REVIEW

Applications of Lipase
Neena N. Gandhi*
Department of Chemical Engineering, Faculty of Engineering, University of Waterloo, Waterloo ON N2L 3G1, Canada

ABSTRACT: Lipases are endowed with a substrate specificity that surpasses that of any other known enzyme. This confers on these enzymes an application potential that is literally boundless. Lipases can be employed in the production of pharmaceuticals, cosmetics, leather, detergents, foods, perfumery, medical diagnostics, and other organic synthetic materials. This review attempts to present a comprehensive discussion on the present status of this unique group of enzymes in industry, as well as the actual potential. It represents an endeavor to provide a sincere answer to the question, “What can be done with this enzyme?” as well as, “Can lipase be utilized for this purpose?” It is intended that the manuscript will cover or at least mention all known applications, based on the exploitation of a particular type of reaction catalyzed by lipases. An attempt will be made to cover as large a number of references as possible, so as to further underline the importance and significance of lipase action for industry. JAOCS 74, 621–634 (1997). KEY WORDS: Applications, esterification, esters, hydrolysis, lipases, transesterification.

(d) Acidolysis
RCOOR′ + R′′COOH ⇒ R′′COOR′ + RCOOH [5]

Lipases (E.C. 3.1.1.3) are enzymes that are primarily responsible for the hydrolysis of acylglycerides. However, a number of other low- and high-molecular weight esters, thiol esters, amides, polyol/polyacid esters, etc. are accepted as substrates by this unique group of enzymes. The wide berth for employment in a variety of reactions, endowed by this broad substrate specificity, is further enlarged by the fact that lipases are capable of catalyzing the reverse reaction of synthesis just as efficiently. In fact, some lipases are better suited for synthesis than for hydrolysis applications! The two main categories in which lipase-catalyzed reactions may be classified are as follows: (i) Hydrolysis:
RCOOR′ + H2O ⇔ RCOOH + R′OH [1]

(ii) Synthesis: Reactions under this category can be further separated: (a) Esterification
RCOOH + R′OH ⇔ RCOOR′ + H2O [2] [3] [4]

(b) Interesterification
RCOOR′ + R′′COOR* ⇔ RCOOR*+ R′′COOR′

(c) Alcoholysis
RCOOR′ + R′′OH ⇔ RCOOR′′ + R′OH
*E-mail: nngandhi@chemical.watstar.uwaterloo.ca. Copyright © 1997 by AOCS Press

The last three reactions are often grouped together into a single term, viz., transesterification. Most applications where the potential for lipase is implicated presently entail the modification and/or derivatization of fats and oils and related substances by using classical chemical procedures. For instance, medium-chain triglycerides are manufactured by esterification of acid with glycerol at high temperatures (200–250oC) and pressures and/or in the presence of an inorganic catalyst (1). This chemical reaction is tedious, nonselective and consumes a large amount of energy. The product obtained has to be purified further, by alkali washing, steam refining, molecular distillation, ultrafiltration, activated carbon treatment, etc. Another process of industrial importance is the hydrolysis of vegetable oils, such as olive oil or coconut oil, to produce fatty acids and glycerol, both of which find widespread applications, especially in soaps and detergents, cosmetics, pharmaceuticals, and food. The current procedure is physicochemical and involves pressures of 3000–5000 kN/m2 and temperatures of 250°C or more. About 96–98% of the hydrolysis of fats takes place in 2 h to yield a sweet water stream of 12% glycerol. The resultant fatty acids are unusable as obtained and need to be redistilled to remove color and by-products. The process is energy-consuming and gives rise to a variety of undesirable side reactions, such as polymerization of highly unsaturated fatty acids and production of ketones and hydrocarbons. The capital investment for this steam splitting process is high because a special splitter column, which must withstand high temperature and pressure as well as corrosive acids, is needed (2). On the other hand, enzymatic fat splitting is carried out at ambient pressure and temperature (40–60°C), allowing for a lower energy cost. The overall cost is also brought down by the fact that reaction vessels need not be highly corrosion-resistant (mineral acids used as catalysts in conventional procedures are very corrosive), nor do they have to withstand severe environmental conditions. The products of such bioprocesses also have better odor and color, and are usually purer (owing to few or no side reactions) than those produced conventionally (3). This has the added advantage of lower cost resulting from the reduced amount of downstream processing required. The lower temperatures employed ensure minimal thermal degradation (4,5). Enzymes used typically for this purpose are lipases.
JAOCS, Vol. 74, no. 6 (1997)

