, and have simple growth rcquinments and short generation times. Microorganisms ouch as bacteria, yeast, and fùngi have ken used widely in the development of a large number of products including yeast biornass, citric acid, monosodium glutamate, lactic acid, xanthan and vitamin BI2. Moreover, microbial fermentation is also used in the production of complex flavor compounds that can not be produced chemically. Some of these flavor compounds include lactones, esters, ketones, aldehydes, aicohols, and fatty acids

(Harlander, 1994).

Enzymes isolated fiom different microbial sources have been broadly used in the food industry instead of using whole microbid 41s. fhey have ken used in wine and juice production (pectinases), hi@ fructose corn symp (glucose isomerases), lactose reduction (lactases), extraction of edible oils (cellulases and pectinases) and aspartame production (thennolases). They are also used in the production of naturai food flavon replacing the convernional chernical synthesis of artificial flavon (Godtfiedsen, 1990).

More specifically, lipases are extensively used in the dairy industry for flavor development.

Initially, the traditional sources of lipases for cheese flavor development are animal pancreatic glands (bovine and porcine) and pregastnc tissues of young mminants (kid, larnb, calf). More recently, microbial lipases are increasingiy used in the cheese manufacture industry. Current applications include flavor enhancement and acceleration of cheese ripenhg, the manufacture of enzyme-modified cheese (EMC) and the lipolysis of butter fat and cream (Vulfson, 1994). 2.2. hdomonasfngi

2.2.1. Physicai Fanres

The bacterium is a member of the genus Pseudomotuzs that include P. fluorescens,

P. putida. P. aemgino.sat P. ceprica and othen. The name Pseudomonus means in Latin

'fdse monad' and fiagi means 'of the strawberry'. fseudomottm fiagi is an aerobic,

Gram-negative, psychrotrophic bactenum. It can grow at low temperature of 7°C or below irrespective of its optimal temperature of growth (Stead, 1986). It is rd-shapeb

0.5-1.0 pm in diameter by 1.5-5.0 pm in Length. The bacterium is motile by one polar flagellum. Ah, it is shown to be catalase positive (a test for the presence of cytochrome c using p-aminodimethylaniBne)ne) and possesses no sheath around its ceii wdl. It produces no pigments. It has been isolateci fiom milk and other dairy products, dairy utensils, mat, water, and soi1 (Buchanan and Gibbons, 1974).

Pseudomotnas nrains have ban recognized as major spoiiage microorganisms in many different foods including milk, fish and meat produc& Specifically, Pseudomorms fiagi was associated with the production of hity off-flavon in dairy produas. Pereira and Morgan (1958) indicated that the fniity arorna of milk cultures of P. fiagiwas due to the production of ethyl esters. Reddy et ai. (1968) showed that the fniity aroma could be enhanced by the addition of 0.2% ethanol to the milk culture medium. This culture was found to wntain ethyl butyrate and ethyl hexanoate in significant leveis contributing for the miity aroma. Morin (1991) reporteci that the supplementation of skim milk medium with valeric acid and ethanol has highly increwd the production of ethyl valerate ester. Therefore, the ability of P. fiagi to produce desirable aroma has ïnitiated

researchers to exploit the micrwrganism using available waste by-products. Whey, a

waste product fiom the dairy indu-, has ban regarded as a serious environmental

pollutant since 6-9 liters of whey is produced for each kilogram of cheese manufactured

(Macrae et al-, 1993). Generally, whey contains 93% water, 5.1% lactose, O.% protein,

0.5% ash and 0.3% milk fat. The composition of whey allows for its utilization as a cheap

medium in the production of flavors by P. fiagi It was found that P. fiagi cm be grown on supplemented whey medium for the generation of flavors. When ethanol and five short- chain fatty acids (C3, C4, CS, C6, C7) were added to the whey medium at O h, the production of flavor metabolites has greatly increased aAer 72 h as opposed to unsupplemented whey medium (Raymond et al, 199 1).

2.2.3. Mechanism of Flavor Roàùction by P. fragi

The mechanism governing the production of the hity aroma in milk and milk products by the action of P. fiagi is not yet known. However, one theory suggens that flavor production by P. frugi may involve one or two enzymes, namely a lipase and an esterase. The lipase enzyme is believed to catalyze the hydrolysis of tnacylglycerols found in milk to produce free fatty acids, which are then estenfied to the supplemented ethanol to yield the hity ethyl esten (Morgan, 1976). But it is not clear whether the lipase enzyme is solely responsible for the hydrolysis and esterification reactions or there may be a second enzyme, an esterase, catalyzing the synthesis of ethyl esters. More recently, a mdy wss carried out on the optimization of the growth

conditions and the lipase activity of Pseudomor~~~aemgirmsc~ EF2 (Gilbert et al.,199 1b).

Both esterase and lipase activities were monitored throughout the growth period. It was

found that the esterase activity varied approximately in parallel with the lipase activity under al1 the growth conditions tested, indicating that a single enzyme rnay be responsible

for both activities, and this was later confirmeci by purifjmg the enzyme (Gilbert et al.,

1991a).

2.3. Lipases

Lipases were first isolated from milk. Raw milk provides a suitable medium for the growth of microbial flora, induding pathogenic strains. With the introduction of bulk refrigerated storage in the 1950s, the growth of the microflora in milk can no longer be sustained, except for some psychrotrophic bacteria, e.g. Pseudomouurç &a@ and

Pseridornonas fluorescems. Although pasteurization can elirninate ail microbial growth in milk, many species of psychrotrophic bacteria can produce kat-stable extracellular lipases that are capable of withstanding high pasteurization temperatures.

2.3.1. Nomenclaîure

Lipases belong to a group of enzymes known as the ester hydrolases, or esterases, classified as enzyme class 3.1 according to the classification recommended by the Enzyme

Commission of the International Union of Biochemistry. In a narrower sense, lipases hydrolyze esters of fatty acids and are therefore carboxyl ester hydrolases and classified as

3.1 .l. They are further classified according to the substrates on which they act. Most of the lipases act on esters of glycerol, which is the molecular backbone d most of the lipid material occumng in nature. Glycerol ester hydrolases are classified as class EC 3.1.1.3

(Sneath et al,, 1984).

2.3.2. Sources of Lipases

Lipases are widespread in nature and they are produced by animals, plants and microorganisms. Some animal lipases include roammalian pancreatic lipases, rnammalian gastric, pregastric, lingual lipases produced by the stomach lining of ruminants, nonmammalian digestive lipases of fish and invertebrates, lipoprotein lipases, tissue lipases, and milk Lipases (Mukhejee and Hills, 1994).

In plants, lipases are most abundant in oilseeds and, to a lesser degree, in cereal gains. Oilseeds such as soybean, wttonseed, corn dower, coconut, sesarne and peanuts are rich in triacylglyerols that serve as an energy source for the germinating seeds. Cereal grains contain less amount of triacylglycerols and they indude wheat, oat, rye, bariey, and rice (Mukhejee and Hilfs, 1394).

As for microorganisms, interest in rnicrobial lipases has recently increased due to their high growth rate compared to animals and plants, and the production of large quantities of the enzyme in short periods of time. Microbial lipases include bacterial lipases such as Pseudonronas, Staphy/ococcus, Achromobacter, Bacillus. Acir~tobacter,

Chromobacterium, Luctobacihs. Cotynebacterim, Micrococcus, Propionibacteriurn~

Strepfococcus, yeast lipases produced by Candida, and fiingal lipases produced by Rhizops, Penicilium, Aspergillus, Geotrichurn. Mucor, 5bccharomyces. Streptomyces, and other strains (Godtfiedsen, 1990).

2.3.3. Ckaracîcristics of Lipases

Lipases are triacylglycerol hydrolases and their natural substrates are tnacylglycerols of long-chah fatty acids. Triacylglycerols are the main constituents of oils and fats and are mostly insoluble in water- A triacylglycerol consists of thee fatty acid esters attached to a glycerol molecuk which is a trihydroxy compound with two pnmary alcohols and one secondary alcohol. Whereas, fatty acids are carboxylic acids with hydrocarbon chain lengths of 3-36 carbons. Lipases can catalyze the hydrolysis of tnacylglycerols at the interface between the insoluble substrate phase and the aquwus phase containing the soluble enzyme. This propeny distinguishes lipases fiom esterases that catalyze the hydrolysis of soluble esters of glycerol in preference to insoluble esters.

The reaction products of triacylglycerol hydrolysis by lipases are diacylglycerols, monoacylglycerols, glycerol, and free fatty acids (Fig. 1). However, not al1 lipases give complete hydrolysis of their substrates, although long incubation periods can result in complete hydrolysis (Macrae, 1983). Triacy lgl ycerol Free Faüy Acids

Figure1 . Complete hydroiysis of ~acylgiyuxolscatalyzed by a lipase

Lipases can also catalyze the reversible reactions in water-restricted environments.

They catalyze the synthesis (esterification) and group exchange of esters

(tramesterification) as weii as the resolution of racemic mixtures into optically active alcohols and acids.

RCOOH + R'OH = RCOOR' + H20 Free Fatty Acid Al cohol Ester Water Figurez. Li pax-catalyzed esterification reaction

R'COOH + R~COHC %

Free Fatty Acid Triacylglycerol Triacy lgl ycerol Free Fatty Acid Figure3. Lipase-cataiyzed tramesterification reaction

11 2.3.4. Mctio~of Micmbiaf -ases

Extracellular lipases norrnally appear in the culture medium when the bacterial ceiis

reach the end of their logarithmic growth phase (Jaeger et al., 1994). Lipase production by

rnicroorganisms can be stimulateci by the addition of iipids or triacylglycerols to the

culture medium. However, the presence of mono- or disaccharides or glycerol can

suppress the production of iipases. For instance, lipase production by Geotrzchum

cdidùm growing on glucose only ouxirs afta the corsumption of gluwse in the

medium (Iwai et a', 1973). On the other hand, small amounts of these simple sugars may

be required to initiate growth. In addition to triacylglycerols, detergents, such as Tweens

and Spans, and fatty acids, such as oleic acid and butyric acid, can stimulate lipase

production. Peptone, soya bean meal, yeast extract and casein hydrolysate are commonly

used as niuogen sources (Macrae, 1983).

