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KMID : 0545120160260101717
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 10 p.1717 ~ p.1722
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Enhancing the Thermal Resistance of a Novel Acidobacteria-Derived Phytase by Engineering of Disulfide Bridges
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Tan Hao
Miao Ren Yun
Liu Tian Hai
Cao Xue Lian
Wu Xiang
Xie Li Yuan
Huang Zhong Qian
Peng Wei Hong
Gan Bing Cheng
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Abstract
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A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.
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KEYWORD
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phytase, acidophilic, acidostable, site-directed mutagenesis, disulfide bridge, thermostable
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