Chem. Eng. Comm., 1996, Vols. 152-153, pp.365-404 © 1996 OPA (Overseas Publishers Association)Reprints available directly from the publisher Amsterdam B.V. Published in The Netherlands underPhotocopying permitted by license only license by Gordon and Breach Science Publishers SA

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LIPASE-CATALYZED BIOCHEMICALREACTIONS IN NOVEL MEDIA: A REVIEW

MAYANK T. PATEL*, R. NAGARAJAN** and ARUN KILARA***

The Pennsylvania State University, University Park. PA 16802

(Received September 15, 1995; in final form January 11.1996)

Lipids in biological matter are mostly triacylglycerols (TAG). Lipolytic enzymes, primarily lipases, areindispensable for bioconversion of such lipids from one organism to another and within the organisms. Inaddition to their biological significance, lipases are very important in the field of food technology, nutri­tionaland pharmaceutical sciences, chemical and detergent industries, and clinical medicine because of theirability to catalyze various reactions involving a wide range of substrates. Conventionally, lipases have beenviewed as the biocatalysts for the hydrolysis of TAG (fats and oils) to free fatty acids, monoacylglycerols(MAG), diacylglyoerols (DAG), and glycerol. The main advantages of lipase catalysis are selectivity, stereo­specificity, and mild reaction conditions. Despite these advantages and the fact that enzymatic splitting offats for fatty acid production was described as early as in 1902,the lipase-catalyzed process has not replaoedthe commercial physicochemical process for the continuous splitting of TAG utilizing super-heated steam.The limited exploitation of lipase technology may be attributed to high enzyme cost, large reaction volume,requirement for emulsification of substrate, and risk of microbial contamination. Many of these limitationsoriginate from the fact that lipases are employed mainly in water-rich reaction media where the solubility ofthe substrate TAG is very small. To circumvent this problem and to realize the full potential of lipase.researchers have explored newer approaches by manipulating the conditions under which the lipases act.Many of these novel approaches for lipase catalysis have been the outcome of the discovery that enzymescan be active in water-poor, non-polar media (Hanhan, 1952; Misiorowski and Wells. 1974; Zaks andKlibanov, 1984). Also, the finding that lipases can act in organic solvents has led to an expansion of theirapplicability in a wide variety of chemical reactions. Lipase catalysis in some of the well established reactionmedia has previously been reviewed (Brockerhoff and Jensen, 1974; Brockman. 1984; Lilly el 01.. 1987;Halling. 1990; Inada et 01., 1990; Maleata er01., 1990). The present review is intended to present a compila­tion and comparison of novel reaction systems used for lipase catalysis. This review describes briefly thegeneral characteristics of lipase reactions, applications of lipase in various fields, and conventional lipasetechnology. The lipase-mediated biochemical reactions, particularly the hydrolysis of TAG in novel reactionmedia is discussed in greater detail.

K EYWOR OS Lipase catalysis Enzymes in organic media Hydrolysis of triacylglycerols Lipaseapplications Lipase in novel media Lipase-catalyzed reactions

I. GENERAL CHARACTERISTICS OF LIPASE CATALYSIS

Lipases (triacylglycerol acylhydrolase, E.c. 3.1.1.3) can be viewed as a special classof esterases. Esterases preferentially catalyze hydrolysis reactions of soluble esterswhereas lipases are distinguished by their ability to catalyze the hydrolysis of insol­uble long-chain fatty acid esters. Although lipases do exhibit some activity toward

• Present Address:James Ford Bell Technical Center, General Mills Inc., Minneapolis, MN .

•• Department of Chemical Engineering.Author to whom correspondence should be addressed.

*.* Department of Food Science.

365

366 M. T. PATEL etal.

soluble substrates, the rate of hydrolysis is generally slow compared to the rate ofhydrolysis of triacylglycerol emulsions. The natural substrates of lipase are triacyl­glycerols of long-chain fatty acids (fats and oils) which on hydrolysis yield diacyl­glycerols, monoacylglycerols and, ultimately, glycerol, with fatty acids beingreleased at each step (Fig. I). Depending upon the origin of lipases, the fatty acidsare released either randomly from any position or preferentially from a specificposition.

Interfacial Catalysis

Interfaces provided by either aggregated or dispersed substrate in the aqueous me­dium constitute the primary site for lipase catalysis. Lipase hydrolyzes triacyl­glycerols at the interface between the insoluble substrate phase and the aqueousphase, in which the enzyme is solubilized (Entressangles and Desnuelle, 1968; Brock­man et al., 1988). The presence of aggregated substrates providing an interface forthe reaction has been demonstrated in experiments involving water-soluble substra­tes such as triacetin and tripropionin (Entressangles and Desnuelle, 1968). It wasshown that, for a lipase but not for an esterase, a stepwise increase in activityoccurred only when the solubility limit of the monomeric substrates was surpassed,i.e., above the critical micellar concentration of the substrate.

The hydrolysis of triacylglycerols by lipase occurs in a heterogeneous systemcomprised of multiple phases. In such systems, the substrate, products, and enzymepartition themselves among the bulk and surface phases. This distribution is notfixed but changes as lipolysis proceeds (Patton and Carey, 1979; Brockman, 1984).These features lead to difficulties in the kinetic analysis of the reaction using enzymekinetic models developed for reactions in homogeneous aqueous systems (in whichboth the enzyme and substrate are soluble). Benzonana and Desnuelle (1965) havepointed out the importance of interfacial area rather than the bulk concentration ofsubstrate and have shown that lipolysis will apparently conform to the Michaelis­Menten equation when the substrate concentration is expressed on the basis ofinterfacial area rather than the bulk system volume.

H20 R1 H20 R2 H20 R3

{R1l t {OH l t {OH l t {OHR2 ~R2 ~HO ~HO

Lipase Lipase Lipase

R3 ~ ~ 00

Trlacylglycerol Diacylglycerol Monoacylglycerol Glycerol

FIGURE I Lipase catalyzed hydrolysis of triacylglycerol 10 glycerol and fatty acids. RI, R2 and R3representIatty acids at the sn-I, 5n-2and 5n-3 position, respectively, on the glycerol moiety.

LIPASE CATALYZED REACTIONS 367

The lipase reaction can be envisaged as a two-step process: adsorption of theenzyme at interfaces and catalysis by the adsorbed enzyme. The first step whichprecedes the formation of enzyme-substrate complex is the adsorption of lipase ontothe surface of substrate phase in a non-specific manner. The second step of thecatalytic reaction proceeds at the interface and, ultimately, the enzyme is regen­erated with the liberation of the product. It has been proposed that adsorption oflipase at the interface could occur independently of the catalysis in the interfacialregion (Brockman et al., 1973; Momsen and Brockman, 1981; Brockman, 1984;Sugiura, 1984). This implies that lipases possess a molecular structure capable ofbinding to the substrate-containing interface at a site that is functionally distinctfrom the catalytic site of the enzyme. Through this binding, presumably, the activesite is localized and oriented in proximity to the ester bonds of triacylglycerol andthe catalytic reaction thus occurs. Brockman et al. (1973) suggested that the simplepartitioning of a lipase and its substrate between the bulk phase and lipid-waterinterface can stimulate lipolysis by increasing their concentration in the surfacephase. It has been proposed that adsorption may also change the intrinsic catalyticactivity of the enzyme, either as an immediate consequence of adsorption, or as aslow, post-adsorptive conformation change (Verger, 1980; Brockman, 1984). Onecould speculate that if lipase catalysis should occur at the interface, the enzymeshould have greater surface activity compared to other enzymes which act in bulkphases. This is confirmed by the fact that the surface tensions of 0.001 weightpercent solutions of various lipases are in the range 56-66 dyne/ern compared to69-73 dynes/em obtained for other enzyme solutions (Sugiura, 1984).

Recent interest in lipase catalysis in organic solvents or in non-polar environ­ments (Butler, 1979; Fukui and Tanaka, 1982; Klibanov, 1986, 1989; Slater, 1988;Yamane et al., 1988; Andersson and Hahn-Hagerdal, 1990) demands a better under­standing of the interfacial nature of lipolysis, given the absence of a bulk aqueousphase and the importance of a true interface in the majority of such reactionsystems. Expression and maintenance of catalytic activity by lipase in non-polarsystems may be attributed to possible conformational changes analogous to the"interfacial activation" through change in conformation occurring at interfaces inaqueous systems.

Reversibility of Lipase Reactions

The natural function of Iipases is to catalyze the hydrolysis of TAG. However, thisreaction is reversible and these enzymes, under appropriate conditions, can catalyzethe formation of TAG and partial acylglycerols from glycerol and free fatty acids.The water content of the hydrolytic reaction system is a crucial factor that controlsthe equilibrium between the forward and reverse reactions. The reaction in reversedirection during hydrolysis also depends upon the enzyme source (Okumura et al.,1981). The reversible nature of the lipase reaction not only offers the possibility offormation of esters (esterification reaction), but also offers the possibility of transferor exchange of the acyl moiety (interesterification) from one molecule to another.Figure 2 schematically depicts various types of reactions catalyzed by lipases. In

368 M. T. PATEL et al.

(1) Hydrolysis of ester (Hydrolysis)

Esler

12] Synthesis of ester (Esterification)

A·OH

Alcohol

+ HOOC-R'

Acid

A·OH

Alcohol

HOOC-A'

Acid Ester

(3) Tmnsfer or Exchange of acyl moieties (Transesterlficalion)

Inleresterlflcatlon

R1·00e-R'1 + R,;.oOC-R'. -> Rt-oOe-R'2 + R•.()OC.R',

Ester Ester Ester Esler

Alcoholysis

R,-QH

Ester AlCOhOl Alcohol Ester

A'-oOC-A,. HOOC-A, -, A'-oOC-R,. HOOC-A,

Esler Acid Ester Acid

FIGU RE 2 Schematic of lipase catalyzed hydrolysis, esterification and transesterification reactions.

addition to these reactions, lipases can also catalyze thiotransesterification (Zaksand Klibanov, 1985; Nagao and Kito, 1990), aminolysis (Kirchner et al., 1985; Zaksand Klibanov, 1985), and oligomerization (Abramowicz and Keese, 1989; Gereshand Gilboa, 1990) under suitable conditions.

Lipase Distribution and Specificities

Lipases are widely distributed in various animals, microorganisms, and plants(Brockerhoff and Jensen, 1974; Shahani, 1975; Brockman, 1984). Lipases from differ­ent sources may have different properties in terms of substrate specificity, thermaland pH stability, and optimum conditions for activity, such as pH, temperature,substrate concentration and ionic strength. Characteristics of various lipases fromanimals (Brockerhoff and Jensen, 1974; Shahani, 1975; Hamosh, 1984; Olivecronaand Bengtsson, 1984; Verger, 1984), plants (Brockerhoff and Jensen, 1974; Shahani,1975; Huang, 1984), and microorganisms (Brockerhoff and Jensen, 1974; Shahani,1975; Macrae, 1983a; 1wai and Tsujisaka, 1984; Sugiura, 1984) have been compre­hensively reviewed.

LIPASE CATALYZED REACTIONS 369

Examples of practical importance are porcine pancreatic and ruminant lingual(pregastric) lipases (animallipases); castor bean(Ricinus communis), rape seed (Brass­ica napus), and oat caryopses (Avena sativa) lipases (plant lipases); and Chromobac­terium viscosum, Pseudomonas fluorescens, P. [raqi, Humicola lanuqinosa, Aspergillusniger, Rhizopus delemar, R. arrhizus, R. javanicus, Geotrichum candidum, Mucormiehei, Penicillium cyclopium, and Candida cylindracea lipases (microbial lipases).

