[High expression of a heat-stable phytase in Pichia pastoris].
It is difficult to obtain naturally occurring phytase having the required 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 from 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 thermostable phytase remained 40% active after exposure at 90 degrees for 80 min.
Peng
Ri-He
RH
Agro-biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai 201106, China.
Xiong
Ai-Sheng
AS
Li
Xian
X
Fan
Hui-Qin
HQ
Yao
Quan-Hong
QH
Guo
Mei-Jin
MJ
Zhang
Si-Liang
SL
D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*
D010832:6-Phytase