Apart fiom the secreted extracellular lipase, a considerable amount of lipase

remains attached to the ceU walls. The presence of the bound Lipase may inhibit the

production and reduce the yield of the extracellular lipase. However, the addition of

rnagnesium ions or lecithin to the culture medium was found effective in increasing the lipase production by releasing the enzyme fiom the ce11 walls. In one study, treatment of the culture medium with nometabolkable polysaccharides such as hyaluronate and alginate could influence the release of cell-bound lipase of Pseudomonas ueruginosa

(Jaeger and Winkler, 1984; Wingender and Winkler, 1984). 2- 3.5. Lipase Kindics

Conventional enzyme kinetics has been described for reactions in aqueous solutions. It involves the formation of an enzyme-substrate complex in which the catalysis takes place. Then the wmplex is dissociated to yield the produa and the free enzyme, which is recyclecl to catalyze another reaction. The kinetic parameters mosz often used to describe enzymes are the Kmand V,. The K,,,is the Michaelis constant and it represents the substrate concentration at which the reaction velocity is half-maximal. If the K, value is low, then a low substrate concentration is needed to achieve the maximal cataiytic eficiency of the enzyme. The magnitude of Kmvaries \ijith the identity of the enzyme and the nature of the subarate. It is also a function of temperature and pH. On the other hand,

V,, is the maximal velocity of the reaction, and it occurs at high substrate concentration when the enzyme is saturated. But in most cases the initial rate of the reaction is more important parameter than the maximal rate of reaction. The initial velocity (vo) is taken as the velocity measured before more than 10% of the substrate has been converted to product. The use of the initial velocity rather than velocity, minimizes such complicating factors as the effects of reversible reactions, inhibition of the enzyme by the product, and progressive inactivation of the enzyme (Voet and Voet. 1990). The best method used to determine the values of C, and K,, is to use the Lineweaver-Burk plot with Ilvo plotted against i/[S] to give a iinear equation. The slope of the line is KJV,, IIV- is the y- intercept, and the extrapolated x-intercept gives - VK,.

Lipase kinetics in panicular differs fiom normal enryme kinetics in that lipases tend to be activated by interfaces. Interfaces occur in nature or can be prepared experimentally as emulsions, micelies or liposomes, and monolayen. In tipolysis, the reaction is heterogeneous in which the substrate is insoluble in water and the enzyme is water- soluble. Sarda and Desnuelle (1958) have shown thaî the activity of a pancreatic lipase increased sharply when the solubility limit of the substrate was exceeded and an emulsion was formed. While esterases follow normal Michaelis-Menten kinetics with soluble substrates, lipases are more active with insoluble substrates and have slight Michaelis-

Menten-type activity wit h soluble esters. However, lipases fiom Pseudomonas aeruginosu

(Jaeger et al., 1993) and Bacrifus subrilis 168 (Lesuise et ai., 1993) do not show activation in the presence of emulsified substrates; instead, their activity continuously increases indicating that these enzymes are able to degrade both emulsions and soluble monomenc substrat es, whereas true esterases degrade only monomeric substrates.

Moreover, fiirther studies by Benzonana and Desnuelle (1965) revealed that the size of the emulsion droplets is important. The sdler the dropiets were, the larger was the surface area available for the lipase and the higher the activity. A plot of the reaction rate and substrate concentration, expressed as surf'ace area, could be found to follow the

Michaelis-Menten kinetics. The only drawback with the interfacial K.is that the concentration is expressed as moles per unit area instead of per unit volume, which makes it difficult to interpret (Martinelle and Hult, 1994). This disadvantage can de resolved by using water-soluble substrates. Lipolysis in a homogeneous aqueous phase eliminates from the kinetic analysis dl the complications that arix from the presence of an interface

(Brockerhoff and Jensen, 1974). Another alternative- is to use a well emulsified and homogenized substrate-enzyme mixture and the substrate concentration can be expressed in terms of molarity and the lipase kinetics can be wnsidered to follow normal Michaelis-

Menten kinetics.

Recent elucidation of lipase stmctures has greatly assisted in understanding the kinetics of lipases. Some of the lipase structures that were detennined include those fiom

Rhiromucor miehei, Geotrïchum candiidum and human pancreas. The crystal structure of a lipase fiom Rhizomucor miehei revealed that the active site is covered by a helical lid and is displaced exposing the catalytic site to the hydrophobic interface (Brady et al.,

1990). Moreover, Verger and de Haas (1976) assumed that lipases undergo a conformationai change concornitantly with, or der, the binding of the lipase to the surface of the insoluble substrate. They have proposed a kinetic model for the hydrolysis of the insoluble lipids by lipases. The model is comprised of an adsorption process of the lipase to the interface, foilowed by the binding of the substrate to the enzyme and then by catalytic reaction in a Michaelis-Menten fashion.

2.3.6. Lipase Substrates

A number of triacylglycerols have been used to assay for lipase activity. Triolein

(C 18: 1, C 18: 1, C 18: 1), an insoluble liquid triacylglycerol, is the most cornmonly used substrate. However, a cheaper alternative is olive oil, which contains over 70% oleate residues. Sina most lipase substrates are insoluble in water, stable emulsions should be prepared by vigorous mechanical or ultrasonic rnixing of the substrate with an emulsifier such as gum arabic. Solid triacylglycerols such as tripalmitin and tristearin do not react as readily with the lipase enzyme, probably due to stearic hindrance rather than any inherent inability to bind to the enqme (Stauffér, 1989). Another lipase substrate that is used more often is tributyrin (C4:0, C4:0, C4:O). It is more convenient to use than olive oii or triolein because it is quite soluble in water and no emulsifier is needed. Also the products of tributyrin hydrolysis are water-soluble and do not inhibit the reaction by accumulating at the water-oil interface. However, tributyrin is not a good lipase substrate since it can be hydrolyzed by esterases and lipase activity should be confirmed with more suitable substrates (Brockerhoff and Jensen, 1974).

2.3.7. Assay of Lipase Activity

2.3.7.1. Titrimetric MétM

A very popular method to assay fiee fatty acids released by lipase action is the titrimetric method. In the fixed-tirne assays, the reaction is incubated for a period of time then stopped by the addition of a mixture of organic solvents, e-g., ethanol, acetone and ethyl ether. The released fatty acids are titrated to an endpoint against a dilute base solution like sodium hydroxide (NaOH) or potassium hydroxide (KOH) by using either an indicator like phenolphthalein or an automatic burette with a pH probe. The lipase activity is then expressed in units of activity and one unit is de6ned as one pnole of fatty acid liberated in one minute.

A more reliable assay is the continuous titrimetric assay or the pH-stat, where the release of fiee fatty acids is continuously monitored throughout the incubation period and the pH of the reaction mixture is kept constant by the addition of a base solution (Stauffer,

1989). The rate of addition of the base is a direct measure of the rate of fne fatty acid formation. The reaction is usually carried out at a basic pH around 8.0-9.0. At pH below

8.0 less fatty acids are in their ionized form (Mattson and Volpenhein, 1966). This method

is very valuable in the investigation of lipase kinetics, as the initial rate of the reaaion can

be deterrnined directly .

2.3.7.2. Spectrophotornetric Method

It is also known as the colorimetnc method and it is used to assay the lipase

activity. This technique is based on the hydrolysis of chromogenic substrates like p-

nitrophenyl esters of stearate, palmitate, and butyrate. pNïtrophenyl esters can be used for

lipase and esterase assays depending on the chah length of the ester. Once the

chromogenic substrate is cleaved, it releases p-nitrophenol, which is a yellow compound.

The degree of hydrolytic activity can be related to the intensity of the yellow color formed

and can be measured with a spectrophotometer. A unit of lipase activity corresponds to one pnole of p-nitrophenol formed in one minute.

Another variation of the method is the use of a difKerent chromogenic substrate

Iike P-naphthyl esters of C2:O to C 18: 1 fatty acids. The assay mixture contains a buffer, P- naphthyl substrate and an enzyme solution. Afier incubation, the color is developed by adding Fast Gamet GBC preparation or fast blue BB salt. The absorbance is measured at

540 nm using a spectrophotometer. A unit of lipase activity is expressed as the amount of enzyme that causes an increase in absorbance at 540 nrn of O.O5/h (Gobbetti et al., 1996;

Bard and Fox, 1997). A third method that has been used to a lesser degree is the fluorimetnc method.

Fatty acids that yield a fluorescent product upon hydrolysis are sometimes used to achieve greater assay sensitivity, three times more sensitive than titration assays. A comrnon substrate is the ester of 4-methylumbeWerone (Stau&r, 1989). ïhe lipase activity is measured by this method using umbelliferyl oleate in buffer as a standard substrate. At time zero the lipase is added to this mixture and the fluorescence emission (470 nm) is recorded over 2 min using a fluorescence spectrophotometer (Cornmenil et a!. , 1995).

2.3.8. Lipase Specz;fici@

Specificity is the comparative difference in the rates of catalysis of certain reactions. In order to study lipase enzyme specificity, several factors have to be controlled.

First, the molecuiar properties of the lipase preparation which cm possess more than one lipase with different types of specificity and so pudication of the enzyme is essential for the determination of the specificity. Secondly, the structure of the substrate which determines the identity of the product and the type of specificity. Thirdly, the factors affecting binaing of the enzyme to the substrate kedng or sonication of the culture medium and the addition of stabilizers and emulsifiers to create a stable emulsions and to provide the maximum suiface area for the enzyme ta fùnction (Jensen et ai., 1983). There are three major types of lipase specificity and they are positional specincity, fatty acid specificity and stereoselectivity. 2.3.8.1. Positioml Spcificiîy

Microbial lipases can be divided into two groups depending on their positional specificity. The fira group is nonspecific and releases fatty acids fiom al1 three positions of the glycerol moiety. These lipases cataiyze complete hydrolysis of tnacylglycerols. The second group is 1,3-specific catalyzing the release of fatty acids at the 1 and 3 positions of the glycerol moiety, giving rise to 6ree faîty acids, 1,2 (2,3)-diacylglycerols, and 2- monoacylglycerols as the reaction products. Because 1,2 (2,S)-diacylgîycerols and 2- monoacylglycerols are chernically unstable and undergo acyl migration to give 1.3- diacylglycerols and 1 (3)-monoacylglycerols, prolonged incubation of a fat with 1.3- specific lipase will result in complete hydrolysis of the triacylglycerols. Until now there is no report of a lipase with specificity towards the secondary position of the glycerol moiety. The positional specificity of a lipase can be detennined by analysis of the products forrned by lipolysis of triacylglycerols using thin-layer chromatography (TLC) on silicic acid, which separates 1,2-diacylglycerols fiom 1,3-diacylglycerols and 2- monoacy lgl ycerols fiom 1-rnonoacylglycerols (Macrae, 1983 ).

