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Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis

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Abstract

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 Å space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX®. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.

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Acknowledgments

We thank Dr. Kashiwagi and Dr. Nio for helpful discussions. We also thank Dr. Onoe and Ms. Tagami for technical assistance. This work was done by in part of the finance from the Global Center of Excellence Program (Project No. B01: Catalysis as the Basis for Innovation in Materials Science) from the Ministry of Education, Culture, Sports, Science, and Technology-Japan. Pacific Edit reviewed the manuscript prior to submission.

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Authors and Affiliations

  1. Institute of Life Sciences, Ajinomoto Co., Inc, 1-1, Suzuki-cho, Kawasaki-shi, 210-8681, Japan

    Keiichi Yokoyama, Takefumi Nakamura, Nobuhisa Shimba & Eiichiro Suzuki

  2. Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, N13W8, Kita-ku, Sapporo, 060-8628, Japan

    Hiroe Utsumi, Daisuke Ogaya & Seiichi Taguchi

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  1. Keiichi Yokoyama
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  2. Hiroe Utsumi
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  3. Takefumi Nakamura
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  4. Daisuke Ogaya
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  5. Nobuhisa Shimba
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  6. Eiichiro Suzuki
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  7. Seiichi Taguchi
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Correspondence to Keiichi Yokoyama or Seiichi Taguchi.

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Yokoyama, K., Utsumi, H., Nakamura, T. et al. Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis. Appl Microbiol Biotechnol 87, 2087–2096 (2010). https://doi.org/10.1007/s00253-010-2656-6

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  • DOI: https://doi.org/10.1007/s00253-010-2656-6

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