High Expression of a Heat-stable Phytase in Pichia pastoris

( 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 over expression
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 ℃ for 80 min.