Intracellular And Periplastic Expression Of Aerococcus Viridans Pyruvate Oxidase In Escherichia Coli

Pyruvate oxidase is an important enzyme used as a reagent in kits or biosensors for biochemical analyses.However,the yield of pyruvate oxidase from wild type Aerococcus viridans and Lactobacillusplantarum was low(120 U/L).This study use Escherichia coli BL21(DE3)as the host to enhace the yield of pyruvate oxidase using recombinant genetic technology.The mian results are followings:(1)According to the gene sequence of pyruvate oxidase derived from Aerococcus viridans by NCBI,using synthetic genome as template obtained pyruvate oxidase gene fragment,which was inserted into different vectors pET25b-pod,pET28a-pod and pET32a-pod,respectively.Three recombinant pET vectors were constructed for pyruvate oxidase expression in E.coli.The fermentation of different recombinant Escherichia coli was optimized.The isopropyl-?-d-thiogalactoside(IPTG)concentration and induction temperature were optimized,with the result that the highest pyruvate oxidase yield(4106.9 U/L)of the recombinant E.coli pET28a-pod was obtained under conditions of 25?,0.5 mmol/L IPTG,0.5 OD600,and 24 h of induction after 2h of cultivation.The activity of other Escherichia coli pET25b-pod and pET28a-pod was 650.1 and 1812.6 U/L.Then the pyruvate oxidase of the recombinant E.coli,after cell lysis by sonication,was purified using a Ni-NTA agarose column.A total of 88.89%of pyruvate oxidase was obtained after washing with 100 mmol/L imidazole solution.Subsequently,9.99%of pyruvate oxidase was acquired after washing with 500 mmol/L imidazole.(2)During optimisation of recombinant E.coli cultivation,96.91%of pod enzyme activity was lost at the condition of 25 mL working volume in 250 mL flasks due to production in inclusion body formation.This result showed that Na2SO3 and glycerol addition had no significant influence on recombinant pyruvate oxidase.After 64 h induction,the lost of pyruvate oxidase activity was 11.95%under the condition of pH 6.0 maintainence.This result indicated that less working volume changed growth rate of recombinant Escherichia coli,then it caused pH change,and pyruvate oxidase was decreased.Finally,glycerol addition increased the cell density and volumetricpyruvate oxidase activity to 21243.26 U/L at pH 6.0 after a 64 h induction.(3)SecB,as a chaperone,was coexpressed to promote the secretion of pyruvate oxidase,and then enhanced the pyruvate oxidase leakage through glycine addition.Further,Cu2+ was used to improve the stability of pyruvate oxidase.We constructed secretion vector pRD-pod and three vectors under different promoters controlled pRD-pod-T7-secb,pRD-pod-lac-secb,pRD-pod-Bla-secb respectively to investigate the effect of SecB protein content on secretion of pyruvate oxidase.During the IPTG concentration optimization,the recombinant E.coli pRD-pod-Bla-secb reached 646.9 U/L.Based on this results,we found that under conditions of 25?,0.05 mmol/L IPTG,0.5 OD600 and pH 7.0,the activity of pyruvate oxidase reached 795.7 U/L.Finally,the secretory activity of pyruvate oxidase reached up to 2926.3 U/L under conditions of 3%glycine and 0.02 mmol/L CuSO4.This study constructed Escherichia coli BL21(DE3)by genetic technology,enhancing the yield of pyruvate oxidase by culture conditions.Finally,we found secretory expression of pyruvate oxidase in recombinant vectors.