title: Redirecting catalysis from proteolysis to perhydrolysis in subtilisin Carlsberg.
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).
authors.affiliation: RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Dragana
authors.initials: D
authors.lastname: Despotovic
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vojcic
authors.forename: Milan
authors.initials: M
authors.lastname: Blanusa
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Martin
authors.initials: M
authors.lastname: Zacharias
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D000144:Acids, Acyclic / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004952:Esters / Q000378:metabolism; D019143:Evolution, Molecular; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering; D059748:Proteolysis; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000144:Acids, Acyclic; D001426:Bacterial Proteins; D004952:Esters; D013381:Subtilisins
keywords: Directed evolution; Enzyme promiscuity; Perhydrolysis; Protein engineering; Subtilisin Carlsberg