Other Lipases

The Bacillus subtilis lipase A (BSLA) was the subject of two short directed evolution studies [19,47]. In one case systematic saturation mutagenesis at all of the ISlpositions of BSLA was performed [19]. Using meso-l,4-diacetoxy-2-cyclopentene as the substrate, reversed enantioselectivity of up to 83% ee was observed. In another study synthetic shuffling (Assembly of Designed Oligonucleotides) was tested using BSLA [47]. 

The Bacillus subtilis lipase A (BSLA) is an unusual lipase because it lacks the so-called lid structural unit 13(3). Moreover, it is a small enzyme composed of only 181 amino acids. The initial results of an ongoing study are remarkable and illustrate the power of directed evolution 45,137). The desymmetrization of meso-, 4-diacetoxy-2-cyclopentene (15) was chosen as the model reaction, and the MS-based ee assay using an appropriately Ds-labeled substrate (Section III.C) provided a means to screen thousands of mutants. 

The WT lipase leads to an ee value of only 38% in favor of the (If ,45) enantiomer. The application of low-error epPCR increased the enantioselectivity slightly, but high-error rate epPCR turned out to be more successful, with several mutants showing ee values of 54-58% (45,137). The results are in line with the experience gained in the Pseudomonas aeruginosa lipase project (Section IV.A. 1). Of course, a library produced by high mutation rate can also contain hits that have only one amino acid exchange, and this was indeed observed in several cases. 

In this ongoing project, a different strategy was also tested, namely, systematic saturation mutagenesis at all of the 181 positions (45,137). In such an approach, all 181 mini-libraries are screened (each about 200 clones for the purpose of 

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Relevant heparin-binding enzymes not involved in the coagulation cascade are, for example, elastase, cathepsin G, superoxide dismutase, lipoprotein lipase and other lipases. The plasma clearing properties of heparin are associated with its binding to lipoprotein lipase and hepatic lipase when the enzymes are released from the surface of endothelial cells [11] and have been studied in view of a potential impact on the regulation of atherosclerosis. 

Imbalance in the stoichiometry of polycondensation reactions of AA-BB-type monomers can be overcome by changing to heterofunctional AB-type monomers. Indeed, IIMU has been subjected to bulk polycondensation using lipases as catalyst in the presence of 4 A molecular sieves. At 70 °C, CALB showed 84% monomer conversion and a low molecular weight polymer (Mn 1.1 kDa, PDI 1.9). No significant polymerization was observed with other lipases (except R cepacia lipase, 47% conversion, oligomers only) and in reference reactions with thermally deactivated CALB or in the absence of enzyme. Further optimization of the reaction conditions (60wt% CALB, II0°C, 3 days, 4 A molecular sieves) gave a polymer with Mn of 14.8 kDa (PDI 2.3) in 86% yield after precipitation [42]. 

A related approach is the enantiotopos-selective ester hydrolysis with PLE or other lipases of mejo-diesters 16-19 and of mesodiacetates 20-24. 

Purification of the Lipase Components. Two lipase components, designated A and B, respectively, have been purified from the crude C. antarctica lipase preparation by well-known chromatographic techniques (8). The crude lipase preparation may contain other lipases, but we consider lipases A and B to be the important ones. 

Two different types of enzymatic time-temperature integrators are described. The first, under the tradename of I-point, is based on a lipase-catalyzed hydrolysis reaction (125). The lipase is stored in a nonaqueous environment containing glycerol. The indicator contains two components that are mixed when the indicator is activated. The operating principle is as follows Upon activation, two volumes of reagents are mixed with each other. Lipase is thereby exposed to its substrate, here a triglyceride. At low temperatures there will be almost no hydrolytic reaction. As the temperature increases, hydrolysis accelerates and protons are liberated. A pH indicator is dissolved in the system. The indicator is selected to shift color after a certain amount of acid has been liberated by the enzyme-catalyzed process. Since the catalytic activity is influenced both by temperature and time, this indicator strip is said to be a time-temperature integrator. 

Lipases can be divided into two groups according to their positional specificity. Some lipases hydrolyze only the ester bonds in positions I and 3 of glycerol, i.e., the primary esters. This is true for pancreatic lipase (Table II). Other lipases, such as many microbial lipases, hydrolyze all three ester bonds. In this case, the primary esters are probably hydrolyzed faster than the secondary ester. 

Pancreatic lipase can be considered a model for all other lipases. Most if not all of these enzymes seem to be nonspecific carboxyl ester hydrolases of the serine histidine type. Their specificity consists, by definition, in their ability to hydrolyze insoluble substrates, but apart... 

