Expression Of Ascorbate Peroxidase Of Chinese Kale In E. Coli And Its Enzymatic Characteristics Study

Ascorbate peroxidase (APX, EC1.1.1.11) is a kind of peroxidase whose electron donor is the ascorbic acid. APX is one of the antioxidative enzymes in reactive oxygen species scavenging in the living cells. APX is very unstable in vitro, which cause difficulty in purification and its characteristic studies. The cloning and expression of plant recombinant APX in E. Coli is one ways to solve the above problem, however, the activity of the recombinant APX is low due to the lack of heme. In this paper, the recombinant plasmid pET-28a-Ba-APX was constructed and expressed in E. coli. Through optimization of culture conditions, the high-level expression was realized and the enzymatic properties of purified recombinant protein were studied.Taking cDNA as a template, the target gene (Ba-APX) from the Chinese kale seedlings was amplified. The similarities of the Ba-APX with the Brassica oleracea, Brassica rapa, Brassica napus and Brassica juncea were99%,98%,97%and92%, respectively. The pET-28a-Ba-APX was constructed and transformed it into the E. coi/BL21successfully.Through optimization of the IPTG concentration, culture time and temperature, the optimal conditions were found to be0.2mmol/L IPTG,23℃and10h. Under these optimized condition, the specific activity increased from18.7U/g to52.7U/g,which was3times compared with that of the control. Further, exogenous glutamate, glycine,5-aminolevulinic acid, FeCl2and CaCl2could improve the recombinant APX specific activity. The optimal concentrations of glutamic acid and glycine were both90μmol/L, and the specific activity were increased from16.1U/g and15.5U/g to47.0U/g and32.0U/g,which were increased1.5times and1time than no addition exogenous respectively. In addition,the optimal concentratioon of5-aminolevulinic acid was150μmol/L, and the specific activity was increased from10.9U/g to53.5U/g, which was4times more than the control.The recombinant APX was purified by affinity chromatography and the enzymatic properties were studied. The optimal reaction temperature and pH were40℃and6.5, respectively. The KmH2O2vmax and KmAsA were1.5×10-4mol/L,1.1×10-6mol/(L·min) and1.3×10-3mol/L, respectively.In order to understand the mechanism that exogenous additives improved the recombinant APX activity, the full wavelength absorption of the purified recombinant proteins was measured. The results showed that the heme saturation of the samples adding0.2mmol/L glutamic acid, glycine,5-aminolevulinic acid or FeCl2were0.29,0.28,0.43,0.28, which were increased17%,21%,79%and13%compared with that of the control, respectively. The activity of the recombinant APX was positive related to the heme saturation.