Directed evolution of the transglutaminase from Streptomyces mobaraensis and its enhanced expression in Escherichia coli
2020
Xue, Ting | Zheng, Xuehai | Su, Xiaomei | Chen, Duo | Liu, Kui | Yuan, Xue | Lin, Ronghua | Huang, Luqiang | He, Wenjin | Zhu, Jinmao | Chen, Youqiang
Transglutaminase-catalyzed reactions can be used widely to modify the functional properties of food proteins, biopharmaceuticals and in tissue engineering. Transglutaminase-producing organisms obtained from natural screening have a low ability to produce enzymes, and the obtained enzyme generally has low activity and poor substrate specificity, which limit its industrial applications. Of 100 isolates collected from five air-dried soil samples, 20 exhibited the typical growth characteristics of Actinomycetes. Of these 20 isolates, S-1and S-2 resulted in 0.47 and 0.30 U/mL transglutaminase production, respectively. Based on phenotypic and the 16S rRNA gene-sequence data, the isolate S-1 was confirmed as Streptomyces mobaraensis. We produced Transetta (DE3)/PET-32(a)-YC2 mutants in Escherichia coli exhibiting improved MTGase activity and production from the screened microbial transglutaminase-producing strain by directed evolution of the MTGase gene using epPCR combined with construction and overexpression of the PET-32(a)-YC2. The activity of Transetta (DE3)/pET-32a-YC2 TGase (3.03 U/mL) in E. coli growth supernatant was 1.5 and 1.8-fold above that of the control Transetta (DE3)/pET-32a-MTGase strain (2.02 U/mL) and Transetta (DE3)/pET-32a strain (1.68 U/mL), respectively. Under the optimized conditions, the content of target protein and MTGase activity by the MTGase gene expression in Transetta (DE3)/pET-32a-YC2 (26.2% and 4.99 U/mL) were 2.07 and 1.65-fold greater than control through optimization of different parameters. These results suggest that directed evolution of the MTGase gene from Streptomyces mobaraensis can effectively enhance the MTGase activity and protein expression in E. coli. This method of enhanced expression of active MTGase in E. coli may be valuable for food and other industrial applications.
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