Gene Cloning, Expression And Characterization Of Phytases From Pectobacterium Wasabiae And Pedobacter Sp.

Phytases (myo-inositol hexakisphosphate phosphohydrolases, EC. 3.2.1.8), a group of enzymes that can hydrolyze phytate to lower myo-inositol phosphates and inorganic phosphate, have been extensively studied as a new type of green feed additive in recent years. Addition of phytase in animal feed allows monogastric animals to utilize the phytate phosphorus in feed with increased nutritive values, and decreases cost, reduces the environmental pollution, thus phytase has been widely applied in feed and foodstuff industry. In this study, novel genes encoding phytase with advantage properties were cloned and expressed from microorganism strains isolated from special environments.A two-step PCR approach including degenerate PCR and thermal asymmetric interlaced (TAIL) PCR, and a genomic library screening approach were used to clone phytase genes from bacteria of different sources. The five phytase genes, named as phyMJ11, phyMY8, phy206B, phyB189 and phyS221, were cloned from Pedobacter sp. MJ11 and Flavobacterium sp. MY8 which were isolated from the root soil of Saussurea sp., Pseudomonas sp. 206B from the pond sediment, the plant rotten pathogen of Pectobacterium wasabiae B189, and a protease active strain of Streptomyces fradiae var. k11 (S221), respectively. The sequence analysis and structure prediction indicated that phy206B and phyB189 belonged to the histidine acid phosphatase (HAP) family, and phyS221, phyMJ11 and phyMY8 belonged toβ-Propellar Phytase (BPP) family. Using BLAST, the deduced amino acid sequences of these phytases showed 40–70% identities to the previously reported genes, suggesting the novelty of these phytases. In addition to the gene cloned from Pseudomonas sp., the other genes are all first reported from the four genera.The phytase from P. wasabiae B189 showed the highest amino acid sequence identity of 48.5% to PhyM from Pseudomonas syringae. The phytase gene phyB189 was expressed in Escherichia coli BL21 (DE3), purified and characterized. The purified recombinant phytase, r-phyB189, had a molecular mass of 45 kD. The optimum pH and temperature were 5.0 and 50°C, respectively. The Km and Vmax values were 0.17 mM and 1,714μmol·min^–1·mg^–1, respectively, with a specific activity of 1,072 U·mg^–1 on the substrate phytate.The phytase from Pedobacter sp. MJ11 showed the highest amino acid sequence identity of 63.0% to the putative phytase from Desulfuromonas acetoxidans. The phytase gene phyMJ11 was also expressed in Escherichia coli BL21 (DE3) and purified. The molecular mass of the purified recombinant phytase was 37.3 kD, and the optimal activity was showed at pH7.0 and 45°C at the presence of 1 mM Ca²⁺. The specific activity was 24.35 U·mg^–1 with phytate as substrate. The Km value was 1.28 mM, with a Vmax of 71.9μmol·min^–1·mg^–1. The recombinant phytase, r-PhyMJ11, had better efficacy than other commonly used phytases in phytate-phosphorus hydrolysis of soybean meal at neutral pH and 20°C, indicating the potential application in aquaculture.