Lipase substrate specificity

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

The i-poly(3HB) depolymerase of R. rubrum is the only i-poly(3HB) depolymerase that has been purified [174]. The enzyme consists of one polypeptide of 30-32 kDa and has a pH and temperature optimum of pH 9 and 55 °C, respectively. A specific activity of 4 mmol released 3-hydroxybutyrate/min x mg protein was determined (at 45 °C). The purified enzyme was inactive with denatured poly(3HB) and had no lipase-, protease-, or esterase activity with p-nitro-phenyl fatty acid esters (2-8 carbon atoms). Native poly(3HO) granules were not hydrolyzed by i-poly(3HB) depolymerase, indicating a high substrate specificity similar to extracellular poly(3HB) depolymerases. Recently, the DNA sequence of the i-poly(3HB) depolymerase of R. eutropha was published (AB07612). Surprisingly, the DNA-deduced amino acid sequence (47.3 kDa) did not contain a lipase box fingerprint. A more detailed investigation of the structure and function of bacterial i-poly(HA) depolymerases will be necessary in future. 

Second, esterases have broad (or even very broad) and overlapping substrate specificities. For example, carboxylesterase (EC 3.1.1.1) also catalyzes reactions characteristic of a number of other hydrolases. The discovery that individual isoenzymes of carboxylesterases may be identical to or closely related to acylglycerol lipase, acylcamitine hydrolase, and palmitoyl-CoA hydrolase (see Sect. 2.4.3) has increased the confusion surrounding esterase classification [59], Many esterases are able to hydrolyze amides, thiolesters,... 

A number of rat liver carboxylesterases identified by their pI values are listed in Table 2.6 [73] five nonspecific carboxylesterases were purified from rat liver and were characterized according to their p/ values [61]. They appeared to be isoenzymes, since they had similar substrate specificities toward phenyl and naphthyl esters and monooleylglycerol. Subsequent studies, however, revealed different specificities with respect to their physiological substrates. The pI 5.2 and 5.6 enzymes were shown to be acylcamitine hydrolases (EC 3.1.1.28), and a p/ 6.0 enzyme an octanoylglycerol lipase. The p/... 

Selected entries from Methods in Enzymology [vol, page(s)] Detergent-resistant phospholipase Ai from Escherichia coll membranes, 197, 309 phospholipase Ai activity of guinea pig pancreatic lipase, 197, 316 purification of rat kidney lysosomal phospholipase Ai, 197, 325 purification and substrate specificity of rat hepatic lipase, 197, 331 human postheparin plasma lipoprotein lipase and hepatic triglyceride lipase, 197, 339 phospholipase activity of milk lipoprotein lipase, 197, 345. 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

FI. Fielding, C. J., Human lipoprotein lipase. I. Purification and substrate specificity. Biochim. Biophys. Acta 206, 109-117 (1970). 

G3. Ganesan, D., Bradford, R. H., Ganesan, W., McConathy, W. J., Alaupovic, P., and Hazzard, W. R., Substrate specificity and polypeptide activation of postheparin plasma lipoprotein lipase in type III hyperlipoproteinemia (broad disease). CireukUion 46, Suppl. II, 248 (1972). 

K5. Kom, E. D., Clearing factor, a heparin-activated lipoprotein lipase. II. Substrate specificity and activation of coconut oil. J. Biol. Chem. 215, 15-26 (1955). 

An efficient method to prepare enantiomerically enriched hydroperoxides is the enzymatic kinetic resolution of racemic hydroperoxides using different kinds of enzymes (mainly lipases, chloroperoxidase, horseradish peroxidase). However, the scope of these reactions may be limit by the narrow substrate specificity of the enzyme. 

Lipase has been used in organic solvents to produce useful compounds. For example, Zark and Klibanov (8) reported wide applications of enzymes to esterification in preparing optically active alcohols and acids. Inada et al (9) synthesized polyethylene glycol-modified lipase, which was soluble in organic solvent and active for ester formation. These data reveal that lipases are very useful enzymes for the catalysis different types of reactions with rather wide substrate specificities. In this study, it was found that moditied lipase could also synthesize esters and various lipids in organic solvents. Chemically moditied lipases can help to solve today s problems in esteritication and hopefully make broader use of enzymatic reactions that are attractive to the industry. 

In the absence of this information, early attempts at rationalization of the experimental results were based on a detailed investigation of enzymatic substrate specificity. For instance, acylation of enantiomeric methyl glycopyranosides by different lipases was focused on the characterization of the reaction outcomes (percentage of the formed regioisomers after the complete disappearance of starting materials or after a fixed reaction time), and the results obtained were interpreted on the basis of the relative orientation of hydroxyls at C-2, C-3, and C-4 [97]. 

The location of the acyl chain is of primary importance in the binding process because of its size. Due to the movement of lid during interfacial activation, a hydrophobic trench is created between the lid and enzyme surface. The trench size is ideal to accommodate the acyl chain. Interactions between the non-polar residues of the trench and the non-polar acyl chain stabilize the coupling. It has been postulated that the configuration of the trench is responsible for substrate specificity. This hypothesis seems plausible since lipases usually discriminate against certain acyl chain lengths, degrees of unsaturation, and location of double bonds in the chain. Any of these factors could affect the interaction between the acyl chain and the trench. 