2.3.8.2. Fatiy Acid Specrficity

This type of specificity depends on the substrate used and on the fatty acid composition of the substrate. In detennining the fatty acid specificity of a lipase, monoacid t nacylgl ycerols of various fany acid chah lengths have been used. Unlike tnacy lglycerols of unsaturated fatty acids and short- and medium-chah saturated fatty acids, triacylglycerols of long-chah saturated fatty acids are solid at normal reaction conditions and are hydrolyzed slowiy by rnicrobial lipases (Sugiura and Isobe, 1975). Moderate dflerences in fatty acid specificity have been found due to variations in the source of the

microbial lipase. But generaîly, most rnicrobial lipases show specificity toward the longer- chah fatty acid esters and little specificity when incubated with natural oils and fats.

Exceptions ocair when marine oils and rnilk fats are used as substrates since they contain a high amount of polyunsaturated fatty acids and short-chah fatty acids, respectively.

A unique type of fatty acid specificity was found for Lipases fiom Geotrichum candidum. These lipases were known to have a very special kind of substrate specificity towards cis-9 unsaturated fatty acids (Jensen, 1974). Other lipases fiom Mucor liplyticus were found to possess different substrate specificities and physicai properties. The lower rnolecular weight enzyme ~tdyzedthe hydrolysis of triacylglycerols of medium- and long-chain fatty acids but showed no activity toward tributyrin. The higher molecular weight enzyme could cataiyze the hydrolysis of tributyrin as well as the medium- and long- chah fatty acids (Nagaoka and Yamada, 1973).

2.3.8.3. Stereoselectivity

Stereoselectivity is a term used to describe a type of enzyme specificity that occurs when two or more stereoisomers produced in a reaction in which one stereoisomer is produced in predominance over al1 others. It has been reported that some lipases possess stereoselectivity and they can release fatty acids of one enantiomeric form more rapidly than the other, e.g. an intracellular lipase fiom PeniciIIium cyclopium (Druet et al., 1992).

This kind of specificity is extremely usehl in the rcsolution of the racemic mixtures to produce a single isoma in preference to the racemate. However, very few lipases are known to possess stereoseiectivity and more investigation is needed.

2.3.9. Ptrrificdn of lipases

The purification of enzymes and other macromolecules is often achieved through chromatographic separation that include ion-exchange, size-exclusion, hydrophobie interaction and affïnity chromtographies. Ionexchange chromatography (IEC) is often used in the fira sep of the purification scherne, although in some procedures it is used more than once. The separation of the proteins by EC depends on the dserences in the net charge on the proteins, at a given pH, which bind to the ion-exchanger by electrostatic attractions. There are two groups of ion-exchangers: anionexchanger (positively-charged matrix) and cation-exchanger (negat ively-charged matrix). The most cornmonly used anion-exchangers are the diethylaminoethyl (DEAE) substituents and the quatemary amino groups (Mono-Q) which are attached to hydroxyl groups on the matrix.

Carboxylmethyl (CM) and sulfopropyl (SP) groups are widely used as cation-exchangers.

The matrix is made up of cellulose, silica, agarose, dextran or any other synthetic polymer

(Taipa, et al., 1992).

The second most fiequently used method is size-exclusion chromatography (SEC) or gel filtration. It invoives the separation of proteins according to their molecular size.

The gel consias of an open, cross-linked, three-dimensional molecular network cast in bead form. The pores within the beads are of such sizes that allow some smaller protein molecules to penetrate while excluding the larger ones. Thus the srnalier molecules take longer to elute f?om the wlumn than the larga ones and hmce Mirent molecular süe

proteins are separated. Cross-linLed dextran (Sephadex) and cross-iinked polyacrylamide

(Biogels) have bem used widely as si~emclusionadsorbents. Recently, more ngid cross-

linked gels have been introduced such as Sephacryl, Superose, Superdex, Trisacryls and

Toyopeari (Scopes, 1987).

Hydrop ho bic-interaction chromatography (HIC) is aiso employed in many

purification techniques. HIC exploit the variability of extenial hydrophobic amino acid

residues on different proteins. In an aqueous solution, the hydrophobic patches on the

proteins tend to interact with the hydrophobic groups on the matrix. Hydrophobie

interactions can be arengthened by high salt concentrations or higher temperatures and

are weakened by low salt concentrations and the presence of miscible organic solvents or

detergents (Scopes, 1987). The most commonly used hydrophobic adsorbents consist of

shon aliphatic chains (C4-C 10) or a benzyl (phenyl) group attached to hydroxyl groups on the matrix (Taipa, et al., 1992).

Other chromatographic techniques are also used in a srnaii number of purification

schemes. One technique is affinity chromatography and it is more specific and expensive than the other chrornatographic techniques mentioned before. Another method is adsorption chromatography using hydroxyapatite. Liquid-liquid extraction using 2-phase system and reverse phase HPLC are also used but to a lessa extent (Taipa, et al., 1992). 2.3-10. In- Application of Lipases

The annual sales of Iipases account for 20 million US dollars, corresponding to less than 4% of the world-wide enzyme market, which was estimated at 600 million US dollars

(Adige and Pitcher, 1989). The sales of other hydrolytic entymes such as proteases and carbohydrases are currently at lest ten times larger than lipases. On the other hand, there are two main reaSOns explaining the misconception of economic value of Lipases. First, lipases have been extensively investigated as a route to novei biotransformation. Secondiy, the diversity of the current industriai applications of lipases exceeds that of proteases and carbohydrases. Thus, the signincance of lipases rests on their potentiai rather than their current level of use (Vulfson, 1994). Nonetheless, lipases are still used in many difFerent industries.

Lipases are used in the production of soaps catalyzing the hydrolysis of fats and oils to yield the corresponding fatty acids and glycerol. The lipase-catalyzed hydrolysis of lipids occurs at moderate temperature and pressure with lower energy consumption and absence of side reactions as opposed to the costiy chernical reaction, Colgate-Ernery process of stem fat-splitting. Lipases are also utilized in the leather manufacture industry for the removal of residual fats associated with the animal skins. Furthermore, they are utilized in the waste treatment plants for the removal of fats forming on the sufiace of aerated sewage tanks. They are also added to detergent formulations for the removal of fat stains fiom fabrics and dishes (Ghandi, 1997).

Other applications include the use of lipases in the food industry. Lipases are extensively used in the dairy industry for flavor development of cheese, the acceleration of cheese ripening and the lipolysis of butterfat and crum. They an also used in the preparation of enzyme-modifieci cheeses (EMC) by producing a ~n~en~atedflavor to be used as an ingredient in other food formulations such as dips, snacks, dressings, soups, sauces, etc. (Ghandi, 1997). The aroma and texture of these milk produas are a result of fat, protein and lactose metabolism in W. Therefore, enzymes such as lipases and proteases are now used to accelerate the maturation of cheese and to produce the characteristic flavors. In this process, milk fat and proteins are hydrolyzed by these enzymes producing fkee fatty acids, amino acids and peptides which act as flavors and flavor precursors. Lipases are also used for the production of fat-fiee mats, the hydrolysis of triacylglycerols for increasing the monoacylglycerol content of bread dough and reducing the staling effkct and the formation of mono- and diacylglycerols to be used as food emulsifiers. They are employed in the synthesis of stnictured triacylglycerols by modivng the fatty acid composition to produce valuable and more nutritious oils such as cocoa butter, milk-fat substitutes and triacylglycerols with medium-chain and polyunsaturated fatty acids (Macrae, 1983).

There are ail1 other potential applications of lipases that are currently under development. However, it is suspected that the employrnent of Lipases in the near fùture will undergo a drarnatic increase and will exceed that of proteases and carbohydrases. 3. MATERIALS AND METHODS

3.1. Bacttrirl Stnin

The bacterial strain Pseudomonas fiagi CRDA 037 was obtained corn the

Agriculture and Agri-Food Research and Development Center (CRDA St-Hyacinthe,

QC).

Triacetin (C2:O), tnbutyrin (C4: O), tricaproui (&:O), tricaprylin (C8:0), trimyrstin

(C l4:O). tripalmitin (C 1610). tristearin (C 18:0), triolein (C 18:l), butyric acid and bovine serurn albumin (BSA) were obtained fiom Sigma Chernical Co. (St-Louis, MO).

Anhydrous ethanol, acetone, hydrochloric acid (HCI), sodium chloride (NaCl), sodium hydroxide (NaOH) and Tween 20 (polyoxyethylene-sorbitan monolaurate), an emulsifier, calcium chloride (CaCI2), potassium chloride (KCl), rnagnesium chloride (MgCI2), sodium deoxycholate and ethylenediamine tetraacetic acid (EDTA) di-sodium salt were purchased fiom Fisher Scientific (Fair Lawn, NJ). Arnmediol (2-amino-2-methyl- 1,3-propandiol) used for the preparation of Arnmediol-HCI buffer, ferrous chloride, Ellman's reagent (5'-

5'-dithiobis-(2'-nitrobenzoic acid), N-bromosuccinimide and diisopropyl fluorophosphate

(DIFP) were obtained from ICN Biochemicals Inc. (Auror* OH). Ethyl ether, phenol reagent which was used for the detemination of protein content, sodium phosphate monobasic and dibasic which were used in the preparation of phosphate buffer and femc chloride were al1 bought fiom ACP Chernicals Inc. (Montréai, QC). Filter membranes and chromatography columns were obtained fiom Arnersham Phannacia Biotech (Uppsala, Sweden). Whey powda, used as a fermentation medium, was obtained âom Saputo (St-

Hyacinthe, QC). Brain Heart Infiision @HI), used as a medium for the subcultures, was bought fiom DIFCO laboratones (Detroit, MI). Food grade ethanol (94%)- uscd in fermentation, was purchased from Sociite des Alcools du Québec (Montréal, QC).

3.3. Biomrss Production

Three successive subcultutes of P. fiagi CRDA 037 were incubated in BHI (Brain

Heart Infiision) broth at 30°C and 200 rpm. An inoculum of 0.01% (vlv) was used to initiate each subculture. The third subculture was used to inoculate (1%, v/v) the culture medium which consisted of whey (6%, w/w),1000 ppm butyric acid (O. 1%, v/v) and food grade ethanol(0.2%, vlv). The biomass production of P. fiagiCRDA 037 was perfonned at the Agriculture and A@-Food Research and Development Center (CRDA, St-

Hyacinthe, QC) in a 150-L (working volume of 100 L) continuously stirred fermentor.

