Iterative multitarget evolution dramatically enhances the enantioselectivity and catalytic efficiency of Bacillus subtilis esterase towards bulky benzoate esters of dl-menthol

Abstract

Structure-based directed evolution has been successfully applied to BSE_V4, a variant of Bacillus subtilis esterase, for enantioselective hydrolysis of DL-menthyl esters with elevated thermostability. BSE_V4 displayed only 90% enantiomeric excess (ee) in preparing L-menthol from DL-menthyl benzoate, whereas the quadruple mutant BSE_V7, produced by iterative saturation mutagenesis, showed >99% ee. Partial deconvolution of BSE_V7 by generating the four respective single mutants indicated that each of them only improved the enantioselectivity slightly, implying pronounced synergy when the mutations act together. BSE_V9 and BSE_V11, produced by site-directed mutagenesis and random mutagenesis, respectively, showed 2.5- and 20-fold higher activities than BSE_V7 while retaining high E-values. With merely 0.1 g L⁻¹ of BSE_V11 loading (cell-free extract), 130 g L⁻¹ of DL-menthyl benzoate was asymmetrically hydrolyzed within 6 h, resulting in an enantiopurity of >99% ee and a space-time yield of 138 g L⁻¹ d⁻¹. This suggests that it is feasible to establish an efficient bioprocess by simultaneously addressing multiple issues of biocatalyst performance using the iterative multi-target evolution approach.