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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 screening 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 succinyl-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.
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Citations
Cited by
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Packer, M. S., & Liu, D. R. (2015) · Nature Reviews Genetics · doi:10.1038/nrg3927 · Cited by 991
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Stepankova, V., Bidmanova, S., Koudelakova, T., Prokop, Z., Chaloupkova, R., & Damborsky, J. (2013) · ACS Catalysis · doi:10.1021/cs400684x · Cited by 659
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Biswas, S., Khimulya, G., Alley, E. C., Esvelt, K. M., & Church, G. M. (2021) · Nature Methods · doi:10.1038/s41592-021-01100-y · Cited by 422
Zaccolo, M., & Gherardi, E. (1999) · Journal of Molecular Biology · doi:10.1006/jmbi.1998.2262 · Cited by 200
Highlights in Biocatalysis – Historical Landmarks and Current Trends
Bornscheuer, U. T., & Buchholz, K. (2005) · Engineering in Life Sciences · doi:10.1002/elsc.200520089 · Cited by 80
Metadata
- Authors
- You, L., & Arnold, F. H.
- Venue
- "Protein Engineering, Design and Selection"
- Publisher
- Oxford University Press (OUP)
- Published
- 1996
- Volume
- 9
- Issue
- 1
- Pages
- 77-83
- ISSN
- 0269-2139 (ISSN-L)
- 1741-0126
- 1741-0134
- OpenAlex
- W2106861615
- Cited by
- 277
- References
- 0
- FWCI
- 4.33
- Type
- journal article
- Language
- en
- Open access
- closed
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- You, L., & Arnold, F. H. (1996). Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. "Protein Engineering, Design and Selection". https://doi.org/10.1093/protein/9.1.77
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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