The medium was prepared according to the procedure described by Schuepp et al. (1997).

The medium was adjusted with sodium hydroxide (NaOH) to pH 8.0 and sterilized by autoclaving at 121°C for 10 Mn. The sterilized medium was adjusted to pH 6.5, inoculated with the third subculture and incubated at 12°C and 100 rpm for 72 h.

3.4. Extraction and Partial furification of the Crude Extracelluar Lipase

The extraction and partial purification of the crude extracellular lipase extract were preformed according to the procedure developed by Schuepp et al. (1997). The culture medium was centrifuged (39,000 xg; 20 min) at 4OC using a flow-through centrifige to separate the cells fiom the culture medium. The Jupematant was collected as a source of the cmde extracellular Lipase, whereas the precipitated cds were discarded. The crude

lipase extract was fieeze-dried and stored as a powder at -20°C.

Partial purification of the cmde extracellular lipase extract was performed

according to the procedure outluied by Schuepp et ai. (1 997). The cmde lipase extract

was diluted with sodium phosphate buffer solution (0.05 M, pH 8.0) to a protein

concentration of 15 mg/rnL. The solution was stirred for 1 to 2 h at 4°C and centrifuged

(39,000 xg, 15 min) at 4OC, using the Avanti-J25 I centrihge (Beckrnan Instrument, San

Ramon, CA). Solid ammonium sulfate was added at 20% of saturation and the

precipitated fraction was removed by centrifbgation (39,000 xg, 15 min). The supernatant

was fbrther treated with ammonium sulfate salt at 4% of saturation and the precipitated

fraction was recovered by centrifugation (39,000 xg, 15 min). The precipitated protein

fraction was suspended in a minimal arnount of sodium phosphate buffer solution (0.05 M,

pH 8.0) and dialyzed against a dilute solution of the sarne buffer solution (0.01 M, pH 8.0)

using a dialysis membrane (SpectraIPor Membrane; MWCO: 12,000- 14,000, Fisher

Scientific). The dialyzed enzymatic fiaction was then lyophilized and stored at -20°C and

used as the partially purified extracellular lipase fiaction.

3.5. Purification of the Extracellular Lipase

The pmially purified extracellular lipase extract was fbnher purified by successive preparative ion-exchange (IEC) and size-exclusion (SEC)chromatographies using the fast protein liquid chromatography (FPLC)system (Amersharn Phannacia Biotech). 3.5.1. Ion-Exchange Chromatogrephy

The IEC was carried out on a Source 15 Q, HR 10/10 column (Amersham

Pharmacïa Biotech) using the FPLC system. The column was equilibrated with one column volume (8 mL) of buffer B (Ammediol-HCI, 0.02 M, pH 9.0) wntaining 1 M

NaCl, then with five column volumes (40 mL) of buffer A (Ammediol-HC1, 0.02 M, pH

9.0). Mer equilibration of the column, samples cbnsisting of 1 mL of enzyme solution (50 mg protein) were injected. A gradient elution systcrn was employed and was made of buffer A (100%) and was increased to 1Wh bder B in 17.5 min, at a flow rate of 2 mUmin. The eluted protein fiactions were monitored with a UV detector at 280 nm and colleaed using an automatic fraction collector. The active -ions were dialyzed against a dilute buffer solution of sodium phosphate (0.001 M, pH 8.0) and fieeze-dried.

The purified fraction with the highea lipase specific activity obtained nom IEC was further purified by SEC on preparative Superdex 75, HR 16/50 colurnn (Amersham

Phannacia Biotech). The column was equilibrated with two column volumes (200 mL) of sodium phosphate buffer solution (0.05 M pH 8.0). and a sarnple of 1 rnL of enzyme solution (50 mg protein) was injected. The fiow rate was 1 mL/min and the eluted protein fiactions were monitored with a UV detector at 280 nm and collected using an automatic fraction collector. The fractions were diaiyzed against a dilute sodium phosphate buffer solution (0.001 M, pH 8.0) and fieeze-dried. 3.5.3. Re-Sïze-~f~~îonCkmma!grqp hy

The purifid fkactions with the highest lipase specific activity obtained fiom SEC were re-chromatographd using the sarne colurnn (Superdex 75, HR 16/50). The column was equilibrated with two column volumes (200 mL) of sodium phosphate buffer solution

(0.0 1 M, pH 8.O), and a sample of 1 mL of enzyme solution (10 mg protein) was injecteci.

The flow rate was 1 mL/min and the eluted protein fractions were collected. The hctions were dialyzed against a dilute sodium phosphate bufkr solution (0.001 M, pH 8.0) and freeze-dried.

3.6. Protein Determination

The determination of the protein content of the samples was performed according to the method described by Hartree (1972). Bovine xrum albumin (Sigma Chernical Co.) was used as the protein standard.

3.7. Measuremtnt of Lipase Activity

3.7.1. Rkodamine B Test

Qualitative tests were carried out on Rhodamine B agar plates to detect for lipase activity in the culture medium. The plates were prepared according to the method of

Kouker and Iaeger (1987) by king 100 mL of agar with few drops of Rhodomine B, a fluorescent dye, and 3 mL of olive oil, which is used as a lipase substrate. mer 24 to 48 h of incubation penod, at rwm temperature, the plates were examined under UV light for orange fluorescent zones using Spectroline model CX-20 ultraviolet fluorescence analysis cabinet (Spectronics Corporation, Westbury, NY).

The lipase activity of the crude enzymatic extract was also measwed spectrophotometrically according to a modification of the method of Wuikler and

Stuckman (1979). This technique is based on the hydroiysis of a chromogenic substrate such as p-nitrophenyi ester of stearic acid. Once the chromogenic substrate is cleaved, it releases p-nitrophenol, which is a yellow compound. The degree of hydrolytic activity can be related to the intensity of the yellow color fomed and can be meanired with a spectrophotometer model DU650 (Bechan Instrument).

Solution A was composed of 230 mg of sodium deoxycholate and Il 1 mg of gum arabic diswlved in 100 mL of sodium phosphate buffet solution (0.05 M, pH 8.0).

Solution B contained 10 mg ofp-nitrophenyl stearate in 10 mL of isopropyl alcohol. The reaction mixture consisted of 2.16 mL of solution A and 0.24 rnL of solution B. The reaction was initiated when 0.1 m.of the enzyme suspension (3 mg protein) was added to the test samples and 0.1 mL of the buffer solution was added to the blanks. The sarnples were incubated at 40°C for 60 min with sbaking at 120 oschnie. At the end of the reaction, a yellow color was fond which comsponded to the released p-nitrophenol molecules and the absorbante was determined spectrophotometrically at 410 nm. To detennine the lipase activity, the absorbame of a wide range of pnitrophcnol concentrations was plotted to construct a standard curve. One unit of Lipase activity corresponds to one mol ofp-nitrophenol forrned in one minute.

The released fice fatty acids fiom the lipase-cataiyzed hydrolysis of triacetin were titrated using the DL 53 automatic titrater (Metler Toledo, AG, Switzerland). The reaction mixture was based on that of Schuepp et a' (1997) with a minor modification. It consisted of O. 1 m.KCl(0.5 M), 1 rnL of NaCl (0-005 M), 50 pL of Tween 20, 18.8 pL of triacetin (100 pnol), 5 rnL of Amrnediol-HCI buffer solution (0.05 M, pH 9.0). The reaction was initiated by the addition of 0.4 rnL of the partiaily punfied Lipase (12 mg protein) or 0.1 rnL of the purified lipase (0.2 mg protein). The volume of the reaction mixture was completed to 10 mL with deionized water (Millipore Corp., Bedford, MA). A tnplicate number of blanks were run in tandem under the same conditions except that the blanks were stopped at O min, afier the addition of the enzyme. The Erlenmeyer flasks (50 rnL) containing the samples were incubated at 3S°C in a reciprocal shaking (120 odmin) water-bath (Precision Scientific Inc., Chicago, IL) for 30 min for the paxtiaily purified lipase, and for 15 min for the purified lipase fiactions. The reaction was stopped by the addition of 10 mL of a mixture of ethanoVethyl ether (1 :3, v/v) and the released fatty acids were titrated with a standardized solution of NaOH (0.05 M).

To caiculate the moles of free fatty acids @TA) produced by the lipase enzyme, the following equations were used Net volume of NaOH titrated (mL) = Volume of sample - Volume of blank

molof FFA produced = molof NaOH titrated

= WaOH] (mmoVmL) x net m.of NaOH

Lipase activity = mm01 of FFNreaction time (min) x 1000

= pmol of FFAhnin

Specific activity = pmol of FFA/rnin/mg of protein

3.8. Molecular Wcight Dtttrmination

3.8.1. Gel Elecîmpkorais

Polyacrylamide gel electrophoresis (PAGE) was carried out on the enzymatic fractions using the Phastsystem Unit (Arnersham Phannacia Biotech). Native-PAGE minigels (5 x 4 cm) of 12.5% polyacrylarnide were precast as outlined in the Pharmacia manual (1992). The gels were run on the Phastsystem for 30 min at a constant current of

10 using PhastGel native buffer strips (Arnersham Pharmacia Biotech); the buffer stnps consisted of 0.88 M L-alanine, 0.25 M Tris, pH 8.8 in 2% Agarose. Low-rnolecular weight standards (Amersham Pharmacia Biotech) were run in tandem with the sarnple fiactions and consisted of phosphoryla~b (94.0 kDa), bovine serum albumin (67.0 Da). ovalbumin (43.0 kDa), carbonic anhydrase (30.0 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). The electrophoretic separation was perfonned according to the Pharmacia instruction manual (1992). Afker the separation of the proteins, the minigels were transferred to the development compartrnent of the

Phastsystem unit. Silver staining was carried out as outlined in the manual. The molecular weights of the purified lipsse fiactions were estirnated by size- exclusion chromatography on Superdex 75, HR 16/50 column (bed volume of LOO mL) equilibrated with 200 mL of sodium phosphate buffer (0.01 M, pH 8.0). A mixture of protein standards (5 mg/mL) including albuMn (67 ma), ovalbumin (43 kDa), chemotrypsinogen (25 kDa) and ribonuclease A (13 kDa) was used to connnict the standard curve. The void volume (Vo) was determined by using Blue Dextran dye (1 -25 mg/mL). The lipase fractions (10 mg) were sepanitely applied on the column with a flow rate of 0.7 mWrnin. A standard curve was wnstructed by plotting the elution volume (V,) of the protein standards againn their Km values. A linear cuwe was obtained and the straight-line equation of the cuwe was used to estimate the molecular weights of the lipase fractions. The Km value was calculated using the following equation

Km = (vC-vo)/(Vrvo) where V, is the elution volume of the sarnple

Vo is the void volume

Vbis the bed volume of the column

3.9. Determination of pH and Tcmpenture Optima

For the determination of the optimum pH, the following bufFer solutions were used: citrate-phosphate (pH 3.0 to 7.0), sodium phosphate (pH 6.0 to &O), Ammediol-

HCl (pH 8.0 to 10.0), phosphate-NaOH (pH 1 1.O to 1 1-5) and hydroxide-chloride (pH 12.0). The optimum temperature for lipase activity was determinecl by incubating the

reaction mimn at different temperatures ranging corn 25 to 90°C increasing in intervals

of 5°C.

