Overexpression and Characterizaion of appA Phytase Expressed by Baculovirus-infected Silkworm

An Escherichia coli strain with high phytaseactivity was screened from pig excreta. The phytase gene,appA, was amplified by PCR technique. To obtain largeamounts of appA phytase, the appA gene was subcloned intothe baculovirus transfer vector pVL1393 under the control ofthe Polyhedrin promoter. The recombinant baculovirusharboring the appA gene was obtained after co-transfectionand screening. The early 5th instar larvae of silkworm wereinfected with the recombinant virus. Using this system, theappA phytase was overproduced up to 7,710 U per mlhemolymph. SDS-PAGE analysis revealed the baculovirusderivedappA phytase to be approximately 47 kDa in size.The optimal temperature and pH of the expressed phytasewere 60oC and pH 4.5, respectively. The enzymatic activitywas increased by the presence of 1 mM Ca2+, 1 mM Mn2+, or0.02% Triton X-100. KCI Citation Count: 3