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such as dehydration. Lipases are utilized in activated sludge and other aerobic waste processes. Lipases may also be used to hydrolyze wax esters. the breakdown of the fat may be more important. and D. . properties. phospholipases.622 REVIEW A large amount of work has been carried out on the catalysis of reactions by lipases. Japan) under the trade name Lipase-MY and is employed in sewage disposal plants in the United States (7). This paper is intended to be the next step toward convincing skeptics of what is actually possible. Some of these were handled in an earlier review (Gandhi. fewer working hazards. This is generally difficult to achieve by conventional means owing to the high temperatures and pressures used. Such removal by chemical processes. Joshi. 74. Vol. which could lead to undesired oxidation of the lipids. Review of lipase applications is not a new idea. The latter is manufactured by Meito Sangyo Co.18).21. A sample of various hydrolytic JAOCS. this manuscript intends to impart a flavor of the enormous input into this field. such as kerosene. This is amply illustrated by the production of ricinoleic acid. Leather manufacture. While justice cannot be done to all these workers for obvious reasons. castor bean (process already operative on a commercial scale) (16). Godfrey and Reichelt (14) have also discussed some applications of lipases in industry. Macrae and Hammond (8). Unlike the above. This skimmed fat-rich liquid is digested with lipases (24). Mukesh. such as liming. Lipases used for this purpose include those from Candida rugosa (3).N. degreasing can be carried out with enzymes rather than expensive and hazardous solvents. and it would not be possible to do justice to the several hundred papers published annually in this frontline area during the last 20 yr or more. where thin layers of fats must be continuously removed from the surface of aerated tanks to permit oxygen transport (to maintain living conditions for the biomass). Seitz (7). covering just a small area in this vast field offered by lipases. In addition. the list is by no means complete. who also reviewed certain aspects of lipase applications. During processing of hides and skins. It may also not be inappropriate to mention here that every year. Sawant.22). and engineering aspects. more than 700–800 research papers and patents are published/issued. Two other publications (12. who discussed industrial applications of microbial lipases. This document therefore attempts to underline the importance of the enzyme lipase in almost every conceivable field. Lipases may also assist the regular performance of anaerobic digesters (14). rugosa. and lipoxygenases—sources. N. viz. The latter is also considered superior owing to moderate reaction conditions. the enzyme is used so as to make use of the ability of lipases to break down lipids. lipases. absence of side reactions. probably due to ignorance about certain intricacies of enzyme reactions. Yamane (11) has discussed various aspects of enzymes that are useful in the lipid industry. such as p-nitrophenyl acetate (20). interesterification. However. Waste treatment. and found that the quality was comparable. 6 (1997) systems employed by workers in this area is presented in Table 1. which discussed the merits of using enzymatic (lipase-catalyzed) processes over conventional chemical reactions. one important step is the removal of residual fats and protein debris that are associated with the hide and the hair. This reaction is frequently used for lipase assays. as shown below. Nor would this fall within the scope of this review. The enzymatic method compares well with the chemical reaction (Colgate-Emery process of steam splitting) in terms of cost and it yields products with better odor and color (2). It cannot be produced from castor oil by the conventional steamsplitting process owing to side reactions. the large amount of work on lipases warrants a constant reviewing of the status to enable interested readers to be updated of the latest status. This point will be emphasized and illustrated through numerous examples and references. While these are some of the prominent reviews that have appeared in the literature since 1974. reactions catalyzed. Enzymatic hydrolysis occurs at ambient or moderate temperatures and pressures and can therefore be used to obtain fatty acids from unstable oils that contain conjugated or highly unsaturated fatty acids (17. S. despite the vast application potential expounded for lipases. The product of interest may be either a specific acid or alcohol that is formed or.B. submitted for publication). For instance. is not efficient (7). This employs the “constructive” consequences of the ability of lipase to hydrolyze lipids so as to obtain fatty acids and glycerol. Muthukumaran and Dhar (23) have compared suede clothing leathers obtained from wooled sheep skins after Rhizopus nodosus lipase-catalyzed degreasing with those produced by treatment with solvents. Lipolysis. Thus. lower power consumption. Also. no. (Nagoya. who wrote about hydrolysis of glycerides by lipases. such as that from C.. These can be avoided by using a lipase. The main objective of this manuscript is to give an overview of the numerous applications of lipases in almost every foreseeable area—ranging from utilization as a research tool in lipid structural analysis to cleaning and to mediating the synthesis of important organic products and/or their intermediates. etc. for hydrolysis of castor oil (19). Iwai and Tsujisaka (9) have listed a few applications of lipase-catalyzed hydrolysis. J. It has now become common practice to utilize a mixture of lipases and proteases for this purpose (known in technical jargon as the bating process) (13. both of which have important industrial applications. etc. fatty acids are used in soap production (15). such as that from castor seed. The literature records attempts by Nielsen (6). a valuable starting material for a variety of technical products. whereas Macrae (10) has reviewed the importance of biotechnology vis-à-vis the fats and oils industry. the present scenario is rather dismal. giving reference to some applications of lipase-catalyzed reactions..B. and Pseudomonas fluorescens (17).13) merit mention here for their discussion of certain industrial applications for lipases. some applications. HYDROLYSIS This refers to the splitting of a fat/ester into its constituent acid and glycerol/alcohol in the presence of water.

