TY - JOUR
T1 - Cloning and sequencing of a Bacteroides rumincola B14 endoglucanase gene
AU - Matsushita, O.
AU - Russell, J. B.
AU - Wilson, D. B.
PY - 1990
Y1 - 1990
N2 - Bacteroides ruminicola B14, 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 (> 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. ruminicola 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 β-glucanases from Ruminococcus albus and Clostridium thermocellum.
AB - Bacteroides ruminicola B14, 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 (> 3 mmol of reducing sugar per mg of protein per min), but its synthesis was repressed by sucrose. B. ruminicola 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 β-glucanases from Ruminococcus albus and Clostridium thermocellum.
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U2 - 10.1128/jb.172.7.3620-3630.1990
DO - 10.1128/jb.172.7.3620-3630.1990
M3 - Article
C2 - 2361940
AN - SCOPUS:0025294603
VL - 172
SP - 3620
EP - 3630
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 7
ER -