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目的:使用表达蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)的大肠杆菌重组工程菌E.coli BL21/pET-spase,作为全细胞催化剂,合成2-O-D-吡喃葡糖基-L-抗坏血酸(Ascorbic acid 2-glucoside,AA-2G)。通过反应条件的优化研究,提高AA-2G的收率。方法:分别考察菌体量、缓冲液pH、蔗糖浓度、维生素C浓度、反应时间和温度对AA-2G合成反应的影响,再组合上述最佳条件进行反应。AA-2G的产量使用高效液相色谱法进行定量。结果:最佳反应条件为:菌体量15 mg/mL,缓冲液pH 4.5,蔗糖浓度100 g/L,维生素C浓度175 g/L,反应时间20 h,温度37℃。在此条件下,AA-2G产量达到了35.7 g/L。结论:以蔗糖为底物,使用SPase合成AA-2G的研究报道较少。本研究通过优化此方法的反应条件,让AA-2G的产量得到了大幅提高。同时本研究中成功地采用了大肠杆菌工程菌作为全细胞催化剂,这比传统的使用粗酶液的方法更省时省力,有良好的应用潜力。
OBJECTIVE: To synthesize 2-OD-glucopyranosyl-β-glucopyranosyl-β-glucuronosyltransferase by using recombinant E. coli BL21 / pET-spase expressing sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) Ascorbic acid 2-glucoside (AA-2G). Optimize the reaction conditions to improve the yield of AA-2G. Methods: The effects of microbial biomass, buffer pH, sucrose concentration, vitamin C concentration, reaction time and temperature on the synthesis of AA-2G were investigated respectively. Then the above optimal conditions were combined for reaction. The yield of AA-2G was quantified using high performance liquid chromatography. Results: The optimal reaction conditions were as follows: bacterial cell volume 15 mg / mL, buffer pH 4.5, sucrose concentration 100 g / L, vitamin C concentration 175 g / L, reaction time 20 h and temperature 37 ℃. Under this condition, AA-2G production reached 35.7 g / L. Conclusion: There are few reports about the synthesis of AA-2G by using sucrose as substrate. In this study, by optimizing the reaction conditions of this method, AA-2G production has been greatly improved. At the same time, Escherichia coli engineering bacteria was successfully used as whole cell catalyst in this study, which is more efficient and labor-saving than the traditional method of using crude enzyme solution and has good potential for application.