Characterization And Expression Of A Novel Phytase Gene YkAPPAS From Yersinia Kristensenii In Pichia Pastoris

In grain,phytic acid is the main storage form of inositol and phosphorus.In plate fooder,the organic phosphate compounds that the form of phytic acid salt referred to as phytic acid phosphorus.Animals can absorb phosphorus from fooder only when the phytate phosphorus is hydrolyzed into the form of inorganic phosphate.Phytase,a phosphate monoester hydrolase,is a new type of green additive in recent years.It is through the hydrolysis reaction,the phytic acid or phytic acid salt can decomposed into phosphates(or salt)and inositol.A lot of studies have demonstrated that using phytase as additives in the food,the absorption of phosphorus can be improved,so only small amount of inorganic phosphorus needed to be added in fooder and the proportion of inorganic phosphorus in animal waste is reduced,which caused environmental problems by excessive phosphorus.In addition of phytase in feed could improve the utilization rate of various trace elements and protein by decomposed anti-nutritrant phytic acid.However,the widespread natural phytase have some trouble for practical production,for example,its expression level is low,and extraction is difficult.So to construct genetic engineering strain as a miniature bioreactor to express the enzyme becomes most important to solve this problem.We can improve the expression level,proteinase resistance,and thermal stability of natural phytase secretion through genetic engineering technology.Pichia pastoris,which is easy operation and can be fermented at high-density,is a good strain to produce recombinant phytase.With the continuous development of microbial engineering technology,to search for the structure and function of the phytase is gradually maturity and deepening.In this study,we modified a phytase gene YkAPPA that coming from Yersinia kristensenii,and successfully expressed the enzyme in Escherichia coli and Pichia pastoris.At the same time,the recombinant phytase related enzymology characteristics and kinetic parameters are analyzed,the main research results are as follows:1.According to the phytase gene sequence(GeneBank: JQ394763.1)of Yersinia kristensenii and codon usage bias of the Pichia pastoris,we optimize the target gene without changing the amino acid sequence.Using the about 60 bp length primers,the phytase gene YkAPPAS was synthesized by the PCR-based two-step DNA synthesis method-PTDS,The length of recombinant phytase YkAPPAS gene is 1266 bp,which was consist of yeast optimal codons and the content of GC was adjusted to 51.9%.The recombinant phytase has two conservative region:the substrate binding site RHG×R×P and the catalytic center HD.The synthetic gene was ligated to vector pMD18-T,cloned and sequenced,then connect the sequenced synthesized gene was inserted into yeast expression vector pYPX88-YkAPPAS.After linearization,the phytase expression vector was transformed into Pichia pastoris GS-115 by electransformation method.After and fermention screening with highly active transformant strain,the production of phytase can reached 106.73?g/mL after induced with 1% methanol.Six histidine tags were added in the 5 'end of the recombinant phytase YkAPPAS gene,so this recombinant phytase can be purified through ammonium sulfate precipitation firstly and Ni ion affinity chromatography.SDS-PAGE showed that the molecular weight of recombinant phytase YkAPPAS is about 46 kDa,which was consistent with the bioinformatics prediction.The result showed that the YkAPPAS has no glycosylation.2.The characteristics of recombinant phytase showed that its optimum pH is pH4.5and pH6,and the activity of pH3-pH12 is over 30%;the optimum temperature is 45?and the activity was reduced to 56.75% at 90? for 5min,and reduced to 47.55% at 100? after treated for 5min.EDTA and Mn2+ have certain activation effect on the activity of enzyme but Cu2+,Al3+,Pb2+ and Zn2+have certain inhibitory effect to the enzyme activity;SDS can inhibit the enzyme activity strong.The related enzyme activity data show that genetic engineering strain can solve some problemes on low production and low activity for natural phytase.