Improvement Of The Thermostability Of Aspergillus Niger N25 Phytase By Using Site-directed Mutagenesis

Phytase is an important industrial enzyme. It has been widely used as a feed supplement to improve the bioavailability of phytate phosphorus to simple-stomached swine and poultry. While, the poor thermostability and low activity is a bottleneck in production and application of phytase, so the reconstruction of phytase through protein engineering technology has important scientific and practical value.The mutants of I44E and T252R of phytase (phyAm) were constructed by in vitro site-directed mutagenesis with long-distance inverse PCR. The code substitution was based on the bias code of yeast. The recombinant plasmid pPIC9k-phyA44, pPIC9k-phyA252 and pPIC9k-phyA44-252 were constructed and transformed into GS115 strain through electroporation after linearization. By riddling of phenotypes and phytase activities, we selected three high activity strains of mutant transformants which are PP-NPm-44, PP-NPm-252 and PP-NPm-44-252.The mutant and WT phytase yest strains cultured in BMGY/BMMY medium, the expression product was purified and characterization. The results showed that:(1) SDS-PAGE analysis suggust that the expression product could be secreted and over-expressed,the size of enzyme protein was 70.15KD. (2) The results showed that the optimum temperature and the optimum pH value did not have the significant change compare with the wild phytase, were 55℃and pH5.5, respectively. (3) The phytase activity of PP-NPm-44, PP-NPm-252 and PP-NPm-44-252 reached 70293U/mL, 72647U/mL and 81227U/mL in BMGY/BMMY culture medium after being induced with 4d, which was the 1.1,1.14 and 1.28 times higher than PP-NPm-8(63667U/mL), and meanwhile the specific activity was increased 1.09,1.04 and 1.27 times, respectively. (4) The results of thermostability showed that:while three mutants all showed improved thermostability, the best is the double mutant(PP-NPm-44-252), which retained about 36% and 47% greater activity after incubating at 80℃and 90℃for 10min and had a 1.2℃higher melting temperature than that of wild-type phyA. (5) From the study of the effect of metal ions on phytase activity, only Cu2+, Zn2+, Ag+, SDS made consumedly decrease of the phytase activity, especially Cu2+made the enzyme activity dropped to 46.24%; Mg2+, Mn2+, Co2+ had significantly activation effect, while mostly metal ions had no or a bit of effect on enzyme activity.In this research, the highly expressive phytase was acquired, and it has better thermostability comparing with natural one. What is more, structure analysis of mutant and natural phytase explored the relationship between structure and function. This research provided a solid fundation to production of genetically engineered bacteria.