Generally, lipases from various sources exhibit differences in substrate specificities.The substrate specificity of a lipase can be related to its positional or regio-selectiv­ity, i.e., ability to hydrolyze or synthesize ester bonds at only sn-1(3) or both sn-1(3)and sn-2 positions; to its fatty acid selectivity, i.e., preference for particular chainlength, level of unsaturation or structure of fatty acid; to its stereo-selectivity, i.e.,ability to act on either the R- or S-isomer of asymmetric compounds, and fortriacylglycerol, the (hypothetical) ability to hydrolyze ester bonds at only the sn-l orsn-3 position. A few examples of such lipase selectivities are listed in Table I.

Specificity of lipases seems to be related to their origin and also to the isolationand purification process. For a lipase acting in a heterogeneous system, substratespecificity may depend on both the chemical structure of substrate and the physicalproperties of interface or emulsion. Although most studies emphasize the chemicalspecificity of lipases (Yamaguchi et al., 1973; Sugiura et al., 1974; Umemoto andSato, 1978; Chander et al., 1979; Adams and Brawley, 1981), there is evidence sug­gesting that physical factors such as reaction temperature and surface pressure cancontrol specificity (Sugiura, 1984). With increasing research efforts in the area oflipase catalysis in non-aqueous media, newer or altered specificities of the lipasesmay be discovered.

Mechanism of Lipase Action

Little effort has been expended on the elucidation of the nature of active site and theexact mode of lipase action. Investigation on Pseudomonas and Chromobacteriumlipases by chemical modification techniques suggested that histidine may be in­volved in the catalytic action (Sugiura, 1984). Pancreatic lipase is reported to be a'serinehistidine' enzyme (Brockman et aI., 1973; Brockerhoff and Jensen, 1974;Shahani, 1975). This observation is based on the photooxidative inactivation of thelipase and inhibition of the lipase by high concentration of diisopropyl fluorophos­phate (DFP) and diethyl-p-nitrophenyl phosphate.

Recently, Brady et al. (1990) reported the three-dimensional structure of Mucormiehei lipase. The x-ray structure revealed that a trypsin-like catalytic triad formsthe catalytic center of this lipase. The triad is close to the surface, buried under thehead of a long chain folded onto the triad. The residues forming the catalytic triadin lipase are Ser 144, His 257, and Asp 203. It is proposed that the lipase may beworking in two stages: the loop is removed or displaced, possibly through inter­facial activation, then the ester bond is subsequently hydrolyzed by an acid-basemechanism very similar to that for serine proteases. The loop may also serve as adevice to inhibit the proteolytic activity of the triad, thereby protecting the lipaseitself.

370 M. T. PATEL et al.

TABLE 1

Substrate Selectivities of Lipases.

Selectivity Preference,Specificityor Discrimination

Enzyme Source(Reference)

[I] Regio-selectivity

Non-specific

Position-specific

No specificity

Preference for 5n·1and sn-3over 5n·2

Candida cylindraceaSonnet, 1988Geotrichum candidumTahoun, 1987; Sonnet, 1988Chromobacterium viscasumSugiura and Isobe, 1975Penicillium cyclopiumIwai and Tsujisaka, 1984

Pancreatic lipaseBrockerhoffand Jensen, I974; Verger, 1984AspergillusnigerOkumura et 01., 1976;Iwai and Tsujisaka, 1984Rhizopus delemarOkomura et 01.,1976;Iwai and Tsujisaka, 1984

[2] Fatty acid selectivity

Chain length

Level of saturation

Preferenceforshortchain

Preference formedium chain

Preference forlong chain

Preference forsaturated

Preference forunsaturated

PreferCI8:3>CI8:2>CI8:1 >CI8:0

Specificity for C 18w-9 cis

Penicillium caseico/urnAlhir er 01., 1990PenicilliumcyclopiumOkumura et 01., 1976;Iwai and Tsujisaka, 1984Pseudomonasfrag;Nishio et 01., 1987b

Rhizopus arrhizusSonnet, 1988Mucor mieheiSonnet, 1988Aspergillus nigerIwai and Tsujisaka, 1984;Sonnet, 1988Chromobacterium lipase BSugiura and Isobe, 1975

Geotrichum candidumIwai and Tsujisaka, 1984Candida cyUndraceaSonnet, 1988

Fusariumoxysporum f.Kwon er 01., 1987a

Chromobacterium lipase BSugiura and Isobe, 1975; Sugiura, 1984

Rhodotorula pilimanaeMuderhwa et 01., 1986

Geotrichum condidumTahoun, 1987

LIPASE CATALYZED REACTIONS

TABLE I (Continued)

371

Fatty acid structure Specificity forTrans-6 over Cis-6

Discriminatesagainst Cis-4 andCis-6

Rape seed (Brassica napus)Hills et al., 1990

Rape seed (Brassica napus)Hillsetal., 1990

[3] Stereo-selectivity

Enantio-selectivity Specificity for R·alcohols

Specificity for R·esters

Specificity for S­ester

Mucor mieheiSonnet, 1988Pseudomonas jragiNishio et al., 1989

Pseudomonas fiuorescensXie et al., 1987, 1988CandidacylindraceaXie et al., 1988

Pseudomonas aeruginosaHamaguchi et al.; 1986

II. APPLICATIONS OF LIPASE

Various lipases, alone or in combination with other enzymes, have been used indairy and other food processes, and lipases produced in situ by microorganisms areimportant in making foods palatable and acceptable. Manufacturers of lipases havesuggested that the enzymes may be used in detergents, leather processing, pharma­ceuticals, cosmetics, etc. Industrial applications of lipases in those fields have beenpreviously reviewed (Posorske, 1984; Rattray, 1984; Kilara, 1985; Nielsen, 1985;Welsh et al., 1989; Mukherjee, 1990; Nagao and Kito, 1990).

Current focus on the use of lipases is for the processing of fats and oils; fattyacid production, glyceride synthesis, and interesterification. Standard technologyfor fatty acid production involves high-temperature and high-pressure counter­current steam splitting (Sonntag, 1988). This reaction can also be achieved en­zymatically and has been the subject of many publications (Linfield et al., 1984b;Hoq et al., 1985b; Tahoun et al., 1987; Buhler and Wandrey, 1988; Hirano, 1988).The rate and degree of hydrolysis varies with the reaction conditions, but undercertain conditions palm oil could be completely hydrolyzed within 3 hours (Khoret al., 1986).

From a commercial viewpoint, lipase-catalyzed hydrolysis of fats for the produc­tion of fatty acids and glycerol appears to be less economical than conventional fatsplitting, mainly because of the relatively high cost of lipase preparations (Buhlerand Wandrey, 1987). However, if one considers the production of specific productsof high commercial value, such as polyunsaturated (w-3) fatty acids by hydrolysis ofmarine oils catalyzed by non-specific lipases, such processes could be economicallyattractive. Polyunsaturated (w-3) fatty acids, which are important as dietetic prod­ucts (Lawson and Hughes, 1988), cannot be obtained by conventional steam split­ting without substantial decomposition. Similarly, y-linolenic acid, a useful dietaryconstituent of certain seed oils, such as evening primrose oil (Mukherjee and

372 M. T. PATEL et al.

Kiewitt, 1987), can be prepared as a concentrate together with linoleic acid bylipase-catalyzed hydrolysis under mild conditions (Hills et al., 1989). Furthermore,very-long-chain monounsaturated fatty acids, such as gadoleic, erucic and nervonicacids, which are of interest to the oleochemical industry (Princen and Rothfus, 1984),can be obtained by partial hydrolysis of some seed oils, such as mustard (Sinapisalba), using sn-I ,3-specific lipases. Partial hydrolysis of oils catalyzed by lipases forthe production of monoacylglycerols (Holmberg and Osterberg, 1988), that are use­ful as emulsifiers, is another possible application of enzymatic hydrolysis of fats forthe preparation of products of reasonable commercial value.

A second area of potential application of lipases is the reversal of the hydrolysisreaction (Hoq et al., 1984; Hills et al., 1989) to synthesize glycerides. By decreasingthe amount of water, it is possible to shift the equilibrium position toward thesynthesis. Using lipases from A. niger and C. rugosa under low water conditions andan excess of fatty acids, various acylglycerols have been synthesized. The type ofproducts obtained could be controlled by using enzymes with regiospecificity. Iflipases derived from R. delemar or A. niger were used, only mono- and diacylglycer­ides were formed; triacylglycerides could not be synthesized under these conditions(Tsujisaka et al., 1977). In general, the esterification process proceeds slowly but hasthe advantage of taking place under mild conditions and without having to resort tothe use of fatty acyl halides.

Lipase-catalyzed reactions can be employed for the "deacidification" of fats andoils by esterifying the undesirable fatty acids that are present in unrefined oils. Theesterification can produce either di- and monoacylglycerols that also occur in suchoils or glycerol and acylglycerols. This is an alternative approach to conventional"fat refining" by alkali neutralization or distillative deacidification (Schuch andMukherjee, 1989). The commercial immobilized lipase preparation from Mucormiehei, Lipozyme (Novo Nordisk, Copenhagen), catalyzes the esterification of agreat variety of carboxylic acids, including short-chain, long-chain, and branched­chain acids to different types of alcohols, ranging from short-chain and long-chainalkanols to cyclic alcohols (Miller et al., 1988). In these studies almost quantitativeesterification of long-chain fatty acids to long-chain alcohols has been obtained byremoving the water formed under vacuum. Moreover, high rates of esterification areattained using water-immiscible solvents as reaction media rather than water-mis­cible solvents. A further interesting application of Lipozyme is the esterification offatty acids to 1,2 (2,3)-isopropylideneglycerols and subsequent mild acidic hydroly­sis of the isopropylidene derivative to yield monoacylglycerols as the sole reactionproduct (Ibrahim et al., 1988; Miller et al., 1988). Products containing high levels ofmonoacylglycerols have been prepared by lipase-catalyzed esterification of glycerolwith rice bran oil containing large proportions of un-esterified fatty acids (Kosugiet al., 1987). Lipase-catalyzed esterification or transesterification reactions have beenused for the preparation of short-chain and medium-chain esters, such as ethylpropionate, ethyl octanoate, isobutyl acetate, isoamyl acetate, phenyl 2-methylpen­tanoate and esters of terpene alcohols, such as menthol and geraniol (Lazar, 1985;Marlot et al., 1985; Gillies et al., 1987; Langrand et al., 1988). Such substances areused as flavoring agents. Another possible application of lipase-catalyzed esterifica­tion reactions is the synthesis of fatty acyl esters of carbohydrates, which can be

LIPASE CATALYZED REACTIONS 373

used as emulsifiers. Esters of sucrose, glucose, fructose, and sorbitol are reported tohave been prepared in good yields by reactions catalyzed by microbial lipases, suchas the lipase from C. cylindracea (Seino et al., 1984).

A third area is interesterification which is the process of exchanging some of thefatty acids on the triacylglyceride molecule for other fatty acids. When ester ex­change is catalyzed chemically, it results in a random distribution of the entire fattyacid pool at each position on the triacylglyceride (Sonntag, 1988). When a lipase isused as the catalyst, production of a variety of desirable acylglyceride mixtures maybe possible due to the specificities for either fatty acid chain length or position(Macrae, 1983b, 1989). Under moderate reaction conditions, the reaction can stillproceed at rates that are acceptably rapid for industrial processing. Fats and oilswith modified physical properties, particularly their melting characteristics, are com­mercially attractive because of potential applications.

