Takaha T, Yanase M, Takata H, Okada S, Smith S M
Institute of Cell and Molecular Biology, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, United Kingdom.
J Biol Chem. 1996 Feb 9;271(6):2902-8. doi: 10.1074/jbc.271.6.2902.
Potato D-enzyme was purified from recombinant Escherichia coli, and its action on synthetic amylose (average Mr of 320,000) was analyzed. D-enzyme treatment resulted in a decrease in the ability of the amylose to form a blue complex with iodine. Analysis of the products indicated that the enzyme catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic alpha-1,4-glucan (cycloamylose). Confirmation of the cyclic structure was achieved by demonstrating the absence of reducing and nonreducing ends, resistance to hydrolysis by glucoamylase (an exoamylase), and by "time of flight" mass spectrometry. The degree of polymerization of cycloamylose products was determined by time of flight mass spectrometry analysis and by high-performance anion-exchange chromatography following partial acid hydrolysis of purified cycloamylose molecules and was found to range from 17 to several hundred. The yield of cycloamylose increased with time and reached >95%. D-enzyme did not act upon purified cycloamylose, but if glucose was added as an acceptor molecule, smaller cyclic and linear molecules were produced. The mechanism of the cyclization reaction, the possible role of the enzyme in starch metabolism, and the potential applications for cycloamylose are discussed.
从重组大肠杆菌中纯化出马铃薯D-酶，并分析了其对合成直链淀粉（平均分子量为320,000）的作用。D-酶处理导致直链淀粉与碘形成蓝色复合物的能力下降。对产物的分析表明，该酶催化直链淀粉上的分子内转糖基化反应，生成环状α-1,4-葡聚糖（环糊精）。通过证明不存在还原端和非还原端、对葡糖淀粉酶（一种外切淀粉酶）水解的抗性以及通过“飞行时间”质谱法，证实了环状结构。通过对纯化的环糊精分子进行部分酸水解后的飞行时间质谱分析和高效阴离子交换色谱法，确定了环糊精产物的聚合度，发现其范围为17至数百。环糊精的产率随时间增加，达到>95%。D-酶对纯化的环糊精不起作用，但如果加入葡萄糖作为受体分子，则会产生较小的环状和线性分子。讨论了环化反应的机制、该酶在淀粉代谢中的可能作用以及环糊精的潜在应用。
