Beldman G, Searle-Van Leeuwen M F, Rombouts F M, Voragen F G
Eur J Biochem. 1985 Jan 15;146(2):301-8. doi: 10.1111/j.1432-1033.1985.tb08653.x.
Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial cellulase preparation derived from Trichoderma viride, using gel filtration on Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and affinity chromatography on crystalline cellulose. Molecular masses were determined by polyacrylamide gel electrophoresis. One group of endoglucanases (Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more random in their attack on carboxymethylcellulose than another group (Endo III, Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo III was identified as a new type of endoglucanase with relatively high activity on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption affinities on a column of crystalline cellulose and could be eluted by a pH gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by high-pressure liquid chromatography of the products obtained from incubation with H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily active on newly generated chain ends. Exo I was essentially another type of exoglucanase which in the first instance was able to split off a cellobiose molecule from a chain end and then hydrolyse this molecule in a second step to two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately active on CM-cellulose, crystalline cellulose and xylan and highly active on p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.
从来源于绿色木霉的商业纤维素酶制剂中，通过使用Bio-Gel凝胶过滤、DEAE-Bio-Gel A阴离子交换、SE-Sephadex阳离子交换以及结晶纤维素亲和层析，分离得到六种内切葡聚糖酶（内切葡聚糖酶I、II、III、IV、V、VI）、三种外切葡聚糖酶（外切葡聚糖酶I、II、III）和一种β-葡萄糖苷酶（β-葡萄糖苷酶I）。通过聚丙烯酰胺凝胶电泳测定分子量。一组内切葡聚糖酶（内切葡聚糖酶I、II和IV），分子量分别为50000、45000和23500，它们对羧甲基纤维素的作用比另一组（内切葡聚糖酶III、V和VI，分子量分别为58000、57000和53000）更具随机性。内切葡聚糖酶III被鉴定为一种新型内切葡聚糖酶，对结晶纤维素具有较高活性，对羧甲基纤维素具有中等活性。外切葡聚糖酶II和III，分子量分别为60500和62000，在结晶纤维素柱上表现出明显的吸附亲和力，并且可以通过pH梯度洗脱到碱性区域。通过对用磷酸膨胀的纤维素孵育所得产物进行高压液相色谱分析判断，这些酶是纤维二糖水解酶。得出的结论是，这些外切葡聚糖酶主要对新生成的链端具有活性。外切葡聚糖酶I本质上是另一种外切葡聚糖酶，首先它能够从链端裂解出一个纤维二糖分子，然后在第二步中将该分子水解为两个葡萄糖单元。β-葡萄糖苷酶I是一种新型芳基-β-D-葡萄糖苷酶，对纤维二糖没有活性。该酶的分子量为76000，对羧甲基纤维素、结晶纤维素和木聚糖具有中等活性，对对硝基苯基-β-D-葡萄糖和对硝基苯基-β-D-木糖具有高活性。
