State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Appl Microbiol Biotechnol. 2013 Apr;97(8):3467-74. doi: 10.1007/s00253-012-4292-9. Epub 2012 Jul 26.
Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.
环糊精（CD）是由葡萄糖单元组成的环状α-1,4-葡聚糖，在食品、制药、化妆品、农业、化工等领域有广泛的应用。CD 通常由淀粉经环糊精葡萄糖基转移酶（CGTase）生产。本研究采用普鲁兰酶与α-CGTase 协同作用，开发了一种从淀粉中同时转化生产 CD 的方法。本研究克隆并在大肠杆菌 BL21(DE3)中表达了嗜热真菌的普鲁兰酶。该酶的生化特性研究表明，重组酶的最适温度和 pH 分别为 50℃和 5.5，在 30℃、40℃和 60℃下，相对酶活分别保持在 60%、85%和 78%。当重组普鲁兰酶与α-CGTase 同时用于转化马铃薯淀粉（15%，w/v）为 CD 时，发现最适条件为：每克底物添加 10 Uα-CGTase 和 48 U 普鲁兰酶，反应温度为 30℃，pH 5.6。在此最佳条件下，24 h 后 CD 的总收率达到 84.6%（w/w），比单独使用α-CGTase 提高了 31.2%。这是首次报道α-CGTase 和普鲁兰酶同步生物转化生产 CD，是迄今为止报道的最高效率的 CD 生产方法。
