Directed evolution of a subtilisin Carlsberg variant towards improved perhydrolytic activity for industrial application

Adapting biocatalysts to non-conventional substrates and environments is of high academic and industrial interest. Directed evolution and rational design methods offer opportunities to tailor protein properties by generating diversity on gene level and screening for new variants on the protein level. Subtilisin Carlsberg is one of the first described serine proteases from the subtilisin family. Due to its stability under production conditions and broad specificity, this enzyme has become industrially important, especially in the detergent industry. Subtilisin Carlsberg also catalyzes ester perhydrolysis, generating peroxycarboxylic acids, compounds well known as bleaching and disinfection agents. The bottleneck for most experiments focused on finding and improving peroxycarboxylic acid producing enzymes is the availability of a medium and high-throughput screening system. To overcome this obstacle, a sensitive, fluorescent assay with detection limit below 1 µM of peroxyacetic acid and suitable for detection of perhydrolytic activity without previous enzyme purification was developed. The assay was optimized for screening in 96 and 384-well microtiter plate system, with the possibility to further upscale throughput by using flow cytometry screening technology. The screening platform was developed in this work to improve protease and likely other hydrolases (e.g. lipases or esterases) having perhydrolytic activity or find new enzymes with peroxycarboxylic acid production by screening of metagenome libraries. Subtilisin Carlsberg was furthermore the first protease engineered through directed evolution towards increased perhydrolytic activity which offers novel opportunities for bleaching applications. The protease variant, T59A/G166L/L217W, identified in the current work showed a 9.4-fold increased specificity constant of perhydrolytic activity compared to the wild type. Position 166 is part of the substrate binding pocket and changes in the side-chain volume at this position were found to modulate substrate ...