The Reserch Of Improving Activity Of Phytase By Error-prone PCR

Phytases belong to the family of histidine acid phosphatases which catalyze the release of phosphate from phytic acid. As a kind of feed additive phytases have been added to the feed to meet the phosphorus requirements of some monogastric animals like poultry, pigs and it play an important part in reducing phosphorous contamination as well. But at present, the activity of phytase still need to improve to meet the needs. Directed evolution mimics the process of Darwinian evolution in a test tube, and it's has become a effective strategies to engineer enzymes for the requirements of industrial, medical and research application.The building of effective high-throughput screening method is the key factor of success for directed evolution of phytase. This research stablished a high-throughput screening method for phytase engineering. On the basis of general Molybdenum-Vanadium method for detecting phytase activity, the microplate method was developed for the determination of trace activity of phytase, and the microculture method was developed for culture of phytase on the basic of flask culture. The results shows that both of the microplate method and Molybdenum-Vanadium methods had similar accuracy,sensitivity and stability, and the microplate method can greatly improve the efficiency and lower the cost of experiments. The samples gained from microculture had enzyme activity and their activity were lower than the flask culture samples,but what we focused is the enzyme activity comparion between mutant strain and original strain in preliminary high-throughput screening, so the microplate method is feasible.This research engineed the activity of phytase with ep-PCR which is a mature technology of directed evolution. After optimization,the concentration of Mn²⁺ is 01.mmol/L and the concentration of Mg²⁺ is 7mmol/L in ep-PCR reaction system. Structured ep-PCR products into plasmid vector pPIC9K and transformed the recombinant palsmid into Pichia pastris GS115 through electroporation after linearization. After high-throughput screening we acquired a mutant strain PP-NP^ep-6A (82034U/mL) which has 29% higher phytase enzyme activity than original strain PP-NP^m-8 (63667U/mL) . And the optimum temperature for catalytic reaction of mutant phytase is 55℃,the optimum pH is 5.6. After DNA sequencing we found that 9 bases have changed in mutant sequence,and the change result in 4 amino acid change which were Glu156Gly. Thr236Ala, Gln396Arg, Leu406Thr. After protein secondary structure prediction we found that mutant phytase has the similar physicochemical property and isoelectric point compared with original phytase. After protein three dimensional structure prediction we found that mutant phytase has the same backbone and 3D structure with orignal phytase,and the active centre has no change. The mutation of 4 amino acid may lead to micro-variation on protein structure and increase the flexibility of enzyme constitution, that may result in the increasing of enzyme activity.This research acquired a mutant strain which has higher enzyme activity through ep-PCR,that will laid the foundation for further studiy on directed evolution of phytase.