Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase

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2024年10月1日

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Mototaka Suzuki,
Masayo Date,
Tatsuki Kashiwagi,
Kazutoshi Takahashi,
Akira Nakamura,
Masaru Tanokura,
Eiichiro Suzuki,
Keiichi Yokoyama

摘要

Abstract

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T₅₀ (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T₅₀ of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (k_cat/K_m) at 37.0 °C was increased from 3.3 × 10² M⁻¹ s⁻¹ for the wild type to 5.9 × 10² M⁻¹ s⁻¹. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency.

Key points

• Improved heat resistance is essential to broaden the application of MTG.

• The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability.

• X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.