S. Karita et al., CLONING AND SEQUENCING OF A NOVEL ENDO-1,4-BETA-GLUCANASE GENE FROM RUMINOCOCCUS-ALBUS, Journal of fermentation and bioengineering, 76(6), 1993, pp. 439-444
The gene encoding an endo-1,4-beta-glucanase from Ruminococcus albus F
-40 was cloned in Escherichia coil JM109 using pBR322. The nucleotide
sequence of the 1,798 bp PstI-PvuII fragment which includes a cellulas
e gene was determined. There was a single open reading frame (ORF) con
sisting of 936 bp encoding a peptide of 312 amino acid residues with a
molecular weight of 35,766. The N-terminal amino acid sequence determ
ined for the enzyme expressed in E. coli was identical to that deduced
from the beginning of the ORF. A putative ribosome-binding site and a
promoter were located upstream of the ORF. Activity was expressed fro
m this fragment when it was subcloned in both orientations in pUC118 a
nd pUC119, indicating that its own promoter functioned in E. coli. The
amino acid sequence of the endoglucanase deduced from the nucleotide
sequence showed 44% homology with CelA from Butyrivibrio fibrisolvens
A46, suggesting that this enzyme was a member of family A2. The enzyme
purified from E. coli exhibited the highest activity against carboxym
ethyl cellulose (CMC) at 40 degrees C and pH around 7.0.