Heterogeneous Expression And Characterization Of The Recombinant Phytase

Phytase can catalyze phytic acid into inorganic phosphorus and inositol by hydrolysis. Phytase used as food additive and feed additive can improve the utilization efficiency of nutrients and reduce the pollution of phytate phosphorus to the environment. The phytase appA from Escherichia coli has a great potential for industrial applications due to the strongest ability to hydrolyze phytate. However, an important question for large-scale production is how to improve its thermal stability.To improve thermostability of phytase appA we have obtained a genetic modified phytase with the name of appAM8 previously. The result showed that phytase appAM8 expressed in Pichia pastoris had higher thermal stability than that of wild-type appA. To study the mechanism of thermostability of phytase appAM8, the recombinant phytase was expressed in Pichia pastoris (P-appAM8) and Escherichia coli (E-appAM8), the objective of the present study was to compare the enzymological property of phytase expressed in different host cells. The results suggested:1) P-appAM8 and E-appAM8 had the same optimum pH of 4.5 and an optimum temperature of 65 ℃. There were no significant differences in pH between appAM8 and appA-WT. The optimum temperature of appAM8 raised 5℃ compared with appA-WT; 2) the molecular weight of E-appAM8 and P-appAM8 was estimated to be 47 kDa and about 53 kDa respectively by SDS-polyacrylamide gel electrophoresis. The protein which expressed in P. pastoris was modified by glycosylation, showing two diffuse bands on SDS-PAGE; 3) when heated to 60℃-80℃, the heat tolerance of E-appAM8 was decreased obviously compared with that of P-appAM8. The phytase activity of E-appAM8 remained at 4% after treatment at 70℃ for 15 min. while phytase activity of P-appAM8 reached 50% under the same condition, suggesting that glycosylation of phytase improved its stability; 4) The Km of E-appAM8 and P-appAM8 was 0.245 mmol/L and 0.36 mmol/L, and their Vmax was 3196 U/mg and 3333 U/mg, respectively. The results suggested that glycosylation plays an important role in the thermal stability by comparing the property of phytase appAM8 in different hosts. Point mutations of appAM8 also affect its stability.In addition, two residue substitutions (Q258N, Q349N) were introduced to appA to improve its thermal stability. Using yeast expression system the mutant phytase appA-2QN produced in shaking-flask culture showed an increased thermal stability. To increase its expression level and lower production costs, an approach of the high cell-density fermentation of recombinant yeast GS115/appA-2QN was carried out in this study. During fermentation the concentration of glycerin was controlled to promote the cell growth and achieve the high cell-density fermentation. The method of chromotropic acid spectrophotography was used to determine the concentration of methanol during fermentation. The results showed that the cell density reached OD600nm=313 after 147 h fermentation in a 5 L fermentor. After inducing of 102 h with methanol at a concentration of 2.5%, the expression level of phytase reached 7.6 g/L. The phytase activity was 2.03×105 U/mL. These results indicated that the cell density and the expression level of target protein were improved by high density fermentation significantly. The recombinant yeast strain has favorable expression stability.