Gene Cloning, Expression And Characterization Of Four Phytases From Enterobacteriaceae

Phytases(myo-inositol hexakisphosphate phosphohydrotases) hydrolyse phytate to lower myo-inositol phosphates and inorganic phosphate. As a type of animal feed additives, addition of phytase in animal feed allows monogastric animal to utilize the phytin phosphorus in feed and reduces the environmental phosphorus pollution. Phytase has wide commercial applications in industry processes such as foodstuff and pharmaceutical industry. Microorganism is an important source of phytase. Screening of new and good nature phytases from the microorganism is an important research direction.A pair of degenerate primers(FI and RI) was designed through identity analysis of published amino acid sequences of phytase. Using primers, fragments of three phytase genes were obtained by PCR method from genome of strains Hqfnia alvei B125, Dickey a dadantii B187 and Dickeya paradisiaca B188. The full-length of three genes were obtained by method of TAIL-PCR and named as B125appA, B187appA and B188appA. A novel phytase gene revealed by genome analysis of Yersinia pestis was obtained by PCR, named as Y1appA. The highest identity of nucleic acid was 75% among these four genes(B187appA and B188appA), the lowest identity of nucleic acid was 48%(B188appA and B125appA, B188appA and Y1appA).In comparison with the sequences of previously isolated phytases by BLAST search, the deduced amino acid sequence of B125appA showed 95% identity to phytase from Obesumbacterium proteus, the deduced amino acid sequence of B187appA and B188appA showed 53% and 52% identities to a phytase from Klebsiella pneumoniae, respectively. The result proved that B187appA and B188appA are two new phytase genes. The deduced amino acid sequence of Y1appA showed 81% identity to phytase from Yersinia intermedia.The gene B125appA, B187appA and B188appA were expressed in Escherichia coli BL21(DE3). The gene y1appA was expressed in Pichia pastoris. Recombinant proteins were purified and their enzymatic properties were determined.The optimum pH for the B125r-APPA was 4.5 and the optimum temperature was 60℃. The relative phytase activity was above 80% after treated in buffers of pH2.0-pH 10.0. The specific activity of B125r-APPA was 356.7 IU/mg. The K_m and V_max values for B125r-APPA were 0.49 mmol/L and 238 IU/mg, respectively.The phytase of B187r-APPA displayed maximum activity at 55℃and pH5.5. The enzyme displayed 60% of the initial activity in pH4.0-pH 9.0. The specific activity of B187r-APPA was 642.64 IU/mg. The K_m and V_max values for B187r-APPA were 0.249 mmol/L and 833 IU/mg, respectively.The phytase of B188r-APPA had two pH optima(pH4.5 and pH5.5) and an optimum temperature of 55℃. The enzyme displayed over 60% of the initial activity in pH2.0-pH 9.0.The specific activity of B188r-APPA was 768.8 IU/mg. The K_m and V_max values for B188r-APPA were 0.399 mmol/L and 666 IU/mg, respectively.The phytase of Y1r-APPA exhibited maximum activity at pH4.5 and 55℃. The enzyme showed good pH stability, which the relative phytase activity was above 80% after treated in buffers of pH2.5-pH10.0. The specific activity was 2346.09 IU/mg. The K_m and V_max values for Y1r-APPA were 0.208 mmol/L and 4403.35 IU/mg, respectively.To our knowledge, this is the first report about finding phytase or phytase gene from Hafhia and Dickeya.