3.10. Lipase Stability

The lipase stability was tested by incubating the purified fiactions at room temperature (25OC) for 72 h. The activity of the purified lipase fiactions was measured every 12 h by the titnmetric method.

3.11. The Effkct of Inhibitors and Activators on the Lipase Activity

The effect of a number of different types of inhibitors and activators on the activity of the punfied lipase fractions was investigated. Six inhibitors, including ferrous chloride

(FeClz), femc chloride (FeCl,), sodium deoxycholate, diisopropyl fluorophosphate,

Ellman's reagent and N-bromosuccinirnide, and the aaivators, including calcium chloride (CaC12), magnesium chloride (MgCl*) and ethylene diamine tetraacetic acid

(EDTA) were used throughout this study. Two concentrations (1 and 10 mM) of each inhibitor or activator were assayed. The lipase activity was measured by the titnmetnc rnethod.

3.12. Substnte Specificity

The specificity of the punfied extracellular lipase fractions was assayed using triacylglycerols of different fatty acid chain lengths as substrates. Triacetin (C2:0), tnbutyrin (C4:0), tricaproin (C6:0), tricaprylin (C8:0), trimyrstin (C 14 :O), tripaimitin (C 16:0), tristearin (Cl 8:O) and triolein (Cl 8: 1) were used as substrates. A 100 pmol of

each substrate was used and 100 pL of Tween-20 was added to ensure complete

emulsification of the substrate. The reaction mixture was sonicated for 2 min and 2-sec

pulses with 1-sec pauses using the sonicator, Ultrasonic Processor 2020 XL (Heat

Systems, Fanningdale, NY). The reaction was initiated by the addition of the enzyme

extract and was incubated at 35°C for 15 min with wntinuous shaking at 120 osdmin-

The lipase activity was determined by the titrirnetric rnethod.

The lipase activity of the purified enzyme extracts was also tested with selected

edible oils including butter and olive, canola and fish oils. The reaction mixture, containing

200 mg of the oil, 100 pL of Tween-20 and 100 pL of punfied lipase -ions (0.2 mg

protein), was homogenized by sonication as described above and the enzyme assay was

conducted by the titrimetric method.

3.13. Analysis of Frcc Fatty Acids on Gis-Liquid Chromatography

The specificity of the punfied lipase fiactions towards four edible oils, butter and olive. canola and fish oils. was determined by the analysis of the released fatty acids on gas-liquid cliromatography (GLC). The hydrolysis of the edible oils was camed out as descnbed above. At the end of the incubation perîod, the reaction was aopped by the addition of 20 mL of hexane. The free fatty acids were extracted in the hexane layer, which was separated and concentrated to 6 rnL under a gentle stream of nitrogen gas. The extracted free fatty acids were methylated acwrding to the method of Badings and De

Song (1983). One rnL of HCVmethanol mixture (20:80, v/v) was added to the hexane extract and the mixture was incubated at 85°C for 15 min with wntinuous shaking. The rnimre was centrifùged (1,000 xg, 5 Mn) and the supernatant was analyzed by GLC.

Free fatîy acid analysis was perfonned on GLC using a Varian Mode1 3400 gas chromatograph (Varian Associates, Sumyale, CA). The system was equipped with a column injector, a flame-ionization detector (Fm)and a capillary colurnn (Omegawax

320, 30 m x 0.32 mm interna1 diameter, coated with polyethylene giycol; Supelco,

Oakville, ON). The flow rates for the amer gas (helium), hydrogen and air were 1.8, 30.0 and 300.0 mUmin, resptctively. The temperatures of the injector and the deteaor were

230 and 200°C, respectively. Mer an isothermal period of 5 min, the column temperature was raised fiom 40 to 200°C at a rate of 8OC/min. 4. RESULTS AND DISCUSSION

4.1. Purification of the Estircdulrr Lipase

The panially purifieci extracellular lipase extract 0,obtained by ammonium sulfate precipitation (Schuepp et al., 1997), was subjected to further purification using successive chromatographie techniques including ion-exchange (IEC) and size-exclusion

(SEC)chromatographies. The pufication of fiaction FII by IEC resulted into two major fractions, FTIIa and FiIld, and two minor ones, fractions Fmb and Fmc (Fig. 4). The results (Table 1) indicate that fiaction Fmd has the highest total and specinc lipase activities, with a purification-fold of 12 and a recovery of 185%.

Fraction FNd was subjected to further purification on SEC and the separation profile shows (Fig. 5) the presence of one major peak (FIVa) and three shoulders (',

FIVc and FIVd). Fractions FlVa and FIVb have the highest total activity with a purification-fold of 80 and 117, respectively. These two fiactions were reapplied separateiy on the same SEC column and chromatographed under alrnost the same conditions. Figures 6 and 7 show the chromatograms of re-size-exclusion chromatography of fiactions FNa and FIVb, respectively. Fractions, FIVa' and FM' showed a purification-fold of 169 and 195 and a recovery of 34 and 600/0,respectively.

The results indicate that the highest increase in purification-fold was reached with

SEC with an increase of 6.7 to 9.8 times compared to an increase of 2.7 times for IEC. On the other hand, re-chromatography on SEC increased the specific activity by 0.6 to 2.1 which is lower than that in the previous step. The increase in spdcactivity throughout 5 10 15 20 25 Elution The(min)

Figure 4. Profile of the purification of Pseudornonasfi.agzextracellular lipase extract by ion-exchange chromatograpby on Source 15 Q: absorbance at 280 nm (- ), specific activity ( --a.----) and NaCl gradient (- - - - ). FNa

......

25 50 75 Elution Time (min)

Figure 5. Profile of the purification of the Iipase fraction FIIId, obtained from IEC, by size- exclusion chrornatograpghy on Superdex 75: absorbante at 280 nrn (- 1 and specific activity ( -.-.--- ). Elution Tirne (min)

Figure 6. Profile of the purification of Monma, obtained fiom SEC, by re-chromatography on Superdex 75: absorbance at 280 nrn ( --) and specific activity ( --O-- ). 50 75 Elution Tirne (min)

Figure 7. Profile of the purification of fiaction FIVb, obtained fiom SEC,by re-chromatograpghy on Superdex 75: absorbame at 280 nm (- ) and specific activity ( +). Table 1. Purification scheme of the extracellular lipase from I?wtrdomo~~asfragi CRRDA 037.

Sample Protein Total weight content protein Specific Total Recovery Purification Fraction (44) (mg/m8 sam~le) (mg) act ivit y activityd (%) (fold)

P SEC (FIV)" N FIVa FIVb FIVc FlVd Re-SEC (FIV')' FIVa' FM'

'crude extracellular lipase produccd from Pseudornonar~gi. b~allypurikd extracellular lipase fraction produccd by ammonium sulfatc prccipiistion at 204% of saturation. Cloncxchnngechmmatography of thc parîially puriW extnecllular lipase fracîion on Source 15 Q-HR 10110 (Amershana Phamucia Biotah). d~ize~~~~usionchmmatography of fraction Fllld, obcained by IEC, on Supcrdex 75-HR 16/50 (Amnham Phrnnacin Biotech). '~echmmatographyon sirecxclusion mlumn of fraction FlVa and FIVb, obtained from SEC, on Superdcx 75-HR 16/50 (Amerrham Phannacia Biotech). f~pccificactivity is expresscd as one pmol of fmfatty acid produccd pcr one min per one mg pmtein x 10.'. g~otalactivity is expressed as one pmol of frce fatty acid proâucod pcr one min. the purification proces could be attributed to the removd of inhïbitory nuteriais such as proteases and 0thdebris that mask the lipase activity (Pabai, 1997).

Many extracellular Lipases from Pseudomolyls species have ban purifieci ushg difEerent chromatographie techniques including EC and SEC. Bard and Fox (1997) reported the purification of a lipase enzyme fiom Pseudomoms tofizsii on IEC and SEC using DEAE-52Cellulose and Sephadex G-150, respectively, with a final purification-fold of 1,000 and an increase of 8.7 times in specific activity. Gilbert et al. (199 1) showed that the purincation of a lipase fiom Pseudomonas aerugimsu by IEC foliowed by SEC resulted in an increase of 5.4 times in speciac aaivity. A lipase from Pseudomonasputido

3SK was purified by LEC and SEC on DEAE Sephadex A40 and Sephadex G-100, respectively, with a purication-fold of 3.3 and 21, respectively, and an increase of 6.4 tirnes in purity (Lee and Rhee, 1993). Two other lipases fiom Pseudornonusj?uorescens were purified with purification-fold and recovery of 10-folds and 5% yield (Lee et al.,

1993) and 12-folds and 235% yield (Pabai, 1997). In addition, lipases fiom PseuCt0mom.s aeruginoa were also p~rifiedwith an overall purification factor of 1265 (Stuer et ai.,

1986) and 60 (Shabtai and Daya-Mishne, 1992).

4.2. Chanctcrization of the Purifid Lipase Fractions

4.2.1. Molec~lar Wdght Estimation

4.2.1.1. Gel Electrophoresis

Native polyacrylamide gel electrophoresis (PAGE) was carrieci out on the lipase

Eractions, FII, Fmd, ma'and FfVb' as well as the protein standards in order to estimate the molecuiar weights (MW) and to usesthe degne of purity of the fractions. Figure 8 shows the elestropherograrn of the native-PAGE. A standard me@ig. 9) was constructeci by plotting the relative mobility &) against the log of MW of the protein standards. A linear cuve was obtained and the straight-line equation of the curve was used to estirnate the MW of the lipase hctions.