Possible applications of lipases in the preparation of products resembling cocoabutter from inexpensive starting materials have been briefly explored. Such prod­ucts, which are of interest to the confectionary, pharmaceutical, and cosmetic indus­tries, have been prepared, for example, by interesterification of olive oil with stearicacid catalyzed by an immobilized sn-l,3-specific lipase from Rhizopus delemar(Yokozeki et al., 1982a). Cocoa butter substitutes have also been prepared usingimmobilized sn-l,3-specific lipases, e.g., by interesterification of a palm oil fractionwith myristic acid using the lipase from Aspergillus sp. (Wisdom et al., 1984) or byinteresterification of olive oil with palmitic acid using Lipozyme from Mucor miehei(Nielsen, 1985). Linolenic acid is an undesirable constituent of soybean oil due to itsoxidative instability. Recently, the linolenic acid content of soybean oil has beensubstantially reduced by interesterification of this oil with lauric, palmitic or oleicacid at low temperatures (10°C) using Lipozyme and, to a lesser extent, with pan­creatic lipase (Kaimal and Saroja, 1988). Triacylglycerols enriched with w-3 polyun­saturated fatty acids have been obtained by interesterification of cod liver oil withfatty acids or ethyl esters using Lipozyme (Haraldsson et al., 1989). Triacylglycerols,contained in common fats and oils, as well as wax esters of jojoba oil have beentransesterified with various sugar alcohols in pyridine using porcine pancreaticlipase or Chromobacterium viscosum lipase to yield primary monoesters of sugaralcohols having excellent surfactant properties (Chopineau et al., 1988).

III. CONVENTIONAL REACTION SYSTEM FOR LIPASE CATALYSIS

As mentioned earlier, conventionally, lipases have been employed for hydrolyticreactions mainly in water-rich media. In such systems, the insoluble substrate isdispersed in the enzyme-containing aqueous solution and a lower phase ratio be­tween substrate and water is used. Since lipase acts only at the interface, the reactionrate is a function of the interfacial area between the substrate and the aqueousphase. Accordingly, substrates must be dispersed in as fine an emulsion as possible.Simple agitation or stirring is not sufficient for substrates such as olive and milktriacylglycerols. Emulsification is done by homogenization or sonication of the

374 M. T. PATEL et al.

mixture of water and triacylglycerols in the presence of additives such as polyvinylalcohol, polyethylene glycol, gum arabic, Triton X-100, and lecithin (Yamaguchiet al., 1973; Omar et al., 1987; Tahoun, 1987; Alvarez and Stella, 1989; Phillips andPretorius, 1991).This requires mechanical energy to increase the interfacial area andthe emulsifier to stabilize the resulting emulsion. Vigorous agitation of emulsifiedmixture is also required in the course of hydrolysis to constantly renew the surfaceof the oil droplets. The system also needs sodium ions to suppress enzyme inhibitionby surface charge effects and calcium ions to accept a charged fatty acid; whichnormally inhibits lipase (Brockerhoff and Jensen, 1974; Shahani, 1975; Nishio et al.,1987b). Also, the volume fraction of substrate and water is critically important to thecourse of hydrolytic reaction. If a very large amount of water is used, a high degreeof hydrolysis may be achieved at the expense of lower yield on a volume-time basis.On the other hand, with a small amount of aqueous phase, the reverse reactionbecomes significant due to increased concentration of glycerol and decreased avail­ability of water needed for the hydrolysis.

The aqueous emulsion reaction system is schematically shown in Figure 3. Thefeatures of this system are that it is heterogeneous and is an oil-in-water emulsion inwhich the water occupies a high volume fraction. The Figure shows action of solubleor immobilized enzymes in the presence of continuous mixing. In a typical hydroly­sis run, oil or melted fat is dispersed in an aqueous solution containing emulsifiersand electrolytes. The dispersion is then homogenized or sonicated. A buffered en­zyme solution is added and the mixture is stirred at a constant temperature, until'the substrate is completely hydrolyzed or maximum degree of hydrolysis is achieved.The amount of enzyme used will depend on the reaction time desired for a degree of

-...e- .e. I I Aqueous Phase

~ Substrate

@ Enzyme Support

• Enzyme

FIGURE 3 Schematic of aqueous emulsion reaction systems. The substrate is emulsified in water usingsurfactants and other additives. The enzyme can be simply dissolved in the water phase or be immobi­lized on a support. The water volume fraction is large and continuous mixing is maintained to ensure thedispersion of the substrate phase in water.

LIPASE CATALYZED REACTIONS 375

hydrolysis. The enzyme is denatured even at room temperature by Cu, Fe, and Niions (Linfield, 1988), therefore stainless steel or glass-lined equipment must be used.Until recently, the majority of scientific reports describing lipase-mediated hydroly­sis in aqueous emulsion system were confined to academic biochemical studies(Brockerhoff and Jensen, 1974; Brockman, 1984; Linfield, 1988). Also, there areseveral studies reported in which aqueous emulsion systems have been used tocharacterize the lipases from new sources (Nishio et a/., 1987a; Yamamoto andFujiwara, 1988; Jacobsen et a/., 1989; van Oort et aI., 1989; Alhir et a/., 1990;Sugihara et a/., 1990; Torossian and Bell, 1991).

Successful applications of lipases in the conventional emulsion systems have beendelayed due to the following drawbacks: poor reproducibility, poor process control,lower volume-time yield, high energy requirement for preparation of substrate emul­sion and continuous agitation in the course of hydrolysis, high enzyme cost becauseof no enzyme reuse, difficulty in product separation, incomplete hydrolysis due tosubstrate/product inhibition, risk of microbial contamination, and incompatibilityfor commercially important synthetic reactions.

IV. NOVEL REACTION SYSTEMS FOR LIPASE CATALYSIS

In the last two decades, considerable research efforts have been made at the indus­trial and academic levels for the commercial utilization of lipase, particularly ofmicrobial origin, using novel reaction media and by finding newer applications forlipase catalysis. In contrast to the conventional water-rich emulsion system, lipasecatalysis in a majority of the new reaction systems is carried out either in thepresence of limited amount of water or practically in the absence of water. Thesemedia are either microaqueous at molecular level or microaqueous at phase level.The water-insoluble liquid substrate or the substrate dissolved in varying amountsof organic solvent, constitutes the bulk of the reaction medium. Lipase is used in freeform, or in immobilized form, or is kept physically separate from substrate bymicroporous membranes.

The different kinds of reaction media used in various studies can be categorizedbased on the nature of phase heterogeneity and the type of phases as follows:

A. Macroheterogeneous systems:1. Liquid-liquid (immiscible solvent + water) systems2. Liquid-liquid-solid (immiscible solvent + water + solid) systems3. Solid-liquid (solid + immiscible solvent) systems4. Solid-liquid (solid + miscible solvent) systems

B. Microheterogeneous systems:1. Enzyme solubilized in mixture of water and miscible solvent2. Enzyme (chemically modified) solubilized in immiscible solvent3. Enzyme solubilized in immiscible solvent by surfactant or co-solvent

C. Specialized reaction systems:1. Organogels or microemulsion-based gels (MBG)

376 M. T. PATEL et al.

2. Various regions of surfactant/solvent/water ternary systems, such as Winsor1Il, lyotropic liquid crystals, and bicontinuous microemulsion systems

3. Supercritical fluids

Each of these systems has advantages and drawbacks relative to one another.Several advantages can be realized through the use of low-water content, organicmedia for lipase-mediated biochemical reactions. Firstly, the high solubility oflipophilic substrates in organic media could substantially reduce the volume of thereaction mixture needed to produce a given amount of product, resulting in im­proved volumetric productivity. Since the substrate is primarily present in the or­ganic phase and the reaction product is transferred back into the organic phase,problems of substrate and product inhibition are reduced, if not, eliminated. Also,product recovery from the organic solution where it is relatively more concentratedis easy and less costly, compared to the product recovery from dilute aquoeussolutions. Secondly, in reactions such as esterification and transesterification, wherewater is a product of the reactions, low-water environment shifts the equilibriumtoward synthesis rather than hydrolysis. Thirdly, the insolubility or physical confine­ment of the enzyme in organic media permits recovery and reuse of the enzyme afterfiltration. Further, in several instances, it has been observed that the use of alow-water environment freezes the enzyme in a catalytically active conformation andimproves its thermal stability as compared to that in aqueous media. Finally, thetechnological problem of bacterial contamination and undesirable water-dependentside reactions can be reduced with the use of organic media. Each of the novelreaction media listed a bove is discussed in this section.

A. Mucroheteroqeneous Systems

I. Liquid-Liquid (Immiscible Solvent + Water) Systems

This type of biphasic reaction system is composed of a water-immiscible organicphase and the aqueous phase. The aqueous phase contains the enzyme and polarreactants such as glycerol or alcohols in the case of esterification reaction. Theorganic phase can be either a solution of the reactant in the organic solvent or theliquid reactant alone without any solvent. Such biphasic systems not only permithigh substrate and product solubility, but also drive the equilibrium-controlledreactions to completion. Since the enzyme and the product are located in differentphases, product recovery is facilitated and enzyme can be recovered for reuse. Theschematic of this type of reaction medium is shown in Figure 4. Such a systemrequires continuous mixing. Some of the factors governing the choice of solvent insuch systems are high solubilizing capacity for the reactants and products, lowsolubility in water (octanolwater partition coefficient more than 4), minimum de­naturing effect on enzyme, and low toxicity. Generally, solvents or lower polarity areless harmful to enzymes than are the more polar solvents (Carrea, 1984). Generally,iso-octane and II-hexane have been found to be most suitable solvents for enzymaticreactions.

LIPASE CATALYZED REACTIONS 377

I I Aqueous Phase

W- Organic Phase

• Enzyme

FIG lJRE 4 Schema lie of macro heterogeneous liquid-liquid (immiscible solvent + water) systems. Theorganic liquid phase can be the non-polar substrate or a solution of the substrate in a water-immisciblesolvent. The aqueous phase contains tbe dissolved enzyme and can also include the polar reactant (ifpresent).

Hydrolytic Reactions: Lipase-mediated hydrolytic reactions have been successfullyconducted in this type of macroheterogeneous reaction system. Kinetics of microbiallipase-mediated hydrolysis of olive oil (Kwon and Rhee, 1987) and beef tallow(Mukataka et al., 1985) have been investigated using this type of biphasic reactionmedium. The rate equation deviated from Michaelis-Menten kinetics when the bulkconcentration of oil was considered; however, the rate data obeyed the kineticequation when the interfacial area between the two-phases was taken into consider­ation. The stability of lipase in this medium is dependent on the substrate concentra­tion (Kwon and Rhee, 1986). Also, the hydrolysis rate in this medium increases inproportion with the olive oil content up to 90% v]v, as compared to 5% in aconventional aqueous emulsion system (Kwon et al., 1987a). The enzyme stabilityincreased in proportion with concentration of oil in organic solvent. Mukataka et al.(1987) studied hydrolysis of palm oil and beef tallow by C. cylindracea lipase forpractical applications in this medium using a high substrate concentration. For bothpalm oil and beef tallow, a percentage of hydrolysis higher than 98% was achievedin the 20% iso-octane system at a substrate concentration of 50%. However, whensubstrate concentration was higher than 50%, the final value of hydrolysis de­creased. Approximately 60% of lipase activity was recovered by ultrafiltration. Khoret 01. (1986) established optimum conditions for lipase-catalyzed hydrolysis of palmoil in this system using a single-variable approach. Lipase from C. rugosa (c.cylindracea) exhibited optimum activity at 37°C and at pH 7.5. The optimal oil-to­hexane ratio was 2:1. The use of biphasic system is particularly helpful for thehydrolysis of animal fats (solid fat). Because of their high melting points, hydrolysisof animal fat in aqueous systems is more difficult than that of liquid oils. In thepresence of 5-10% iso-octane, beef tallow and pork lard at 50% concentration werehydrolyzed below their melting points up to 94-97% in 24 hr using C. rugosa lipase(Virto et 01., 1991).

