Improve The Enantioselectivity Of Lipase A Of Bacillus Subtilis By Site-directed Mutagenesis

An important feature of lipases is the fact that they exhibit good to excellent enantioselectivity. They can be used in direct asymmetric (trans)esterifications, hydrolysis and synthesis. In addition, they have the preference to react with one enantiomer of racemic mixtures. However, there are few lipases that have attractive industrial potentials because the enantioselectivity of most natural lipases are very low. Protein engineering has been used to change the characters of lipases for many years. Furthermore, as the knowledge of the structure and catalysis mechanism of lipases is known, with the development of directed evolution and in vitro high-throughput screening and computational design, more and more lipases with new characters have been exploited and applied.We had cloned, expressed and characterized Bacillus subtilis lipase A (LipA). LipA has a small molecular mass (19.3 kDa), and contains 181 amino acid residues. LipA represents one of the few examples of lipase that does not show interfacial activation in the presence of oil-water interfaces. The absence of interfacial activation , together with its small size, suggests that LipA does not have a lid. The active site residue Ser is located in the consensus sequence Ala-X-Ser-X-Gly where the alanine replaces a glycine found in most of the bacterial lipases. LipA was classified as a lipase rather than an esterase, because it is able to hydrolyse sn-1 and sn-3 glycerol esters with long fatty acid chains. Its highest activity is on glycerol esters with medium-length (C8) fatty acid chains. LipA contains putative N-terminal signal sequences of 31 amino acid residues. The X-ray structure of LipA had been reported The second B. subtilis lipase, LipB, consists of 182 amino acid residues, and is 74% identical with LipA, was reported by Eggert in 2000. The conserved pentapeptide of LipB is also Ala-X-Ser-X-Gly. To investigate a potential function of this structural motif, LipB variant A76G was construted by site-directed mutagenesis thereby restoring the canonical lipase consensus motif by Eggert and coworkers. The variant lipase displayed a marked temperature sensitivity at 45℃, with maximum activities against p-nitrophenyl-esters with chain lengths of C8 and C14 and against the triacylglyceride tricaprylin (C8:0). However, the most obvious difference in catalytic activities of wild-type LipB and A76G were found when monoolein was used in a monolayer assay. The specific activity of variant A76G was at least 1000-fold higher than that of the wild-type LipB. To investigate whether Ala residue at position 75 is essential for LipA, its variant A75G was constructed. The variant A75G was found to be much unstable and 35% activity lower than that of the wild-type LipA. However, other details were not been reported. As the homology of LipA and LipB is very high and the core of the LipB molecular are mostly identical to those of LipA, the replacement of alanine to glycine of LipA was thinked to influence the enantioselectivity. In this report, the LipA variant A75G was constructed by site-directed mutagenesis. LipA and variant A75G were used in the resolution reaction. The results showed that the enantioselectivity of variant A75G was higher than LipA when catalyzed the resolution of racemic mandelic acid . The enantiomeric ratio (E) improved from 4.9 to 9.7.Although the enantioselectivity of A75G was improved, the E value was still low for an acceptable resolution. Improving enantioselectivity based on the structure-function relationship was a convenient and reasonable method. According to the crystal structure of LipA, the residue Asn18 might contribute to the enantioselectivity. Saturation mutation in this site and high-throughput screening based on GC was used to screen the mutants which had good enantioselectivity. The best mutant, A75G/N18D, was obtained. The E value of A75G/N18D was 19.1. To comparison the contribution of every residue, the mutant LipA/N18D was constructed. The E value of N18D was 15.4, lower than that of A75G/N18D. These results indicated that the residues A75 and N18 were important for the enantioselectivity.The wild-type LipA and variants were characterized. In addition, the computer simulation was carrying out to elucidate the reaction mechanism of enzymes and substrates.