Directed evolution, enzyme performance

The use of molecular biology methods, described in Section 5.3 seems to be especially worthwhile as it offers novel possibilities of optimization on process adjustment. Directed evolution leads to the formation of new biocatalysts with improved characteristics (selectivity, activity, stability, etc.). Incorporation ofnon-proteinogenic amino acids makes it possible to reach beyond the repertoire of building blocks used by nature. The prospect of bioconjugate preparation offers the possibility to form functional clusters of enzymes and to perform multiple synthetic steps in one pot. 

Mathur, E.J., Toledo, G., Green, B.D. et al. (2005) A biodiversity-based approach to development of performance enzymes applied metagenomics and directed evolution. Industrial Biotechnology, 1, 283-287. 

For some recent reviews on enzyme modification see (a) Reetz, M.T., Evolution in the test-tube as a means to creat selective biocatalysts. Chimia, 2007,61, 100-103 (b) Sylvestre, J., Chautard, H., Cedrone, E. and Delcourt, M., Directed evolution of biocatalysts. Org. Proc. Res. Dev., 2006, 10, 562-571 (c) Hibbert, E.G. and Dalby, P.A., Directed evolution strategies for improved enzymatic performance. Microb. Cell Fact., 2004, 4, 29 (d) Otten, L.G. and Quax, W.J., Directed evolution selecting today s biocatalysts. Biomol. Eng., 2005, 22, 1-9. 

Based on these arguments, we developed the simple directed evolution strategy illustrated in Fig. 1 [2, 6], In comparison to the enzyme performing a function for which it is selected in nature, the new job is performed poorly indeed. Beneficial mutations will be identified during screening the products of... 

Just as nature uses evolution to naturally select enzyme variations that provide an advantage to the host, directed evolution of the amino acid substitution utilize techniques to screen or select for mutant enzymes that perform better than wild-type enzymes. Unlike site-directed mutagenesis, directed evolution techniques do not require a detailed understanding of the enzyme in order to identify useful variants. The techniques of applied molecular evolution however, do require a screening or selection step to identify the individual mutants of interest (Fig. 12.2). 

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