Characterizations Analysis Of Phytase From Yersinia Rohdei Heterologous Expression In Pichia Pastoris

Phytic acid and its salt sare widely present in the plant tissue and the corresponding food products, accounting for 1-3 % of the total plant. Which accounted for 60-90 % of total phosphorus plant, and phyticis the main storage form of phosphorus and inositol.Phytate may also form complexes with proteins, in the form of phytate phosphorus not only cannot take advantage of monogastric animals to digest, but also ananti-nutritional factors will inhibit the absorption and utilization of nutrients.Phytase widely founding animals, plants and micro-organisms（fungi and bacteria）,phytases are enzymes capable of hydrolyzing phytic acid to less-phosphorylated myo-inositol derivates and inorganic phosphate. Phytases belong to the phosphate monoester hydrolase, histidine acid phosphatase an important member of a family of enzymes. Phytase enables the utilization of phosphorus in plant feed increased by60 %,40 % reduction in fecal excretion of phosphorus, phytase in the feed cannot only improve the utilization of phosphorus in plant feed and reducing feces phosphorus pollution to the environment, but also by reducing the anti-nutritional effect of phytate,adding animal protein and some metal ions absorption capacity. So these are important to improving live stock production efficiency and reduce the environmental pollution.Since most phytase low expression in the original strains, and enzymology properties less prominent is not suitable for large-scale industrial production, using genetic engineering strain Pichia pastoris GS-115 strains as the host system, in order to improve the yield of phytase and now become widely used ways.Pichia pastoris eukaryotic expression system relative to the prokaryotic expression system with advantages of high levels of expression, fermentation technology is mature,secreted expression of the desired product, don’t form irreducible endosomes, and easy separation and purification etc. Pichia pastoris has been successful many applications in the expression of exogenous gene research. Alcohol oxidase promoter 1is a Pichia pastoris expression in the most widely used promoter, which was induced by methanol is very strong, can be used to facilitate expression of the gene encoding a foreign protein. Pichia pastoris expression system with high biological safety, has been widely recognized, in cluding the USFDA, including. Pichia pastoris strain GS-115 cells themselves secreted small amount of other proteins, ease separation and purification of heterologous proteins, can be high cell density fermentation, and the presence of secreted protein post-translational processing modifications, bringing them closer to natural conformation and activity of the protein, and therefore more suitable for expression of eukaryotic genes.In this study, according to published NCBI database phytase gene sequence of Yersinia rohdei（Gen Bank Accession No: ABU98779）, the use of codon bias optimization target gene sequence of Pichia pastoris, Yersinia rohdei synthesized by the PCR-based two-step DNA synthesis method（PTDS） phytase gene（Yr APPA）. Will contain the desired gene recombinant expression vector YR3345 electroporation in to GS115 strain invivo and secreted expression, by Vanadium–Molybdenum Yellow spectrometric method activity identification, SDS-PAGE and other methods later, the table can be obtained Yr APPA positive yeast strains. Experimental study the expansion of culture and induced by methanol, recombinant phytase, a crude enzyme solution after Ni²⁺-NTA agarose affinity column purification, related enzymatic characteristics and kinetic parameters of the positive recombinant Pichia strain was obtained. Experimental study the expansion of culture and induced by methanol, the crude enzyme solution after Ni²⁺-NTA agarose affinity column purification n related enzymatic characteristics and kinetic parameters of the positive recombinant Pichia pastoris strain.The results showed that: Km and Vmax values of recombinant phytase were 0.37mg/m L and 6410 μmol min-1 mg^-1, the enzyme relative molecular weight of 47 KDa.Enzyme optimum temperature and p H were 55 ℃ and 4.5; the impact studies of metalions and the chemical reagents（2 mmol） to enzymes that are Zn²⁺,Fe³⁺ and Cu²⁺inhibited the enzyme activity, while Mg²⁺,Cr³⁺,Co²⁺ and Ni+ ions inhibition only ten percent of the enzyme, has little effect on the enzyme activity. At different p H buffers37 ℃ for 3 h incubation, the residual activity of the recombinant phytase at p H 3-7.5range relative activity were more than 60 %. There atively wide range of p H acidic and alkaline, have a good adapt ability. The thermal stability of the enzyme was incubated at different temperatures for 30 min measured residual activity: at 60 ℃ for 10-30 min incubation the activity is relatively stable, the relative activity were about 40 %, in the65 and 70 ℃ relative activity was about 20 %; the refore enzyme stability at 60 ℃ more than 70 and 65 ℃. Pepsin and trypsin phytase showed at endency to inhibit recombinant phytase, recombinant phytase inhibited of trypsin is stable butin hibition of pepsin change is relatively large.In recent years, recombinant phytase is widely used as an additive used in feed industry, which needs to be able to secrete high levels of expression and easy product separation and purification of the expression system. However, naturally occurring strains of phytase expression and enzyme activity is low, it is difficult to obtain a large number of products, is not conducive to large-scale commercial production. The use of Pichia pastoris expression system as a bioreactor, to large-scale production of high fermentation activity of recombinant phytase and reduce production costs. The first experiment will Yersinia rohdei phytase in Pichia yeast expression of trans forming obtain thermally stable and relatively wide p H range of recombinant phytase. Taken together, these studies for the industrial production of recombinant phytase and applications provide a potential alternative.