Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants

For synthetic applications of proteases, such as for peptide coupling, a co mbination of high esterase and low amidase activities is required. While ac hieving such specificity has been a long-standing goal, the decreases in am idase activity achieved to date have often also been accompanied by decreas es in esterase activity. In the current study, a strategy of combined site- directed mutagenesis and chemical modification was applied to the serine pr otease subtilisin Bacillus lentus (SBL) to improve its esterase-over-amidas e specificity. Using the crystal structure of SBL as a guide, the N62, L217 , S166, and M222 active site residues were chosen for mutagenesis to cystei ne and subsequent modification by alkyl methanethiosulfonate reagents. An i nitial rapid, combinatorial screen of the chemically modified mutant enzyme s (CMMs) generated and, of their parent cysteine mutants, identified 25 pro mising candidates which were then subjected to detailed kinetic evaluations . Of these CMM and mutant enzymes, 20 exhibited an improvement, of up to 52 -fold, in esterase-over-amidase activity compared to the wild type (WT). Fu rthermore, these increased esterase-to-amidase ratios were not gained at th e expense of esterase activity, which was improved up to 3-fold higher than that of the WT in absolute terms. The general success of this approach is evident from the fact that, of the 25 CMMs and cysteine mutants evaluated, 19 displayed higher esterase activity than the WT, whereas only 3 had bette r than WT amidase activity.