Matsushita O, Russell J B, Wilson D B
U.S. Department of Agriculture Agricultural Research Service, Ithaca, New York.
J Bacteriol. 1990 Jul;172(7):3620-30. doi: 10.1128/jb.172.7.3620-3630.1990.
Bacteroides ruminicola B(1)4, a noncellulolytic rumen bacterium, produces an endoglucanase (carboxymethylcellulase [CMCase]) that is excreted into the culture supernatant. Cultures grown on glucose, fructose, maltose, mannose, and cellobiose had high specific activities of CMCase (greater than 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. rumincola did not grow on either ball-milled or acid-swollen cellulose even though the CMCase could hydrolyze swollen cellulose. The CMCase gene was cloned into Escherichia coli, and its nucleotide sequence contained a single open reading frame coding for a protein of 40,481 daltons. The enzyme was overproduced in E. coli under the control of the tac promoter and purified to homogeneity. The N-terminal sequence, amino acid composition, and molecular weight of the purified enzyme were similar to the values predicted from the open reading frame of the DNA sequence. However, the CMCase present in B. ruminicola was found to have a monomer molecular weight of 88,000 by Western immunoblotting. This discrepancy appeared to have resulted from our having cloned only part of the CMCase gene into E. coli. The amino acid sequence of the CMCase showed homology to sequences of beta-glucanases from Ruminococcus albus and Clostridium thermocellum.
反刍月形单胞菌B(1)4是一种非纤维素分解瘤胃细菌，它能产生一种内切葡聚糖酶（羧甲基纤维素酶[CMCase]），该酶会分泌到培养上清液中。在葡萄糖、果糖、麦芽糖、甘露糖和纤维二糖上生长的培养物具有较高的CMCase比活性（每分钟每毫克蛋白质产生的还原糖大于3 mmol），但其合成受到蔗糖的抑制。即使CMCase能够水解膨胀的纤维素，反刍月形单胞菌也不能在球磨纤维素或酸膨胀纤维素上生长。CMCase基因被克隆到大肠杆菌中，其核苷酸序列包含一个单一的开放阅读框，编码一种40,481道尔顿的蛋白质。该酶在tac启动子的控制下在大肠杆菌中过量表达并纯化至同质。纯化酶的N端序列、氨基酸组成和分子量与从DNA序列的开放阅读框预测的值相似。然而，通过蛋白质免疫印迹法发现反刍月形单胞菌中存在的CMCase单体分子量为88,000。这种差异似乎是由于我们只将部分CMCase基因克隆到了大肠杆菌中。CMCase的氨基酸序列与来自白色瘤胃球菌和嗜热栖热梭菌的β-葡聚糖酶序列具有同源性。
