DOI 10.1515/hsz-2012-0210 Biol. Chem. 2012; 394(1): 79–87 Ljubica Vojcic a , Dragana Despotovic a , Karl-Heinz Maurer b , Martin Zacharias, Marco Bocola, Ronny Martinez and Ulrich Schwaneberg* Reengineering of subtilisin Carlsberg for oxidative resistance Abstract: Mild bleaching conditions by in situ production of hydrogen peroxide or peroxycarboxylic acid is attrac- tive for pulp, textile, and cosmetics industries. The enzy- matic generation of chemical oxidants is often limited by enzyme stability. The subtilisin Carlsberg variant T58A/ L216W/ M221 is a promiscuous protease that was improved in producing peroxycarboxylic acids. In the current article, we identified two amino acid positions (Trp216 and Met221) that are important for the oxidative resistance of subtilisin Carlsberg T58A/L216W/ M221. Site-saturation mutagenesis at positions Trp216 and Met221, which are located close to the active site, resulted in variants M4 (T58/W216M/ M221) and M6 (T58A/W216L/M221C). Vari- ants M4 (T58/W216M/ M221) and M6 (T58A/W216L/M221C) have a 2.6-fold (M4) and 1.5-fold (M6) increased oxidative resistance and 1.4-fold increased k cat values for peroxycar- boxylic acid formation, compared with wild-type subtili- sin Carlsberg. Keywords: oxidation; peroxycarboxylic acid; promiscuity, protease; protein engineering. a These authors contributed equally to this work. b Present address: AB Enzymes GmbH, Feldbergstraße 78, D-64293 Darmstadt, Germany *Corresponding author: Ulrich Schwaneberg, Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany, e-mail: u.schwaneberg@biotec.rwth-aachen.de Ljubica Vojcic: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany Dragana Despotovic: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany Karl-Heinz Maurer: International Research Laundry and Home Care, Biotechnology, Henkel AG & Co. KGaA, D-40191 Düsseldorf, Germany Martin Zacharias: Physics Department (T38), Technische University Munich, D-85748 Garching, Germany Marco Bocola: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany Ronny Martinez: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany Introduction Gentle bleaching under mild pH conditions can be achieved through in situ generation of low concentra- tions of peroxycarboxylic acids or hydrogen peroxide. Mild bleaching is especially attractive for cosmetics, therapeutics, washing, and disinfection applications (Rüsch gen. Klaas et al., 2002; Wieland et al., 2009). Per- oxycarboxylic acid with a concentration around 1.5 mm is sufficient for bleaching in liquid detergent formula- tions. The common bleaching agent hydrogen peroxide is produced at high local concentrations by spontaneous decomposition of percarbonate and perborate combined with tetraacetylethylenediamine or nonanoyloxybenze- nesulfonate (Wieland et al., 2009). Because of the hydro- gen peroxide production conditions, it is not possible to maintain a low and constant level for mild bleaching. Peroxycarboxylic acids are more desirable in cleaning compositions because they are strong oxidizing agents with superior performance compared with hydrogen per- oxide (Swern, 1949). Enzymatic in situ production of per- oxycarboxylic acids by the hydrolysis of esters or amides in the presence of hydrogen peroxide is used in appli- cations requiring mild bleaching conditions (Hofmann et al., 1992). Enzymatic generation of chemical oxidants is, in general, limited by the oxidative inactivation of the enzyme producer (Jori et al., 1968; Stauffer and Etson, 1969; Omenn et al., 1970; Kuroda et al., 1975; Simat and Steinhart, 1998). Among the 20 amino acids, Met, Cys, and Trp are most prone to oxidation (Dakin, 1906), espe- cially if they are located close to the active site (Estell et al., 1985). Systematic studies on the resistance of enzymes toward hydrogen peroxide were performed, for example, for chymotrypsin (Stauffer and Etson, 1969), lysozyme, ribonuclease A (Jori et al., 1968), and subtilisin from Bacillus amyloliquefaciens (Estell et al., 1985). Various mechanisms for understanding oxidative resistance have been proposed: (i) electronic changes such as sul- foxidation of Cys or Met close to or in the active sites and (ii) conformational changes in the protein structure. These conformational changes can be caused by the formation Brought to you by | RWTH Aachen Authenticated | 134.130.177.132 Download Date | 2/22/13 2:16 PM