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Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Engineering / Faculty of Engineering >
Peer-reviewed Journal Articles, etc >
Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis
| Title: | Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis |
| Authors: | Yokoyama, Keiichi Browse this author | | Utsumi, Hiroe Browse this author | | Nakamura, Takefumi Browse this author | | Ogaya, Daisuke Browse this author | | Shimba, Nobuhisa Browse this author | | Suzuki, Eiichiro Browse this author | | Taguchi, Seiichi Browse this author →KAKEN DB |
| Keywords: | Transglutaminase | | Streptomyces | | CORYNEX® | | Screening | | Solvent-accessibility | | Specific activity |
| Issue Date: | Aug-2010 |
| Publisher: | Springer Berlin / Heidelberg |
| Journal Title: | Applied Microbiology and Biotechnology |
| Volume: | 87 |
| Issue: | 6 |
| Start Page: | 2087 |
| End Page: | 2096 |
| Publisher DOI: | 10.1007/s00253-010-2656-6 |
| Abstract: | Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. To improve MTGT, a novel method of rational mutagenesis, called WASH-ROM (Water Accessible Surface Hot-space Region Oriented Mutagenesis), was first attempted. Based on the three-dimensional structure of MTG, 151 point mutations were selected at 40 different residues bearing high solvent accessibility surface area, within a 15 Å of the active center site nucleophile, Cys64. Among them, 32 mutants showed higher specific activity than the wild type enzyme. We found that beneficial mutations are distributed in two regions and with distinctive amino acid substitutions. Next, random mutagenesis was applied to the entire MTG region by developing a new plate assay-based screening system, using Corynebacterium glutamicum as the secretion host strain. This in vivo screening system allowed us to readily distinguish the change in enzymatic activity upon mutation by monitoring the intensity of enzymatic reaction-derived color zones which appeared around the recombinant cell colonies on the plate. From the library of 24,000 clones, 10 mutants were finally selected as beneficial enzymes exhibiting higher specific activity than wild type. Notably, most of the mutations differed from those obtained by WASH-ROM, except for H289Y. Beneficial mutations were distributed in two other regions as well. Furthermore, we found that the FRAP-S199A mutant (FRAP: N-terminal four amino acid residues extension) showed the highest specific activity (45 U/mg: 1.7 times higher than the wild type enzyme). Through these different mutation approaches, various beneficial positions leading to increased specific activity of MTG were surveyed. |
| Rights: | The original publication is available at www.springerlink.com |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/46867 |
| Appears in Collections: | 工学院・工学研究院 (Graduate School of Engineering / Faculty of Engineering) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 田口 精一
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