The partidly purified lipase fiaction FI1 showed two major bands of 8.3 and 16.2 kDa and two rninor bands of 27.6 and 75.2 kDa. Fraction FïIId fiom IEC showed two major bands and three minor bands with estimated molecular weights of 7.8, 16.2, 27.6,

3 8.5 and 75.2 kDa. Schuepp et al. (1997) reported the preance of three protein bands for the partiaily purified extracellular lipase extract with MW of 14.1, 25.5 and 67.0 kDa.

These results were in close agreement with the ones obtained in this study. The obtained bands of MW of 75.2 and 16.2 kDa are probably corresponding to the bovine senun albumin and a-lactalbumin, respectively, since they are found abundantly in whey medium and have MW of 69.0 and 14.4 kDa, respectively. Moreover, it is noted that the partially purified lipase (FD) showed a lower number of bands than the fiaction hmIEC (Fmd) which may be due to the fact that fiaction FI1 was more diluted than the other one.

The results also show the presence of three protein bands for the purified Mon

FWa' with estimated MW of 16.2, 25.8 and 38.5 kDa, and one major and one minor protein bands with estimated MW of 1 5.2 and 25.8 kDa for the purified fiaction FIVb'. It is assumed that the bands of MW of 25.8 and 27.6 kDa that were visualized in al1 the purified fractions are most probably corresponding to the lipase enzyme since most lipases nom Pseudomoms species range in MW betwecn 25 and 65 kDa (Nishio et 01.. 1987; STD FIVb' FNa' FFmd FI1 STD

Figure 8. Electrophoretic profile of the lipase Fractions fiom P. fragi CRDA 037on native polyacryiamide gel (native-PAGE): Standard (STD) protein markers (STD), partially purified lipase fraction 0,purified lipase Eraction obtained by ion-exchange chromatography (FIIId), purified lipase fiactions obtained by re-size exclusion chromatography (FTVa' and FIVb'). -

carbonic anhydrase

soybean trypsin inhibitor

0-2 0-4 0.6 O .8 Relative Mobility (Rf)

Figure 9. Standard curve for moldarweight estimation by native polyacrylamide gel electrophoresis Yamamoto and Fujiwara, 1988; Iiauni et al,,1990; Kordel et al., 1991; Lin et ai., 19%).

One exception is a lipase isolated fiom Pseudo~tlo~wlsjiagiAFO 3458 with a MW of 14.6

kDa (Kujimiya et of., 1986). The electropherograms show the presence of dark smears of

high MW proteins in al1 the lanes containing the lipase fiactions; it is believed that these

dark protein smears correspond to some kind of lipase aggregation. It was reported by

Stocklein et ai. (1993) that dunng native PAGE, the purified lipase migrated as a di- and

tetramer with estimated MW of 52 and 100 kDa, respectively, as was demonstrated by

activity staining .

The localization of the lipase bands wss attempted by overlaying the gel on a

Rhodamine B plate. However, it was not possible to detect any fluorescence in any of the

bands. This can be attributed to the low arnount of protein used in gel electrophoresis (1

to 2 mg/mL) which was insufficient for detection by the Rhodarnine B test.

4.22.2. Si*-ErcIusion Chromafography

The MW of the purified lipase fiactions, FIVa' and FIVb', were also estirnated by

SEC on Superdex 75, HR 16/50 column using a mixture of protein standards. A standard curve (Fig. 10) was prepared by plotting the K, value against the log of MW of the protein standards. The MW of fractions FIVa' and FIVb' were estirnateci to be 88.9 and

56.1 ma, respectively. The estimated MW obtained by this method were higha than those obtained by gel electrophoresis. One possibity is that the lipase hctions could be fonning higher MW aggregates. In decd, many lipases fiom Pseudomoms spccics have been found to form aggregates of various si= (Fox et al., 1989; Stcpaniak and Serhaug, ribonuclease A

chemotrypsinogen A

0.2 -

0.1 - bovine senun altumin

I I I 4.2 4.4 4.6 4.8 5.0 5.2 5.4

Log Molecular Weight

Figure 10. Standard curve for molecular weight estimation by size-exclusion chromatography 1989;Lin et aL, 19%). Mormver, a dernumkr of protein paLs could k detected by this method which may be due to the fact that the dadon technique of ske-excIusion is less sensitive than thaî of gel ~1ectrophoresis.

The prexnce of multiple forms of lipases was rcported by several investigators.

Two forms of lipases fiom Rhizomucor mieki (Boel et al., 1988) and Geotnëhum candzdum (Sugihara et al., 1996) wcrt found to be due to the dierence in the degree of glycosylation or to the synthesis of tmiy âiiermt lipases, respectively. Another lipase f?om Penicillium canrembertii was purifid in four active fiactions (Isobe et al., 1992).

These fiactions possessed the same polypeptide backbone but were dflerent in their carbohydrate composition.

4.2.2. Eflect ofpH on Lipase Adviîy

The eEect of pH on the activity of the purified extracellular lipase fiactions wu investigated using a number of buffer solutions ranging fiom pH 3.0 to pH 12.0. The results (Fig. 1 1) show that the paiially purified MonFIi, the IEC-fiaction Fmd and the SEC-fiactions FIVa, FIVb, RVa' and Fm'have the highest activity at pH value of

9.5 to 10.0.However, low entymatic activity was determined at pH values less than 7.0; this low activity could be due to the incornpiete ionization of the fret fatty acids (Macrae,

1983) rather than to the inactivity of the lipase -ions.

Most Psetldomorms species have maximum lipase activity in the pH range of 7.0 to

9.0, including enzymes fiom P. pti& (Pabai, 1997). P. j71101escens BW%CC 1 (Pabai,

1997), P. aervgîmsa EF2 (Giibe~et al.. 1991b) and P. toksii (Bad and Fox. 1997). Figure 11. Effect of pH on the Mivity of the partiaily purified lipase fiaction FlI(f ), fraction FïIïd ( +) obtained hmion-exchange chnnnuognphy, MonsFIV. (-+) and FIVb ( - ) obtained fiom sizeexclusion chrornatography and MonsFWa '(l-) and FIVb' ( +) obtained fbm re-size exclusion chromatography The lipase extract fiom P. fiagi 22.398 demonstrate an optimum pH of 9.0 to 9.5 (Nishio et al., 1987). Whereas that from Pseudomoms psetlhlmligenes F-111 showai its highest activity in the pH range of 6.0 to 10.0 (Li et al., 19%). Schuepp et ai. (1 997) showed that the partiaily purificd extracellular lipase from P. fiagi CRDA 037 has a pH optimum of 9.0 which is relatively close to the present results (Fig. 11). The slight difference in optimum pH could be due to experimental manipulation or to dinercnces in culture preparation.

4.2.3. Eflect qf Te-ure or, Lipase Arncnvi!y

The optimum temperature for the purified extracellular Lipase hctïons, FII, FIIId,

FNa' and FIVb', was determined by inmbating the reaction mixtures at difFerent temperatures fiom 25 to 90°C. The results (Fig. 12) indicate that the Lipase activity is increasing with elevated temperatures, reaching its maximum at 70 to 80°C for the partially purified fiaction FII and IEC-fiaaion FKIId, and 80°C for the purified fiactions

FIVa' and FTVb' obtained fiom rc-SEC. Moreover, a minor temperature optimum was also observed for dl the lipase frictions at 40°C.

Schuepp et al. (1997) reporteci an optimum temperature of 35°C for the partially purified extracellular lipase fiom P. fiagi CRDA 037 which is in agreement with the resutts obtained in this study; however, these authors did not report a major temperature optimum of 80°C, since their studies on the optimum temperature of lipase activity was limited to a range of temperature between 10 and 60°C. Other lipases from Pseudononm species, including P. frrgi 22.398 (Nishio et ai., 1987). Psedmonas sp. (Yamamoto and Temperature ('C)

Figure 12. Effkct of temperature on the activity of the partidly purified lipase monFII Ce ), fiactîon FIIId (+ ) obtained hmion-exchange chromatography and MonsFIVa' (+) and FIVb' (u)oôtained hmre-size+xclusion chromatography Fujiwara, 1988). Psetlh~ilt~~sp. KWI-56 (Iizumi et ai., 1990) and P. mephitic4 var. iipoI'ic(~ (Kosugi and Kamibayashi, 1971) exhibitcd high temperature optima rangiag fiom 60 to 80°C. In addition. lipases fiom P. aen@nosu EF2 (Gilbert et al., 1991b) and

P. fluorescens a102(Kogirna et al.,1994) have the highest lipase advity at SS6C.

Lipases are considerd quite thermostable enzymes, which nuke them very attractive toois for indumial applications. Moreover, psychroaopbic bactena Ne P. fiagi, which were origidy isolateci fiom miik, can produce heat stable Lipases that can withstand the high pasteurization ternperatures; this phenornenon may explain the fact that pasteurized rnilk can still dcvelop off-fiavors due to the action of these thermostable ii pases (Stead, 1986).

4.2.4. Kinetic Pwwneîtrs

Kinetic studies were conducteci with the purifid Lipase fiactions using triacetin

(C2:O) as a substrate. The results were plotted in Lmeweaver-Burk double reciprocal plots

(Fig. 13) and the Km and V,, values for each fiaction were calculateci fiom the linear regressions of the plots. Table 2 shows a su- of the dinerent kinetic parameters of the purified lipase fkactions. The experimentd findings indicate that the purification of the extracellular lipase from P. fiagi CRDA 037 by EC and SEC resulted in an ovedl decrease in the Km values for the purified lipase fiactions Fractions FIVa and FIVb obtained by SEC have Km values of 5.%2 and 3.744 rnM, respectivveiy, which were lower than that of fiaction Fmd obtained fiom IEC. These results suggest that the enzyme atnnity towards its substrate was enhancd with the increase in the degnc of azyme Figure 13. Lineweaver-Burk double reciprocal plots of the parîially purifid lipase Mon FII ( +), fiaction FIIId ( ++ ) obtained fiom ion-exchange chromatography, fiactions RVa ( +) and FIVb ( ) obtained fkom size-exclusion chromatography and fiactions FiVal ( * ) and FIVb' (Q ) obtained fkom te-size-exclusion chromatography using triacetin as a substrate Table 2. Kinetic parameters of the purified extracellular lipase fractions from PseudontorasJragi CRDA 037.

