Bolam D N, Ciruela A, McQueen-Mason S, Simpson P, Williamson M P, Rixon J E, Boraston A, Hazlewood G P, Gilbert H J
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
Biochem J. 1998 May 1;331 ( Pt 3)(Pt 3):775-81. doi: 10.1042/bj3310775.
To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLACBD) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellum endoglucanase (EGE). The two CBDs exhibited similar Ka values for bacterial microcrystalline cellulose (CELECBD, 1.62x10(6) M-1; XYLACBD, 1.83x10(6) M-1) and acid-swollen cellulose (CELECBD, 1.66x10(6) M-1; XYLACBD, 1.73x10(6) M-1). NMR spectra of XYLACBD titrated with cello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The Ka values of the XYLACBD for C6, C5 and C4 cello-oligosaccharides were estimated to be 3.3x10(2), 1.4x10(2) and 4.0x10(1) M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLACBD to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates. Pseudomonas CBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLACBD and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.
为研究纤维素结合结构域（CBDs）的作用模式，荧光假单胞菌纤维素亚种木聚糖酶A（XYLACBD）和纤维素酶E（CELECBD）的II型CBD被单独表达或与嗜热栖热放线菌内切葡聚糖酶（EGE）的催化结构域融合表达。这两种CBD对细菌微晶纤维素（CELECBD，1.62×10⁶ M⁻¹；XYLACBD，1.83×10⁶ M⁻¹）和酸溶胀纤维素（CELECBD，1.66×10⁶ M⁻¹；XYLACBD，1.73×10⁶ M⁻¹）表现出相似的Ka值。用纤维寡糖滴定XYLACBD的核磁共振谱表明，当CBD结合纤维六糖、纤维五糖或纤维四糖时，三个色氨酸残基的环境受到影响。XYLACBD对C6、C5和C4纤维寡糖的Ka值分别估计为3.3×10²、1.4×10²和4.0×10¹ M⁻¹，这表明CBD可以容纳至少六个葡萄糖分子，并且对不溶性纤维素的亲和力远高于可溶性寡糖。将CELECBD或XYLACBD与EGE的催化结构域融合增强了该酶对不溶性纤维素形式的活性，但对羧甲基纤维素无活性。当分别以不溶性纤维素和纤维素/半纤维素复合物为底物，将CBD与EGE或XYLA的催化结构域一起孵育时，未观察到纤维素酶活性增加。假单胞菌CBDs不会像一类称为扩展蛋白的植物细胞壁蛋白那样诱导分离的植物细胞壁伸展，也不会使纤维素纸条变弱。XYLACBD和CELECBD不会从棉花表面释放小颗粒。讨论了这些结果与II型CBDs作用模式的相关性。
