Expression, purification and characterization of a phyAm-phyCs fusion phytase |
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| Authors: | Li-kou Zou Hong-ning Wang Xin Pan Guo-bao Tian Zi-wen Xie Qi Wu Hui Chen Tao Xie and Zhi-rong Yang |
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| Affiliation: | [1]College of Life Science, Bioengineering Research Center for Animal Disease Prevention and Control, Sichuan University, Chengdu 610065, China; [2]Faculty of Biotechnology of Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan 611830, China; [3]College of Earth Science, Chengdu University of Technology, Chengdu 610059, China; [4]College of Life Science, Sichuan Agricultural University, Ya ＇an 625014, China |
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| Abstract: | The phyAm gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichia pastoris in order to expand the pH profile of phytase and decrease the cost of production. The fusion phytase phyAm-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is (25.4±0.53) U/ml at the flask scale and (159.1±2.92) U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 °C and an optimal pH at 5.5~6.0 and its relative activity remains at a relatively high level of above 70% in the range of pH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 °C to 95 °C for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). After deglycosylation by endoglycosidase H (EndoHf), the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those of phyCs and phyAm. |
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| Keywords: | Expression PhvA^m PhvCs Fusion ohvtase Pichia pastoris |
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