以纤维寡糖为底物，通过稳态动力学评估黑曲霉β-葡萄糖苷酶的亚位点结构。

Subsite structure of the beta-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates.

作者信息

Yazaki T, Ohnishi M, Rokushika S, Okada G

机构信息

Department of Agricultural and Biological Chemistry, College of Agriculture, University of Kyoto Prefecture, Japan.

出版信息

Carbohydr Res. 1997 Feb 20;298(1-2):51-7. doi: 10.1016/s0008-6215(96)00287-x.

DOI:10.1016/s0008-6215(96)00287-x

PMID:9076931

Abstract

The beta-glucosidase from a commercially available preparation from Aspergillus niger was highly purified. The Michaelis constant Km and the molar activity K0 for cello-oligosaccharide substrates Gn (n = 2-6) were obtained by steady-state kinetic analysis on the beta-glucosidase-catalyzed hydrolysis at 25 degrees C and pH 5.0. Stoichiometric production of Gn-1 by the beta-glucosidase reaction for Gn was confirmed by HPLC techniques. Based on Km and K0 for Gn, subsite affinities (Ai, i = 1-6) were estimated as follows (kcal/mol): A1 = 1.3, A2 = 5.2, A3 = 0.65, A4 = -0.10, A5 = -0.65, and A6 = -0.26, of which A1-A3 are much higher than those of the beta-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the alpha-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for beta-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.

摘要

对市售黑曲霉制剂中的β-葡萄糖苷酶进行了高度纯化。通过在25℃和pH 5.0条件下对β-葡萄糖苷酶催化水解进行稳态动力学分析，获得了纤维寡糖底物Gn（n = 2 - 6）的米氏常数Km和摩尔活性K0。通过HPLC技术证实了β-葡萄糖苷酶对Gn反应生成化学计量的Gn - 1。基于Gn的Km和K0，亚位点亲和力（Ai，i = 1 - 6）估计如下（千卡/摩尔）：A1 = 1.3，A2 = 5.2，A3 = 0.65，A4 = -0.10，A5 = -0.65，A6 = -0.26，其中A1 - A3远高于威克汉姆念珠菌β-葡萄糖苷酶的相应值。该亚位点结构与黑曲霉α-葡萄糖苷酶的结构非常相似，而k0对n的依赖性是β-葡萄糖苷酶的高度特征，且随n降低，表明特定亚位点之间存在一些相互作用。