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食品与发酵工业  2017, Vol. 43 Issue (6): 49-    
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环糊精葡萄糖基转移酶生产α-熊果苷的反应条件优化及分子改造
张文蕾,宿玲恰,陶秀梅,吴敬
江南大学,食品科学与技术国家重点实验室,江苏无锡,214122;江南大学,生物工程学院工业生物技术教育部重点实验室,江苏无锡,214122
Optimization of conditions for production of α-arbutin by cyclodextrin glucosyltransferase and its molecular modification
ZHANG Wen-lei,SU Ling-qia,TAO Xiu-mei,WU Jing
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摘要 选择4种环糊精葡萄糖基转移酶(CGTase)分别为来源于Paenibacillus macerans的α-CGTase,Bacillus circulans 251的β-CGTase,Bacillus stearothermophilus NO2和Anaerobranca gottschalkii的α/β-CGTase,研究不同种类的CGTase生产α-熊果苷的情况.以麦芽糊精为供体,对苯二酚(Hydroquinone HQ)为受体,通过CGTase和淀粉葡萄糖苷酶的两步酶法反应催化合成α-熊果苷.分别优化不同类型的酶合成α-熊果苷的条件,发现Anaerobranca gottschalkii来源的CGTase在如下最优条件下获得的HQ摩尔转化率最高,为25%:以葡萄糖当量(DE)值为9% ~ 13%的50 g/L麦芽糊精作为供体,8 g/L HQ为受体,缓冲液pH6.0,在40℃下反应24 h,沸水浴灭活后,加入糖化酶处理,高效液相色谱检测产物.为了进一步提高CGTase对底物的转化率,利用定点突变技术对A.gottschalkii CGTase进行分子改造,得到1个突变体Y299A,在最优的反应条件下突变体的HQ转化率可达40%.
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Abstract: α-CGTase of Paenibacillus macerans,β3-CGTase of Bacillus circulans 251,α/β-CGTase of Bacillus stearothermophilus NO2 and Anaerobranca gottschalkii were selected to study the effects of cyclodextrin glucosyltransferase (CGTase) on production of α-arbutin.α-arbutin was synthesized by a two-step enzymatic reaction system of CGTase and amyloglucosidase.The optimum conditions for the synthesis of α-arbutin were optimized,and the highest conversion rate of hydroquinone (HQ) was 25% by CGTase from Anaerobranca gottschalkii.The optimum conditions for enzyme conversion were as follows:50 g/L maltodextrin with glucose equivalent (DE) of 9%-13%,8 g/L hydroquinone,buffer pH 6.0,reaction at 40 ℃ for 24 h.The production of α-arbutin was determined by high performance liquid chromatography.In order to further improve the conversion rate of HQ,site-directed mutagenesis on CGTase under the optimal reaction conditions was carried out,and the hydroquinone conversion rate of the mutant Y299A was 40%.
               出版日期:  2017-06-25      发布日期:  2017-12-01      期的出版日期:  2017-06-25
基金资助: 国家杰出青年基金(31425020)
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