Constructing And Expression Of Trehalose Synthase Gene In Bacillus Subtilis

Trehalose was extensively used in food, medicine and husbandry due to its special biological function and unique practical application. Although the development prospect was very promising, but the application was constrained by underproduction to some extent. The research explored by many scientists indicated that, the process of transferring maltose into trehalose by trehalose synthase in one step was highly recognized. But the trehalose has been impacted by lack of trehalose synthase.To expand the application of trehalose in the field of medicine and food safety, genetic engineering technique was adopted to clone trehalose synthase gene in the Pseudomonas putida P06 in this thesis. And then the target gene was inserted into expression vector pHT01, finally, the recombinant expression plasmid pHT01-TreS was constructed. The recombinant plasmid was transformed into Bacillus subtilis WB800n, and the IPTG gradient screening was used to select high copy transformants and some colonies were isolated at the concentration of 0.5mM IPTG. The recombinant strain was induced by 0.5mM IPTG for successful intracellular expression in Bacillus subtilis. The enzyme activity indicated that the trehalose synthase was effectively expressed in Bacillus subtilis, and the strain can produce crude trehalose synthase to transfer maltose into trehalose.Changing the cell disruption method, initial culture medium, induction conditions, protease test cultivation in fermenter, the optimum conditions for enzyme expression was obtained (IPTG 0.5mM, induction temperature 37℃, induction pH was 7.5, glucose 1.5%, peptone 1.0%, yeast extract 2.0%,muriate 1.0%, disodium hydrogen phosphate 0.6%, monometallic sodium orthophosphate 1.2%, magnesium sulfate 0.1%, ammonia sulfate 0.75%). Bacillus subtilis WB800n was cultured under optimization of fermentation medium and induction condition, the enzyme activity was up to1854U/g in shake flask culture increased by 98.3%. Compared with Escherichia coli cultured in both shake flask and high density fermentation, the result showed that enzyme activity expressed by Bacillus subtilis was upside potential. And Bacillus subtilis did not produce toxin and was much safer than Escherichia coli, so the application prospect was very broad. Although there are many problems about Bacillus subtilis expression of exogenous protein of trehalose synthase to be solved, but Bacillus subtilis was an internationally recognized safety strain, which has great advantage in the production of food and medicine grade.