| 余 磊,李骥璇,王忆茗,郑桂兰,王洪钟.蔗糖磷酸化酶全细胞催化AA-2G的条件优化[J].现代生物医学进展英文版,2017,17(14):2601-2605. |
| 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 |
| Received:October 20, 2016 Revised:November 15, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.14.001 |
| 中文关键词: 蔗糖磷酸化酶 AA-2G 全细胞催化 |
| 英文关键词: Sucrose phosphorylase AA-2G Whole-cell catalysis |
| 基金项目:国家自然科学基金项目（21476124） |
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| 中文摘要: |
| 摘要 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。 |
| 英文摘要: |
| ABSTRACT 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. |
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