Porcine pancreatic lipase catalyzes the transesterification reaction between tribu-tyrin and various primary and secondary alcohols in a 99% organic medium (Zaks, 1984). Upon further dehydration, the enzyme becomes extremely thermostable. Not only can the dry lipase withstand heating at 100 °C for many hours, but it exhibits a high catalytic activity at that temperature. Reduction in water content also alters the substrate specificity of the lipase in contrast to its wet counterpart, the dry enzyme does not react with bulky tertiary alcohols. 

Various lipases and esterases have been used for the enantioselective esterification of alcohols and hydrolysis of esters. For example, Burkholderia cepacia lipases (PS, Amano Enzyme Inc.) and Candida antarctica lipase (CAL, Novozymes) have been widely used for its wide substrate specificities, high activities and chemo, regio and enantioselectivities. Fundamentals and some selected applications are shown in this section. The origins and abbreviations of lipases introduced here are as follows. 

Pancreatic carboxytester lipase, secreted by the pancreas as an active enzyme without proteolytic activation, displays broad substrate specificity and has therefore received many names in the literature carboxylesteraae, bile salt-stimulated (or activated or dependent) lipaae (due to its absolute requirement for bile salts to hydrolyze insoluble substrates), carbaxylester lipase or hydrolase, cholesterol... 

H, BrockerhofF, Substrate specificity of pancreatic lipase influence of the structure of forty adds cm the reactivity of esters. Bbdiin. Biophys. Acta 212 92 (1970). 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 19.1, Reaction 2). These enzymes also catalyze transesterilications and the desymmetrization of mew-substrates (vide infra). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities.160 In addition, they have enhanced stability in organic solvents, require no co-factors, and have a broad substrate specificity, which make them some of the most ideal industrial biocatalysts. Alteration of reaction conditions with additives has enabled enhancement and control of enantioselectivity and reactivity with a wide variety of substrate structures.159161164... 

Wang, C.-S., Kuksis, A., Manganaro, F., Myher, J.J., Downs, D., Bass, H.B. 1983. Studies on the substrate specificity of purified human milk bile salt-stimulated lipase. J. Biol. Chem. 258, 9197-9202. 

Some element reactions for BDF production can be applied widely to oil and fat processing. Since enzyme-catalyzed reactions proceed efficiently under mild conditions, they are suitable for the treatment of materials including unstable compounds. Furthermore, enzymes can convert only a desired compound to its other molecular form because of the strict substrate specificity compared with chemical catalysts. We hope that much attention will be focused on the superiority of enzyme, and that lipase reactions will be applied more and more as the practical process in the oil and fat industry. 

Specificity of lipases may be expressed in a number of different ways—substrate specific, regiospecific, nonspecific, fatty acyl specific, and stereospecific. Examples of these specificities have been presented by Villeneuve and Foglia (1997) (Table 10-6). 

To investigate the lipase enantiomeric specificity, HPLC was used to monitor the enantiomeric ratios of products 22C and 22A. From these analyses, 99% and 98% ee of ester products 22C and 22A, respectively, were obtained and shown to have -configuration. This revealed that lipase-catalyzed transesterification is an efficient resolution technique since only two products were selected from 20 p-nitroalcohol substrates. 

To investigate the lipase enantiomeric selectivities, chiral HPLC was used to monitor the enantiomeric ratios of the individual ester products 27A-E. All ester products, except the disfavored product 27A, were asymmetrically resolved by the lipase transesterification process. Interestingly, the highest enantiomeric ratio of the ester products was recorded for the preferred product 27D (83% ee). This indicated that not only the lipase substrate selectivity but also the lipase enantiomeric specificity could be controlled in the dynamic cyanohydrin system. 

Lipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non-aqueous media) [1], regioselective acylations of poly-hydroxylated compounds, and the preparation of enantiopure amino acids and amides [2, 3]. Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specificity, and exhibit, in general, a high enantioselectivity. All these features have contributed to make hpases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents. 

Enzymes are active in organic solvents at low water contents. Porcine pancreatic lipase in glycerin tributyrate (tributyrin) shows, for 0.015% water in the tributyrin—pentanol reaction mixture, a rate of transesterification comparable to the value in aqueous solution (Klibanov, 1986 Zaks and Klibanov, 1984). The water content of the protein in the above reaction mixture was 0.01—0.03 h. This is below the level expected for the onset of enzyme activity in protein—water powders. Nonaqueous solvents can produce change in the substrate specificity of an enzyme (Zaks and Klibanov, 1986 Zaks and Klibanov, 1988a) and possibly can lock the enzyme into a more active conformation (Russell and Klibanov, 1988). The dependence of the catalytic activity on added water has been measured for several enzymes in several solvents (Zaks and Klibanov, 1988b). 

The substrate specificity of lipase has been investigated in my laboratory. An ester such as a glyceride is likely to be hydrolyzed by nucleophilic attack ... 

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