Linfield and co-workers (1984a,b, 1988) investigated the hydrolysis of fats and oilsusing approximately equal proportions of enzyme solution as an aqueous phase and

378 M. T. PATEL etal.

liquid substrate as an organic phase. A 95-98% hydrolysis of tallow, coconut oil,and olive oil at 26-40 °C was achieved in 72 hr using C. rugosa lipase. The kineticsof lipolysis were found to be approximately of first order in the substrate concentra­tion. From the hydrolysis data, it was empirically found that the percentage of freefatty acids formed was a linear function of the logarithm of reaction time andlogarithm of enzyme concentration. They also observed that addition of hydrocar­bon solvents and nonionic surfactants in this system led to an adverse effect in thehydrolytic reaction. Omar et al. (1987) studied hydrolysis of tallow by Humicolalanuginosa lipase in a similar biphasic system of liquified fat and aqueous enzymesolution. They observed that beef tallow was hydrolyzed up to 65% in 72 hr at45°C in a system containing about equal proportion of fat and buffer. Addition ofn-heptane in this system increased the degree of hydrolysis up to 95%.

A technologically feasible biphasic system for the recycling of lipases in continu­ous fat hydrolysis has been developed (Buhler and Wandrey, 1987, 1988). It exploitsthe fact that lipases accumulate at the phase boundary. The process employs twostirred-tank reactors and two continuously operating centrifuges. The operatingconditions were selected such that about 90% of the pure aqueous phase containingglycerol and about 90% of the pure fat phase containing fatty acids were separated.It was possible to recycle about 90% of the enzyme together with the interfaciallayer. The feasibility of such a process was demonstrated with a continuous hydroly­sis of soybean oil up to 98% and a volume-time yield of 11.4 kg fatty acid per literper 24 hr.

Transesterification Reactions: There have been few studies in which reversal of hy­drolytic reactions, i.e., esterification and transesterification have been addressed.Kwon and Rhee (1985) have evaluated different organic solvents for their effects onthe stability and activity of R. arrhizus lipase for interesterification of fats and oil inbiphasic systems. Di-iso-propyl ether and iso-octane were found to be superior forthe interesterification reaction. Glycerolysis reactions have been studied in somedetail for the production of monoacylglycerols from beef tallow and palm oil usingthis type of reaction medium (McNeil et al., 1990; McNeil and Yamane, 1991;McNeil et al., 1991). The systems were composed of 18.3% glycerol, 0.7% water, and81% fat or oil (molar ratio of glycerol to fat was 2). Of the several enzyme tried,lipase from Pseudomonas fiuorescens was the most effective, giving the highest yieldof monoacylglycerols. Also, combinations of lipases from different sources weremore effective than a specific lipase alone. The yield of monoacylglycerols wasgreatly influenced by the reaction temperature. At higher temperatures (48-50 "C), ayield of approximately 30% monoacylglycerols was obtained; while at lower tem­peratures (38-46 0C), a yield of about 70% monoacylglycerols was obtained(McNeil et al., 1990). The authors designated the temperature below which a highyield of monoacylglycerols could be obtained as the critical temperature (1;) In afurther investigation (McNeil et al., 1991), it was observed that the value of 1;depended on the fat type and varied between 30°C and 46 °C for naturally occur­ring oils. Based on this observation, McNeil et al. (1991) developed a lipase-me­diated glycerolysis process employing two-step temperature programming during

LIPASE CATALYZED REACTIONS 379

reaction. With an initial temperature of 42 DC for 8-16 hr followed by incubation at5 DC for up to 4 days gave a yield of about 90% monoacylglycerols from beef tallow,palm oil, and palm stearin.

Despite some advantages, the use of lipase in a two-phase system cannot be ext­ended to catalyze the esterification, since the location of enzyme is not different fromthat in an aqueous emulsion. A real obstacle to the use of lipase even for hydrolysisin a two-phase system is the small interfacial area. This causes the reaction to be masstransfer-limited. The observation that water droplets formed by agitation are muchlarger than oil droplets formed by emulsification indicates that increase of interfacialarea is still desirable (Han et al., 1988). However, vigorous agitation to reduce thedroplet size may lead to deactivation of lipase due to shear force. This mayalso increase the energy cost. Therefore, methods are needed that could retain theadvantages of a two-phase reaction system while alleviating its drawbacks.

2. Liquid-Liquid-Solid (Immiscible Solvent + Water + Solid) Systems

This type of macroheterogeneous medium is characterized by existence of multiplephases including an aqueous liquid phase, an organic liquid phase, and a solidphase. The aqueous phase may be either a solution of polar reactant in water or thepolar liquid reactant by itself. The organic phase may be either a solution of non­polar reactant in a solvent or the reactant in the absence of any solvent. The solidphase may be either a lipase immobilized on solid support or a microporous mem­brane containing the enzyme. Compared to the biphasic systems, two additionalbenefits may be gained with this reaction medium. Firstly, as the biphasic systemscause denaturation of the enzyme because of adsorption at liquid-liquid interface,immobilization of the biocatalyst on a solid support may alleviate this problem.Secondly, the reuse of the enzyme through recycling is greatly facilitated. The sche­matic of this type of reaction medium is shown in Figure 5.

Hydrolytic Reactions: This type of reaction medium is suitable, particularly, forhydrolytic reaction of lipase because of the presence of significant amount of waterin the systems. Also, inhibition against the enzyme is less as the product is transfer­red into the organic phase. Most studies of the hydrolysis of triacylglycerols in thistype of medium have used membrane-type bioreactors with few exceptions. Kwonel al. (1987b) studied hydrolysis of triacylglycerol in iso-octane-water system usinglipases from C. rugosa and R. arrhizus immobilized by entrapment with photo-crosslinkable resin prepolymer. The ratio of solvent to water phase was 8:2 with 10% v/volive oil in iso-octane. Lipase entrapped with hydrophobic gel (ENTP-3000)exhibited the highest activity. As the degree of hydrophobicity of the immobilizationmatrix increased, the Michaelis-Menten reaction velocity Vmax of the lipase entrap­ped was increased. In comparison to free enzyme, the initial hydrolysis rate with theimmobilized lipase was lower. However, both free and immobilized lipases showedalmost the same degree of hydrolysis after 25 hr of reaction. Similarly, hydrolysis oftriolein has been studied using immobilized C. cylindracea lipase on hydrophobic

380 M. T. PATEL etal.

I I

•

Aqueous Phase

Organic Phase

Enzyme Support

Enzyme

MicroporousMembrane

•••

•

FIGURE 5 Schematic of liquid-liquid-solid (immiscible solvent + water + solid) macroheterogeneousreaction system. The aqueous phase contains the immobilized enzyme while the organic phase containsthe substrate. The membrane can either separate the enzyme containing phase from the organic phase orserve to immobilize the enzyme itself.

and hydrophilic zeolites in a system containing 1.5 g of enzyme preparation in amixture of 21% (v/v) of buffer (Lie and Molin, 1991). Triolein was hydrolyzed up to70% with the lipase immobilized on hydrophobic zeolite; whereas the hydrolysiswas completely blocked by hydrophilic zeolite.

Membrane bioreactors offer promising possibilities for an efficient integration ofbioconversion and separation processes. The possibility of performing biocatalyticconversions between two immiscible phases separated by a membrane makes thistechnology, particularly, interesting for the oils and fats industry. Recently, severalapproaches involving a membrane bioreactor (MBR) have been tried. Membranebioreactors can be operated in different ways. For the hydrolysis of fats and oils,mainly microporous (0.1-0.4 urn) hydrophobic membranes have been used (Hoqet al., 1985a, b, 1986; Taylor et al., 1986). The lipase is adsorbed onto polypropylenemembranes and the interfacial enzyme concentration is dependent on the purity of theenzyme separation used (Hoq et aI., 1985b). Using hydrophobic membranes, the en­zyme must be immobilized on the membrane side that will be exposed to the aqueousphase. The membrane will be saturated with oil, which results in a very poor diffusionof the substrate water and of the product glycerol through the membrane and, conse­quently, in low reaction rates if the enzyme is immobilized on the oil-phase side of the

LIPASE CATALYZED REACTIONS 381

membrane. By properly balancing the pressure on either side of the membrane, it ispossible to keep the two phases completely separated. Separation of the resultingglycerol solution and free fatty acids is realized in the membrane unit. Using athermostable lipase immobilized on a hydrophobic membrane, Taylor et al. (1986)succeeded in passing tallow through the membrane. The two phases were separatedby settling the mixture after which the aqueous phase could be recycled.

Alternatively, the enzyme can be immobilized (via ultrafiltration) onto a hy­drophilic membrane on the oil side of the reactor (Pronk et aI., 1988). For such aconfiguration, the following mechanism is required for the lipolysis reaction. First,water must diffuse through the membrane to the enzyme active site. Lipolysis thentakes place and the resulting glycerol molecule must diffuse back through the mem­brane to the aqueous effluent stream. Failure of these two diffusion processes to takeplace would result in a rapid build-up of the glycerol concentration in the vicinity ofthe enzyme and, consequently, decrease the reaction rate. Pronk et al. (1988) demon­strated a quantitative diffusion of glycerol through the membrane during the totalrecycle lipolysis of olive oil. Whether the reaction rate is transport-controlled orkinetically controlled is not yet known, but in either case it would be dependent onspecific enzyme activity, membrane thickness and morphology.

In most cases, the stability of the immobilized lipases was high. Half-lives in theorder of several months have been reported. It is generally accepted that lipolysiscan be described by Michaelis-Menten kinetics if the substrate concentration isreplaced by the interfacial surface area (Macrae, 1983a). In an MBR, this area isdetermined by the total membrane area. Kloosterman et al. (1988) added enzymedissolved in a small amount of water to the oil phase, so that the co-substrate waterand interfacial area (emulsion) become available to the enzyme. This approachresulted in high rates of hydrolysis. In the membrane unit, glycerol moves to theaqueous phase and water to the oil phase. Using this approach, only 5-10% (v/v)water had to be added to the oil phase to obtain a maximum rate of hydrolysis at40°C. Whether this route offers better opportunities for commercialization in rela­tion to the immobilized enzyme system will depend on the stability/reusability of theenzyme and on the reaction rate of both systems.

Esterification Reactions: During esterification, water is produced while glycerol isconsumed. If esterification reactions are conducted in emulsion systems, then wateractivity is increased during the reactions. In contrast, using an MBR, there areseveral approaches to control water activity. Hoq et al. (1984) advocated the use of amicroporous hydrophobic membrane onto which lipases could be adsorbed. Theadsorbed lipases desorbed relatively easily in glycerol solutions (97%). Therefore,lipase was added to the glycerol solution, which was pumped across the membranesurface. In this way, an equilibrium was established between surface-adsorbed en­zyme and enzyme in free solution. On the opposite side, free fatty acid (oleic acid)was circulated through the reactor. The water activity in this system was controlledby circulating the glycerol solution through a continuous dehydrator.