Detergents. etc.REVIEW TABLE 1 Hydrolysis Catalyzed by Lipases Lipase Rhizopus delemar + Penicillium + Rhizopus niveus Candida rugosa. Trex n-Propylibuprofenate Olive oil Soybean oil Butteroil Butteroil Tuna oil Olive oil Cumin seed oil Butter oil Tributyrin Soybean phosphatidylcholine Lesquerella fendleri oil Olive oil Fish oil 2-Naphthyl acetate. the food processing industry. fluorescens. the leather industry.. P. delemar Porcine pancreatic C. with reasonable results—up to 90% removal. 74. niveus C. no. etc. in isooctane Hybrid membrane-emulsion reactor Spiral-wound membrane reactor Flat-sheet membrane reactor Discrimination against docosahexaenoic acid Biphasic isooctane-aqueous system Kinetics determined Lecithin-isooctane reverse micelles Kinetics determined Solvents more polar than hexane better Different reaction systems compared Cells as lipase source in organic-aqueous two-phase systems Lipase discrimination against docosahexaenoic acid Interfacial kinetics determined Stereospecific hydrolysis. 6 (1997) . rugosa R. pork lard Fish oil Triacetin Oleyl oleate Tributyrin Acyl nucleosides Olive oil Palm oil Glycidol esters Methyl-2-chloropropionate Higher hydrolytic rate for neutral lipids than phospholipids AOT-isooctane reversed micelles Kinetics studied in a flat-plate immobilized lipase reactor Effect of amines on hydrolysis investigated Reactions in isooctane at temperatures lower than the melting points of substrates Docosahexaenoic acid concentration due to discrimination Hollow fiber reactor Thermodynamics and kinetics studied Various immobilization supports for lipase tested Regioselective deacylation Hydrolysis enhanced by dimethyl β-cyclodextrin Monoglyceride preparation in microemulsions Enantiomers of glycidol-starting materials for manufacture of cardiovascular β-blockers. rugosa Substrate Soybean oil Fish oil Soybean oil Cooking oil. septic tanks. rugosa. rugosa Mucor miehei M. miehei C. such as abattoirs. rugosa C. M. etc. amylase. and poultry waste processing (14). cleaning of holding tanks. rugosa R. protease. rugosa Black cumin seed C. M. rugosa P. Beef tallow. Rhizopus arrhizus Pseudomonas putida C. Vol. rugosa C. delemar Pseudomonas C. grease traps. rugosa C. etc. rugosa C. In line with the two examples above is another important industrial area where lipases are employed for their lipid degradation potential. miehei. inorganic nutrients. rugosa C. cellulase. Effluent treatment is also necessary in industrial processing units. miehei C. viz. Ap- JAOCS. rugosa M. porcine pancreatic. In fact. etc. caprate. miehei. wheat bran. (ii) sewage treatment. rugosa C. rugosa C. niger C. is employed for this purpose. a step in the synthesis of a selective leukotriene antagonist Effect of branching and stereobias studied 623 Reference 2 95 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 Neutral glycerides and phospholipids Olive oil. laurate. Carbon tetrachloride allows for stereospecific hydrolysis 172 173 174 175 176 177 178 179 180 181 182 183 184 185 Effective breakdown of solids and the clearing and prevention of fat blockage or filming in waste systems are important in many industrial operations. rugosa A. rugosa R. Hydrophobic diester Methyl-branched octanoicacid thiol esters Remarks Combined lipase system Concentration of polyunsaturated fatty acids Membrane reactor was used Electrically enhanced dispersion Enantioselective hydrolysis in AOTmicroemulsions Lipase immobilized on Sephadex. rugosa. Examples include: (i) degradation of organic debris—A commercial mixture of lipase. fluorescens C. washing and cleaning. triolein Beef tallow Tallow. cod liver oil. Aspergillus nige C. etc. Tschoke (25) was granted a patent for enzymatic treatment of fats in wastewater treatment plants whereby scum that contains mainly triglycerides at the plant entrance is hydrolyzed by immobilized lipase-generating bacteria. lipoprotein lipases Rat hepatic lipase Chromobacterium viscosum Thermomyces lanuginosus C. Amberlite.

