Department of Forest Products and Technology, Chonnam National University, Gwangju, 500-757, Republic of Korea.
J Ind Microbiol Biotechnol. 2012 Jul;39(7):1081-9. doi: 10.1007/s10295-012-1110-4. Epub 2012 Mar 7.
The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.
来自栓菌（Gloeophyllum trabeum）的内切葡聚糖酶（Cel5B）被克隆并在毕赤酵母 GS115 中表达，没有信号肽，丙氨酸残基 22 被突变为谷氨酰胺。Cel5B 的 DNA 序列有一个 1077bp 的开放阅读框，编码一个由 359 个氨基酸残基组成的蛋白质，分子量为 47kDa。根据序列相似性，Cel5B 显示出活性位点残基 Glu-175 和 Glu-287。通过定点突变将这两个残基突变为丙氨酸后，均完全丧失了水解活性。纯化的重组 Cel5B 在 pH3.5 和 55°C 时，在羧甲基纤维素和滤纸底物上的比活性分别比其他内切葡聚糖酶和纤维二糖水解酶高 80-1000 倍和 13-70 倍。Cel5B 在酸性条件下表现出双功能特性。通过 Lineweaver-Burk 作图确定的酶动力学特性表明，Cel5B 是一种催化效率高的纤维素酶。这些结果表明，Cel5B 在酸性条件下具有高效的内切葡聚糖酶和外切葡聚糖酶活性，是木质纤维素生物转化的良好候选酶。
