Department of Chemistry, Regional University of Blumenau, Rua Antônio da Veiga, 140, 89010-971 Blumenau, SC, Brazil.
Enzyme Microb Technol. 2012 Oct 10;51(5):280-5. doi: 10.1016/j.enzmictec.2012.07.010. Epub 2012 Jul 31.
This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30 min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20 min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.
本研究描述了一种使用滤纸作为唯一底物选择性测定内切（EG）和外切（ExG）纤维素酶活性的方法。该方法基于酶的作用模式，其中 EG 活性主要形成不溶性还原糖，而 ExG 活性形成可溶性还原糖。该方法是使用滤纸作为底物进行水解，使用三种含有内切葡聚糖酶（EG）、主要外切葡聚糖酶（ExG）或同时含有内切和外切葡聚糖酶活性的Hypocrea jecorina 纤维素酶制剂进行开发。水解实验，通过评估总还原糖、可溶性还原糖和不溶性还原糖（RS）的形成来跟踪，结果表明，在水解的最初 30 分钟内主要形成不溶性还原糖，而在此初始水解阶段后，可溶性还原糖的形成显著增加，因此可以分别测量 EG 和 ExG 活性。在不同反应时间从反应产物中获得的 FPA 活性表明，EG 活性（FPA(insol)）应在水解 10-20 分钟之间进行测量。所提出的方法可以评估任何纤维素酶制剂中总纤维素酶活性的 EG 和 ExG 活性贡献，并计算内/外活性比。