are extensively used for accelerating the maturation of cheese and for the production of typical flavors (13). All three are ingested in substantial quantities by the body. toffees. the powdered yeast had a beefy/blue cheese-like flavor. the Aspergillus oryzae-derived lipase. for example. Lipases have also found application in soybean milk preparation. Besides carbohydrates and proteins. JAOCS. such as milk chocolate. lipases active under such conditions are preferred (28.40).24. Such flavors can be obtained by the use of cultured broths or by lipases. (vii) removal of dirt/cattle manure from domestic animals by lipases and cellulases (48). toffee. such as cutinases. Haas and Lugay (63) reported that. butter and margarine. Vol.60). snack foods. surfactant systems used in laundering frequently contain lipases (39) in association with other enzymes. blue cheese flavor development is due to enzymes from Pencillium roqueforti (58). degreasing. such as cheese. utilis in the presence of beef extract/butteroil and lipase to a total mass of 800 kg. (iii) liquid leather cleaner (45). cheese substitutes. such as cheese dips. In coffee whiteners. which allows for smaller amounts of surfactants and operation at low temperatures (49). and (iv) miscellaneous. Both act as flavors as well as flavor precursors (56). On the other hand. and caramel fudges. Formation of monoand diglycerides also allows for improvement of egg-white whipping properties (62). Flavor production in dairy and related industries. . and their digestion. etc. lipases assist in imparting a rich creamy flavor (14). (iv) contact lens cleaning composition to degrade deposits on the lens (46). cheese dips. Lipases function in the removal of (fat) stains from fabrics and are important components of detergent mixtures (27–35). Lipase-containing mixtures for this use have been patented by Fukano and Abe (41). exhibit a characteristic “piccante” flavor with a peppery characteristic. During this process. According to Posorske (13). salad dressings. sweet doughs. Such cheeses. Lipases used for this purpose include those from Candida (36) and Chromobacterium viscosum (37). Apart from detergents for cleaning fabrics. Because laundering is generally carried out in alkaline media. The lipase component causes an increase in detergency and prevents scaling. cake mixes. sauces. and butter creams is appreciable. caramels. This is another related industry with enormous potential for lipase application. Biolipolysis is already being used for the production of fat-free meats (7. (iii) dairy. (38) for the removal of olive oil from cotton fabrics. The superiority of detergents that contain lipase over those that do not was proven by Fujii et al. In the chocolate industry. resulting ultimately in products of better flavor and acceptability than their untreated counterparts (57). coffee whiteners. and soups. such as cake and cookie mixes. it greatly increases its palatability.624 REVIEW plications of lipases and other enzymes in textile detergency were the subject for Nagayama’s review (26). 74. Lipase-treated milk fats are used in the production of butter/margarine flavors (52). The flavor developed will depend on the enzyme used. the other major group of biomaterials consists of lipids. Therefore. and lactose metabolism in milk (28). cheese made with commercial animal rennets (pancreatic extracts) extracted from empty. lipases also improve flavors of rice and alcoholic beverages. Other related applications for lipase that have obtained recognition through patents include: (i) a bleaching composition (43). etc. Margrove and McDonough (51) have used modified butterfat to promote flavor in cream that is used to improve skim-milk cheese. followed by spray-drying (in the absence of a centrifugation step). In summary. The use of lipases in flavor development for dairy products. Shay et al. Butterfat modified by PGE is utilized to impart dairy flavor character to a wide range of processed foods (54). Van Dijk (42). no. (ii) decomposition of lipid contaminants in drycleaning solvents (44). vegetable fermentation. primarily provolone and Romano. (v) clearing of drains clogged by lipids in food processing or domestic/industrial effluent treatment plants (24). Foods. such as apple wine. In the absence of these flavor-enhancing additives. the other common commercial application for detergents is in dishwashing. products flavored by addition of lipolyzed materials include: (i) bakery/cereal products. Apart from milk products.30. smoked carp processing. etc. washed vells or fermentation-derived rennets do not exhibit “piccante” flavor (55). and its deficiency would pose dire consequences to health. including margarines. creams and cream centers. and water reconditioning by using lipases along with oxidoreductases. the palatability of dog food can be improved by partial hydrolysis of beef tallow with lipases. In contrast. Fat removal during fish processing can be done with lipases (7). Therefore. For instance. enzymes. such as lipases and proteases. is well established (50–55). (ii) candy/confectioneries. (vi) degradation of organic wastes on the surface of exhaust pipes/sewage pipes. After heat treatment (at about 82°C) of the biomass. cheese. the yeast had a bland flavor. protein. toilet bowls. absorption and assimilation constitute a major function. when a component consisting of protein and emulsified fat is treated with a mixture of pancreatic lipase and a protease and incorporated into dog food. The aroma and texture of these milk products are a result of fat. 6 (1997) Enzyme-modified cheeses can be used in cheese spreads. which was 15–20% greater in the former product than in the latter. pregastric esterase (PGE) is used in rennet paste preparation to curdle milk in Italy. pancake mixes. (viii) washing. Partial hydrolysis of triglycerides to increase the monoglyceride content by addition of lipase to bread dough reportedly leads to retardation of staling (61). They enhance the buttery character of toffees and caramels and reduce excessive sweetness. and meat curing by virtue of their lipolytic ability (7). Medical applications. (59) carried out continuous fermentation of C. (47). The primary enzyme for fat metabolism is lipase. the free fatty acid contribution to flavor of milk chocolate. popcorn oils. there is formation of free fatty acids and soluble peptides and amino acids.

