蔗糖磷酸化酶全细胞催化AA-2G的条件优化

Optimization of Whole-cell Synthesis of AA-2G by Sucrose Phosphorylase

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： Sucrose phosphorylase （EC 2.4.1.7, Sucrose phosphorylase, SPase） was produced by recombinant strain E. coli BL21/pET-spase. This recombinant strain was used as Whole-cell catalysis to synthesize 2-O-a-D-Glucopyranosyl-L-ascorbicacid （Ascorbic acid 2-glucoside, AA-2G）. Reaction conditions were optimized to get higher production of AA-2G. Methods： The effects of wet cell weight, pH value of buffer solution, sucrose concentration, Vitamin C concentration, reaction time and temperature on production of AA-2G were investigated. High-performance liquid chromatography was used to determine the production of AA-2G. Results： The optimal conditions for synthesis of AA-2G were wet cell weight of 15 mg/ml, buffer solution pH value of 4.5, sucrose concentration of 100 g/L, Vitamin C concentration of 175 g/L, reaction time of 20 h, and temperature of 37 ℃. Under these conditions, the production of AA-2G was 35.7 g/L. Conclusion： Synthesis of AA-2G from sucrose by SPase is still under basic research. No industrial application is reported yet. In this paper, reaction conditions of AA-2G synthesis were optimized, which led to higher production of AA-2G. Whole cells of recombinant E.coli were successfully used as catalysis. Since whole-cell catalysis is easier to prepare than crude enzyme, its potential of application is promising.