Phytase Gene Expresstion In Lactobacillus Casei And Analysis Of Its Biochemical Characters

In this experiment,recombinant expression vector pIAβ8-phyA which contains phytase gene from Aspergillus ficuum was transformed into Lactobacillus casei (L. casei) by electroporation , and then the expressed phytase characteristics and incubation condition were analysized.The transgenic L. casei was encapsulated by the different embedding materials to increase its availability and living ability.The ability of the transgenic L. casei to resist the antibiotics was also studied for its application in animal husbandry.The competent cells of L. casei were prepared with MRS incubation medium,and then the recombinant expression vector pIAβ8-phyA was transformed into the competent L. casei by electroporation.Through the screening by antibiotics,the positive colonies with phytase activity were selected and incubated.The maximal phytase activities in the supernatant and cells were 21.31 and 4.57 U/ml(P<0.05),respectively.The phytase proteins from the supernatant and cells of the transgenic L. casei were subjected to polyacrylamide gel electrophoresis (SDS-PAGE) for protein analyses,which indicated that the molecular weight of the expressed phytase protein was 39.2 kDa.The phytase was used for thermostability and pH determination,the result demonstrated that the optimum pH and temperature of phytase were 5.0 and 70℃(P<0.05),respectively.The analysis of liquid fermentation showed that the pH value of the media got to the lowest value of 3.35 after 72 h incubation,cell dry weight reached the peak of 3.9 mg/ml after 96 h incubation,and cell optical density reached the peak of 5.89 after 144 h incubation,and the living cells reached the peak of 1.1×109 CFU/ml after 48 h incubation,respectively (P<0.05).Through the above analysis,the optimum harvest time was at the 4 th day of anaerobic incubation for the transgenic L. casei.The transgenic L. casei was encapsulated by the different embedding materials,such as agar,sodium alginate,polyvinyl alcohol and gelatin,and then the thermostability and availability of the embedded transgenic L. casei were determined.The results indicated that the best embedding conditions were 3% sodium alginate and frozen drying,and the living cells were kept at 7.55×105 CFU/ml.After embedding with the agar,the embedded L.casei had good thermostability at 85℃,and the living cells got to 1.68×107 CFU/ml.In order to know the relationship between probiotics and antibiotics ,chlortetracycline and colistin sulfate were used to determine their effects on L. casei growth. The results indicated that the 0.1% chlortetracycline and 0.05% colistin sulfate had significant resistant effect on L . casei growth,respectively(P<0.05).This study also showed that the L. casei and its supernatant had the effect to inhibit proliferation of the pathogenic Escherichia coli.