Cloning And Heterologous Expression Of Phytase Gene From Aspergillus Terreus And Bacillus Subtilis

Phytate and phytic acid are the major phosphorus storage form contained in food or feeds of plant origin. Phytases are myo-inositol hexakisphosphate phosphohydrolase that catalyze the stepwise removal of inorganic orthophosphate from phytate. Addition of phytase into feed can result in the following advantages: increasing the absorptive efficiency of phosphate in animal body, decreasing the phosphate pollution in environment, eliminating the chelate reaction between phytate and metal ion so that phytase can improve the utilization efficiency of the feed. Hence the study on phytase, especially phytase produced by microorganism, has been paid much attention by scientists in China and abroad.Phytase production on a large scale are restricted by low level of phytase gene expression in wild strains. The objective of this study is to clone phytase gene from wild strains and then obtain its expression in heterogeneous yeast and escherichia strains. The main results of this study are as the following:1. About 1.4kb phyA fragment absent signal sequence and intron of Aspergillus terreus was amplified by PCR with primers designed according to published phyA sequences. It was further cloned and sequenced. The recombinant expression plasmid pPIC9K-phyA was constructed and transformed into Pichia pastoris GS115. The phytase gene was expressed in Pichia pastoris GS115 detected by PCR and hydrolytic zone on a solid phytate-containing medium . The enzyme activity in supernatant from flask fermentation after 132 hours induction reached to 167U/ml. Its enzyme activity was active under pH 2.0-8.0 and up to the the highest under pH5.5.The optimum temperature for enzyme activity was 55 ℃.2. A pair of primers were designed based on the phyC sequence of Bacillus stubilis. The phyC structure gene encoding phytase but without signal peptide sequences was obtained by polymerase chain reaction. The non-fusion fragment amplified was subcloned into a expression vector pET-17b with T7 promoter and then transformed into E. coli BL21(DE3). The phyC gene expression was detected by PCR and the hydrolytic zone observed on a solid phytate-containing medium.