Bacillus lipase

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]. 

Massing U, Eibl H (1994) Substrates for phospholipase C and sphingomyelinase from Bacillus cereus. In Woolley P, Petersen SB (eds) Lipases. Their structure, biochemistry and application. Cambridge University Press, Cambridge, p 225... 

Figure 8. Enzymatic preparation of (S)- and (R)-furyl methyl carbinol. TADH, Thermoanaerobium brokii alcohol dehydrogenase (NADPH was regenerated by glucose/glucose dehydrogenase from Bacillus cereus obtained from Amano.) CCL, lipase from Candida cvlindraceae ChE, cholesterol esterase from Pseudomonas.

Lipase, which is highly useful for kinetic resolution, however, has a limitation for use in DKR in that it carmot be used for (S)-configuration products. For this purpose, subtiHsin, a protease from Bacillus licheniformis, can replace lipase since it provides complementary enantioselectivity (Scheme 1.4). Subtilisin, however, has been much less frequently employed in resolution compared to lipase because it displays poor catalytic performance in organic media. Subtilisin is inferior to lipase in several properties such as activity, enantioselectivity and stability. Accordingly, the use of the enzyme usually requires some special treatments for activation and stabilization before use. For example, the treatment of subtilisin with surfactants has enhanced substantially its activity and stability up to a synthetically useful level. 

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. 

In addition to cutinases, various lipases, such as from C. antarctica, Candida sp. [13, 47], Thermomyces lanuginosus [2, 14, 15, 55, 56], Burkholderia (formerly Pseudomonas) cepacia [57] and esterases from Pseudomonas sp. (serine esterase) [58] and Bacillus sp. (nitrobenzyl esterases) [59, 60], have shown PET hydrolase... 

Other similar lipase/esterase resolution processes have been developed such as the use of Bacillus that esterase to produce the substituted propanoic acids that are precursors of non-steroidal anti-inflammatory drags, snch as naproxen and ibuprofen etc., and the formation of chiral amines by Celgene. Other methods start from prochiral precursors and have the advantage that enantioselective synthesis allows the production of particular isomers in yields approaching 100%, rather than the 50% yields characteristic of resolution processes. For instance Hoechst have patented the production of enantiomers using Pseudomonas fluorescens lipase to either acylate diols or hydrolyse diacetate esters. 

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. 

Oligonucleotide primers for amplification of the wild- type lipase gene of Bacillus subtilis... 

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). 

In addition to the conversion of dextran with organic acids after in situ activation, transesterification is an interesting synthesis tool for the introduction of sensitive carboxylic acid moieties, which is illustrated for the acrylation of dextran. Thus, dextran is acylated with vinyl acrylate in the presence of Proleather FG-F and lipase AY, a protease and lipase from Bacillus sp. and... 

Several psychrotrophic bacteria produce extracellular phospholipases, the most prevalent in milk being pseudomonads (particularly P. fluorescens), Alcaligenes, Acinetobacter, and Bacillus species (Fox et al., 1976 Owens, 1978a Phillips et al., 1981). Most of these produce phospholipase C, some produce phopholipase Ai and some produce both types (Deeth, 1983). Ser-ratia spp. have been shown to produce only phospholipase A (Deeth, 1983), while P. fragi has been reported not to produce phospholipases (Kwan and Skura, 1985). Phospholipase C from some pseudomonads has been purified and characterised (Doi and Nojima, 1971 Sonoki and Ikezawa, 1975 Stepa-niaketa/., 1987a Ivanov etal., 1996). Like the lipases, many of these enzymes have considerable heat stability and are not destroyed by pasteurization... 

Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM (2008) Thermostable Bacillus subtilis lipases in vitro evolution and structural insight. J Mol Biol 381 324—340... 

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. 

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. 

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

Bacillus thermocatenulatus lipase epPCR/SDM visual inspection 17-fold increase in phospholipase activity 39... 

Kauffmann, I., Schmidt-Dannert, C. (2001), Conversion of Bacillus stearother-mophilus lipase into an efficient phospholipase with increased activity towards long chain fatty acyl substrates by directed evolution and rational design, Protein Eng., in press. 

Bacillus stearothermophilus lipase 1J13 Aspa 19 HiSa 5 HisL 150 Asp2ba... 

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