Malburg L M, Iyo A H, Forsberg C W
Department of Microbiology, University of Guelph, Ontario, Canada.
Appl Environ Microbiol. 1996 Mar;62(3):898-906. doi: 10.1128/aem.62.3.898-906.1996.
Two adjacent, highly homologous endoglucanase genes, celD and celE from Fibrobacter succinogenes S85, which were separated by an AT-rich 223-nucleotide intergenic region were characterized. The celD gene codes for endoglucanase D (EGD), a protein of 668 residues with a molecular mass of 71.7 kDa, while the celE gene encodes endoglucanase E, a protein of 467 amino acids with a molecular mass of 50.7 kDa. Both gene products belong to family 9 of glycosyl hydrolases. EGD displays an array of serine-rich periodic sequences (SRPS) near its C terminus which separate the catalytic domain from a basic terminal domain (BTD) rich in positively charged amino acids. Endoglucanase E has a BTD which is homologous to that of EGD, but it lacks the SRPS and 151 residues present at the N terminus of EGD. The SRPS structures may function as flexible linkers which facilitate interactions between the BTDs and acidic membrane proteins from F. succinogenes S85. The recombinant EGD showed pH and temperature optima of 5.5 and 35 degrees C, respectively. The enzyme cleaved barley-beta-glucan, carboxymethyl cellulose, and acid-swollen cellulose with specific activities of 19.1, 11.5 and 1.7 micromol x min-1 x mg of protein-1, respectively. There was a rapid drop in viscosity during hydrolyses of carboxymethyl cellulose, which is characteristic of an endoglucanase. Glucose was the main hydrolysis product of acid-swollen cellulose. Monospecific polyclonal antibodies against EGD detected the expression of a 68-kDa cellulose-inducible protein corresponding in size to the recombinant EGD in the culture fluid of F. succinogenes S85 and several larger proteins. The celE gene appeared to have little activity when expressed from the beta-galactosidase promoter in pBluescript in Escherichia coli; however, reverse transcriptase PCR analysis with internal primers for the gene revealed that a cellulose-inducible message was made in F. succinogenes, thereby documenting expression of the gene.
对来自产琥珀酸丝状杆菌S85的两个相邻且高度同源的内切葡聚糖酶基因celD和celE进行了表征，它们被一个富含AT的223个核苷酸的基因间隔区隔开。celD基因编码内切葡聚糖酶D（EGD），这是一种由668个残基组成、分子量为71.7 kDa的蛋白质，而celE基因编码内切葡聚糖酶E，这是一种由467个氨基酸组成、分子量为50.7 kDa的蛋白质。这两种基因产物都属于糖基水解酶家族9。EGD在其C末端附近显示出一系列富含丝氨酸的周期性序列（SRPS），这些序列将催化结构域与富含带正电荷氨基酸的碱性末端结构域（BTD）分隔开。内切葡聚糖酶E有一个与EGD的BTD同源的BTD，但它缺乏SRPS以及EGD N末端存在的151个残基。SRPS结构可能作为灵活的连接子，促进BTD与产琥珀酸丝状杆菌S85的酸性膜蛋白之间的相互作用。重组EGD的最适pH和温度分别为5.5和35℃。该酶分别以19.1、11.5和1.7 μmol·min⁻¹·mg蛋白质⁻¹ 的比活性切割大麦β-葡聚糖、羧甲基纤维素和酸膨胀纤维素。在羧甲基纤维素水解过程中粘度迅速下降，这是内切葡聚糖酶的特征。葡萄糖是酸膨胀纤维素的主要水解产物。针对EGD的单特异性多克隆抗体在产琥珀酸丝状杆菌S85的培养液中检测到一种68 kDa的纤维素诱导蛋白的表达，其大小与重组EGD相对应，还检测到几种更大的蛋白质。当celE基因在大肠杆菌的pBluescript中由β-半乳糖苷酶启动子表达时，似乎几乎没有活性；然而，用该基因的内部引物进行逆转录酶PCR分析表明，产琥珀酸丝状杆菌中产生了纤维素诱导的信息，从而证明了该基因的表达。
