定量薄层层析法用于内切β-1,3-葡聚糖酶筛选纯化过程中酶活力测定

Determination of enzymatic activity during endo-β-1,3-glucanase screening and purification processes using quantitative thin layer chromatography

根据寡糖与显色剂反应后的呈色物质在540nm处吸光值强度,建立了一种用于分析水解液中寡糖浓度的定量薄层层析法。应用阴离子交换层析结合两步分子筛层析技术在β-葡聚糖酶样品(来源于Trichoderma reesei)中纯化得到一种内切β-葡聚糖酶(EndoG,23.6ku),内切酶纯化44.5倍,酶活回收率12.4%。底物特异性及水解产物分析表明该酶属于内切β-1,3-葡聚糖酶,初步分类为EC3.2.1.39。经测定,EndoG降解热凝胶主要生成昆布二糖和昆布三糖,最适反应条件为pH5.0,50℃。由于EndoG对水不溶性底物热凝胶具有较高的水解活性,对该酶的深入研究可以为提高热凝胶利用率和应用范围提供帮助。

A quantitative thin layer chromatography method was established in this study to determine the concentration of oligosaccharides in curdlan hydrolyzate according to the absorbance of color materials at 540nm. Then, based on this method, an endo- β-1, 3-glucanase （ EndoG ） was purified to unity from Trichoderma reesei product by means of anion exchange chromatography combining with two-stage size exclusion chromatography process.The purification fold of 44.5 and enzymatic yield of 12.4% were achieved at the final step.The molecular weight of EndoG was 23.6ku.According to the substrate specificity and hydrolysis pattern, EndoG was classified into EC3.2.1.39. Results indicated that laminaribiose and laminaritriose were the major products released from curdlan hydrolyzate and the optimal pH and temperature were 5.0 and 50℃, respectively.A further investigation of EndoG will favor the utilization and application of water-insoluble curdlan due to the definite hydrolysis activity of EndoG on curdlan.