PH Specific Km vm~ Enzyme Fraction optimum act ivitya (mM) (UImg) efficiency (1 O-'lb

FII" 10.0 2.60 8.06 0.087 I .O8

FIII~~ 10.0 7.12 8.46 O. 167 1.97

FIVa* 10.0 49.63 5.90 1.053 17.84

FIVb" 10.0 73.14 3.74 1.339 35.76

~l~a~9.5 105.50 3.85 2.781 72.16

FIVb" 10.0 121.60 5.49 2.093 38.15

-p "Specific aaivity is dcfined as one volof frez fa@ acid produad pr one min pr one mg protein x 10.'. b~nzymcclfieiency is calculaied as V,IK,. C Pariially purificd exltaccllular lipax fraction produccd by ammonium sulfatc prccipiîation at 2040%saturation. dhrified lipase fraction obtained by prcpamtive ionexchange chmmatography of the parîially purifiai lipase. ePurified lipase fradion obîained by prcparative sizcexclusion chromalography of fraction FIlld. fh~allipase fiadion obiainui by n~hmmatographyon sizecxclusion colwnn of fwion FIVa. 'Purifial lipase fraction obisincd by rebmmatography on size+xclusion cdumn of filion FIVb. punty. However, MonFIVb' obtiind by RSEC has a K, duc of 5.487 mM adis

higher than that of fiaction RM; these rcsults mry indiutte tbat FIVb' could have more

aflinity towards a substrate othcr than triacetin It was reportcd that the partiaiiy purificd

Lipase from P. ficgi CRDA 037 (Schuepp et d., 1997) bas a Kmvalue of 7.1 rnM which is

close to that (8.057 mM) obtained in this study. LQctobocillus plantamm purihed lipase

has a Km value of 2.3 1 mM using tnbutyrin (C4:O) as a substrate (Gobbctti et aL, 19%).

Two other purified Lipases fkom P. fiagi(Mencher and Mord, 1967) and P. jluorescem

(Fox and Stepanie 1983) bave Km values of 0.9 and 3.65 mM, respoctively, using

tributyin as a sub~te.

The maximum velocity rates, V-, wae dso calculateci for each hction and wae

expressed in units of specific activity (mM/mg proteidmin or U/mg). The purification procedure resulted in an increase in the Y, vlaues which indicate a concomitant incruse

in the degree of purity of the enzymatic fractions. Hence, the V- value for fiaction FIIId obtained fiom IEC (0.167 U/mg) is twia as that (0.087 U/mg) for the partially purined fraction FKi (Table 2). In addition, the enzyme efnciency (Y-/Km) was increased as the purification proceeded fiom 1.1 for haion FII to 72.4 and 38.2 for fiactions FIVa' and

FlVb', respectively .

4.2.5. Lipase Slobili4

The effect of storage time on the stabiility of the purifid lipase fiadons was investigated. Lipase fiactions, FIVa' and FIVb', wae incubatecl at rmom tempenhire

(25OC) for 72 h. The nsults (mt shown) indiute tht the lipue fhctions were @&y losing their activity; however, more than W/o of the activity was retained ova a paiod of

36 h. The loss of lipase activity may be due to the donof prottiws, prcsat in the

enzymatic extract, that are most active at room temperature (Sapes, 1987). A purifid

lipase fiom Bohytis cinereu (Comrnénil et al., 1995) demonstrateci considerable stabiiity

at room temperature &er incubation for 48 h. Bard and Fox (1997) reported that the

lipase extract Born P. ~ol~iwas abk to retain most of its activity for a period of 48 h at

2 1OC. A high stability under 40°C was also reported for the Lipase fiom Chromobucterizun viscosum (Castellar et a!., 1996). For othcr Lipases fiom Pseuabmoms species, the stability at high temperatures (40 to 70°C) was found to last for ova few hours (Gilbert,

1993).

4.2 6. Tke Effect of Inkibiîom und Acîivaîors on the Lipase A&@

The effects of a nurnber of inhibitors and activators, including FeClt, FeC13, sodium deoxycholate, diisopropyl fluorophosphate, Eiiman's ragent, N-bromosuccùiimide, CaClz,

MgC12 and EDTA, on the activity of the purifie- lipase fiactions FWa' and FIVb' were investigated and the results are shown in table 3.

4.2.6.1. Reversible No,j-Spec~jicinhibitors

Compounds that do not aa directly at the active site, but inhibit the lipase actiuity by changing the conformation of the lipase or the interfaciil surface prophes, are defimd as non-specific inhibitors (Patkar and Bjorkling, 1994). 42.6.1.1. Fe- and Femè Chlorides

Ferrous chloride (FeC12) was found to suongly inhibit the purifid lipase fiactions at 10 mM of concentration, but showed sorne activation effect at the lower concentration of 1 mM. As for femc chlonde (FeCl3), similar inhibitory &kt was found, with wmplete inhibition at 10 mM concentration. A large nwnber of purined microbiai lipases were inhibitcd by metal ions includig iron. The partidy purifid Lipase fiom P. fiagi CRDA

037 was found to be strongly inhibiteci by both iron ions (Schuepp et d.,1997). Simiiar effects were documentecl in other studies done on lipases fiom Pseudomo~species

(Nïshio et a/., 1987; Lin et al., 19%; IWmi et al., 1990; Yamamoto and Fujiwara, 1988).

Salts of iron (Fe II and Fe m) were shown to inhiiit rnany lipases but the mechanism of inhibition is still not clear. However, it is believed that the effect of metal ions on the erqme and the reaction systern is manifold. Some metal ions can bind to the enzyme changing its activity by stabiiization or destabiition of the protein conformation.

They may also act as scavengers of free fatty acids (Patkar and Bjorkling, 1994).

4.2.6.1-2. Mium Deoxychola te

Sodium deoxycholate of both concentrations, 1 and 10 mh4, caused partiai inhibition of the lipase fkactions. FIVa' and FIVb', with residual activity rangin8 fiom 54 to 65%. On the other hand, the activation effect of deoxycholate salt could also be observed if' a lower concentration of that sait was used. The partially purifieci lipase fiom

P. fia* CRDA 037 was found to bc inhibited by sodium dwxycholate (Schuepp et al.,

1997). The purifieci lipase from Psadosnon~ssp. KWI-56 was aloo inhibited by sodium Table 3. Effect of metai salts and chernid reagmts on the dvity of the purificd lipase hctions, ma'and FIVb', fkom PSetcctDm~l~l~fiagzCRDA 037.

Relative activity (%)O

Concentration Substance (mM) FIV~'~ FIVb"

Sodium deoxycholate 1 .O 10.0

Diisopropyl fluorophosphate 1 .O 10.0

Eiiman' s ragent 1.O 10.0

EDTA 53.2 (0.239)~ 1 529 (o.10s)~

"~herelative activity is exprtssed as a perccntage of the activity obtaiaed with a metal sa11 or a chemical reagent to rhat obtailiad without any metal salt or a chemical reagtnt bEnzyme fiaction obtaincd by rc-sizecxclusioa chramatography of fraction FIVa on Superdcx 75-HR 16/50. '~nzymefraction obtained by resizecxclusion chromatography of fraction FiVù on Superdex 75-HR 16/50. d Relative standard deviation caiculated as (standad dcviation/mean) x 100. deoxycholate with a rtsidud activity of 37% (IWmi et al., 1990). Another lipase fiom P. fluorescens BW96CC1 was inhibitcd by this sait at concentrations of 10 and 20 rnM

(Pabai, 1997).

Sodium dtoxycholate belongs to a group of nonspecific Lipase inhibitors known as the bile salts which are choleic acid derivatives of cholestrol. Bile salts are hown to either activate lipases at low concentrations or inhibit thmi at higher concentrations. The inhibitory effect of bile salts was explaineci by two hypotthtsts- One explanation stated that bile salts accumulate at the wbstrate dace and inhibit the lipase adsorption to the intefiace (Borgstrom and Erlansen, 1973). The other interpretation was that bile salts fom a complex with the lipaw, which has low affinity towards the büe-salt-covered airfice

(Momsen and Brockman, 1976).

4.2.6.2. Irresible Spec~ficInhibitors

4.2.6.2.1. Diisopropyl Fiuorophosphate

The addition of 1 mM of diisopropyl fluorophosphate (DIFP) to the reaction medium caused partial inhibition of the purified lipase fiactions FlVa' and FNb'. While 10 mM of DIFP was sufficient to wmpletely inhibit MonFIVb' but that concentration was not enough to inhibit Mion FNa' which retained around 16% of its activity. The partially punfied extracellular lipase fiom P. fiagîCRDA 037 was completely inhibited by

DIFP at a concentration of 0.2 pM (Schuepp et al., 1997). While the purineci lipase fiom

P. fluorescens BW96CC1 was inhibited by Wh udg 20 rnM of DIFP (Pabai, 1997). Lu and Liska (1969) found that the exvacellular lipase nom P. fiagi no. 3 wis inhibited by 37.5% with DIFP at a ooncmvation of 5 mM. Another lipase from P. pSetcLjOaic~Iigenes

F-1 1 1 was greatly inhibited by 1 mM of DIFP (Lin et ai., 1996).

It is known that the catalytic triad of lipases consists of glycine, histidine, serine or aspartic acid. And it is also known that DIFP is used to inhibit serine esterases by reacting with the serine residue at the active site (Patkar and Bjorkling, 1994). The extracellular iipase fiom P. fiagi IF0 3458 was found to possess a Gly-His-Ser amho acid sequence at the catalytic site and was inhibited by DIFP (Kugirniya et al., 1986).

Chernical modifiers are used to modq or ruct with certain amino acid residues that are believed to be involved in enzymatic catalysis (Patkar and Bjorkling, 1994).

4.2.6.3.1.EIIman 's Reagent

A total inhibition of the hydrolytic activity of the puritied lipase fiactions FIVa' and FNb' was obtained by 10 rnM concemtration of EUman's reagent. The partidly purified lipase fiom P. @agi CRDA 037 was also strongly inhibited by EUman's reagent

(Schuepp et al., 1997). Pabai (1997) also found that the same reagent inhibited the puritied lipase f'rom P. fluorescens by 20% at a concentration of 20 mM. It is believed that the inhibition of the lipase enqme may be due to structural changes in the conformation of the enzyme. Ellman's reagent (5,5-dithiobiI-2-nitrobell~oicacid)) is used to modifL the cysteine groups in order to daennine whether the lipase fiactions are sulfhydryi enzymes.

A direct relation was found betwecn the modification of one sulfliydryl group and the 10s of activity of a rabbit gastric lipase (Moreau et ai., 1988). The lipase fiom P. fiagiIF0

3458 was found to possess two cysteine residues (Kugimiya et ai., 1986). These findings

suggest that the sulfhydryl group is essential for iipast cataiysis.

