title: Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.
abstract: The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, People's Republic of China. yelidan@zju.edu.cn.|Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang University, Hangzhou, 310027, China. yelidan@zju.edu.cn.
authors.forename: Lidan
authors.initials: L
authors.lastname: Ye
mesh_terms: D010832:6-Phytase / Q000096:biosynthesis / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D015971:Gene Expression Regulation, Enzymologic; D005818:Genetic Engineering / Q000379:methods; D010843:Pichia / Q000235:genetics*; D013696:Temperature
chemical_list: D010832:6-Phytase
keywords: Alkaline phytase; Bacillus subtilis; Enzymatic property; Expression host; Glycosylation; Pichia pastoris