Characteristics Of The Mannitol Dehydrogenase In Pathway Of Leuconostoc Highly Producing Mannitol

Mannitol, a six carbon acyclic suger alcohol, have been widely used in fodder as sweetening agent.Mannitol is mainly produced by extraction method, chemical synthesis method, enzyme conversion method and microbiological fermentation method etc. Microbiological fermentation method would be the main trend of mannitol production in the future owing to high productivity and avoiding any by-products production. At present, a variety of microorganisms have been reported they can produce mannitol, including molds, yeasts and bacteria, among them, the heterofermentative lactic acid bacteria is a kind of main bacteria. Leuconostoc acts as one of heterofermentative lactic acid bacteria, the metabolic pathway of synthesizing mannitol is catalyzing fructose to produce mannitol under the role of mannitol dehydrogenase.In order to verify the mannitol dehydrogenase characteristic for better application in mannitol production, Leuconostoc pseudomesenteroides was used as the research object in this study, mannitol dehydrogenase gene was obtained from Leuconostoc pseudomesenteroides and expressed in E. coli BL21(DE3). The results revealed that Leuconostoc pseudomesenteroides genomic DNA was used as template, a large number of mannitol dehydrogenase genes were amplified by PCR, and PCR product was 1.107kb. The recombinant plasmid pETDuet-1-mdh was transformed into E. coli BL21 (DE3) for translation and analyzed successful expression of recombinant protein by SDS-PAGE. Results indicated that the protein molecular weight was about 36.007 kDa, which was consistent with the expected molecular weight and meant successful expression and higher expression quantity of recombinant protein.For optimizing the mannitol dehydrogenase gene expression condition of IPTG induction, the recombinant mannitol dehydrogenase gene expression was induced by different concentrations, times, and temperatures of IPTG. Results suggested 0.5 mM IPTG induced 16 h under 30℃ was benefit for mannitol dehydrogenase expression.Optimum reaction temperature, pH, thermal stability and chemical inhibitors of mannitol dehydrogenase were determined for futher understanding the characteristics of mannitol dehydrogenase. The results showed:Optimum temperature and pH were 30 ℃ and 7, respectively. Mannitol dehydrogenase enzyme reaction could be promoted by adding Ca2+ and urea, and mannitol dehydrogenase activity was increased with little difference. However, activity was inhibited by adding Fe2+, Ba2+, Zn2+, Cu2+, Na+, and SDS, and Na+and SDS had stronger inhibitory effect on enzyme activity among them. Enzyme was incubated at metal bath for 10 min from 30℃ to 80℃, about 25% activity lost at 30℃ and 40℃,32% lost at 50℃,47% activity lost at 60℃, about 60% lost at 70℃, and all enzyme activity lost at 80℃ for 10 min with zero of residual activity.The experimental results provide a theoretical basis for Leuconostoc application in mannitol production.