4.2.6.3.2.N-Bromostrccinimide

At 10 mM concentration, N-bromoniccinimide (NBS) was a very potent inhibitor

of the purified lipase fiaction FIVb'. Fraction FIVa' was also inhibited but ta a lesser

degree and had a residuai activity of 2W. The partially purifid Iipase fiom P. fiagi

CRDA 037 was also strongly inhibited by NBS (Schuepp et al., 1997). Pabai (1997)

found that the purified lipase fiom P. fluorescens was inhibited by 20% using NBS at

concentration of 20 mM. A study by Sugiura et ai. (1977) showed that the lipase corn P. fluorescens was panially inactivated by NBS. NBS has been used for the oxidation of

tryptophan and tyrosine residues in proteins (Liu et al., 1977). Tryptophan is the most

hydrophobic amino acid and may be located in the hydrophobic region of the lipase where

the formation of the enzyme-substrate complex is believed to take place (Patkar and

Bjorkhg, 1994). Hence, the inhibition of the puritied lipase fiactions by NBS may

indicate that tryptophan residues are essentiid in maintainhg the lipase activity.

4.2.6.4. Lipase Activators

4.2.6.4.1. Calcium and Magnesium Chlorides

The hydrolytic activity of the purified Lipase fiaction ma' was increased by 132

and 149% with the addition of 1 and IO rnM of CaC12, respectively, whereas that of

fiaction FWb' was only activated by the use of 10 rnM of CaCl2. The addition of 1 and 10 m.of MgClz, increaseû the enzyme activity for Monma' by 20 and 53%, respectively. However, the Mivity of fiaction FIVb' was not affected by the addition of

10 mM of MgClz. These results are in agreement with those reported in the Litcrame. The

Lipase 6rom P. aemgimsu EF2 (Gilbert et aL, 1991 b) was activated by ca2', whereas that from Luc!o&ciiius pimtamm was modcrately stimulated by 10 mM ca2+ and hfg2+

(Gobbetti et ai-, 1996). The activity of the extracellular lipase fiom Pylhium uirimum was stimulated in the presence of ca2' and ~g*'(Modar and Weete, 1993). Ions of caicium and magnesium have an activating effect on Lipases; they act in removing the reaction products (fiee fatty acids) fiom the interface as insoluble salts, muiimitùig the product inhibition effect and providing an activating action (Macrae, 1983). It was reported that the addition of ~a"to the reaction medium does not affect the hydrolysis rate for tnacylgiycerols of short- and medium-chah fatty acids, but can promote the hydrolysis of the longer-chah ones (Nishio et al.,1987).

4.2.6.4.2. Ethylene Diamine Tetraacetic Acid

The results show that the addition of 10 mM of EDTA (ethylene diamine tetraacetic acid) to the reaction mixture resulted in the activation of the lipase hctions

FIVa' and FIW' by 37 and 53%. respectively. The literature reported that the extracellular lipase enzymes produced hmP. toImi (Baral and Fox, 1997) and those tiom Pseudomonus sp. (Yamamoto and Fujiwara, 1988) were strongly inhibited by

EDTA. The sarne inhibitory effect of EDTA was dso doairnented by Dring and Fox

(1 983) and Botoglu et ~~(1984).However, lipases fiom P. jlvarescens BW96CC 1

(Pabai, 1997) and P. uemginosa EF2 (Gilbert et ai', 199 1b) were activated by EDTA. It is weil hown that EDTA acts as a metal chelator that attracts the divalcnt donsor mctal ions away tiom the enzyme. Hence, its activahg cffkct on lipase fiactions could be due to its ability to chelate metal ions that inhibit these enzymes. These Wigsrnay imply that lipases are not metalloproteins since they are activated by the metal chelator EDTA.

The spdcity of the lipase fiactions towards a number of triacylglycerols with a wide range of fatty acid chah lengths (C2 to C 18: 1) was investigated. The results indicate that the partially purified lipase -ion FII and -ion FXiId obtained by IEC have low aaivity towards triacylglycerols with fatty acids of chah lengths longer than C4 (results not shown).

The purified lipase fiactions, FIVa' and FIVb', obtained by re-SEC have the highest specificity towards tnacetin (C2:O); howevcr, longer-chah triacylglycerols, including trimyristin (C 14:0), tripaimitin (C 16:O). tristearin (Cl 8:O) and triolein (C 18: l), were also hydroiyzed by these lipase fiactions. The results (Fig. 14) indicate that fiaction

FIVa' showed the highest activity towards triacetin followed by tristearin (C18:O) and then by tripalmitin (C16:O). However, fiaction FIVb' exhibited higher specificity towards tnacetin followed by trimyristin (C14:O) and then by tnolein (Cl 8: 1). These results suggea that the punfieû lipase fractions, FIVa' and FIVb', fiom P. fia@ CRDA 037 have a wide range of specificity towards triacylglycerols of dinerent faîty acid chab Iengths.

Schuepp et al. (1997) studied the substratt specificity of the partially purifid Carbon Number

Figure 14. Substrate specificity of the purified lipase fractions FNa' a ) and FIVb' a ) obtained tiom re-size-exclusion chromatography towards trïacylglycerols extracellular Lipase fkom P. fia@ CRDA 037 using four triacylglycerols (triacetin, tributyrin, trimrystin and triolein). T)ic highest iipase activity was observai wiîh trirnyrstin followed by triacetin then tributyxin. Nishio et al. (1987) found that the purified extracellular lipase hction from P. fiagi 22.398 showed the highest activity with tributyrin foliowed by tricaprylin (C8:O). Whcreas the purified lipase enzyme fiom P. fluorescens BW96CC1 exhibited preferentiai specificity for triacylglycerols of long-chain fatty acids fkom C 12:O to C 18:1 (Pabai, 1997). Furthemore, lipases fiom P. aemginosa

(Jaeger et al., 1993) and Baciffus subtiifs 168 (Lesuise et al.,1 993) were able to degrade both emulsified long-chain fatty acid esters as well as the soluble monomeric esters.

4.2.7.2. Edible Oils

The specificity of the puntied lipase frpaons, FWa' and FIVb', towards a number of edible oils, including butter and olive, canola and fish oils, was also examined. The results (Fig. 15) demonstrate that the lipa~ehctions FIVa' and FIVb', obtained by re-

SEC, were capable to catalyze the hydrolysis of these oils to almost the rame extent.

Butter contains 13% short-chain fatty acids (C4:O to C12:0), 26V0 of palmitic acid

(C16:O) and 28% of oleic acid (C18:l) (Gurr, 1992); it was hydrolyzed by both lipase fiactions since they show some degree of specificity toward tnacyiglycerols with short- chain fatty acids, such as triacetin, tributyrin and tricaproin, as well as triacylglycerols with long-chain fatty acids, such as tnmyrstin, tripalmitin and tnstearin.

The hydrolysis of canola and olive oils that contain 60 to 70'36 oleic acid (C18:1)

(Eskin et of., 1991; Gurr, 1992) by the lipase fiactions was possible since both fhctiom, Butter Olive oil Canola oil Fish oil

Substrate

Figure 15. Specificity of the purified lipase MonsFIVd (0) and FIVb' (.) obtained from re-sizeaclusion chromatography towards fair edible oils ma' and FIVb'. exhibitcd hi$ spedicity towards triacyIglycefols of long-chain f%ty acids such as tripalmitin, triste- and triolein. Most Mcrobid lipases. such as P. pseudmiwIigenes F-1 1 1 (Lin et ai-,19%). Ps~&monas sp. KWI-56 (Iizumi et al.,

1990), Pseudomoms sp. f-B-24 (Yamaxnoto and Fujiwara, 1988) and Peneciilîum erpansum (Stocklein et al., 1993) are capable to hydrolyze olive oil.

Fractions FIVa7 and FIVb7 were dm capable to hydrolyze fish oil, which consirs of more than 55% of polyunsaturated long-chah fany acids (Gurr, 1992). These mults indicate that the purified lipases are able to catalyze the hydrolysis of esten of polyunsaturated long-chah fatty acids. Lipases fkom P. pseudmiculigenes F-1 1 1 (Li et al., 1996), P~~O~OIICIFsp. KWI-56 (Iiauni et al-, 1990), Peniciliium eqxmsurn

(S tockiein et al.,1993) and Penicilllium abeunum (Sugihara et al., 1996) were also able to hydrolyze fish oils. including whale and sardine oils. Tuna oil was also hydrolyzed by the lipase fiom Penicîllium abeunum (Sugihara et al., 19%).

The GLC analysis (ïable 4) demonstrate that both fiactions FIVa' and RM', were capable to hydrolyze the four edible oils, butter and olive, canola and fish oils.

Fraction ma' showed specificity towards saturated fatty acids of chah lengths from

C12:O to C18:O. in addition, the fatty acid C20:4 was also releosed by fiaction FIVa'.

However, fiaaion FIVb7 exhibited no fatty acid specificity and was able to catalyze the release of short-chah fatty acids nich as C8:0, and C10:O as well as long-chah saturated and unsaturated fatty acids includùig C 1&O, CM:1, C 16:07 C 16:1, C 18:07 Cl 8: 1, Cl 8:3, CC, *. l m C20:O and C20:4. Tbese results suggest haî the two purifieci lipase fractons Ma' and

FIVb' demonstrate differcnt fatty acid specificities; fiaction FiVa' is mon spedc towards the long chain saturatcd fatty acid esters, while fiaction FIVb' is a non-specific lipase. S. CONCLUSION

The purification of the partidy purifieci extraceliular lipase fiom P. fiagi CRDA

037 by IEC and SEC resulted into two purified Lipase Wons with up to 200-fold increase in specific activity. The rdtsshowed that the purifieci lipase &actions exhibited ceriain sirnilarïties in their properties. Both purifieci fractions are alkaline lipases and very thermostable enzymes. Inhibition studies on the purified lipase fiactions suggen that they are both sehe esterases and sulfhydryl-containing enzymes. Furthemore, the purified fiactions are able to hydrolyze short- and long-chah fatty acid esten of triacylglycerols as well as a selected number of edible oiis, including butter and olive, canola and fish oh.

GLC analysis also revealed that the two purifiecl Lipase fractions exhibited different specîficity towards fatty acid esters.

Analysis by native polyacrylamide gel electrophoresis demonstrated slight difference in the estimateci molecular sizes of the purifieci lipase fractions. The results suggested that the two fiactions could be lipase isoentymes.

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