( Agro-biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China;
¹Bioengineering Department of East China University of Science and Technology, Shanghai 200237, China )

Abstract

It is difficult to obtain naturally occurring phytase having the requir ed thermostability for application in animal feeding. The 1.3 kb thermal stable phytase gene (fphy) of Aspergillus fumigatus was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris. Though the nucleotides of synthetic fphy share only 74% homology with the natural gene, the amino acid sequences coded by both genes were identical. After being cloned into the yeast expression vector (pPIC9) and inserted into the chromosome of Pichia pastoris by homologous recombination, phytase was expressed in the yeast and secreted f r om the cell. The strains for phytase overª²expression were selected out by SDS-PAGE and enzyme analysis. After fermentation in 5 L fermention tank and induced by 0.5 % methanol for 60 h, about 5.6 g purified phytase was obtained per liter culture fluid. The activity of phytase was 130 000 u per microlitre fluid. The thermo stable phytase remained 40% active after exposure at 90 ¡æ for 80 min.