| D-Mannose is used as a popular nutritional and health-beneficial food supplement all over the world. Reports have shown that D-mannose has beneficial effects on regulating blood sugar and against intestinal diseases. It is also widely used as a starting material for the synthesis of biological agents, vitamins and mannitol. D-mannose isomerase(MIase) is an important enzyme which is utilized for the conversion from D-fructose to D-mannose. Compared with other methods, enzymatic reaction is generally processed at gentle temperatures, pressures, and pH levels, which is much safer and more efficient than chemical synthesis or extraction methods. The aim of this research is to obtain a highly producing MIase strain with good enzyme properties.A MIase-producing bacterium Pseudomonas sp. SK27.016 was isolated from rotten fruits and deposited at China Center for Type Culture Collection(CCTCC) under the accession number M 2014411. When using D-fructose as substrate, analyses of purified product spectrum after enzyme reaction by HPLC, LC-MS, 1H NMR and FT-IR data revealed that the main product was D-mannose.To purify MIase, the crude cellular lysate of Pseudomonas sp. SK27.016 was undergone of stepwise purification protocols, including ammonium sulphate precipitation, HiTrap Q HP and HiTrap Phenyl FF [HS]. The molecular mass of enzyme was estimated as 45 kDa by SDS–PAGE.Using D-fructose as substrate, the enzyme showed maximum enzymatic activity under the condition of 40 °C and pH 8.0. Besides, the enzyme retained more than 90% of its initial activity after storage at 4 °C for 6 months. MIase was not a metal-required enzyme, bivalent metal ions like Zn2+?Ni2+ and Cu2+ significantly inhibited the enzyme activity, respectively. Using D-fructose as substrate, the 224.27 mM of Km, 21.67 s–1 of kcat, and 96.63 mM–1 s–1 of catalytic efficiency(kcat/Km) values were also estimated, respectively.By comparing the enzymatic property of Pseudomonas sp. SK27.016 MIase with the reported MIases, a MIase gene yihS from E. coli BL21 was transducted into plasmids pET-20b(+) and pET-28a(+), and expressed in E. coli BL21, respectively. The two transformants can both express active MIase. E. coli BL21/pET-28a(+)-yihS was an efficient system for intracelluar over-expression of MIase. Maximal expression was observed at 28 °C after 6 h incubation with 1 mM IPTG added, and its maximum intracellular MIase activity reached 4.8 U/m L.Then, the recombinant strains B. subtilis 168/pMA5-yihS and B. subtilis WB800/pMA5-yihS were constructed to express the MIase gene yihS. B. subtilis WB800/pMA5-yihS could achieve secretory expression of MIase without any inducer during fermentation process. When the fed-batch fermentation was performed, the enzyme activity of the recombinant MIase reached 51.2 U/m L after 39 h fermentation in 3.0 L Biotron-Li Flus GM bioreactor, which was 10.7-fold than that of the MIase expressed in E. coli. These results indicated that the MIase gene was successfully expressed in B. subtilis.The crude enzyme(1.3 L fermentation supernatant) was concentrated to approximately one fifth of the original volume, and then the concentrated solution with D-mannose producing activity was freeze-dried to produce 6.7 g of tan powder, which offered an alternative approach for D-mannose production. With 1g/L lyophilized MIase powder, a D-mannose yield of 150 g/L was obtained from 600 g/L D-fructose after 2 h, with the highest turnover yield of approximately 25% and a STY(space time yield) of 75 g D-mannose/L/h.The enzyme was further purified to electrophoresis homogeneity by DEAE Sepharose Fast Flow column. The molecular mass was estimated to be 275 kDa by size exclusion chromatography and 45 kDa by SDS-PAGE. The recombinant MIase expressed in B. subtilis exhibited optimal activity at pH 7.0, with the optimal temperature of 45 °C. Compared with Pseudomonas sp. SK27.016 MIase, the the optimal pH of recombinant MIase was at neutral, and the optimal temperature was 5 °C higher. Co2+, Ni2+ and Zn2+ showed inhibitory effects. Remarkably, MIase activity was totally inhibited by Cu2+. The apparent Km, Vmax, kcat, and catalytic efficiency(kcat/Km) values using D-fructose as substrate were 203.7 ± 6.7 mM, 0.6 ?M s-1 mg-1, 27.7 ± 0.7 s-1, and 136.0 ± 2.9 s-1 M-1.A constitutive promoter P43 from B. subtilis 168 genome and a yihS gene from E. coli were fused by overlapping extension PCR. The generating P43-yihS expression cassette and lox71-Spc-lox66 unit were together integrated into B. subtilis 1A751 chromosome. And then the antibiotic resistance gene(Spc) was knocked out based on Cre/loxP system. Finally, a food grade recombinant strain B. subtilis/loxP-P43-yihS was constructed. With the action of P43 promotor, the yihS gene was continuously expressed at the logarithmic growth phase and stationary phase, and the value of OD600 could reach to 5.6 after 14-hour fermentation. The enzyme activity of the recombinant MIase was about 3.5 U/m L after 16-hour fermentation, which was lower than that of the recombinant strain B. subtilis WB800/pMA5-yihS, but the recombinant strain B. subtilis/loxP-P43-yihS exhibited improved stability. |
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