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ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2008, Vol. 9 Issue (7): 536-545    DOI: 10.1631/jzus.B0720006
Biotechnology and Biochemistry     
Expression, purification and characterization of a phyAm-phyCs fusion phytase
Li-kou ZOU, Hong-ning WANG, Xin PAN, Guo-bao TIAN, Zi-wen XIE, Qi WU, Hui CHEN, Tao XIE, Zhi-rong YANG
College of Life Science, Bioengineering Research Center for Animal Disease Prevention and Control, Sichuan University, Chengdu 610065, China; Faculty of Biotechnology of Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan 611830, China; College of Earth Science, Chengdu University of Technology, Chengdu 610059, China; 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.

Key words： Expression      PhyAm      PhyCs      Fusion phytase      Pichia pastoris     
Received: 07 December 2007     
CLC:  Q55  
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Zhi-rong YANG
Li-kou ZOU
Hong-ning WANG
Xin PAN
Guo-bao TIAN
Zi-wen XIE
Qi WU
Hui CHEN
Tao XIE
Cite this article:

Li-kou ZOU, Hong-ning WANG, Xin PAN, Guo-bao TIAN, Zi-wen XIE, Qi WU, Hui CHEN, Tao XIE, Zhi-rong YANG. Expression, purification and characterization of a phyAm-phyCs fusion phytase. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2008, 9(7): 536-545.

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http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B0720006     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2008/V9/I7/536

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