Modulation of Esterase and Amidase Activity of Subtilisin Bacillus lentus by Chemical Modification of Cysteine Mutants

Abstract

For synthetic applications of proteases, such as for peptide coupling, a combination of high esterase and low amidase activities is required. While achieving such specificity has been a long-standing goal, the decreases in amidase activity achieved to date have often also been accompanied by decreases in esterase activity. In the current study, a strategy of combined site-directed mutagenesis and chemical modification was applied to the serine protease subtilisin Bacillus lentus (SBL) to improve its esterase-over-amidase specificity. Using the crystal structure of SBL as a guide, the N62, L217, S166, and M222 active site residues were chosen for mutagenesis to cysteine and subsequent modification by alkyl methanethiosulfonate reagents. An initial rapid, combinatorial screen of the chemically modified mutant enzymes (CMMs) generated and, of their parent cysteine mutants, identified 25 promising candidates which were then subjected to detailed kinetic evaluations. Of these CMM and mutant enzymes, 20 exhibit...