McGavin M J, Forsberg C W, Crosby B, Bell A W, Dignard D, Thomas D Y
Department of Microbiology, University of Guelph, Ontario, Canada.
J Bacteriol. 1989 Oct;171(10):5587-95. doi: 10.1128/jb.171.10.5587-5595.1989.
The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.
从产琥珀酸丝状杆菌克隆到大肠杆菌中的cel - 3基因编码EG3酶，该酶具有内切葡聚糖酶和纤维二糖酶活性。该基因有一个1974个碱基对的开放阅读框，编码一个73.4千道尔顿（kDa）的蛋白质。然而，从大肠杆菌渗透休克液中纯化得到的酶为43 kDa。43 kDa蛋白质的氨基末端与开放阅读框编码的蛋白质的第266个氨基酸残基匹配，表明在大肠杆菌中发生了蛋白水解。除了43 kDa的蛋白质外，蛋白质免疫印迹法显示大肠杆菌中有该酶的94 kDa膜状形式，而产琥珀酸丝状杆菌中有一个118 kDa的单一蛋白质。因此，纯化的蛋白质似乎是一种天然蛋白质的蛋白水解降解产物，该天然蛋白质在大肠杆菌中为94 kDa，在产琥珀酸丝状杆菌中为118 kDa。基于DNA序列预期的分子量与体内形式之间的差异可能是由于电泳过程中的异常迁移、天然酶的糖基化或N末端的脂肪酰基取代。两个假定的信号肽切割位点之一与已知的脂蛋白前导序列非常相似。纯化的43 kDa肽对羧甲基纤维素的Km值较高（53 mg/ml），但对地衣多糖和大麦β - 葡聚糖的Km值较低（3至4 mg/ml）。该酶能水解无定形纤维素，水解的主要产物是纤维二糖和纤维三糖。该酶能切割纤维三糖，但不能切割纤维二糖。EG3与嗜热栖热菌的内切葡聚糖酶CelC具有显著的氨基酸序列同源性，并且与嗜热栖热菌的CelA和CelC一样，它有一个假定的活性位点，该活性位点可以在高度保守的氨基酸残基Asn - 44和Asp - 52处与鸡蛋清溶菌酶的活性位点对齐。