Hirashima et al. arrhizus lipase. Joshi.3-specific. These function as lipid biosensors (68. (73). rugosa and A. The preparation of a number of optically active secondary alcohols by enantioselective hydrolysis of racemic esters by lipases/esterases has been described by Roberts (92). Other therapeutic applications of lipases. S. Esterification mixtures generally contain only the substrates and enzyme (solvent option may be foregone).69) and may be used in triglyceride (70) and blood cholesterol determinations (71). Like many other enzymes. They have been reviewed (Gandhi. Thus. Table 2 summarizes some esterification systems in the literature. leaving the triglyceride fraction rich in PUFA). etc. etc. ESTER SYNTHESIS The ability of lipases to catalyze the reverse reaction of synthesis is used in the manufacture of desired products.3) of glycerol preferentially and are said to be 1. R. Patents for lipase as a deinking agent have also been granted to Sharyo and Sakaguchi (87) and Hagiwara et al. Lipases have been used widely for the resolution of racemic acids and alcohols through asymmetric hydrolysis of corresponding esters as a result of the stereospecific characteristics of lipase activity (89–91). Hoshino et al. lipases from Mucor miehei. For instance. The selective preparation of petroselinic (cis-6-octadecenoic) acid from fennel oil was possible by hydrolysis with R. niger lipases toward PUFA (other acids were preferentially formed during hydrolysis. PGE preparations are reported to be useful in the treatment of calf scours and human malabsorption syndrome (54). and porcine pancreas attack at the primary hydroxyl positions (1.83–85). can be used in the treatment of malignant tumors (72). acidolysis and interesterification. such as γ-linolenic and docosahexaenoic acid (80). Berrobi et al. On the other hand. along with proteinases (98). submitted for publication). JAOCS. lipases from C. Thus.REVIEW 625 The corrective measure would be external administration of lipase.66) (the lipase assay is more sensitive than amylase estimation). Because the latter is generally easy to remove. (75) have filed a patent for cosmetic. being an activator of the Tumor Necrosis Factor. Lipase is a component of a hair-waving preparation (74) in which it promotes penetration of the preparation. (i) Mulberry silk refining by means of protease and lipase yields a product of better quality than alkali-refined silk (97).B. transesterification becomes more lucrative when any of these are the desired products. have been found for treatment of gastrointestinal disturbances. delemar. Lipase activity/level determination is also important in the diagnosis of heart ailments (67). J.. this process is quite superior to other synthetic processes. Miyate and Sato (94) have utilized this property for the manufacture of optically active 2-hydroxy-4-phenylbutyrate esters which are useful in the synthesis of drugs. are nonspecific with respect to position. which has a high selectivity for this acid (96). pharmaceutical preparations that contain hyaluronidase and/or thiomucase enzymes in addition to lipases for use in skin inflammations.N. The lipase level in blood serum is a diagnostic indicator for conditions such as acute pancreatitis and pancreatic injury (65. Others reactions. such as oleic acid (79). miehei discriminate against polyunsaturated acids. Chromobacterium viscosum. delemar lipase. (2.2 diacylglycerophospholipids for the purification of plasmalogens with R. Mukesh. lipases may be used as digestive aids (28. lipases may be immobilized onto pH/oxygen electrodes (in combination with glucose oxidase). especially because esterification can proceed efficiently without the use of solvent (99) and because almost complete conversion is possible by adopting means such as continuous water removal. (93) achieved the resolution of acyclic 2-azido alcohols by hydrolysis of racemic butyrates.5 to 58% and reduced residual ink spots from 18 to 7 cm (86). and D. while adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides (81).64). the regiospecificity and substrate selectivity of lipases can be advantageously exploited for their use in structural determination (82) of triglycerides. While esterification produces water and ester (usually the desired product). dyspepsias. Hence. Lipase. Lipases from Brassica napus and M. (88).B. Deinking of wastepaper.79). the hydrolysis of lipids with different types of fatty acids at different positions will lead to hydrolyzates that are specific for a particular type of lipase. corrosive acid catalysts. Fatty acid fractionation. and for the synthesis of a specific and defined set of mono. Structural analysis of triglycerides. transesterification processes. can be carried out by fat removal by using lipase as a component of topical creams (76) or by its oral administration (77). The lipase from Geotrichum candidum is selective toward cis-unsaturated (∆9) fatty acids.and/or diglycerides (71. (ii) a solid mouth deodorant contains lipase. Vol. give rise to alcohol. “Slimming down. cutaneous manifestations of digestive allergies. and water is the only by-product of the reaction. along with other components. such as cilizapril. acid. rugosa. The addition of 200 units of lipase from Pseudomonas species (KWI-56) to a deinking composition for ethylene oxide—propylene oxide adduct stearate improved whiteness of paper from 56. Resolution of racemates. Pharmaceuticals and cosmetics. 6 (1997) . (78) carried out the hydrolysis of acyl bonds at the 1position of 1. Hence. which entail the use of hazardous solvents. no. A large amount of work has been carried out to elucidate the mechanisms and kinetics of lipase-catalyzed esterification and the factors affecting them. Sawant. castor bean.” which is currently in vogue. Foelsche et al. N. Lipases can be classified on the basis of their substrate specificity. etc. (95) developed a bioreactor for enriching triglycerides with n-3 polyunsaturated fatty acids (PUFA) in cod liver and sardine oils by more than 20% of the original by exploiting the inactivity of C. This enables an equilibrium shift in favor of the synthetic reaction. or ester instead of water. 74. such as alcoholysis.

delemar. 2-ethylhexanol. cyclopium. lanuginosa. cyclopium. 2-substituted alcohols (C3–C5) C1–C14. sebacic. solvent-free — Isooctane Cyclohexane Hexane — 192 193 194 195 196 197 198 199 200 201 202 (continued) JAOCS. isopropylidene glycerol. delemar. Melted coconut acids C2–C4 C3–C12 Terpene alcohols. miehei (esterase) Porcine pancreatic C. R. etc. branched and unsaturated. M. miehei P. Vol. cyclopium A. C4 Oleic Erucic. M. etc. rugosa. etc. etc. niveus. niger. R. fructose. succinic. candidum A. C4–C8. G. R. rugosa. Cholesterol — Hexane Buffer (pH 5. 6 (1997) . 2-octyldodecanol. Mucor. oleic. 74. Aspergillus. etc. etc. oleyl. C4 C2. ricinoleic.626 TABLE 2 Esterification Catalyzed by Lipasesa Lipase R. rugosa C. rugosa Acid C4 REVIEW Alcohol C2. β-citronellol. pusillus. etc. rugosa R. javanicus.6) 152 186 187 α-Hydroxycaproic Toluene 188 C3–C6 γ-hydroxy C4–C12 acids Primary straight-chained. oleyl. Hexane Hexane Heptane 189 190 191 M. oleic Oleic C2–C18. A. miehei. Pseudomonas. M. viscosum. miehei Oleic 15-Hydroxy-C15 Vinyl acrylate Oleic C14 C3 C10–C18. isostearic. delemar. antarctica P. C4 C2 Isoamyl C18. delemar. benzyl alcohol. C. prenyl. C. 12-hydroxystearic. P. isopropanol. rugosa M. niger. oleic. Kochlight). etc. 2. rugosa M. etc. P. C4 Sucrose. benzoic. rugosa. miehei Ethyl D-glucopyranoside Isoamyl. miehei.and 3substituted alcohols. amines. C. glucose. C. isobutyric. etc. H. α-linolenic. A. P. Oleic C18 Oleic Oleic Hexane Benzene Isooctane — Supercritical CO2 Hexane. arrhizus. γ-linolenic. cyclic. thiols. niger.5) Buffer (pH 5. M. B. miehei C.4) Cyclohexane 110 113 115 117 C. erucyl. secondary and tertiary. primary alcohols (C1–C12). delemar. sugars. miehei C. linoleic. Torulopsis ernobii. lanuginosa. geraniol — Heptane 119 132 C. Penicillium M. docosahexaenoic. PPL (Rohm. C. methylvaleric C2 C2 C1–C8. tert-butanol. stearic. benzyl. etc. no. farnesol. miehei M. miehei G. niger. Myriococcum. 2-mercaptoethanol. miehei P. rugosa. R. isoamyl Solvent Hexane Reference 104 C12. C4. sorbitol. miehei. etc Glycerol — Buffer + casein 106 108 Water 109 Geraniol. P. phytol. viscosum (in MBG) Oleic. M. M. C4 Oleic Oleic. C. cyclohexanol. etc. roqueforti. etc. rugosa M. fluorescens C. cyclopium A. C6 — Primary straight-chained. P. etc. Alcaligenes M. Pseudomonas. sec-. branched acids. fluorescens C. candidum. etc. R. R. rugosa C. C3–C6. H. rugosa. oleic. fluorescens. oligosporus R. Ethanol — β-Phenethyl. Cholesterol Cholesterol C6 C2 Pentane 144 Heptane Buffer (pH 5. diglycerol.

