Expression Of Phytase Gene PhyA In Yarrowia Lipolytica

Phytase have been became a novel type of green feed additive in recent years, because it can hydrolyze inositol hexaphosphoric acid to myo-inositol and inorganic phosphate, which can increase forage availability, decrease cost, reduce the environmental pollution, therefore, it has been widely applied in animal feed,foods,medicine industry and environmental conservation. However, the thermal stability of the natural enzyme limits its application. The Yarrowia lipolytica can be used as a superordinary expressing host, which has been classified as generally regarded as safely, it was able to grow on carbohydrate and lipid as carbon source, so it is more suitable for different industrial application. This research selected the PT14 (modified pINA1296 vector) and pINA1297 expression vector of Yarrowia lipolytica. So using the expression vectors, we successfully constructed the recombinant strains Yarrowia lipolytica PT14-phyA and pINA1297-phyA. Then we wished to obtain the phytase of mass high activity and good stability. Up to now, there had no reports about using the two expression vectors to express phytase phyA gene.Methods,content,experimental results:1. The clone of phytase phyA gene: the genomic DNA of Pichia pastoris GS115-phyA was obtained by sodium chloride method, the study used it as the template, and designed two couples of primers according to Aspergillus niger NRRL3135 phyA gene sequence, then the research amplified the Aspergillus niger NRRL3135 phyA gene without the signal peptide sequence and intron sequence by the polymmerse chain reaction(PCR), the two kinds of amplified fragments were PT14phyA and 1297phyA, the PT14phyA contained seven encoding genes of LacO, the another had the SfiⅠand KpnⅠrestricted enzyme sites.2. The amplified fragment PT14phyA was cloned into PT14 vector to generate a recombinant vector of PT14-phyA,PT14-phyA was linearized by NotⅠand transfor- med into Yarrowia lipolytica po1g by electroporation method, the recombinant Yarrowia lipolytica po1g was obtained by MD and PPB plates, after induced in PPB medium for 4 day at 28℃, the activity of the expressed phytase phyA achieved 22.75U/mL, the optimal pH and temperature of the expressed phytase phyA was 5.5 and 55℃, the phytase retained 62.47% activity after incubation at 90℃for 10 min, and it was highly resistant to pepsin or trypsin treatment.3. The 1297phyA fragment was seclected into pINA1297 vector to construct a recombinant vector of pINA1297-phyA,pINA1297-phyA was linearizedn by NotⅠand transformed into Yarrowia lipolytica po1h by the lithium acetate method. The positive transformants were obtained by YNBcasa and PPB plates, after cultivated in YM medium for 6 day at 28℃, the activity of the expressed phytase phyA achieved 636.23 U/mL, it had a molecular weight of 130 kDa with SDS-PAGE analysis, but its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytase phyA revealed its optimal pH and temperature was 5.5 and 55℃, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h, moreover, its activity remained about 86.08% after exposure at 90℃for 10 min, it is higher than all of the phytase phyA expressed in other yeasts, and it had 97.08% or 88.85% activity after treatment for 2 hours with pepsin and trypsin respectively. For sodium phytate, the Km value of the expressed phytase was 0.187 mmol/L at 37℃and pH was 5.5。...