Evolutionary molecular engineering by random elongation mutagenesis


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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Figure 1: Distribution pattern of the three enzymatic properties of the two elongation mutant populations prepared from the wild–type enzyme () and its triple mutant (I108T/D130N/I222T) ().
Figure 2: Correlation between two properties of the elongation mutants in the populations prepared from the wild–type enzyme () and its triple mutant ().


  1. 1

    You, L. and Arnold, F.H. 1996. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 9: 77–83.

    Article  Google Scholar 

  2. 2

    Moore, J.C. and Arnold, F.H. 1996. Directed evolution of a para–nitrobenzyl esterase for aqueous–organic solvents. Nat. Biotechnol. 14: 458– 467.

    Article  Google Scholar 

  3. 3

    Zhang, J.–H., Dawes, G., and Stemmer, W.P.C. 1997. Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94: 4504–4509.

    Article  Google Scholar 

  4. 4

    Matsuura, T., Yomo, T., Trakulnaleamsai, S., Ohashi, Y., Yamamoto, K., and Urabe, I. 1998. Nonadditivity of mutational effects on the properties of catalase I and its application to efficient directed evolution. Protein Eng. 11: 789– 795.

    Article  Google Scholar 

  5. 5

    Aita, T. and Husimi, Y. 1996. Fitness spectrum among random mutants on Mt. Fuji–type fitness landscape. J. Theor. Biol. 182: 469–485.

    Article  Google Scholar 

  6. 6

    Trakulnaleamsai, S., Yomo, T., Yoshikawa, M., Aihara, S., and Urabe, I. 1995. Experimental sketch of landscapes in protein sequence space. J. Ferment. Bioeng. 79: 107–118.

    Article  Google Scholar 

  7. 7

    Trakulnaleamsai, S., Aihara, S., Miyai, K., Suga, Y., Sota, M., Yomo, T. et al. 1992. Revised sequence and activity of Bacillus stearothermophilus catalase I (formerly peroxidase). J. Ferment. Bioeng. 74: 234–237.

    Article  Google Scholar 

  8. 8

    Yomo, T., Yamano, T., Yamamoto, K., and Urabe, I. 1997. General equation of steady–state enzyme kinetics using net rate constants and its application to the kinetic analysis of catalase reaction. J. Theor. Biol. 188: 301–312.

    Article  Google Scholar 

  9. 9

    Kobayashi, C., Suga, Y., Yamamoto, K., Yomo, T., Ogasahara, K., Yutani, K. et al. 1997. Thermal conversion from low– to high–activity forms of catalase I from Bacillus stearothermophilus. J. Biol. Chem. 272: 23011–23016.

    Article  Google Scholar 

  10. 10

    Wells, J.A. 1990. Additivity of mutational effects in proteins. Biochemistry 29: 8509–8517 .

    Article  Google Scholar 

  11. 11

    Triggs–Rain, B.L. and Loewen, P.C. 1987 . Physical characterization of katG encoding catalase HPI of Escherichia coli. Gene 52: 121–128.

    Google Scholar 

  12. 12

    Loprasert, S., Urabe, I., and Okada, H. 1990. Overproduction and single–step purification of Bacillus stearothermophilus peroxidase in Escherichia coli. Appl. Microbiol. Biotechnol. 32: 690– 692.

    Article  Google Scholar 

  13. 13

    Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  14. 14

    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  Google Scholar 

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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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Correspondence to Itaru Urabe.

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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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