CLONING AND SEQUENCING OF A NOVEL ENDO-1,4-BETA-GLUCANASE GENE FROM RUMINOCOCCUS-ALBUS

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.