Department of Bioscience and Bioinformatics, Yongin, Gyeoggido, Korea.
Biological and Genetic Resource Assessment Division, National Institute of Biological Resource, Incheon, Korea.
J Basic Microbiol. 2018 Apr;58(4):310-321. doi: 10.1002/jobm.201700436. Epub 2018 Feb 5.
The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters k and K , for cellotetraose at pH 5.0 and 50 °C were 13.3 s and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.
链霉菌 A3(2) 的 sco6546 基因被注释为一种假定的糖基水解酶，属于家族 48。它预计编码一个 973 个氨基酸的多肽（103.4 kDa），带有 39 个氨基酸的分泌信号。在这里，SCO6546 蛋白在链霉菌 lividans TK24 中过表达，纯化的蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中显示出成熟分泌形式的预期分子量（934 个氨基酸，99.4 kDa）。SCO6546 对 Avicel 和羧甲基纤维素表现出高活性，但对滤纸和β-葡聚糖表现出低活性。SCO6546 在 pH 5.0 和 50°C 下对 Avicel 的纤维素酶活性最高，这与对纤维四糖和纤维五糖底物的最大活性条件相似。在 pH 5.0 和 50°C 下，SCO6546 对纤维四糖的动力学参数 k 和 K 分别为 13.3 s 和 2.7 mM。SCO6546 水解 Avicel 生成的产物的薄层色谱（TLC）仅显示纤维二糖，这通过质谱分析得到证实。TLC 分析 SCO6546 生成的纤维寡糖和显色底物水解产物表明，它可以从非还原端将纤维二糖主要水解成纤维二糖。这些数据清楚地表明，SCO6546 是一种外切-β-1,4-纤维二糖水解酶（EC 3.2.1.91），作用于纤维素的非还原端。
