| Phytate(phytic acid) is main storage form of phosphorus beans and cereals’ seeds. But due to the lack of enzymes to break down Phytate in vivo, monogastric animals(poultry, pigs, etc.) have a difficult to effectively utilize phytate, most of phosphorus in the form of phytic acid is excreted by animals directly, to cause serious environmental pollution; meanwhile, phytate is an anti-nutritional factors, and can be synthesized corresponding insoluble complex with a variety of metal ions such as Ca2+, Mn2+, Mg2+, Zn2+, Cu2+, Fe2+ and many proteins, etc. Reducing biologically effective utilization of these nutrients and effective use of nutrients for animals.Phytase(myo-inositol hexakisphosphate phosphohydrolase,EC 3.1.3.8) is the general term of a class of enzymes of catalysis phytic acid and inositol, phosphate acid(or phosphate)(these are hydrolyzates of phytate), belongs to histidine phosphatase family. As monogastric animal feed additives, phytase can effectively improve the utilization of phosphorus in plant feed, thereby increase the absorption of mineral elements for monogastric animals, but also reduce the phosphorus pollution of the environment in animal waste. Variety of microorganisms such as bacteria, yeast, filamentous fungi and so on can produce phytase, but phytase content of natural organisms is extremely low, it is difficult to obtain large quantities of products. Phytase genetic engineering bacteria constructed by genetic engineering techniques can achieve efficient heterologous expression of phytase, by genetic manipulation for phytase gene at the molecular level, improving the enzyme properties of phytase such as p H optimum, thermal temperature resistance, catalytic activity, etc., and improving its effectively biological activity in animals, especially the stability of phytase, etc., this will open up broader prospects for a large-scale production and application of phytase.This article will use phytase gene(Kr APPA) from Kosakonia radicincitans to efficiently express biologically active extracellular phytase(Kr APPAS) in Pichia pastoris expression system. Specific experimental procedure is as follows:Firstly, it will design the new nucleotide sequences of phytase gene, according to the phytase protein sequence of Kosakonia radicincitans and Pichia codon preference, then design a series of primers based on this sequence and synthesize the target gene. Through E. coli clone and sequence identification for the synthesized genes, the results showed that designed gene sequence is fully consistent with the wild type. The nucleotide sequence alignments between the new phytase gene and the wild-type showed that homologies are 77.7%.Connect the sequenced correctly phytase gene Kr APPAS to the p PIC9 K expression carrier, and import gene into Pichia GS115 cells by electric shock method, then screen the highly active recombinant yeast transformant strain, after methanol induction(concentration of 1%) 24 h, the amount of protein reached the highest, approximately 45 μg / ml, phytase activity of fermentation supernatant reached 82.27 U / m L, the specific activity was 1828.18U/mg, thus efficient secretion expression of phytase gene Kr APPAS in Pichia pastoris has achieved. This article has isolated and purified single recombinant phytase protein by ammonium sulfate precipitation and Ni ion affinity chromatography, SDS-PAGE showed a molecular weight is an approximately 45 k Da, and equivalent to the predicted theoretical value of bioinformatics in size, as a result almost no glycosylation phenomenon occured.The results of enzymatic properties showed that Vmax and Km values of recombinant phytase were 1735 μmol? min-1mg-1 and 0.236 m M, the optimum p H value is 3.5, the optimum temperature is 55 ℃, between 30-65 ℃, the relative activity of enzyme is high, the temperature is higher than 60 ℃, and relative activity decreased with increasing temperature. Thermal stability of recombinant phytase is better, and when below 65 ℃ it is very stable, after incubated 30 minutes at 65 ℃ activity of enzyme remained 90%, when the temperature exceeds 70 ℃, the stability of the recombinant phytase declined sharply, and incubated only 15 min to only about 50%. The inhibition of Cu2 + and Pb2 + to recombinant phytase is more intense, when the final concentration reached only 2 m M, the activity of recombinant phytase was about respectively 40% and 70%. Mg2 +, Ca2 +(final concentration is 2 m M) in metal ions have a certain activation on recombinant phytase, especially Mg2+’s activation is most obvious, and can make enzyme increase by about 15%. Recombinant laccase is extremely sensitive to SDS, after processed on final concentration of 2 m M, the residual activity of recombinant phytase is almost zero.In summary, the phytase from Kosakonia radicincitans has had a very highly successful expression in Pichia pastoris, this will provide excellent material factors for structure and function of phytase. This study has obtained an excellent trait’s and a potential commercial value’s phytase- Kr APPAS, identified as HAP phytase family and had the same active site consensus sequence RHGXRXP. Therefore, this study has important theoretical and practical value. |
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