E. Plettner et al., Modulation of esterase and amidase activity of subtilisin Bacillus lentus by chemical modification of cysteine mutants, J AM CHEM S, 121(21), 1999, pp. 4977-4981
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.