Hydrophilic membranes also can be used in cases where the enzyme is physicallyimmobilized via ultrafiltration on the fatty acid/acylglycerol side of the membrane.

382 M. T. PATEL etal.

A glycerol solution with a given water content is pumped along the opposite side ofthe membrane. The water activity of this phase can be controlled either by a mole­cular sieve or by evaporation (Kloosterman et al., 1988). As for lipolysis in theprevious examples, the reaction is limited to the oil-water interface at the membranesurface. Direct addition of the lipase dissolved in a small amount of water to the oilphase also may be considered (Kloosterman et al., 1988). This water phase willequilibrate with the glycerol solution on the opposite side of the membrane. For thisto occur, the glycerol and water activities in the droplets containing enzymes mustequilibrate with those in the glycerol feed solution. Thus, by this approach anemulsion reaction is combined with a membrane process to control the water acti­vity in the vicinity of the enzymes.

3. Solid-Liquid (Solid + Immiscible Solvent) Systems

This type of reaction medium is composed of a large proportion of the organic solventphase containing the non-polar reactant, and a solid phase comprised of either dryenzyme powder or enzyme immobilized on the microporous support. A non-polarliquid substrate may be used as the organic phase without addition of any organicsolvent, as well. The system contains water only at the molecular level. A smallamount of water required for the catalytic activity of lipase is associated with dryenzyme powder or microporous support, and with the organic solvent. Dependingupon the type of reaction, the water is either continuously recharged or removed fromthe system. This type of system is primarily used for the synthetic and transesterifica­tion activity of lipase with a few studies on hydrolytic reactions. The extremely lowwater content in this system facilitates shifting of equilibrium toward synthetic activityof lipases, which is difficult to realize in water-rich systems. The schematic of this typeof biphasic macroheterogeneous reaction medium is shown in Figure 6.

Hydrolytic Reactions: As mentioned earlier, the system is more suitable for esterifi­cation or transesterification reactions. However, hydrolytic reaction of lipases hasbeen investigated in a few studies. Continuous hydrolysis of olive triacylglycerols indi-isopropyl ether using R. arrhizus mycelia as a source of lipase has been studied(Bell et al., 1981). Typically a packed-bed reactor containing 1 g of mycelia fed atI mL/min with a solution of 2.5% (w/v) substrate in the solvent gave a fatty acidyield of 45% at 30°C. The optimum water concentration was found to be 0.17%(wIv). The loss of enzyme activity was 0.6-1.0% /hr at 30°C. Kang and Rhee(1988,1989a, b) have conducted a detailed study on the hydrolysis of olive oil by animmobilized lipase from C. rugosa in an organic solvent. The lipase was immobilizedby adsorption on various supports which could contain water available for thehydrolysis of olive oil in organic solvent. The lipase immobilized on swollenSephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in iso-Oc­tane. The hydrolytic reaction did not follow Michaelis-Menten kinetics. Maximumactivity was obtained at pH 7. The optimum temperature shifted towards highervalues with an increase of olive oil concentration. Bilyk et al. (1991) studied the

LIPASE CATALYZED REACTIONS 383

hydrolysis of different triacylglycerols in hexane using Rhizomucor miehei lipase,immobilized on anion exchange resin in the presence of amines. The presence ofsecondary amines greatly increased the extent of hydrolysis. Lipase, in the absenceof amine, hydrolyzed the tallow up to 76% in water-saturated hexane. The additionof secondary amines to tallow solution in hexane increased the hydrolysis to 95%under similar conditions. Recently, a moistened caryopses of oat (Avena sativa) hasbeen used as a natural lipase bioreactor for hydrolysis of oil (Lee and Hammond,1990). The moistened (about 20% of its weight) caryopses of oat, when immersed inoil or in hexane containing oil, hydrolyzed the oil without any positional specificity.The optimum temperature was about 40°C.

Esterification and Transesterification Reactions: After the first report (Zaks andKlibanov, 1984) that described the catalytic activity of dry lipase in organic media atvery high temperature, several studies were conducted using suspension of solidpowdered enzyme in organic solvent for synthetic reaction of lipases. Boyer et al.(1990) studied the esterification activity of solid Mucor miehei lipase using oleic acidand decanol in heptane. The activation of the enzyme was affected by its swellingwith water. By controlling the water content a conversion of up to 94% was ob­tained in 1.5 hr. Lipase-mediated alcoholysis of sunflower oil in anhydrous petro­leum ether was examined using lipase powder from P.fiuorescens (Mittelbach, 1990).Significant proportions of mono- and diacylglycerols were produced with methanol.Chopineau et al. (1988) studied transesterification reactions between a number ofsugar alcohols and various plant and animal fats in pyridine using solid lipase fromChromobacterium viscosum. The reaction produced highly surface-active primarymonoesters of sugar alcohols and fatty acids. Elliott and Parkin (1991) studied

•

Organic Phase

Enzyme Support

Enzyme

FIGURE 6 Schematic of solid-liquid (solid + immiscible solvent) macroheterogeneous reaction medium.The liquid phase is a solution of the substrate in an organic solvent or the pure liquid substrate. The solidphase consists of dry enzyme powder or enzyme immobilized on a solid matrix. Only molecularlydissolved water is present.

384 M. T. PATEL et al.

acyl-exchange reactions between butteroil and fatty acids in anhydrous media usingsolid pancreatic lipase. Optimum temperature and pH for the reaction were found tobe 70°C and 6.5, respectively.

Several immobilized lipases have been used to catalyze transesterification reac­tions in organic solvents for the modification of fats and oils (Lilly and Dunnill,1987; Sawamura, 1988; Schuch and Mukherjee, 1988; Tanaka et al., 1988; Bloomeret al., 1990; Chang et al., 1990). Macrae (1983b) reported that Rhizopus niveus lipaseadsorbed on kieselguhr had been used for interesterification for 400 hr with almostno loss of activity. The substrates, palm oil midfraction and myristic acid, weredissolved in petroleum ether (b.p. 100-120°C) saturated with water. In a similarexample Mucor miehei lipase on Hyflo Supercel was used to catalyze the interest­erification of palm oil midfraction and stearic acid in packed-bed reactor at 50°C(Macrae, 1985). The enzymatic activity decayed exponentially with a half-life ofabout 60 days. Recently, lipase-mediated reactions in this type of reaction mediumhas comprehensively been reviewed (Malcata et al., 1990).

Solid-Liquid (Solid + Miscible Solvent) Systems

This type of macroheterogeneous biphasic system is characterized by a solid phasecomprised of enzyme adsorbed on a solid support or entrapped in a microporouscarrier, and a homogeneous liquid phase comprised of water and miscible organicsolvent. The mixed liquid phase increases the solubility of polar or non-polar reac­tants in the medium. Solvents used include ethanol, acetone, dimethylsulfoxide,acetonitrile, and tetrahydrofuran. The major criterion for the selection of the organicsolvent is the ability to maintain the catalytically active conformation of the enzymewithout stripping the essential hydration shell. With small additions of these sol­vents, enzymes usually retain their activity, but at higher solvent concentrationsinactivation of enzyme is observed. The schematic of this type of macro­heterogeneous biphasic systems is shown in Figure 7.

The system may be suitable for lipase-mediated synthetic reactions because of theability of this type of medium for solubilizing both polar and non-polar reactants.However, there are no examples in the literature where lipase-mediated reactions

1:]J[fIDIli!iuil Mix Liquid Phase

@ @ Enzyme Support

• Enzyme

FIGURE 7 Schematic of solid-liquid (solid + miscible solvent) macroheterogeneous system. The enzymeis immobilized in a solid matrix or adsorbed on a solid support. The liquid phase is a mixture of waterand a polar organic solvent and contains the substrate.

LIPASE CATALYZED REACTIONS 385

have been carried out in such a system. However, a few other enzymes have beenstudied in this type of system. A column containing immobilized glucose oxidasewas operated continuously for 14 days in a 20% (v/v] acetone solution without anyconsiderable decline in enzyme activity (Alberti and Klibanov, 1982). In anotherexample, Yokozeki et al. (1982b) carried out a transglycosylation reaction usingimmobilized whole cells iEnterobacter aerogenes). This biocatalyst retained its orig­inal activity after 35 days of operation in 40% (v/v) dimethysulfoxide.

B. Microheteroqeneous Systems

1. Enzyme Solubilized in Mixture of Water and Miscible Solvent

This reaction medium is composed of a single liquid phase with different propor­tions of water and a miscible organic solvent, with the substrate and the enzymedissolved in the mixed solvent. Enzymatic reaction in such media offer advantagessimilar to those described in the preceding section. The reaction in this medium isnot limited by mass transfer consideration and reactants of different polarity can beconveniently dissolved. However, there is some degree of difficulty in product separ­ation. Further, an organic solvent at concentrations greater than 50-70% may stripthe hydration shell of the enzyme causing it to denature (Arakawa and Goddette,1985; Guagliardi et al., 1989). However, modest concentrations of some organicsolvents enhance the stability of enzymes (Butler, 1979; Freeman, 1984). The sche­matic of the reaction system is shown in Figure 8.

Hydrolytic Reactions: A reaction system containing lipase from Pseudomonas sp. inwater/acetone solvent mixture was used for resolution of rac-a-acyloxystannanes toprepare optically active a-hydroxystannanes (ltoh and Ohta, 1990; ltoh et al., 1990).A similar solvent system was used for hydrolysis of prochiral diacetates to opticallyactive paraconic acid (intermediate for streptomycin) by pig pancreatic lipase (Moriand Chiba, 1989).

11&1111111%11 Mix Liquid Phase

• Enzyme

FIGURE 8 Schematic of systems with enzyme dissolved in mixture of water and miscible solvent. Thesolvent mixture can dissolve both the substrate and the enzyme.

386 M. T. PATEL et al.

Transesterification Reactions: Lipases have been used in water-miscible solvent sys­tems for transesterification reactions. Pseudomonas fiuorescens lipase dissolved in themixed solvent system of water, tetrahydrofuran and pyrine has been used for re­gioselective esterification of sugars such as methyl rhamnopyranoside (Ciufredaet al., 1990). Lipase from Chromobacterium viscosum has been studied usingwater/acetone solvent system for the regioselective esterification of chloramphenicolat primary hydroxy group (Ottolina et al., 1990).

Water-miscible organic solvent is not used for lipase-mediated reactions withtriacylglycerols. This may be due to the less than desirable stability of the enzyme inthese systems and comparatively lower solubility of the highly lipophilic substrates.

2. Enzyme (Chemically Modijied) Solubilized in Immiscible Solvent

Chemical modification of enzymes has potential biotechnological utility. The struc­tural stability, performance in the hydrophobic environment, and ease of recoveryand reuse of enzyme may be improved by such chemical modifications as reductivealkylation of lipase (Kaimal and Saroja, 1989), polyethylene glycol- modified lipases(lnada et al., 1990), and lipid-coated lipase (Okahata and Ijiro, 1988). Generally, thistype of microheterogeneous reaction system is comprised of water-immiscible or­ganic solvent, in which enzymes are made soluble and stable by chemical modifica­tions. The system contains water only in the molecularly dissolved state. Theessential water is associated with the modified enzyme preparation. The advantageof this reaction system is the enhanced solubility of highly lipophilic substrates andthe enzyme in the medium. The reaction is no longer limited by interfacial area ordiffusion. The schematic of the reaction system is shown in Figure 9.

