State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi 214122, PR China.
Carbohydr Res. 2010 May 7;345(7):886-92. doi: 10.1016/j.carres.2010.02.002. Epub 2010 Feb 4.
The cgt gene encoding alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90h of induction, the activity of alpha-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant alpha-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 degrees C, and half-lives are approximately 8h at 40 degrees C, 1.25h at 45 degrees C and 0.5h at 50 degrees C. The recombinant alpha-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca(2+), Ba(2+), and Zn(2+) in a concentration-dependent manner, while it is dramatically inhibited by Hg(2+). The kinetics of the alpha-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.
来自解淀粉芽孢杆菌 JFB05-01 的 cgt 基因编码的α-环糊精葡萄糖基转移酶（α-CGTase）在大肠杆菌中作为 C 端 His 标记蛋白表达。诱导 90 小时后，培养基中α-CGTase 的活性达到 22.5 U/mL，约比原始菌株高 42 倍。通过镍亲和层析或离子交换和疏水相互作用层析的组合，将重组α-CGTase 纯化为均一性。然后，详细研究了纯化酶的环化活性。它在溶液中是单体。其最适反应温度为 45°C，在 40°C 时半衰期约为 8h，在 45°C 时半衰期约为 1.25h，在 50°C 时半衰期约为 0.5h。重组α-CGTase 的最适 pH 为 5.5，在 pH 6 和 9.5 之间具有较宽的 pH 稳定性。它在浓度依赖性方式下被 Ca(2+)、Ba(2+)和 Zn(2+)激活，而被 Hg(2+)强烈抑制。α-CGTase 催化的环化反应的动力学可以用 Hill 方程很好地描述。
Zotero 插件