| Phytic acid is the main storage form of phosphorus in cereal grains and legume seeds. Phytase is an enzyme that can catalyzes the hydrolysis of phytic acid in such plant seeds to release inorganic phosphorus. Phytase supplementation in animal feed can effectively improve the use efficiency of phytic acid, reduce phosphorus pollution of animal waste and remove anti-nutritional effect of phytic acid to increase the nutritional value of feedstuff. The growing demands for phytase with specific activity and high thermostability for feed pellet production have promoted the development and utilization of the new enzyme resource and genetic and protein engineering research on phytase as well as laid the foundation for better application in the practice production.Phytases from Bacillus strains belong to beta-propeller phytase family, their activity is associated with metal ions. These phytases exhibit specific activity for phytic acid and higher thermal stability that helps combat the enzyme deactivation caused by high temperature during pelletting, have neutral pH optimum (pH6.0-7.5) that effectively make up for the inadequacy of acidic phytase and improve the effectiveness of phytase in animal gastrointestinal tract, and enlarge the application range of phytase. Therefore, bacillus phytases are suitable as feed additives for animals with neutral digestive tracts such as freshwater fish of the family cyprinidae, and can also be effective in neutral intestinal segments of the single stomach animals. Our research group has previously screened two Bacillus strains from rhizosphere soil of soybean. From them two neutral phytase genes were cloned and named as phy(ycD) and phy(ycE), respectively. Prokaryotic expression system was established for these two phytase genes. The phy(ycD) gene was cloned into pET-28-a(+) expression vector carrying the T7lac promoter and designated as pET-28-a(+)-phy(ycD). This constructed plasmid was transformed into E. coli BL21(DE3). The positive transformant containing pET-28-a(+)-phy(ycD) was grown at37℃and induced overnight at16℃by addition of0.5mM IPTG. The his-tagged r-phy(ycD) protein was purified to electrophoretic homogeneity by Ni2+-NTA affinity chromatography. The purified protein migrated on SDS-PAGE as a single band corresponding to molecular mass of～45kDa. The expressed fusion protein was checked using a activity assay, and accounted for more than40%of the total soluble bacterial protein and with a high purity above90%indicating good bioactivity. The enzymatic properties of expressed phytase were analyzed after purification. The optimum enzymatic Ca2+concentration is1mM, the enzymatic reaction pH optimum is about6.5and the optimum reaction temperature was45℃, the Km and Vmax values were0.213mM and92.6nmol mg-1protein s-1. The enzyme activity of the expressed phytase was reduced to30%after incubation with1mM Ca2+at pH6.5and65℃for20minutes when compared to those incubated at37℃. The residual phytase activity was60%under the condition of pH6.5and1mM Ca2+for60minutes at37℃. When incubated under the condition of pH6.5and65℃for different times, the expressed phytase exhibited better thermostability at the concentration of10mM Ca2+than that at the concentration of0,1or5mM Ca2+. The expressed phytase is a Ca2+dependent enzyme,1.0mM Ca2+can significantly improve its thermal stability and pH stability, whereas EDTA〠Zn2+, Ba2+and Fe2+show significant inhibitory effect on the enzymatic activity of the expressed phytase.In conclusion, all these properties of r-phy(ycD) indicated its application potential in aquaculture and laid the foundation for industrial production of neutral phytase products. Thus, the research results of this study can be important for theoretical development and practical application of neutral phytase. |
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