A new approach in biotechnological processes is to use lipase modified withpolyethylene glycol (PEG) which has both hydrophilic and hydrophobic properties.The PEG-lipase is soluble in organic solvents such as benzene and chlorinatedhydrocarbons and exhibits high enzyme activity and stability in organic solvents.The PEG-lipase catalyzes the reverse reaction of hydrolysis in organic solvents: estersynthesis and ester exchange reaction (Inada et al., 1986, 1990; Baillargeon andSonnet, 1988; Takahashi et al., 1988). The PEG-lipase can also be conjugated to

_ Organic Phase

* Modified Enzyme

FIGURE 9 Schematic of micro heterogeneous systems with chemically modified enzyme solubilized inwater-immiscible solvent. The substrate is dissolved in the solvent medium and is directly accessible tothe enzyme.

LIPASE CATALYZED REACTIONS 387

magnetite (Fe30 4 ) . The magnetic lipase catalyzes ester synthesis in organic solventsand can be readily recovered by using magnetic field without loss of enzymic activity(Tamaura et al., 1986; Takahashi et aI., 1988; Inada et al., 1990). The PEG-lipasecatalyzed the ester-exchange reactions between an ester and an alcohol, an ester andan acid and between two esters (Takahashi et al., 1985). These reactions occur notonly in organic solvents but also in hydrophobic substrates in the absence of anysolvent. This suggests that the modified lipase is useful for many practical applica­tions. One of them is the modification and formation of fats and oils. PEG- lipasecatalyzed the interesterification reaction between trilaurin and triolein in the ab­sence of solvents at 58°C (Matsushima et al., 1986). Dilauroyl-monoleoyl glyceroland monolauroyl-dioleoyl glycerol were formed as the products of the reaction. As aresult, the melting temperature of the mixture of two substrates decreased from 33­36°C to 11-13 "C. Similarly, interesterification between hard fats and olive oil byPEG- lipase resulted in modified triacylglycerols with substantially lower (II-14°Cless) melting point (Matsushima et al., 1986; Inada et al., 1990).

Kaimal and Saroja (1989) used reductive alkylation of porcine pancreatic lipasewith butyraldehyde and acetone to enhance activity of the lipase in organic solvent.They found about 50% increase in the maximum reaction rate. The esterificationactivity of C. cylindracea lipase in organic solvent was markedly enhanced by thereduction of disulfide bonds with dithiothreitol without appreciable loss of hyd­rolytic activity (Kawase and Tanaka, 1988, 1989). A new type of organic solvent­soluble lipase preparation was developed using lipid-coating of the enzyme(Okahata and Ijiro, 1988). The lipid-coated lipase was prepared by mixing aqueoussolutions of enzyme and synthetic dialkyl amphiphiles; it was insoluble in water butsoluble in most organic solvents and showed a high activity for the synthesis of di­and triacylglycerols from monoacylglycerols and fatty acids in non-aqueous andhomogeneous organic solvent systems. In another study, the lipid-coated lipasevigorously catalyzed the reaction of enantioselective esterification of racemic alco­hols in this type of reaction system (Okahata et al., 1988).

3. Enzyme Solubilized in Immiscible Solvent by Surfactant or Co-Solvent

These microheterogeneous systems are characterized by having a continuous or­ganic liquid phase containing non-polar reactants, and a dispersed microaqueousliquid phase containing polar reactants and enzyme solution. As in the case ofbiphasic macroheterogeneous systems, the enzyme in this system is also spatiallyseparated from the organic solvent, but it is free from a diffusioned limitation. Thereason is that the separation between aqueous and organic phase occurs at the'molecular level' rather than at the 'phase level'. Unlike the macro heterogeneoussystems these systems are also optically transparent and thermodynamically stablemixtures of water and immiscible solvent. In this type of microheterogeneous sys­tem, enzyme is solubilized in an immiscible organic solvent either by micellar sol­ution of surfactants (named as water-in-oil microemulsion or reverse micellar me­dium) or by hydrogen-bonded aggregates of water and polar co-solvent (named asdetergentless microemulsion or ternary system of hydrocarbon/polar-solvent/water).

388 M. T. PATEL et al.

~ Organic Phase

CJ Aqueous Phase

• Enzyme

--- Surfactant

)- Co-solvent

FIGURE 10 Schematic of micro heterogeneous system with enzyme solubilized in immiscible solvent bysurfactant or co-solvent.

Figure 10 shows the schematic of this type of microheterogeneous reaction system.Reverse micellar system is a novel approach to biphasic aqueous-organic biocataly­sis. In such a system, enzyme is solubilized in the microaqueous phase inside thesurfactant micelles. The reverse micelles protect the enzyme from solvent-induceddenaturation and provide the enzyme with the water, essential for its structuralstability. Water-insoluble substrates and products are solubilized in the bulk solvent,providing a large interface for the enzymatic reaction.

Reverse micellar systems have been extensively studied in the last two decades forvarious reasons ranging from fundamental understanding of invivo enzyme action toapplication in the area of protein separation and enzymatic reactions. As mentionedearlier, the scope of this paper is limited to applications of reverse micellar systemsfor lipase catalysis. Different aspects of enzyme catalysis in reverse micellar media,namely, biotechnological applications (Leser et al., 1987; Martinek et al., 1987a, b),fundamentals of micellar enzymology (Levashov et al., 1984; Martinek et al., 1986;Luisi and Steinmann-Hoffman, 1987), and general aspects such as structure of re­verse micellar media, preparation of the system, and experimental procedures (Luisiand Wolf, 1982; Luisi, 1985; Luisi and Laane, 1986; Luisi and Magid, 1986; Laaneet al., 1987) have comprehensively been reviewed. Lipase-mediated reactions in re­verse micellar media have a great potential for food applications. Use of reversemicellar media greatly facilitates the lipolytic reactions. The substrates which haveto be emulsified prior to enzymatic hydrolysis can be solubilized to a large extent inthe oil- continuous phase while the enzyme is hosted in the reversed micellar phase.The enormous interfacial area that is possible in reverse micelles tremendously

LIPASE CATALYZED REACTIONS 389

improves enzymatic activity. Since the substrate is mainly concentrated in the oil­continuous phase, substrate inhibition problems are mitigated. The first report(Misiorowski and Wells, 1974) on the catalytic activity of lipolytic enzyme in reversemicellar media initiated the subsequent research in the field of reverse micellarenzymology. Since then several enzymes including various microbial lipases havebeen studied in reverse micelles.

Hydrolytic Reactions: A reverse micellar system composed of Aerosol-OT (bis (2­ethylhexyl) sodium sulfosuccinate) as a surfactant and iso-octane as a non-polarsolvent is well characterized and widely used system for many enzyme-catalyzedbiochemical reactions. This system has been used in a number of studies of lipase­mediated hydrolysis of triacylglycerols (fats and oils) to glycerol and fatty acids (Hanand Rhee, 1985, 1986; Kim and Chung, 1989) and partial acylglycerols (Holmbergand Osterberg, 1988). Batchwise hydrolysis of olive oil by lipase from C. rugosarevealed that the substrate (5% v/v) could be almost completely hydrolyzed at Rvalue of 10 (R value is the molar ratio of water to surfactant in the reverse micelle)and at Aerosol-OT concentration of 100 mM. At the end of the reaction, fatty acidsproduced could be recovered with high yield by adding water and acetonitrile,centrifuging the mixture and collecting the upper phase (Han and Rhee, 1985). In thehydrolytic reaction of olive oil, lipase activity and stability in reverse micelles werehighly dependent on the R value. The R values which maximized the initial velocityand stability were 10.5 and 5.5, respectively (Han and Rhee, 1986). Lipase in thissystem was not inhibited even at a substrate concentration of 40% (v/v). This was incontrast to the behavior in aqueous macroemulsions where substrate inhibitioncould be observed at 3-5% (w/v) of substrate concentration. The temperature-acti­vity and pH-activity profiles of the enzyme were similar to that in buffer solution.Among the eight organic solvents tested, iso-octane was most effective for the hy­drolysis of olive oil in reverse micelles. Similar results were obtained by Kim andChung (1989) who investigated R. arrhizus lipase-mediated hydrolysis of palm kernelolein in Aerosol-OT/iso-octane reverse micelles. The lipase was the most effective atR = 13 when hydrolysis was carried out at pH 7 and 30°C. Among the variousadditives tested, glycine, histidine, and glycerol increased the initial rate of reaction.In the presence of histidine and casein, the fatty acid production was increasedabout 1.5-fold after 48 hr reaction.

Lipase reaction in reverse micellar system is expected to be affected by the waterconcentration because its content in the system is usually below 2% (v/v) of the totalreaction mixture. In fact, the initial water concentration affected significantly theequilibrium of hydrolytic reaction of lipase. The equilibrium fractional conversion ofester bond to fatty acid and alcohol moiety increased in proportion with the initialwater content in reverse micelles (Han et al., 1987). While hydrolytic reactions inwater are usually considered to be a pseudo-first-order reaction, the low-waterenvironment in reverse micelles necessitated the reaction to be modelled as a two­substrate reaction. Although effect of water on equilibrium and rate parameter maybe inferred from the two-substrate kinetics, other factors such as the amount ofprotein in water pool. ionic strength, pH, ionic species, and temperature have

390 M. T. PATEL et al.

greater influence on the reaction by altering the properties of water. Han et al. (1990)studied the activity of C. rugosa in reverse micelles at various concentrations ofwater and the enzyme. For low water content (below R = 6), the activity increaseswith increasing water content indicating the requirement of a minimum amount ofwater for the full expression of enzymatic activity. The minimal R value for obtain­ing maximal activity depends on the enzyme concentration: the higher the enzymeconcentration, the higher the optimum value of R.

Holmberg and Osterberg (1988) studied hydrolysis of palm oil using sn-I(3)­specific lipase (R. delemar) for preparation of monoacylglycerols in Aerosol-OT/iso­Octane reverse micelles. Monoacylglycerols in 80% yield was obtained in 3 hr at35°C. The molar ratio R was critical in determining the amount of fatty acids andmonoacylglycerols obtained as a results of the reaction. The optimum R was foundto be 12 for monoacylglycerol yield at pH 7.0 and 35 T.

Type of surfactant used in reverse micellar media could greatly influence the lipaseactivity. Enzymatic hydrolysis of palm oil by Rhizopus sp. lipase has been studiedusing different composition of reverse micelles based on non-ionic surfactant, pen­taethylene glycol monododecyl ether in iso-octane (Stark et al., 1990). The rate ofreaction decreased as the water content of the reaction medium was increased. Thenon-ionic surfactant was found to be unsuitable for enzymatic reactions since onlypartial hydrolysis was obtained in all experiments. In another study (Kery et al.,1989), six ethylene oxide-based non-ionic surfactants were tested for their suitabilityto mediate lipase action on olive oil in reverse micellar media. Only with Siovasol El(ricine oil + 20 molecule of ethylene oxide), it was possible by the lipase to catalyzehydrolysis of the substrate over a wide range of iso-octane and water content. Thepossible advantage of this type of system over Aerosol-OT based system is that thestable systems for catalysis can be prepared over a wide range of concentration ofaqueous and non-aqueous phases.

