Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
J Biotechnol. 1999 Jan 22;67(2-3):85-97. doi: 10.1016/s0168-1656(98)00168-0.
We have cloned two endoglucanase genes (egl3 and egl4) from a thermophilic fungus, Humicola grisea. The coding region of the egl3 gene was interrupted by an intron of 56-bp, and the deduced amino acid sequence of the egl3 gene was 305 amino acids in length and showed 98.4% identity with Humicola insolens EGV. The coding region of the egl4 gene was also interrupted by an intron of 173-bp, which contains 34 TTC repeated sequence units, and the deduced amino acid sequence of the egl4 gene was 227 amino acids in length and showed 61.5% identity with H. grisea EGL3. The typical hinge and the cellulose-binding domain were observed in the C-terminal region of EGL3, but they were not observed in EGL4. In the 5' upstream region of both genes, there were a TATA box or its similar sequence, CAAT motifs, and 6-bp sites which are identical or similar to the consensus sequence for binding a catabolite repressor CREA in Aspergillus nidulans. The egl3 and the egl4 genes were expressed in Aspergillus oryzae, and the translation products were purified. The fusion protein, EGL4CBD, which consists of a catalytic domain of EGL4 and the C-terminal region of EGL3, was also constructed and produced by A. oryzae, and purified. These enzymes showed relatively high activity toward carboxymethyl cellulose (CMC) and could not hydrolyze p-nitrophenyl-beta-D-glucoside and p-nitrophenyl-beta-D-cellobioside. The positive effect of substituting the C-terminal region of EGL4 with that of EGL3 was observed in the hydrolysis of CMC.
我们从嗜热真菌灰腐质霉中克隆了两个内切葡聚糖酶基因（egl3和egl4）。egl3基因的编码区被一个56 bp的内含子打断，egl3基因推导的氨基酸序列长度为305个氨基酸，与特异腐质霉EGV的同一性为98.4%。egl4基因的编码区也被一个173 bp的内含子打断，该内含子包含34个TTC重复序列单元，egl4基因推导的氨基酸序列长度为227个氨基酸，与灰腐质霉EGL3的同一性为61.5%。在EGL3的C末端区域观察到典型的铰链区和纤维素结合结构域，但在EGL4中未观察到。在这两个基因的5'上游区域，存在一个TATA框或其类似序列、CAAT基序以及与构巢曲霉中结合分解代谢物阻遏物CREA的共有序列相同或相似的6 bp位点。egl3和egl4基因在米曲霉中表达，并对翻译产物进行了纯化。还构建了由EGL4的催化结构域和EGL3的C末端区域组成的融合蛋白EGL4CBD，并由米曲霉产生和纯化。这些酶对羧甲基纤维素（CMC）表现出相对较高的活性，且不能水解对硝基苯基-β-D-葡萄糖苷和对硝基苯基-β-D-纤维二糖苷。在CMC水解过程中观察到用EGL3的C末端区域替换EGL4的C末端区域具有积极作用。
