Enhancing the thermostability of transglutaminase from Streptomyces mobaraensis based on the rational design of a disulfide bond

Hongjing Wang; Haiqing Chen; Qingbin Li; Fan Yu; Yaru Yan; Shuang Liu; Jian Tian; Jianxin Tan

doi:10.1016/j.pep.2022.106079

Enhancing the thermostability of transglutaminase from Streptomyces mobaraensis based on the rational design of a disulfide bond

Protein Expr Purif. 2022 Aug:195-196:106079. doi: 10.1016/j.pep.2022.106079. Epub 2022 Mar 7.

Authors

Hongjing Wang¹, Haiqing Chen¹, Qingbin Li², Fan Yu¹, Yaru Yan², Shuang Liu¹, Jian Tian³, Jianxin Tan⁴

Affiliations

¹ College of Food Science and Technology, Hebei Agricultural University, Baoding, 071000, Hebei, China.
² Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
³ Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: tianjian@caas.cn.
⁴ College of Food Science and Technology, Hebei Agricultural University, Baoding, 071000, Hebei, China. Electronic address: jianxintan@sina.com.

PMID: 35272012
DOI: 10.1016/j.pep.2022.106079

Abstract

Transglutaminase (TGase), a transferase, is widely adopted in the food industry and other biological fields due to its unique characteristics of modifying proteins by intra- or intermolecular cross-linking. However, obtaining a mutant TGase that is highly thermostable and active would significantly aid in food processing. Therefore, this study sought to improve the thermostability of TGase by introducing an artificial disulfide bridge through a structure-based rational enzyme engineering approach. After the rational screening, six disulfide mutants (E139C/G143C, R146C/E149C, A182C/N195C, L200C/R208C, T223C/F226C, and E139C/G143C+L200C/R208C) of the transglutaminase gene from Streptomyces mobaraensis (Sm-TGase) were selected and constructed by rationally designed mutations in cysteine. Of them, a mutant (E139C/G143C) with enhanced thermostability was selected and characterized for further analysis. The results indicated that the mutant E139C/G143C had a similar specific activity, optimal temperature, and pH but a lower K_m and higher V_max than the wild-type. Its half-life (t_1/2) at 55 °C was 10.7 min, which was 1.69-fold higher than the wild-type, while its melting temperature (Tm) was 3.52 °C higher than the wild-type. These results proved that the introduction of disulfide bonds into TGase by rational design could be an effective approach to improve the thermostability of TGase and other food enzymes for food processing.

Keywords: Disulfide bond; Enzymatic property; Rational design; Thermostability; Transglutaminase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Disulfides / chemistry
Enzyme Stability
Mutation
Streptomyces*
Temperature
Transglutaminases* / genetics

Substances

Disulfides
Transglutaminases

Supplementary concepts

Streptomyces mobaraensis