Sawant. tetrahydrofuran. etc. (119). rugosa. Polyesters. which are excellent substitutes for diesel fuel (112). fragi (PEG-modified) M. etc. submitted for publication). Penicillium cyclopium. soaps. antarctica M. diglycerol 2′-Deoxyuridine Ethyl D-glucopyranoside Sugar acetal Glucose Sorbitol Isosorbide Glycerol Benzene — Hexane Chloroform. Butyl oleate and butyl laurate are also used as plasticizers. All can be prepared efficiently with lipases from M.N. C. Apart from functioning as emulsifiers. lubricants. Aspergillus. Polymers of dimethyl terephthalate. Benzyl benzoate and benzyl salicylate are fixatives for artificial musks. Such glycerides are frequently produced by transesterification by means of lipases. rugosa M. miehei P. Neopentyl polyol esters are important as high-temperature lubricants (113). Sugar ester preparation with lipases has been studied by Adelhorst et al. no. monoglycerides (as also di. sugar esters (115–117) and fatty acyl amino-esters. (114) used porcine pancreatic lipase to catalyze polycondensations to prepare both a series of achiral polyesters from simple alkanedioates and diols and an optically active epoxy-substituted polyester with 95% stereochemical purity. Mukesh. such as 3-stearoyl D-glucose.118). the lipase-catalyzed alcoholysis of oils with methanol and ethanol results in methyl and ethyl esters. geraniol. candidum. Lipases can be used in the synthesis of monoglycerides such as monolaurin.and triglycerides).109. geraniol. dimethyl formamide. miehei R. miehei. Thus. phytol. are mainly used for their flavor and aroma qualities. Candida M. Butyl oleate has applications in flavors (105). and coatings. etc. miehei C.4) DMSO. lacquers. lipases exhibit a much broader substrate spectrum (100. etc.110). Terpenyl esters are used in fragrance preparations (108). Diisodecyl phthalate and ditridecyl phthalate are used in paints. It may also be used in fruit flavors in conjunction with other low-boiling esters (106). DMF — — tert-Butanol — — Hexane 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 DMSO: dimethyl sulfoxide. Joshi. For instance.B. Morrow et al. S. are used in plastics. THF. etc. G. G.. miehei M. R. candidum. linoleic. foods. (99. are also used as base material for edible films or edible lubricants for food processing. ethylene dichloride — — — Acetone/water (1:1) Buffer (pH 5. R. The consequence of this phenomenon is reflected in an application potential for lipases that is much greater than for any other known enzyme! Low. M. G. antarctica Candida M. are useful as bread-softening agents. Whereas esterification refers mainly to the reaction of an acid with alcohol. which find use as food emulsifiers (101). DMF. miehei C. thiol esters. miehei a 627 Acid C4 C12 C2–C5 C4 C4 High free fatty acid rice bran oil Oleic. and laminates. such as O-acyl-L-homoserine. viscosum.101). etc. Another use of such esters is for fuel. (Gandhi. Ethyl butyrate is known to impart a pineapple flavor (107). Certain volatile esters form bases for perfumes. Pseudomonas (PS-30). coatings. delemar. such as geranyl acetate. fatty amides. such as lipase from P. P. peptides. rugosa. such as those derived from octanoic and decanoic acids. 6 (1997) . farnesol. as in salad dressings (12. etc. miehei C. vinyl acetate. (111) reported a process for the preparation of polyol fatty acid esters with mixed groups for cosmetics. cosmetics. mainly for apricot and peach flavors (for fatty-oily notes).103). etc. fluorescens C. Vol. miehei. Solvent Heptane Heptane Reference 203 204 Citronellol. etc. (102. N. Geraniol Geraniol Glycerol Glycerol Acetone-glycerol Glycerol Glycerol Polyglycerol. and D. these esters find widespread use in perfumes. 74. butyl laurate is a component of flavor compositions. M. isoamyl butyrate and benzyl propionate.B.105).117. Sugar acyl esters. Mucor sp. Such esters/monomers of polyesters may be synthesized with lipases. miehei C. Miyamoto et al. J.REVIEW TABLE 2 (continued) Lipase M. Penicillium. Low-molecular weight esters (104. Foods. enabling them to catalyze the synthesis of sugar esters. adhesives. oleic C14 Methyl caprate Oleic Oleic n-3 fatty acid concentrate Alcohol Isoamyl Menthol. arrhizus. fragi. fluorescens (120). pharmaceuticals. butyl acrylate. Pseudomonas (CES).and medium-molecular weight esters. Such biosurfactants are used to stabilize emulsions.. nodosus Aspergillus P. candidum. linolenic C14–C18 C10 C8–C16 Oleic Caproic anhydride C8–C18. For instance. Applications of lipases and/or esterases in ester synJAOCS.

