Schülein M
Novo Nordisk, Novo Allé, Bagsvaerd, Denmark.
J Biotechnol. 1997 Sep 16;57(1-3):71-81. doi: 10.1016/s0168-1656(97)00090-4.
We present the analysis of the activities towards soluble and insoluble substrates of seven cellulases cloned from the saprophytic fungus Humicola insolens. The activity on the soluble polymer substrate carboxymethylcellulose (CMC) was used to determine the pH activity profiles of the five endoglucanases (EG), whereas cellotriose and reduced cellohexaose were used to determine the pH activity profiles of cellobiohydrolase I (CBH) and CBH II. All the EGs show optimal activity between pH 7 and 8.5, while CBH I and CBH II peak around pH 5.5 and 9, respectively. The catalytic activities of five of these cellulases were investigated under neutral and alkaline conditions using reduced cellohexaose as a substrate in a cellobiose oxidase coupled assay. EG I and CBH I both belong to family (7) according to a recent classification of glycosyl hydrolases. They both have activity against cellotriose. Therefore, they were studied using a coupled assay involving glucose oxidase. The activity on insoluble substrate (phosphoric-acid swollen cellulose) was assessed by the formation of reducing groups. The presence of a cellulose binding domain (CBD) lowers the apparent KM. This can be explained by the dispersing action of CBD. However, the CBD also reduces the apparent k(cat) probably by slowing down the mobility. EG I, EG II and EG III show similar activity towards CMC and amorphous cellulose, while EG V, EG VI, CBH I and CBH II have the highest catalytic rate on amorphous cellulose. In summary, Humicola insolens possesses a battery of cellulose-degrading enzymes which cooperate in the efficient hydrolysis of cellulose.
我们展示了对从腐生真菌特异腐质霉克隆的七种纤维素酶针对可溶性和不溶性底物的活性分析。利用对可溶性聚合物底物羧甲基纤维素（CMC）的活性来测定五种内切葡聚糖酶（EG）的pH活性曲线，而纤维三糖和还原型纤维六糖则用于测定纤维二糖水解酶I（CBH）和CBH II的pH活性曲线。所有的内切葡聚糖酶在pH 7至8.5之间表现出最佳活性，而CBH I和CBH II的活性峰值分别在pH 5.5和9左右。在中性和碱性条件下，以还原型纤维六糖为底物，通过纤维二糖氧化酶偶联测定法研究了其中五种纤维素酶的催化活性。根据糖基水解酶的最新分类，EG I和CBH I都属于第7家族。它们都对纤维三糖有活性。因此，使用涉及葡萄糖氧化酶的偶联测定法对它们进行了研究。通过还原基团的形成来评估对不溶性底物（磷酸膨胀纤维素）的活性。纤维素结合结构域（CBD）的存在降低了表观KM。这可以通过CBD的分散作用来解释。然而，CBD也可能通过减缓流动性降低了表观k(cat)。EG I、EG II和EG III对CMC和无定形纤维素表现出相似的活性，而EG V、EG VI、CBH I和CBH II对无定形纤维素具有最高的催化速率。总之，特异腐质霉拥有一系列纤维素降解酶，它们协同作用以高效水解纤维素。
