Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg

Dragana Despotovic, Ljubica Vojcic, Milan Blanusa, Karl Heinz Maurer, Martin Zacharias, Marco Bocola, Ronny Martinez, Ulrich Schwaneberg

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


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).

Original languageEnglish
Pages (from-to)279-286
Number of pages8
JournalJournal of Biotechnology
Issue number3
StatePublished - 10 Sep 2013
Externally publishedYes


  • Directed evolution
  • Enzyme promiscuity
  • Perhydrolysis
  • Protein engineering
  • Subtilisin Carlsberg


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