Cloning And Heterologous Expression Of Themanganese Superoxide Dismutase Gene Fromlactobacillus Casei LC18

Lactobacillus is a probiotic with a variety of benefits, including antioxidative effect to the host. Probitic products have also been studied and applied much more widely. The antioxidantive effect is one of the most important healthe properties and of great research value.Lactobacillus species evolve different mechanisms to defend themselves against oxygen toxicity such as superoxide dismutases (SODs), hydroperoxidases and high intracellular levels of metal ions. The SODs provide a cellular defense mechanism by scavenging O2-, which constitutes one of the major defense mechanisms of cells against oxidative stress. To the best of our knowledge, most of Lactobacillus species have no sod gene; only two kinds of Lactobacillus (Lactobacillus casei and Lactobacillus sake) may have sod genes according to the analysis of whole genome sequences, while no experimental verification for cloning and heterologous expression of Lactobacillus sod gene has been reported.In this study, first we evaluated six strains of Lacbacillus, and selecte Lactobacillus casei Lc2 which has the best antioxidant capacity as the sod gene donor. But after repeated PCR operations, we couldn't get specific results. The primers we used are designed according to the whole genome sequence of standard strain Lactobacillus casei ATCC 334. And maybe the genes of Lc2 changed a lot during the evolution, so the primers couldn't bind the sod gene specifically, or it didn't have sod gene at all. However, it still had high antioxidant abilities, may be because of the high lever of metal ion concentration in its cell or other mechanisms.After that, we used the Lactobacillus casei Lc18 as the donor, we got the sod gene successfully. Then the gene encoding the SOD was cloned from Lactobacillus casei Lc18, using primers designed according to the whole genome of Lactobacillus casei ATCC 334, ligated into the vector pET-28a(+) and heterologously expressed in Escherichia coli BL21(DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The superoxide radical scavenging activity of the recombinant strain was increased compared to that of the control. The SOD was purified by nickel ion affinity chromatography. The purified SOD showed a single band by SDS-PAGE analysis, performed an activity of 39.97 U/mg, The O2- scavenging activity and H2O2 scavenging activity of the recombinant E. coli BLSod was slightly higher than that of E. coli BL21(DE3). The slight increase in hydrogen peroxide scavenging activity may be explained by the positively related interaction between superoxide dismutase and catalase.The data also suggested that the antioxidative effects of disrupted cells were higher than those of intact cells. A similar finding was previously reported by Saide and Gilliland, who found that intracellular extracts of lactobacilli possessed higher antioxidative activity than intact cells. It is possible that the higher antioxidative activity of the intracellular extracts could be due to the better accessibility of antioxidative enzymes to the oxidant substrates.And the purified SOD was speculated to be manganese-containing SOD on the basis of its N-terminal amino acid sequence analysis. This is the first report of sod gene from Lactobacillus spp. being expressed in other prokaryote.