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J. Microbiol. Biotechnol. 2005; 15(3): 466-471

Published online June 28, 2005

Copyright © The Korean Society for Microbiology and Biotechnology.

Overexpression and Characterization of appA Phytase Expressed by Recombinant Baculovirus-Infected Silkworm

Chen, Yin , Zhongze Zhu , Xu'ai Lin , Yongzhu Yi , Zhifang Zhang 1* and Guifang Shen 1*

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, 212018, China, 1Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

Abstract

An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early $5^{th}$ instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were $60^{\circ}C$ and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM $Ca^{2+}$, 1 mM $Mn^{2+}$, or $0.02\%$ Triton X-100.

Keywords: Escherichia coli, appA, phytase, baculovirus, silkworm