Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis
Determination of the action modes of cellulases from hydrolytic profiles over a time course using fluorescence‐assisted carbohydrate electrophoresis
复制标题
DOI：
10.1002/elps.201400563
复制
发表时间：
2015-03
期刊：
影响因子：
2.9
通讯作者：
Qing Zhang;Xiaomei Zhang;Peipei Wang;Dandan Li;Guanjun Chen;P. Gao;Lushan Wang
中科院分区：
文献类型：
--
作者：
Qing Zhang;Xiaomei Zhang;Peipei Wang;Dandan Li;Guanjun Chen;P. Gao;Lushan Wang
荧光辅助碳水化合物电泳（FACE）是一种灵敏、简便的低聚糖分离方法。它依赖于用荧光基团标记低聚糖的还原端，然后是PAGE。低聚糖在碳水化合物聚合物水解后的浓度变化可以使用FACE方法连续定量测量。基于定量分析，我们认为FACE是一种相对高通量、可重复且适合分析纤维素酶作用模式的方法。根据纤维素酶水解谱的时间进程，用表观进程率来表示纤维素酶的不同作用方式。纤维素酶可以很容易地分为外葡聚糖酶、β -葡萄糖苷酶和内葡聚糖酶。此外，来自同一糖苷水解酶家族的内切葡聚糖酶具有不同的表观加工性，表明其作用方式不同。具有相同结合能力和水解活性的内切葡聚糖酶具有相似的低聚糖谱，这有助于它们的分类。对里氏木霉（Trichoderma reesei）的内切葡聚糖酶Cel12A的水解谱进行了分析，发现其含有葡萄糖、纤维素二糖和纤维素三糖，表明其可能具有新的葡糖苷酶活性，与EC 3.2.1.74相对应。对T. reesei Cel12A‐N20A突变体的水解研究表明，FACE方法对检测单位点突变对酶活性的影响足够敏感。
Fluorescence‐assisted carbohydrate electrophoresis (FACE) is a sensitive and simple method for the separation of oligosaccharides. It relies on labeling the reducing ends of oligosaccharides with a fluorophore, followed by PAGE. Concentration changes of oligosaccharides following hydrolysis of a carbohydrate polymer could be quantitatively measured continuously over time using the FACE method. Based on the quantitative analysis, we suggested that FACE was a relatively high‐throughput, repeatable, and suitable method for the analysis of the action modes of cellulases. On account of the time courses of their hydrolytic profiles, the apparent processivity was used to show the different action modes of cellulases. Cellulases could be easily differentiated as exoglucanases, β‐glucosidases, or endoglucanases. Moreover, endoglucanases from the same glycoside hydrolases family had a variety of apparent processivity, indicating the different modes of action. Endoglucanases with the same binding capacities and hydrolytic activities had similar oligosaccharide profiles, which aided in their classification. The hydrolytic profile of Trichoderma reesei Cel12A, an endoglucanases from T. reesei, contained glucose, cellobiose, and cellotriose, which revealed that it may have a new glucosidase activity, corresponding to that of EC 3.2.1.74. A hydrolysate study of a T. reesei Cel12A‐N20A mutant demonstrated that the FACE method was sufficiently sensitive to detect the influence of a single‐site mutation on enzymatic activity.