重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷

Whole-cell biosynthesis of 2-O-α-D-glu-copyranosyl-sn-glycerol by recombinant Bacillus subtilis

摘要:

2-O-α-D-甘油葡糖苷是一种在食品、化妆品、保健品及医药领域有着重大应用前景的高附加值产品，但国内仍未实现2-O-α-D-甘油葡糖苷的工业化生产，且鲜有关于2-O-α-D-甘油葡糖苷合成的相关报道。文中旨在开发一种利用食品安全级重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷的方法，通过构建一株异源表达肠膜明串珠菌蔗糖磷酸化酶 (Sucrose phosphorylase，SPase) 的重组枯草芽孢杆菌Bacillus subtilis 168/pMA5-gtfA，并将其用作全细胞催化剂合成2-O-α-D-甘油葡糖苷，通过优化培养温度、时间及全细胞转化条件，提高其转化合成2-O-α-D-甘油葡糖苷的产量。结果表明，重组枯草芽孢杆菌B. subtilis 168/pMA5-gtfA在30 ℃下培养20 h，菌体裂解物酶活力最大达1.43 U/mL，并且在1 mol/L蔗糖、2.5 mol/L甘油、pH 7.0、菌体OD600为40、30 ℃下全细胞转化反应48 h，共生成2-O-α-D-甘油葡糖苷189.3 g/L，平均转化速率为15.6 mmol/(L·h)，蔗糖转化率约为75.1%，是目前报道的利用重组枯草芽孢杆菌催化合成2-O-α-D-甘油葡糖苷的最高产量，这为2-O-α-D-甘油葡糖苷的工业化生产及应用奠定了理论和实验基础。

Abstract:

2-O-α-D-glu-copyranosyl-sn-glycerol is a high value-added product with prospective application in food, cosmetics, health products and pharmaceutical industries. However, industrial scale of 2-O-α-D-glu-copyranosyl-sn-glycerol has not yet been applied in China, and there are few related reports on 2-O-α-D-glu-copyranosyl-sn-glycerol synthesis. The purpose of this experiment is to develop a method for catalyzing the synthesis of food-grade 2-O-α-D-glu-copyranosyl-sn-glycerol using whole cells of “Generally Recognized as Safe” (GRAS) recombinant Bacillus subtilis. In our work, a recombinant B. subtilis 168/pMA5-gtfA that heterologously expressing Leuconostoc mesenteroides sucrose phosphorylase was constructed and used as a whole-cell catalyst to synthesize 2-O-α-D-glu-copyranosyl-sn-glycerol. Optimizing the culture temperature, time and whole cell transformation conditions has increased the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol. The results showed that 1.43 U/mL of sucrose phosphorylase was achieved in B. subtilis 168/pMA5-gtfA after culturing for 20 h at 30 °C in fermentation medium. The highest conversion rate reached 75.1%, and the yield of 2-O-α-D-glu-copyranosyl-sn-glycerol was 189.3 g/L with an average transformation rate of 15.6 mmol/(L·h) after 48 hours whole-cell transformation with the sucrose concentration of 1 mol/L and the glycerol concentration of 2.5 mol/L at 30 °C, OD600 40 and pH 7.0. This is the highest yield of 2-O-α-D-glu-copyranosyl-sn-glycerol synthesized catalytically by recombinant B. subtilis that was ever reported, and this study provides the theoretical and experimental basis for the industrial production and application of 2-O-α-D-glu- copyranosyl-sn-glycerol.