patented by Frito-Lay (Dallas. Lipases may be used for kinetic resolution of racemic alcohols. and 3-methyl benzyl alcohols by esterification with vinyl esters in toluene or by alcoholysis of the corresponding acetate in n-butanol with microbial lipases (134). may also be synthesized with lipase (136). allowing for the synthesis of peptide precursors of penicillin G and other penicillin analogs from starting materials . sausages. Some of these products that are on the production threshold include (127): (i) olestra—mixture of hexa-.and octaesters. household cleaning products. pharmaceuticals. Optically active natural products. such as menalone-lactone.. which is an important ingredient for chocolates and confectioneries. Along with fatty esters. TX). fluorescens for the preparation of a chiral building block. etc. DE) and CPC International (Englewood Cliffs. such as those from C. napus lipases can be used to selectively enrich the medium with a particular acid. skin preparations. such as γ-linolenic and docosahexanoic acid. Pharmaceuticals and cosmetics. Lipase-catalyzed alcoholysis can be employed in the production of fatty acid alkyl esters that are valuable intermediates in oleochemistry. These esters are useful as intermediates for vitamins. and is a triglyceride esterified with caprylic. whipped toppings. has a good texture (126). miehei. etc. the key intermediate in the synthesis of gypsy moth hormone (133). (121). salad oils. Azuma and Minamii (139) have obtained a patent for manufacture of optically active terphenyl derivatives that can be used as agrochemicals and intermediates for electric materials. 1. Yamamoto and Oda (135) have reviewed certain applications of lipase-catalyzed asymmetric synthesis. NJ). Some examples from the literature are (i) kinetic resolution of 2. formed by esterifying sucrose with long-chain fatty acids. A patent for this process was granted to Kitatsume and Kokusho (138). di-. Transesterification of racemic α-alkyl-substituted primary alcohols is carried out to obtain separate optical isomers (141) that may be used to manufacture antiinflammatory agents. M.3-epoxy-8-methyl-1nonanol. The ability of lipases to act on the amide/peptide bond (C-N) has already been mentioned. etc. unlike proteases. (145) reported the synthesis of this cocoa butter-like fat by Lipozyme (commercially available immobilized preparation of the M.50. etc. antibiotics. and patented by Procter & Gamble (Cincinnati. Mono-. Xier and Sakai (137) used the lipase from P. mayonnaise. capric and behenic saturated fatty acids. Oleyl monooleate is used in bath oils. toiletries. and produced by esterifying alcohols with tricarballyic acid. One such application is in the manufacture of a cocoa butter-equivalent from palm oil (144). makeup. Currently. hair preparations. binaphthol. endo.3-syn-diol. (vi) sorbitol fatty acid esters—developed by Pfizer (New York. no. NY). the fatty acid discrimination property of M. However. It is in great demand because it can substitute for various high-caloric fats and oils. which are not acted upon by these lipases (80). Other benefits offered by these products are high thermal stability. and hydroperoxides by enantioselective acylation. and triglycerides of octanoic and decanoic acids can act as dyes and perfume bases in cosmetics. pharmaceuticals. Fatty acid fractionation/selective extraction/isolation. as explained earlier (146. baked goods. NY). fatty hydroxamic acids. They are also known to dissolve gallstones in humans. prepared by esterification of hydroxyl amine and fatty acids.628 REVIEW thesis and transformation of acyl groups have been reviewed by Antizak et al. As mentioned earlier. and glycerides. pasta. miehei and B. topping sauces. Luck and Bauer (122). These are used in salad dressings. Margarine obtained by transesterification of hydrogenated fish oil with oils that contained unsaturated fatty acids. and Bauky (123). cardiotonics. OH). which allows for high-temperature applications. JAOCS. lipases can act on D-amino acids as well. Stereospecificity of lipases can be used to selectively transesterify particular enantiomers of chiral acids and/or alcohols (129–132) and in the asymmetric transformation of symmetrical compounds to obtain selectively the active isomers (12). Dairy industry and confectioneries.141–143). 6 (1997) Resolution of racemates. Peptide synthesis. Acidolytic reactions have been used in the manufacture of shortenings (125). 74. and (ii) resolution of racemic tricarbonyl chromium complexes of 2-methyl. Mukherjee (124) has discussed certain aspects of hydrolysis and esterification reactions for preparation of diverse products in food and nonfood industries. cosmetic creams and lotions. ice creams. are used as constituents of cosmetics. (iii) methyl glucose polyester—used in cheese. and the fact that the substitute is not absorbed by the body. sugar esters. etc. and pharmaceuticals. cyanohydrins. Vol. Optically active fluoride-containing polyesters are prepared by stereospecific esterification with Alcaligenes and Achromobacter lipases. mayonnaise. hepta. an important lipid product with immense potential for lipase action is the synthetic fat substitute. such as higholeic sunflower oil. Such a defined triglyceride property modification is essentially possible because of the substrate specificity/regioselectivity of lipases. and P.147). (iv) dialkyldihexadecylmalonate—ester of malonic and alkylmalonic acids. and patented by Curtice Burns Foods (Rochester. Chang et al. (128). (v) trialkoxytricarballylate—patented by CPC International. sauces.and exo-brevicomin. Menthyl salicylate is the sunscreen agent in many suntan preparations. The best-publicized use of lipase-catalyzed synthesis lies in the interesterification of fats to produce synthetic triglycerides with desirable characteristics (13. miehei lipase)-catalyzed reaction between cottonseed and olive oils. (vii) caprenin—marketed by Procter & Gamble in 1992. (ii) esterified propoxylated glycerols—developed by ARCO Chemicals (Greenville. fluorescens. ice cream. Synthesis of intermediates for diverse uses. and can be employed in frozen desserts. viscosum. for resolution of racemic carboxylates (with polyethylene glycol as acceptor). 2-methoxy. Sorbitol and sugar monoesters of lauric and stearic acids possess antitumor and plant growth-inhibiting activity.

