Lipase stereospecificity

Zandonella G, Haalck L, Spener F, Faber K, Paltauf F, Hermetter A (1995) Inversion of lipase stereospecificity for fluorogenic alkyldiacyl glycerols. Effect of substrate solubilization. Eur J Biochem 231 50-55... 

Uzawa, H., Noguchi, T., Nishida, Y., Ohrai, H., and Meguro, H. 1993. Determination of the lipase stereospecificities using circular dichroism (CD) lipases produce chiral di-O-acylglycerols from achiral tri-O-acylglycerols Biochim. Biophys. Acta(-Lipids and Lipid Metabolism), 1168, 253-260. 

Fig. 10. Lipase. Stereospecific transesterification of methyl trans- -phenyl glycidate, precursor of the side chain of Taxol [135]...

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

H. Uzawa, Y. Nisbida, H. Ohrui, H. Meguro, A New Approach to Determine the Stereospecificity in Lipase Catalyzed Hydrolysis Using Circular Dichroism (CD) Lipases Produce Optically Active Diglycerides from Achiral Triglycerides , Biochem. Biophys. Res. Commun. 1990, 168, 506-511. 

Lipase catalysed hydrolysis of racemic esters of the important chiron solketal , 1, 2-0-isopropyhdene glycerol, are not very stereospecific due to the fact that they are primary esters. Secondary esters usually show much higher -values. Table 2.1 shows -... 

Lipases still offer the potential for an important range of applications since they are able to carry out the reactions of esterification, transesterification (acidolysis or alcoholysis), inter-esterification, or hydrolysis, often with high specificity or selectivity, suitable for the production of high-added-value molecules as shown in Example 1 above (stereospecific alkylation, acylation, or hydrolysis for the resolution of racemic mixtures of acids, alcohols or esters). 

Other Acylglycerols. If some of the DGs in freshly drawn milk are involved in biosynthesis, it is possible that they are enantiomeric and are probably the sn-1,2 isomer. If so, the constituent fatty acids are long chain. Their configuration can be determined by stereospecific or other analyses, but it is difficult to accumulate enough material for analysis. Nevertheless, Lok (1979) isolated the DGs from freshly extracted cream as the trityl derivatives. Trityl chloride reacts selectively with primary hydroxyls. The stereochemical configuration of the DGs was identified as sn-1,2 therefore, these residual DGs were most likely intermediates of biosynthesis. If the DGs were products of lipol-ysis, they would be a mixture of 1,2/2,3 isomers in a ratio of about 1 2, since milk lipoprotein lipase preferentially attacks the sn-1 position of TGs (Jensen et al. 1983). 

Enzymes can be stereospecific, and lipases as esterases can act as very efficient catalysts even on molecules which ester groups are not glycerides. 

The stereospecificity may be carried, either by the carboxylic acid moiety, or by the alcohol part of the molecule. There is no rule up to now to predict if a given molecule will be a substrate, and if the enzyme will express its stereospecificity toward it. Screening of lipases and esterases is the only method ta select firstly the active enzymes, and secondly the specific ones that give the wanted isomer. 

Rantakyla, M. Alkio, M. Aaltonen, O. Stereospecific Hydrolysis of 3-(4-Methoxyphenyl)glycidic Ester in Supercritical Carbon Dioxide by Immobilized Lipase. Biotechnol. Lett. 1996, 18, 1089-1094. 

The lipase enzyme stereospecifically hydrolyzes the (+) isomer of naproxen ester. The enzyme is immobilized in the wall of an inside-skinned hollow fiber membrane. The racemic d and / naproxen ester mixture, dissolved in methyl isobutyl ketone, is introduced on the shell side of the fiber and an aqueous buffer solution is circulated through the fiber lumen. The lipase enzyme hydrolyzes the d form of naproxen ester, forming ethanol and naproxen d. Naproxen d is a carboxylic acid soluble in aqueous buffer but insoluble in methyl isobutyl ketone. Consequently naproxen d is removed from the reactor with the buffer solution. The naproxen / ester remains in the methyl isobutyl ketone solution. This technique achieves an essentially complete separation of the d and Z forms. In a clever final step... 

Thus, description of a simple change of fatty acid at the primary position required frequent changes of configuration prefixes. Also, the R/S system, like the older D/L one, did not account for the stereospecificity of the acylglycerol derivatives toward lipases (phospholipase A2 in particular). Finally, nonrandom distribution of fatty acids in natural or synthetic enantiomeric acylglycerols could not be systematically correlated by reference to either the R/S or D/L configuration. 

Somerharju, P., Kuusi, T., Paltauf, F., Kinnunen, P.K.J. 1978. Stereospecificity of lipoprotein lipase is an intrinsic property of the active site of the enzyme protein. FEBS Letts 96, 170-172. 

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). 

Bohenin (BOB) is the name given to glycerol 1,3-behenate 2-oleate, which inhibits fat bloom when added to chocolate. It is produced in Japan by enzymic interesterification of triolein and behenic (22 0) acid or ester in the presence of a 1,3 stereospecific lipase. 

The major advantage of lipase-catalyzed reactions over those carried out with chemical catalysts lies in the fact that a wide variety of products having different composition and properties can be prepared, depending on substrate specificity or stereospecificity of the lipase used. Further advantages of lipases-catalyzed reactions include mild reaction conditions leading to reduced energy consumption and less thermal damage of reactants and products. 

If regio- or stereospecific lipases are used to interesterify oil blends, the products formed are different from those obtained by chemical interesterification, and may exhibit better functional properties. For example, interesterification of blends of canola and palm oils, using the in-1,3-specific Rhizopus delemar lipase as a biocatalyst, gave oils with improved fluidity compared with the original blends or chemically interesterified products. 

A process utilizing a stereospecific ester cleavage yielding the product l -glycidylbutyrate in high optical purity has recently been scaled to commercial levels (63). The process catalyzed by porcine pancreatic lipase is shown in Fig. 19. R-glyddylbutyrate is a useful chiral synthon for a variety of commercial products, for example, p-blockers. 

On the other hand, the specificities of hPL and RmL are very low. RmL strongly favors positions 1 and 3 (rather independent of the type of the fatty acid) in triacylglycerols. To date it is the only lipase that has been crystallized in a complex with an inhibitor containing a short aliphatic chain (Brzozowski et al., 1991). However, the low resolution of the study does not allow for a detailed description of the molecular basis of substrate specificity. Also, no structure-based analysis of stereospecificity in lipases is possible results of purely chemical studies (Kaz-lauskas et al., 1991, and references therein Xie et al., 1990) fall outside the scope of this review. 

Lipases can hydrolyze triglycerides into fatty acids and glycerol. They have been used extensively to produce optically active alcohols, acids, esters, and lactones by kinetic resolution. Lipases are unique, in that they are usually used in two-phase systems. A classic example is the use of a lipase for the production of (5, / )-2,3-p-methoxyphenylglycyclic acid, an intermediate for diltiazem. In this process, methyl-/7-methoxyphenylglycidate is stereospecifically hydrolyzed by a lipase immobilized in a hollow fiber membrane reactor. The enzyme is located at the interfacial layer between an organic and an aqueous phase. 

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