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

热稳定性 突变体 合理设计 化学 组织谷氨酰胺转胺酶 半胱氨酸 二硫键 生物化学 野生型 蛋白质工程 生物 基因 遗传学
作者
Hongjing Wang,Haiqing Chen,Qingbin Li,Yu Fan,Yaru Yan,Shuang Liu,Jian Tian,Jianxin Tan
出处
期刊:Protein Expression and Purification [Elsevier BV]
卷期号:195-196: 106079-106079 被引量:5
标识
DOI:10.1016/j.pep.2022.106079
摘要

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 Km and higher Vmax than the wild-type. Its half-life (t1/2) 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.
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