We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C–terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/I222T)—the thermostability of which is much lower than that of the wild type—was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild–type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.
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This work was supported in part by Grants–in–Aid (10450310, 10145234, and 07280101) from the Ministry of Education, Science, Sports, and Culture, Japan, and was performed as a part of the Research and Development Project of Industrial Science and Technology Frontier Program supported by NEDO.
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Matsuura, T., Miyai, K., Trakulnaleamsai, S. et al. Evolutionary molecular engineering by random elongation mutagenesis. Nat Biotechnol 17, 58–61 (1999). https://doi.org/10.1038/5232
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