Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli
Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli
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碱性植酸酶大肠杆菌不同表达宿主的比较研究
DOI：
10.1007/s12010-016-2046-3
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发表时间：
2016-07-01
影响因子：
3
通讯作者：
Ye, Lidan
中科院分区：
文献类型：
--
作者：
Chen, Weiwei;Yu, Hongwei;Ye, Lidan
碱性植酸酶比活力较低，限制了其作为饲料添加剂的应用。大肠杆菌是包括碱性植酸酶在内的蛋白质定向进化的常用宿主，以提高其活性。然而，由于潜在的致病性，它不适合于食品级产品的生产。为了结合不同表达系统的优点，首先在大肠杆菌中通过定向进化获得了枯草芽孢杆菌168(Phy168)碱性植酸酶的突变体，然后转化食品级宿主枯草芽孢杆菌和巴斯德毕赤酵母进行分泌表达。为了考察不同表达系统的适应性，对在不同宿主中表达的Phy168突变体进行了比活力、pH谱、pH稳定性、温度谱和热稳定性的鉴定和比较。枯草杆菌表达的D24G/K70R/K111E/N121S突变株在pH 7.0和60℃时的比活力为30.4U/mg，明显高于巴斯德毕赤酵母和大肠杆菌中的比活力(22.7U/mg和19.7U/mg)。在80℃培养10min后，枯草杆菌表达的D24G/K70R/K111E/N121S在pH 7.0和37℃时的活性保持了约70%，而在巴斯德酵母和大肠杆菌中的表达活性分别只有25%和50%左右。这些结果表明，枯草芽孢杆菌是一种合适的Phy168突变体的表达宿主，在一个宿主中创建突变并在另一个宿主中表达的策略可能是工业化生产具有所需性质的蛋白质的新解决方案。
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.