Efforts have been made to replace biocompatible surfactant and/or solvent in thetypical Aerosol-OT/iso-octane reverse micellar media to develop more practicalreaction systems. Lipase-mediated hydrolysis of triacylglycerols in reverse micellesformed by soybean lecithin in iso-octane has been studied (Schmidli and Luisi,1990). The reaction rate was found to be diffusion-controlled. The maximum ratiowas found to be at low R value of 2.2. The temperature stability of the lipase inlecithin reverse micelles was higher than aqueous solution. The initial rate washighest at 60°C. A solvent-free reverse micellar medium of purified lecithin or crudesoy lecithin preparation as the surfactant and liquid triacylglycerol as the substrateand continuous non-polar phase for lipase-mediated hydrolytic reaction have beendeveloped in our laboratory. In a concurrent study, Chen and Pai (1991) usedsimilar lecithin reverse micellar media for hydrolysis of milk triacylglycerols usingC. cylindracea lipase. The initial rate was maximum at 55°C. The maximum activitywas observed between pH 4 and 6. The molar ratio (R) of 10 produced maximumreaction rates. O'Connor et al. (J991) studied surfactant-free microemulsion media ofn-hexane/iso-propanol/water for C. cylindracea lipase-mediated hydrolysis of methylpalmitate. Since this system does not require the presence of surfactant for itsformation, it offers the advantages of simple product separation and enzyme reuse.However, only 9% of the substrate methyl palmitate ester could be hydrolyzed in

LIPASE CATALYZED REACTIONS 391

this system. Thus, poor intrinsic catalytic activity and stability of enzymes maymake the surfactant-free system less than desirable.

Many of these studies have focused on the initial rate based on 20-60 minreaction time for assessing the influence of reaction parameters on hydrolytic reac­tion. During the hydrolysis of triacylglycerols, water is consumed and proton- dona­ting species (fatty acids) are generated. As a result, the microenvironment in theaqueous pool is continuously changing from the initial conditions. So, for the practi­cal utility of such systems, the relationship of reaction parameters with initial rate aswell as with the extended time-course of the hydrolysis should be examined. Also,studies reported in the literature have been conducted using a single-factor approachwhich does not provide important information regarding interactive effects of reac­tion parameters on hydrolysis. Nevertheless, this reaction system seems to be prom­ising for the enzymatic hydrolysis of fats and oils since it it does not require energyto promote and maintain interfacial area for catalysis. Also, the system is chemicallywell defined. Although it is certain that reverse micelles may open a new way to theindustrial application of lipase, a principal hurdle to the successful application maybe the difficulty in recovering the enzyme, if necessary, from the reaction mixture atthe end of the reaction. Appropriate enzyme reactors which are compatible with theorganic solvent used, and thus permit the continuous operation, may contribute toeconomically feasible applications of reverse micelles.

The emulsion-based conventional enzymatic hydrolysis of fats and oils (triacyl­glycerols) has not been successful as an alternative process to physicochemical hy­drolysis of fats. A reverse micellar system of Aerosol-OTlisa-octane was studied forhydrolysis of various triacylglycerols (TAG) using Rhizapus jauanicus and Candidacylindracea lipases (Patel el al., 1995). The influence of various reaction parameterson the hydrolytic reaction was investigated using multi-variable experiments (Patelel al., 1996a). A solvent-free micellar medium, suited to food application, was devel­oped and investigated using model substrates and milk fat (Patel et al., 1996b). Withthe Aerosol-OT reverse micellar system, up to 98% of substrate could be hydrolyzedby using non-specific lipase. The hydrolysis reaction obeyed Michaelis-Menten kin­etics. Kinetic constants were related to enzyme source, reaction conditions, andphysicochemical characteristics of substrates. The initial rate (I R) and the degree ofhydrolysis (DH) of TAG were most significantly affected by pH, temperature, andmolar ratio of water to surfactant (R). The I R was most influenced by the interactiveeffect of pH with temperature, R, and Aerosol-OT concentration. The DH was mostinfluenced by the interactive effects of reaction temperature with R and pH. Opti­mum reaction conditions for hydrolysis in this system were 22 DC, 140 mM Aerosol­OT, pH 6.8, and R = 14. Hydrolysis of TAG in solvent-free lecithin reverse micelleswas significantly influenced by temperature and R. Both lipases exhibited unusuallyhigh thermal stabilities in this system as evidenced by the significant activity at74 DC and 64 DC, respectively. Unlike the Aerosol-OT system, the optimum reactionconditions in this system were highly dependent on the physicochemical propertiesof substrates (Patel el al., I996c). Degree of hydrolysis was increased 2-fold by main­taining R value or was increased 2.6-fold by gradually increasing the R value of thesystem during hydrolysis. Positional selectivity of the two lipases varied with type ofreverse micelles. The content and the composition of various components in milk fat

392 M. T. PATEL etal.

hydrolysates prepared in the solvent-free lecithin reverse micellar system were sig­nificantly influenced by reaction temperature and R. The optimum reaction condi­tions necessary for favoring the production of short-chain fatty acids, mono- anddiacylglycerols, and specific regie-isomers of partial acylglycerols have been defined.Thus, it may be possible to use a solvent-free system in the modification of milk fatto impart desirable multiple functionality by selecting appropriate reaction condi­tions.

Reverse micellar system of Aerosol-OT in iso-octane can be used for lipase­catalyzed hydrolysis of triacylglycerols (TAG). In this system, TAG at a concen­tration of 20% (v/v) were hydrolyzed up to 50-60% by Rhizopus javanicus lipaseand up to 95-98% by Candida cylindracea lipase. The hydrolysis reaction obeyedMichaelis-Menten kinetics. As evidenced by the linear relationship of initial ratewith enzyme concentration, the reaction rate for TAG hydrolysis in reverse micelleswas kinetically controlled and not limited by mass transfer consideration. The kin­etic parameters for hydrolytic reaction were related to enzyme source andphysicochemical characteristics of substrate. Michaelis constant (K m ) and maximumreaction rate (Vrnaxl for hydrolysis of olive TAG by R. javanicus lipase were signifi­cantly higher than those by C. cylindracea lipase. The kinetic parameters for coconutTAG were lower than those for olive TAG. The K m and Vrnax for TAG hydrolysisincreased with increase in reaction temperature and decreased with increase inreacncn pH. Buffer component may have considerable effect on enzyme activity andR-activity profile of lipases in reverse micelles. When considering the influence ofreaction variables on lipase- catalyzed reactions for practical purposes, the initialrate as well as molar yield over extended period should be studied. The effect of areaction condition, e.g., surfactant concentration, could be different on initial rateand degree of hydrolysis (% of TAG hydrolyzed at 24 hr). Lipase activity wasrapidly reduced in reverse micellar media in absence of the substrate. The effect wassevere at higher values of R. The stability of enzyme in reverse micelles was alsorelated to the source of the enzyme. R. javanicus lipase exhibited more stability thanC. cylindracea lipase did.

The four reaction parameters - pH, reaction temperature, R, and Aerosol-OTconcentration - had significant linear, quadratic and interactive effects on the in­itial rate and the degree of hydrolysis for lipase-mediated hydrolysis of TAG inAerosol- OT/iso-octane reverse micellar media. Reaction temperature and pH hadthe most significant influence on the rate and the degree of hydrolysis; whereasAerosol-OT concentration had the least influence on those parameters. Initial ratewas primarily influenced by the interactive effect of pH with all other variables;whereas degree of hydrolysis was primarily influenced by the interactive effect ofreaction temperature with other variables. Lower-level variable combinations werefavorable over higher- level variable combinations for TAG hydrolysis in reversemicellar media. Regression models, developed for the initial rate and the degree ofhydrolysis as a function of reaction variables, accounted for up to 96% of thevariation in the two responses. The optimum reaction condition ranges were deter­mined based on maximization of both the rate and the degree of hydrolysis. Thedifferences in the physicochemical characteristics of substrates had no significanteffect on the optimum condition ranges. However, noticeable differences were

LIPASE CATALYZED REACTIONS 393

observed for these ranges between the systems with R. jaoanicus and C. cylil1dracealipases. Lipase-catalyzed hydrolysis of TAG in Aerosol-OTliso-octane reversemicellar media was optimum at about 22°C, 140 mM Aerosol-OT concentration,pH 6.8, and R = 14.

The novel solvent-free reverse micellar media composed of lecithin as a surfactant andtriacylglycerols as the continuous non-polar phase is suitable for lipase-catalyzed hy­drolysis of TAG. Reaction temperature, pH, and molar ratio (R) of water to surfactanthad significant linear, quadratic, and interactive effects on the initial rate and the degreeof hydrolysis for R. jaoanicus and C. cylindracea lipase-catalyzed hydrolysis of olive andcoconut TAG. Temperature and R had the most significant influence on the lipasecatalysis. In contrast to Aerosol-O'Tnso-octane system, lipase catalysis in solvent-freesystem was least sensitive to pH. Both enzymes exhibited unusually high thermal stabi­lity in this system. Regression models developed for the initial rate and the degree ofhydrolysis as a function of the reaction variables accounted for up to 98% of thevariation in the two responses. Optimum reaction conditions were determined based onmaximization of both the rate and the degree of hydrolysis. Unlike the Aerosol-O'Tnso­octane system, the optimum reaction conditions were highly dependent on thephysicochemical characteristics of the substrates. The optimum reaction conditions werealso dependent on the intrinsic characteristics of the enzymes. It is possible to achievehigh degrees of TAG hydrolysis by maintaining or gradually increasing the R valueduring hydrolysis by intermittent addition of water.

R. jauanicus and C. cylindracea lipases exhibited considerably different activity inthe three types of reaction media, namely, solvent-based reverse micelles of Aerosol­OT, solvent-free reverse micelles of lecithin, and lecithin-stabilized aqueous emul­sion. The specific activity and normalized initial rate for olive TAG hydrolysis byeither enzyme were higher in the two reverse micellar media as compared with theaqueous emulsion system. The degree of hydrolysis was higher in aqueous emulsionsystem than that in the other two reverse micellar media. However, molar yield inthe latter two systems was much higher than that in the aqueous emulsion systemindicating need for lower reaction volumes for two reverse micellar media. Thedifference in the kinetics of the reactants, intermediates, and products among thethree systems was marginal. Both enzymes exhibited different positional selectivityin different media. R. jauanicus lipase exhibited high degree of selectivity for sl1-1 orsl1-3 position in solvent-based and solvent-free reverse micellar media; whereas therewas no specificity in aqueous emulsion. C. cylindracea lipase exhibited no positionalspecificity in solvent-based Aerosol-O'T reverse micellar media, and some degree ofselectivity for sl1-2 position in solvent-free lecithin reverse micelles and aqueousemulsion. Both enzymes showed remarkable differences among the three reactionmedia in terms of optimum reaction conditions. Both enzymes exhibited unusuallyhigh thermal stability in the solvent-free lecithin reverse micellar medium as com­pared with the other two systems. The optimum R value was lower in the solvent­based Aerosol-OT reverse micellar medium than that in the solvent-free lecithinreverse micellar medium. There was no apparent difference in the pH optimum inAerosol-OT reverse micellar medium and the aqueous emulsion; however, the opti­mum pH in both systems was considerably higher than that in lecithin reversemicellar media.

