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Published January 1996 | public
Journal Article

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Abstract

Sequential rounds of error-prone PCR to introduce random mutations and screenrng of the resultant mutant libraries have been used to enhance the total catalytic activity of subtilisin E significantly in a non-natural environment, aqueous dimethylformamide (DMF). Seven DNA substitutions coding for three new amino acid substitutions were identified in a mutant isolated after two additional generations of directed evolution carried out on 10M subtilisin E, previously 'evolved' to increase its specific activity in DMF. A Bacillus subtilis-Escherichia coli shuttle vector was developed in order to increase the size of the mutant library that could be established in B.subtilis and the stringency of the screening process was increased to reflect total as well as specific activity. This directed evolution approach has been extremely effective for improving enzyme activity in a non-natural environment: the resulting-evolved 13M subtilisin exhibits specific catalytic efficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilide in 60% DMF solution that is three times that of the parent 10M and 471 times that of wild type subtilisin E. The total activity of the 13M culture supernatant is enhanced 16-fold over that of the parent 10M.

Additional Information

© 1996 Oxford University Press. Received September 5, 1995; accepted on October 14, 1995. The authors are grateful to Jeffrey Moore and Huimin Zhao for their assistance with the modeling and Figure 3. This work was supported by the US Department of Energy's program in Biological and Chemical Technologies Research within the Office of Industrial Technologies, Energy Efficiency and Renewables.

Additional details

Created:
August 20, 2023
Modified:
October 25, 2023