A similar resolution has also been achieved on large scale <20040PD22>. The KR of racemic isoxazoline 312 catalyzed by enzymes was studied. The best result was obtained with lipase B from Candida antarctica (CALB), which hydrolyzed the ethyl ester of (—)-312 to the corresponding monoacid (—)-313. The reaction, which was run in 0.1 M phosphate buffer/acetone at room temperature, spontaneously stopped at 50% conversion to yield monoacid (—)-313 and the residual ester (- -)-312 with ees higher than 99% <2004TA3079>. The C-5 epimer of 312 underwent enantioselective hydrolysis (>99% ee) of the methyl ester linked to C-5 in the presence of the protease proleather (subtilisin Carlsberg), whereas CALB and other lipases were not able to resolve it (Equation 53). 

Selective deacylations of primary ester groups in the presence of secondary are also possible using enzymes (i.e., various lipases) [40]. Other lipases show selectivity for the anomeric position (Scheme 3.22). Also, as mentioned above, anomeric esters are more labile than other esters and can be removed selectively by mild base treatment. Furthermore, anomeric silyl ethers can be removed selectively on treatment with mild acid. 

The human pancreatic lipase is homologous to two other lipases playing pivotal roles in lipoprotein metabolism lipoprotein lipase and hepatic lipase. Together these three proteins constitute the human lipase gene family. 

The cDNA-derived amino acid sequences of human gastric lipase (Bodmer et ai, 1987) and rat lingual lipase (Docherty et al., 1985) show clear homology, with 78% identity. There are 379 and 377 amino acids, respectively, and three of the four putative glycosylation sites in the human enzyme were also found in the rat lingual protein. However, with the exception of the G-X-S-X-G consensus sequence motif, there appears to be no homology with any other lipase family. 

HSL cDNA from rat adipocytes was cloned and sequenced (Holm et al., 1988). The predicted protein is 757 amino acids in length and over 82.8 kDa in size. There is no homology with any other protein. The only sequence feature shared by this enzyme with other lipases is the G-X-S-X-G pentapeptide, which occurs within a stretch reasonably consistent with the /3-eSer-a motif. Thus, Ser-423, which is a part of this pentapeptide, is the likely nucleophile. Nothing is known about the other members of a catalytic triad, if indeed it exists in this enzyme. The regulatory serine that undergoes phosphorylation by cAMP-dependent kinase has been identified as Ser-563 (Holm et al., 1988). Nothing more is known about the molecular structure of this important enzyme. 

Enantiotopically selective ester hydrolysis can also be achieved enzymatically (Table 8). Eiflier one ester group in a me.ro-diester (103)-(1(M>) or one acetate in a me.m-diacetate (107) 111) are saponified with PLE or other lipases. In favorable cases enzymatic acylation and deacylation are stereochemically complementary and may thus be combined to gain access to bodi enantiomers, as illustrated by the example in Table 9. ... 

Even though CaLB is the lipase most widely used in ionic liquids, other lipases such as Pseudomonas cepacia lipase (PcL) and Candida rugosa lipase (CrL) have been often used as biocatalyst for ester synthesis in ionic liquid [13, 14]. Nara et al. 

The application of ionic liquids in lipase biocatalysis has not remained entirely restricted to CaLB, PcL or CrL. Other lipases have been used in ionic liquids for ester synthesis such as Candida antarctica lipase A (CaLA) [15,16], Thermomyces lanuginosus lipase [17] (TLL), Rhizomucor miehei lipase (PmL), Pseudomonas fluorescens lipase (PJL) [18], Pig pancreas lipase (PpL) [17] and Alcaligenes sp. lipase (A5 L) [16]. 

The storage stability of other lipases has been also analysed. For instance, the storage stability of PsL in hydrophobic ILs for a period of 20 days at room temperature, measured with the variation of the transesteriiication activity of this enzyme during transesterification of ethyl 3-phenylpropanoate with different alcohols, resulted in an increased yield of 62-98% in [bmim fNTfj ] and 45-98% in [bmim ] [PFg ], respectively, depending on the nature of alcohol used in the tiansesterifica-tion reaction. In these ionic liquids, the operational stability was also measured and found that the P.yL-IL mixture was recycled live times without any decrease in the transesterification activity [13]. 

Other lipases have also been tested in supercritical carbon dioxide for ester synthesis. In this context, the synthesis of terpinyl acetate from a-terpineol and acetic anhydride in supercritical carbon dioxide (scCO ) was carried out using five different lipases as catalysts (Candida rugosa type Vll, Amano PS, Amano AP-6,... 

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