Heterologous Expression And Fermentation Optimization Of D-psicose 3-epimerase

D-allulose is a C-3 epimer of D-frutcose, which is a kind of rare sugar with excellent physiological functions. It has a extremely good application prospect in food, health care and medical fields. At present, an important method for producing D-allulose is enzymatic biotransformation catalyzed by D-psicose 3-epimerase(DPEase). Nowadays, most studies about DPEase are using E.coli as the expression host, while Bacillus subtilis as traditional industrial production bacteria, with strong protein secretion capacity, is a ideal system for protein expression and secretion research. More importantly, it is also a kind of food-grade microorganism. In this research, dpe genes from different microorganisms were cloned and expressed in Bacillus subtilis, thus establishing the foundation for food-grade expression system of DPEase.Firstly, we designed primers and cloned D- psicose 3- epimerase genes from Clostridium bolteae ATCC BAA-613, Clostridium cellulolyticum H10 and Clostridium scindens ATCC 35704. Then P43 promoter was inserted directly upstream of the dpe genes by overlap extension PCR technology to form P43-Cb-dpe, P43-Cc-dpe and P43-Cs-dpe expression cassette; these fragments were subcloned into shuttle vector pMA5 thus generating the recombinant plasmids pMA5-P43-Cb-dpe, pMA5-P43-Cc-dpe and pMA5-P43-Cs-dpe, respectively.Secondly, the recombinant plasmids were transformed into competent cells of Bacillus subtilis WB800 for expression, which constructed recombinant strains B. subtilis WB800/pMA5-P43-Cb-dpe, B. subtilis WB800/pMA5-P43-Cc-dpe and B. subtilis WB800/pMA5-P43-Cs-dpe. The recombinant strain B. subtilis WB800/pMA5-P43-Cb-dpe showed the highest fermentation enzyme activity. After fermentation and protein expression, the recombinant enzymes were purified by nickel-affinity chromatography and analyzed by SDS-PAGE.Then the enzymatic properties of recombinant Cb-DPEase were studied. The optimum temperature and pH for recombinant enzyme were 55℃ and 7.0, respectively. It was highly stable under 50℃ and pH 6.5~7.5. Co2+ and Mn2+ could significantly enhance enzyme activity. In terms of the kinetic parameters, the optimum substrate was D- allulose and its apparent Km value was 26.68 mM, which was much smaller than 61.80 mM of D-frutcose and 323.44 mM of D-tagatose. This suggested the recombinant enzyme’s affinity for D-allulose was higher than D-tagatose and D-frutcose. Meanwhile, D-allulose showed the highest catalytic efficiency and the kcat /Km value was 95.8 min-1 mM-1.After that, the effects of medium compositions and fermentation conditions in shake culture on the production of Cb-DPEase were studied. The optimum culture medium was consisted of 15 g/L sucrose as carbon resource, 20 g/L yeast extract as nitrogen resource and 0.05 mmol/L Mn2+ as the metal ion. The optimum fermentation conditions were as following: fermentation temperature 37℃, initial pH 7.0, inoculums size 3% and bottled fluid volume 10%. Under these conditions, the highest activity was 8.13 U/m L in shake culture at 18 h, and that reached 7.55 U/m L in fermentation tank for 18 h. In the end, the stability of recombinant plasmid was tested, the results showed both segregational and structural stability of double promoters expression vector were fully stable after 100 generations.