Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46.
Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11. Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46. Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside. Nucleotide sequencing of the B. fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da. Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence. EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases. The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp. cellulosa and Cellulomonas fimi.
Hazlewood
G P
GP
Department of Biochemistry, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, UK.
Davidson
K
K
Laurie
J I
JI
Romaniec
M P
MP
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004269:DNA, Bacterial; D002480:Cellulase