394 M. T. PATEL et al.

Lipase-mediated hydrolytic reactions in the solvent-free reverse micellar mediacomposed of natural phospholipids (soy lecithin) as the surfactant and TAG as thecontinuous non-polar phase can be used for the bioconversion of milk fat to value­added food ingredients (Patel et al., 1996d). The reaction parameters had significantlinear, quadratic, and interactive effects on the hydrolytic reaction of R. jauanicusand C. cylindracea lipases and on the free fatty acid (FFA) profile, content andcomposition of monoacylglycerols (MAG) and diacylglycerols (DAG) in milk fathydrolysates prepared by R. javanicus lipase. The initial reaction rate for milk fathydrolysis was dependent on all three variables. The degree of hydrolysis was inde­pendent of the reaction pH. Both enzymes exhibited remarkably high thermal stabil­ity in this reaction system. The content and the composition of various componentsin milk fat hydrolysates prepared by R. jauanicus lipase were most influenced byreaction temperature and R. The optimum reaction conditions necessary for favor­ing production of short-chain FFA, MAG, DAG, and specific regio-isomers ofMAG and DAG have been defined. Thus, it is possible to use this novel system fortailoring the production of milk fat hydrolysates with desired multiple functionalityby selecting appropriate reaction conditions.

This study has demonstrated that the solvent-based and solvent-free reversemicellar systems could be used for lipase-mediated bioconversions of TAG to freefatty acids, glycerols, and other value-added ingredients. However, these reactionsystems cannot be made economically feasible process, unless efficient protocol forproduct separation and enzyme recovery for reuse can be developed. Further re­search to develop such protocols, as described below, will be necessary for thecommercialization of lipase catalysis in reverse micellar media.

Esterification/Interesterification Reactions: Lipase-catalyzed synthesis reactionsnecessitate the use of low-water environments and reverse micelles have been shownto be particularly useful in this regard. Nagao and Kito (1990) discussed in detail thepossibilities of lipase-catalyzed fatty esters for use in the food industry. In thisarticle, they point out the advantages of reverse micelles in facilitating such syn­theses. In general, microemulsion/reverse micelle-based interesterification and tran­sesterification reactions are more efficient and easier to perform than reactions usingimmobilized enzymes.

Holmberg and Osterberg (1987) and Osterber et al. (1989) demonstrated thatmicroemulsions composed of the anionic surfactant AOT in hydrocarbon solventsas well as non-ionic surfactants such as triethylene glycol monodecyl ether in hydro­carbon could be used for the lipase-catalyzed interesterification of triglycerides andfatty acids. The reaction rate in the non-ionic surfactant microemulsion was higherthan that in the AOT reverse micelles. Moreover, addition of water to the reversemicelles composed of the non-ionic surfactant resulted in phase-splitting into anaqueous phase containing the enzyme while the surfactant was concentrated in theorganic solvent phase permitting facile enzyme recovery. Similarly, Bello et al. (1987)studied the lipase-catalyzed interesterification in a quaternary system composed oftriglyceride and fatty acids, water, surfactant Brij 35, and alcohol. As the wateractivity was reduced, interesterification was favored over hydrolysis. At optimum

LIPASE CATALYZED REACTIONS 395

conditions, 40% conversion could be achieved for the interesterification reaction.Abraham et al. (1988) observed that in reverse micelles composed of AOT in hexane,triacetin-tributyrin interesterification was favored over hydrolysis as the degree ofhydration of the micelles was lowered. Hayes and Gulari (1990) conducted a detailedkinetic study of lipase-catalyzed interesterification in Aerosol-OT/iso-octane reversedmicelles. They observed that, while the pH profile was unchanged, the ratio of waterto surfactant had a profound effect on activity as well as stability of the enzyme.

Lipase-catalyzed synthesis of triacylglycerols has also been found to be favorablein reverse micelles. Morita et al. (1984) conducted the synthesis of triacylglycerolby lipase in phosphatidylcholine reverse micelles in n-hexane. Though inother media 1,2 diacylglycerol was hydrolyzed to 2-monoacylglycerol, triacyl­glycerol synthesis took place in reverse micelles. The initial activity of the synthesiswas optimum at R = 10. Lauric, myristic, palmitic, stearic, oleic, and arachidicacids were found to be effective substrates for the synthesis of triacylglycerol.Other studies conducted by Hayes and Gulari (1990) as well as Fletcher andParrott (1987) showed that a 60% conversion of fatty acids to triglycerides couldbe achieved in reverse micelles composed of Aerosol-OT/iso-octane. Legoy et al.(1987) obtained 95% synthesis of ester from heptanol and oleic acid usingC. cylindracea lipase in Brij 35 microemulsions after 14 days of reaction at optimumreaction conditions. Chang and Rhee (1990) studied glycerolysis of triacylglycerolsin Aerosol-OT/iso-octane reverse micellar media using Chromobacterium viscosumlipase. The glycerolysis reaction was maximum at pH 7 and 40°C. The maximumactivity was obtained at R value of 1.21.

Thus, the reverse micellar medium is particularly useful in the synthetic reactionof lipases. The microaqueous nature of the system facilitates shifting of equilibriumtowards synthetic activity.

C. Specialized Reaction Systems

Recently, efforts have been made in the area of enzyme catalysis in non-polar mediato develop reaction systems that not only enhance the catalytic activity of enzymebut also facilitate separation of product as well as recovery of enzyme for reuse. Forlipase catalysis, the efforts are focused in three areas: microemulsion based gels ororganogels, use of other phase regions in the ternary system of surfactant/sol­vent/water, and use of supercritical fluids.

1. Microemulsion-Based Gels

Under certain conditions, it is possible to transform reverse micellar solutions to rigid,optically transparent gel-like structures with extremely high viscosity. These gels serveto entrap biopolymers such as proteins and enzymes with retention of catalytic activ­ity. Microemulsion-based gels have a distinct advantage over conventionalreverse micelles in that they facilitate enzyme reuse and easy product separation.

Recently, Jenta et al. (1991) have conducted a detailed kinetic study of the enzy­matic transesterification activity of Chromobacterium viscosum lipase in gelatin based

396 M. T. PATEL etal.

organogels where octyldecanoate was synthesized from decanoic acid and oc­tanol. Concentrations of the enzyme to the extent of 0.6 mg enzyme/m l, of gelcould be achieved. The catalytic activity of the enzyme was essentially invarianton changing gelatin concentration and water content of gel over a wide range.Though an attempt was made to increase surface area by gel fragmentation,severe mass transfer limitations of the substrate from the gel phase to the oilcontinuous phase still prevailed. This dramatically reduced the catalytic effi­ciency of the gel-entrapped lipase. Scartazzini and Luisi (1988) recently demon­strated that addition of small amounts of water to soybean/lecithin organicsolvent reverse micelles causes a dramatic increase in viscosity and the formationof a rigid, optically transparent gel matrix. Solutions of lecithin in up to 50different solvents are amenable to gelation on the addition of small amounts ofwater which causes an increase in viscosity by a factor of 106

. Since lecithinsolutions do not contain any polymeric material, the formation of such highlyviscoelastic solutions is intriguing and a major research thrust has aimed atelucidating the microstructure of lecithin gels (Schurtenberger et al., 1989; Luisiet al., 1990; Schurtenberger et al., 1990; Capitani et aI., 1991). Scartazzini andLuisi (1990) have investigated the lipase-catalyzed hydrolysis of tricaprylin usingenzyme immobilized in microemulsion gels of soybean lecithin in cyclooctane.The reaction was slow and was carried out over a period of 8 days to obtainmoderate yields. The enzyme was active in the gel for about a month. However,the free fatty acids produced as a result of the hydrolysis reaction acted asco-surfactant and destabilized the gel causing the viscosity to decrease appreci­ably. Eventually, the gel was converted to a viscous solution.

2. Various Phase Regions of Ternary System

Most studies on enzymatic reactions in microheterogeneous media have used theregion on the phase diagram of surfactant/solvent/water ternary system correspond­ing to the formation of reverse micelles. However, it has been shown that it is alsopossible to entrap enzymes in surfactant aggregates where the spherical micellarstructures do not prevail in phase regions such as lyotropic liquid crystalmesophase, biocontinuous microemulsion, and middle phase microemulsion (WinsorIII system). These types of reaction systems utilizing phase regions other than re­verse micellar system not only entrap the enzyme for catalysis but also facilitate theproduct separation and enzyme recovery by temperature and concentration inducedphase changes. Use of Winsor III system was investigated by lipase-catalyzed hy­drolysis of triacylglycerols (Sonesson and Holmberg, 1991). The system was pre­pared by addition of polyoxyethylene ether phosphate surfactant to equal volumemixture of buffer and iso-octane. Since lipase is a membrane protein, 95% of theenzyme was concentrated in the middle phase microemulsion. The rate of enzymatichydrolysis of trimyristin to 2-myristoyl glycerol and fatty acid was comparable tothat in Aerosol-OT/iso-octane reverse micelles. The fatty acids produced remainedin the bottom phase and could be easily separated.

LIPASE CATALYZED REACTIONS 397

3. Supercritical Fluids

Supercritical fluids formed by compression of carbon dioxide has been suggested asan interesting medium for biocatalytic reactions. Such system permits high masstransfer rate and easy separation of reaction product. Recently, a few studies haveexplored the supercritical carbon dioxide for lipase-mediated interesterificationreactions.

The interesterification of trilaurin and myristic acid, catalyzed by a 1,3-specificlipase from Rhizopus arrhizus, was investigated in supercritical carbon dioxideusing a continuous-flow packed-bed reactor containing lipase covalently at­tached to glass beads (Miller et al., 1991). The reaction rate was not influenced bymass transfer limitations over the range of flow rates studied, and lipase retainedfull activity at 1400 psi and 35°C for up to 80 hr. The carbon dioxide watercontent did not affect the intrinsic activity of the enzyme, but a higher waterconcentration caused a greater degree of unwanted hydrolysis. The selectivity ofthe reaction for interesterification over hydrolysis improved at higher pressuresas the extent of hydrolysis reaction was reduced. The activity and stability oflipase in supercritical carbon dioxide were similar to those in organic liquidsolvents. Chi et al. (1988) studied R. delemar lipasemediated hydrolysis and inter­esterification using synthetic fat in supercritical carbon dioxide at 50°C and 29.4MPa. The time-course of interesterification was influenced by the water contentas well as by the kind of reaction medium. The initial velocities of hydrolysis andinteresterification were greater in supercritical carbon dioxide than in II-hexanewhen the water content increased. These few studies show the potential of super­critical fluids as media for biochemical reactions. Further work aimed at devel­oping a supercritical fluid bioreactor will be necessary for the commercializationof this reaction system.

V. CONCLUSIONS

In this review, the general characteristics of lipase catalysis are described. Applica­tions of lipase-mediated hydrolytic and synthetic reaction in various fields are brieflyreviewed. Lipase-mediated bioconversion of triacylglycerols (fats and oils) and othersparingly water-soluble compounds in practically water-free media is of currentinterest for the production of value-added and specialty products. Recently, severalkinds of novel reaction systems have been developed for lipase catalysis in organicenvironments. Lipases in these system offer considerable potential for the industrialcatalysis of fats and oils and related chemicals, and some processes are likely to becommercialized in the near future. Technological feasibility of many of the systemshas been confirmed. Also, the factors influencing the equilibrium positions in thesesystems are reasonably well understood, but knowledge of kinetics is still ratherlimited. Ultimately, economical feasibility has to be determined for the commercialsuccess of the process.

398

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