DISCUSSION More than 95% of the total production of technical enzymes is accounted for by the hydrolases—proteases. and other medical fields. and in processes where high enantio. such as foods. making the enzyme cost low in relation. this would shut out most if not all proposed reactions because their substrates are generally poorly soluble in water. Thorough understanding in these areas would enable the development of “tailor-made lipases” for specific applications in the long run and possibly open up newer vistas. and to the Council of Scientific & Industrial Research. Lipases and a number of other enzymes are active in organic media. The reasoning behind the concept of natural identity is that the metabolism of the compounds that enter the body or come in close contact (such as creams) is in terms of individual chemical species. it also enables facile recovery and reuse of the enzyme between batches and permits continuous operation. of factors affecting lipase activity and stability (Gandhi. and dehydration.. Apart from this. 74. such as oxidation. this is astonishing—a state of affairs that needs to be rectified! Despite the enormous potential for lipase catalysis in industry and research. i. and it would not be surprising if lipases take the top position in the enzyme application area. regardless of whether the source is natural or not. These species can have possible health implications. which are highly nonspecific with respect to the types of reactions they catalyze.e. The second major problem that is perceived is the instability of enzymes. S. lipase technology is chiefly restricted to those operations where the cost of the product is high. J. The latter is a problem frequently faced with the high temperatures and/or mineral acid catalysts. Considering the extremely high versatility of lipases (with respect to the diverse substrates and reactions they catalyze). no. natural ingredients are in high demand.N. prospects for the future are bright. Apart from its effect on stabilization. Thus. or that may entail a number of side-reactions. Hence. manufacture of various products via lipase catalysis will prove to be a boon to industry. The same is true for lipases. either in the raw state or after processing for human consumption” (151). Joshi. ACKNOWLEDGMENTS The author is grateful to Prof. India. N. as evident from the detailed descriptions presented above. the International Organization of the Flavor Industry defines the natural flavoring ingredients as “those obtained by appropriate physical. so that peptide bonds are not broken right after being made (12. Therefore. This is not possible with proteases. and lipases (103).N. S. carbohydrases. 150). Joshi and Dr. enzymatic or microbiological processes from material of vegetable or animal origin. The other opportunity for lipase applications is in aroma and flavor industries. This is easily manipulated by the use of organic media and/or enzyme immobilization (Gandhi. submitted for publication. such as phospholipids. which prevents translation of potential into actual application. Many of the synthetic applications of this enzyme would be out of the question in aqueous media. As of now. necessitates the reversal of the thermodynamic equilibria. Vol. perfumes and cosmetics.B. synthesis. there is the widespread belief that enzymes require aqueous media for activity. Sawant for their help during the preparation of this manuscript. the actual tapping of this potential has been limited. as is possible in organic media. This results in increased cost effectiveness of the entire process because enzyme cost is a major contributor to process economics. J.B. 6 (1997) . Another advantage of using lipases in protein synthesis is that they do not possess amidase activity.100). This. It would not be an exaggeration to say that actual application of this technology is negligible compared to what is possible! This discrepancy is mainly due to problems and suspicions that may or may not be real. pH. and lipases account for a meager 5–10%. Mukesh. In fact. Sawant.. Factors such as immobilization and organic solvents also affect the catalytic efficiency of lipase with respect to its substrates. While the first accounts for almost 60–70% of the technical market. the work of Zaks and Klibanov in the 1980s (148) has shown that this belief is highly misplaced.B. 149). Joshi. Thus. this technology is applicable to high-value fine chemicals. For lipases.. 149. and last but not the least.B. the reversal of hydrolysis. the mechanism by which these factors influence the lipase three-dimensional structure and/or its ability to catalyze reactions. which is possible by the maintenance of low water activity. Mukesh. lipase employment may be attractive in processes that involve thermolabile substrates/products. racemization. However. and D. It also solves the third major problem associated with enzymes. for the research fellowship awarded. dairy. etc. submitted for publication. pharmaceuticals. 99.B. ingredients prepared by using whole cells and/or enzymes may command substantially higher prices (104)! This makes biocatalysis quite attractive.and L-configurations. Sawant. Compounds produced from natural substrates by biological synthesis are accepted as natural (152). JAOCS. compared to the other hydrolases (100). N. and this may be different for different substrates. 25% goes to the carbohydrates. Therefore. while the same materials produced chemically are not. both of which reduce drastically the vulnerability of lipases to deactivation (by heat. Immobilization of enzymes may in fact be considered a panacea for most ills that prevent widespread use of enzyme technology. calls for a greater understanding of all aspects of lipase production and upstream and downstream processing. The production of various ingredients by natural processes assumes great significance in these fields. and D. J.). which will enable greater production owing to finding higher-yielding strains and the new-found ability to manipulate (microbial) genomes. however. This finding has heralded the beginning of a new chapter in lipase technology.B.and/or regioselectivity is required. For instance. S. The same is also brought about by increasing production possibilities—thanks to an improved understanding of fermentation and downstream factors and to the strides made in genetic engineering.REVIEW 629 of both D.

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