| A bacteriocin named plantaricin MG was produced by Lactobacillus plantarum KLDS1.0391 which was isolated from a traditional, naturally fermented cream from Inner Mongolia in China. Plantaricin MG had better inhibitory effect on gram-negative and gram-positive bacteria. The previous study of our project team showed that quorum sensing system mediated by AI-2(autoinducer-2) was existed in L. plantarum KLDS1.0391 and signaling molecule AI-2 played an important role on the regulation of bacteriocin production in co-culture with other lactic acid bacteria. Biosynthesis pathway of AI-2 has been very clear, and AI-2 is synthesized from the metabolite S-adenosylhomocysteine(SAH) via a process catalyzed by Pfs and Lux S. The prokaryotic expression vector p QE-30-lux S and p QE-30-pfs were constructed, and Lux S and Pfs proteins were expressed successfully in E.coli M15 in order to research effect of AI-2 on bacteriocin production of Lactobacillus plantarum KLDS1.0391. The results were helpful for the synthesis of AI-2 in vitro.In this study, induced expression and purification conditions of recombinant protein Lux S and Pfs were optimized in order to improve the expression of bioactive recombinant protein Lux S and Pfs. Then the synthesis conditions of AI-2 in vitro were optimized, and bioactive AI-2 was synthesized successfully in vitro. Effect of AI-2 on the growth and bacteriocin production of Lactobacillus plantarum KLDS1.0391 was studied on the basis of the above results. The expression level of pln EF gene encoding bacteriocin was studied by Real-time PCR. The results were as follows:(1) The factors including media, induction temperature, bacterial biomass before induction, induction time and concentration of IPTG were optimized by single-factor experiment design. The optimum conditions for the induced expression of recombinant protein were determined as follows: LB medium as the induction medium, the OD600 of bacterial before induction with 0.6~0.8, the IPTG concentration of 0.1mmol/L, the induction temperature of 37℃ and the induction time of 12 h.(2) Recombinant protein was purified using Ni-NTA Purification System under native conditions. The optimum Native Elution Buffer was determined by comparing effect of the imidazole concentration and p H of Native Elution Buffer on purification. The optimum binging buffer of protein and resin was determined by comparing effect of different binging buffer on purification. PBS buffer containing 3mol/L Na Cl was used for the binging of Lux S protein and resin. Recombinant protein Lux S was eluted with Native Elution Buffer(p H 6.5) containing 500mmol/L imidazole. PBS buffer was used for the binging of Pfs protein and resin. Recombinant protein Pfs was eluted with Native Elution Buffer(p H 8.0) containing 250mmol/L imidazole.(3) Recombinant protein Lux S and Pfs were expressed and purified under the optimum conditions. The concentrations of recombinant protein Lux S and Pfs obtained in this study were 5.23mg/mL and 6.71mg/m L respectively. The bioactive AI-2 was produced by recombinant protein Lux S and Pfs obtained in this study. The results suggested that recombinant protein Lux S and Pfs were bioactive.(4) The factors including incubation temperature, p H, enzyme concentration and incubation time were optimized by single-factor experiment design. The optimum conditions for the synthesis of AI-2 in vitro were determined as follows: the SAH concentration of 2mmol/L, the Lux S protein concentration of 1.0mg/mL, the Pfs protein concentration of 0.5mg/mL, the incubation temperature of 37℃, p H of 7.5, and the incubation time of 5h. AI-2 was synthesized in vitro under the optimum conditions. The biological activity of AI-2 synthesized was about 1-fold higher than the positive control. The concentration of AI-2 synthesized was about 2mmol/L.(5) In order to research effect of AI-2 on the growth and bacteriocin production of Lactobacillus plantarum KLDS1.0391, Lactobacillus plantarum KLDS1.0391 was cultured in MRS broth supplemented with AI-2 synthesized. The results showed that growth rate of Lactobacillus plantarum had no significant change, and bacteriocin production increased significantly(P<0.01). The increase of bacteriocin production varied with the addition amount of AI-2. Promoting effect of 3%(v/v) AI-2 on bacteriocin production was significantly higher than that of 1% and 5% AI-2 added(P<0.01).(6) After Lactobacillus plantarum was cultivated for 15 h at 37 ℃ in the MRS broth supplemented with 3%(v/v) AI-2, the expression level of pln EF gene encoding bacteriocin was researched by Real-time PCR. The results showed that the expression level of pln EF gene was 1.89-fold higher than the blank control.Conclusion: The induced expression and purification conditions of recombinant protein LuxS and Pfs were optimized, and the recombinant protein Lux S and Pfs with bioactivity was obtained under the optimum conditions obtained in this study. The conditions for the synthesis of AI-2 in vitro were optimized, and AI-2 was synthesized successfully in vitro by Lux S and Pfs proteins obtained under optimum conditions. AI-2 played an obvious positive regulatory role on bacteriocin production, and the regulatory effect varied with the addition amount of AI-2. AI-2 played no significant role on the growth of Lactobacillus plantarum. |
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