Using Genetic Mutation Techniques To Improve PhyA Phytase Expression Level In Pichia Pastoris And Its Thermostability

Phytase, an additive in forage for mono-gastric animal is widely acknowledged for its effect. However, the restrictions in following aspect have affected its application: (1) The yield of phytase genetic engineering strain need to be promoted more; (2) Thermalstability can not satisfied the requirement in forage processing, store and use. Therefore, protein engineering is an effective way to design and reconstruct the natural enzyme, and it provides a new method to improve the yield and thermostability of phytase. Based on the phyA gene that registerd in Gene Bank Based on array analysing of amino acid of registered phyA gene, the novel phytase is aquired through synonym mutant, site mutant and structure extending mutant. And it expresses highly effictively in Pichia pastroi with better thermostability comparing with the natural strain.1. By using long-distance inverse PCR, Arg (cgt) and Arg (egg) in the expression fragment of phytase phyA gene (GenBank : accession numbers AF218813 and AAF25481) were mutated synonymously to Arg (aga), which is a bias code of yeast. The mutated gene phyA^m was cloned into pUC18 vector, and the two mutated sites were confirmed by sequencing. The expression plasmid pPIC9k-phyA^m was constructed and transformed into GS115 strain. By riddling of phenotypes and phytase activities, we selected out two mutated kinds of strains which have high activity. Southern blotting analysis to the two yeast transformants showed that phyA gene was intergrated into the chromosome genome through single-crossover events with one copy of phyA gene. What is more, SDS-PAGE analysis suggested that the expression product could be secreted and over-expressed, and the size of enzyme protein was 70.15KD. The phytase activity of PP-NPm-8 optimized by codons reached 47600 U·ml^-1 in malt wort culture medium after induced for 36h and doubled the original strain PP-NP-2. Moreover, PP-NP^m-8 showed a quite good genetic stability.2. The mutants of F43Y and I354M, L358F of phytase (phyA^m) were constructed by in vitro site-directed mutagenesis with long-distance inverse PCR. The recombinant pPIC9k-phyA^m-1 and pPIC9k-phyA^m-2were expressed in Pichia pastoris. The comparison experiments of mutant enzymes with nature phytase showed that: The optimum temperature of F43Y was increased by 3 °C, and its thermostability wasincreased by 15%, when it was exposed for 10 min at 75 °C. Its specific activity was increased by 11%. The optimum temperature of I354M, L358F wasn't increased, and its thermostability was only increased by 3%. However, the specific activity of I354M, L358F was decreased by 6.5%. After a comparation of natural phytase and mutant phytase, we found that hydrogen bond between Tyr43 and Asn416 promote the thermostability. Moreover, flask culture and phytase activity detection showed that the expression capacity of PP-NP"1"1 and PP-NP1"'2 was consistent with PP-NPm-8.3. The phytase gene phyAm acquired from Aspergillus niger N25 was recombined into E. coli expression vector pET-30b(+), recombined expression vectors pET30b-Fp/i>vlm was served as template to amplify phytase gene, and the PCR product named elongation mutation gene phyAe was expanded with a 13 amino acid sequence from pET-30b-Fp/zy,4m vector at C-terminal of phytase gene phyAm. Furthermore, phyA* gene was recombined into expression vector pPIC9k and expressed in Pichia pastoris. The comparison experiment of mutant enzyme PP-NPe with natural phytase PP-NPm-8 showed that: the optimum temperature of PP-NPe was increased by 3 °C, and its thermostability was increased by 21% when it was exposed to 10 min at 75 °C. Its effective reaction pH range was pH4.6-pH6.6 that the catalysis efficiency was above 70%, and 0.4 of pH value wider than that of wild-type phytase. Stucture prediction has shown out: the elongation of phytase C terminal by adding 13 amino acids caused an a-helix and an irregular coil composed by 4 His. And this a-helix is made of 6 amino acids. Through out the a-helix dipole interation and additional surface salt bridge network, protein thermostability was increased. Meanwhile, elongation mutation had also influenced the electrostatic distribution of surface, which expanded the effective pH range. Moreover, flask culture and phytase activity detection showed that the expression capacity of PP-NPe was consistent with PP-NPm-8.