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Updated: May 9, 2026

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
Published on: January 16, 2016
Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg
Dragana Despotovic1, Ljubica Vojcic, Milan Blanusa
1RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
Abstract:
Enzyme promiscuity describes the ability of biocatalysts to catalyze conversions beyond their natural reactions. Enzyme engineering to promote side reactions is attractive for synthetic and industrial applications. For instance, a subtilisin Carlsberg protease variant (T58A/L216W) catalyzes in addition to its proteolytic activity the generation of peroxycarboxylic acids from corresponding esters in the presence of hydrogen peroxide. In the current study we used a semi-rational design approach to shift the specificity of subtilisin Carlsberg towards production of peroxycarboxylic acid. Among other identified amino acid substitutions, position Gly165 in the S1 binding pocket provided insights in subtilisin Carlsberg's promiscuity by promoting ester perhydrolysis. Catalytic constants of subtilisin Carlsberg for perhydrolysis of methyl-propionate, methyl-butyrate and methyl-pentanoate were increased up to 3.5-, 5.4- and 5.5-fold, respectively, while proteolysis was decreased up to 100-fold for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate (suc-AAPF-pNA).
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