Bacillus subtilis lipase A

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. 

Van PouderoyenG, E ertT, Jaeger KE, Dijkstra BW (2001) The crystal structure of Bacillus subtilis lipase a minimal alpha/beta hydrolase fold enzyme. J Mol... 

In a different ongoing study, a Bacillus subtilis lipase has been chosen as the catalyst in the asymmetric hydrolysis of the meso-diacetate 11 with formation of enantiomeric alcohols 12 (Fig. 11.19) [82]. This reaction does not constitute kinetic resolution and can thus be carried out to 100 % conversion. Screening is possible on the basis of the ESI-MS system [50] (see above) using the deuterium labeled pseudo-meso substrate 13 (Fig. 11.20). The ratio of the two pseudo-enantiomeric products 14 and 15 can easily be determined by integrating the two appropriate MS peaks. 

Esterases are much less tolerant of anhydrous media than lipases. The esterases from Bacillus stearothermophilus (BstE) and Bacillus subtilis (BsE) are exceptional, as these mediated transesterification in hexane at aw=0.1 [66]. Both esterases, if immobilized on Celite 560, mediated transesterification in [BMIm][BF4], [BMIm][PF6], and [BMIm][ Tf2N] at a rate that varied from 20 to 60% of the rate in hexane or ME. 

Apply a standard error-prone PCR (epPCR see Chapter 2) to the wild-type lipase gene from Bacillus subtilis and express conventionally in E. coli [37] initiate by inoculation of the cultures in deep-well microtiter plates (96-well format). Use LB/M9 medium with 100 pL carbenicillin (lOOmgmL-1) per 100 mL of medium and incubate for 5-6 h at 37 °C while shaking. 

A number of steroids have been regioselectively acylated in a similar manner (99,104). Chromobacterium viscosum lipase esterifies 5a-androstane-3p,17p-diol [571-20-0] (75) with 2,2,2-trifluoroethyl butyrate in acetone with high selectivity. The lipase acylates exclusively the hydroxy group in the 3-position giving the 3p-(monobutyryl ester) of (75) in 83% yield. In contrast, bacillus subtilis protease (subtilisin) displays a marked preference for the C-17 hydroxyl. Candida cylindracea lipase (CCL) suspended in anhydrous benzene regioselectively acylates the 3a-hydroxyl group of several bile acid derivatives (104). 

The first step in setting up a successful directed evolution protocol is the development of an efficient expression system using an appropriate bacterial host. This is not a trivial task, in particular when overexpression is to be coupled to enzyme secretion. Fortunately, some proteins can easily be overexpressed and secreted by using commercially available systems [27 - 29], a prominent example being subtilisin of Bacillus subtilis [30]. However, many enzymes of interest are not amenable to such systems examples include a variety of different lipases from Pseudomonas species. 

Fig. 11.19. Desymmetrization of a meso-substrate 11 catalyzed by a lipase from Bacillus subtilis [82],...

Pancreatic preparations have been widely used as digestive aids, because they contain proteases, amylase and lipase. They have been prescribed for patients who have pancreatic disorders or after removal of the pancreas. The various activities present in the pancreatic preparations can be duplicated by in vitro methods from blends of microbial enzymes derived from Bacillus subtilis, Aspergillus flavus-oryzae and Aspergillus niger. Cellulase derived from Aspergillus niger is often added to the microbial preparation. The pancreatic preparations still hold the major share of the market, but this could be a useful application for the right combination of microbial enzymes. 

Shi B, Wu W, Wen J, Shi Q, Wu S (2010) Cloning and expression of a lipase gene from Bacillus subtilis LS1403 in Escherichia coli. Ann Microbiol 60 399 04 Shi H, Shiraishi M, Shimizu K (1997) Metabolic flux analysis for biosynthesis of poly (/1-hydroxybutyric acid) in Alcaligenes eutrophus from various carbon sources. J Ferment Bioeng 84 579-587... 

Excellent yields were achieved in the selective acylation at 0-3 of 6-0-acelyl-l,5-anhydro-2-deoxy-D-arafe/no-hex-l-enitol (25) by lipase mediated acyltransfer from several vinylesters. As shown in Scheme 7, the starting material (25) could be recovered in 80% yield from two of the products (26) by enzymatic hydrolysis. Reports have been published on the lipase mediated selective synthesis of 2-functionalised 3-monoesters (27) of methyl 5-0-decyl-a-D-arabinofuranoside, on the regioselective, lipase-catalysed acylation of methyl a- and -D-arabino- and -xylo-pyranoside, on the regioselective acylation and deacylation of 2 -deoxynucleoside derivatives by use of a Pseudomonas fluorescens lipase and a Bacillus subtilis protease, respectively, and on the regioselective acylation of castanospermin with a variety of enzymes in pyridine. 

The enzymatic activity of lipases is very comparable to that of esterases, with the main difference being the chain length and hydrophobicity of the acid moiety of the substrate. Therefore in fine chemical applications, lipases and esterases are being used as alternatives for several conversions. For instance, for the kinetic resolution of 2-arylpropionic acids such as naproxen and ibu-profen, both a lipase and an esterase have been found that can perform a stereoselective hydrolysis yielding the pharmaceutically preferred enantiomer S-naproxen (Bertola et al. 1992 Hedstrom et al. 1993). High activity and ease of production have made the carboxylesterase from Bacillus subtilis Thai 1-8 the prime choice of industry (Quax and Broekhuizen 1994). 

Enzymes, ) derived from pancreatic extracts of slaughtered animals, were introduced already in 1913 into formulations of d. Only with the invention of proteolytic enzymes, derived from Bacillus subtilis or B. licheniformis, which are stable against alkali and higher temperatures, enzymes received a permanent position in d. Amylases are second in importance and attack starch-based soil. Lipases are used to remove remaining greasy soil. Cellulases are added in special d. to remove cotton fluffs and smoothen the surface of cotton fibers. 

Naproxen, (S)-2-(6-methoxy-2-naphthyl)propanoic acid 126 is a nonsteroidal anti-inflammatory and analgesic agent first developed by Syntex [220,221]. Biologically active desired S-naproxen has been prepared by enantioselective hydrolysis of the methyl ester of naproxen by esterase derived from Bacillus subtilis Thai 1-8 [222]. The esterase was subsequently clone in Escherichia coli with over 800-fold ipcrease in activity of enzyme. The resolution of racemic naproxen amide and ketoprofen amides has been demonstrated by amidases from Rhodococcus erythropolis MP50 and Rhodococcus sp. C311 (223-226). 5-Naproxen 126 and 5-ketoprofen 127 (Fig. 44) were obtained in 40% yields (theoretical maximum yield is 50%) and 97% e.e. Recently, the enantioselective esterification of naproxen has been demonstrated using lipase from Candida cylindraceae in isooctane as solvent and trimethylsilyl as alcohol. The undesired isomer of naproxen was esterified leaving desired S isomer unreacted [227]. 

All of the preceding techniques are extensively used for the production of detergent enzymes such that today the vast majority of industrial enzymes are produced by recombinant techniques. This is done in a limited number of optimized, well-known production hosts. Some of the most frequently used host organisms are the Bacillus species—B. subtilis, B. licheniformis, and B. clausii, which are used for production of proteases and amylases. The lipases and cellulases of fungal origin are produced by cultivation of the filamentous fungi Aspergillus oryzae and Trichoderma reesei. 

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