Gilkes N R, Langsford M L, Kilburn D G, Miller R C, Warren R A
J Biol Chem. 1984 Aug 25;259(16):10455-9.
Three recombinant plasmids, pEC1, pEC2, and pEC3, each containing a unique Cellulomonas fimi chromosomal DNA insert, expressed Cm-cellulase activities in Escherichia coli C600 (Whittle, D. J., Kilburn, D. H., Warren, R. A. J., and Miller, R. C., Jr. (1982) Gene (Amst.) 17, 139-145; Gilkes, N. R., Kilburn, D. G., Langsford, M. L., Miller, R. C., Jr., Wakarchuk, W. W., Warren, R. A. J., Whittle, D. J., and Wong, W. K. R. (1984) J. Gen. Microbiol. 130, 1377-1384). Viscometric and chemical analyses showed that the enzymes encoded by pEC2 and pEC3 behaved as endoglucanases, whereas that encoded by pEC1 behaved as an exoglucanase. The activities of the exoglucanase and the pEC2-encoded endodglucanase were additive on Cm-cellulose as substrate. The pEC1-encoded enzyme also hydrolyzed xylan and p-nitrophenyl cellobioside. Two substrate-bound Cm-cellulases were isolated from the residual cellulose in a C. fimi culture by guanidine hydrochloride elution, affinity chromatography, and polyacrylamide gel electrophoresis. Both were glycoproteins of apparent Mr = 58,000 and 56,000, respectively. The 56-kDa enzyme appeared to be identical with the pEC1-encoded product, suggesting that they arise from the same gene.
三种重组质粒，即pEC1、pEC2和pEC3，每种都含有一个独特的纤维单胞菌染色体DNA插入片段，它们在大肠杆菌C600中表达了羧甲基纤维素酶活性（惠特尔，D.J.，基尔伯恩，D.H.，沃伦，R.A.J.，以及米勒，R.C.，Jr.（1982年）《基因》（阿姆斯特丹）17卷，第139 - 145页；吉尔克斯，N.R.，基尔伯恩，D.G.，兰斯福德，M.L.，米勒，R.C.，Jr.，瓦卡丘克，W.W.，沃伦，R.A.J.，惠特尔，D.J.，以及黄，W.K.R.（1984年）《普通微生物学杂志》130卷，第1377 - 1384页）。粘度测定和化学分析表明，由pEC2和pEC3编码的酶表现为内切葡聚糖酶，而由pEC1编码的酶表现为外切葡聚糖酶。以外切葡聚糖酶和pEC2编码的内切葡聚糖酶为底物时，其活性具有加和性。由pEC1编码的酶还能水解木聚糖和对硝基苯基纤维二糖。通过盐酸胍洗脱、亲和层析和聚丙烯酰胺凝胶电泳，从纤维单胞菌培养物中的残留纤维素中分离出两种与底物结合的羧甲基纤维素酶。两者均为糖蛋白，表观分子量分别为58,000和56,000。56 kDa的酶似乎与pEC1编码的产物相同，这表明它们来自同一个基因。
