Purification and characterization of endoglucanase C of Cellulomonas fimi, cloning of the gene, and analysis of in vivo transcripts of the gene

Abstract

Two nonglycosylated endoglucanases which bind to Sephadex were purified from culture supernatants of Cellulomonas fimi grown on microcrystalline cellulose. Their Mrs were 120,000 and 130,000. The N-terminal amino acid sequences of the enzymes were identical, suggesting that the enzymes were related. A DNA fragment encoding this N-terminal sequence was cloned in Escherichia coli. The nucleotide sequence corresponding to the N-terminal amino acid sequence was preceded by a sequence encoding a typical leader peptide. Transcripts hybridizing to the cloned fragment were detected in total RNA isolated from C. fimi cells grown on carboxymethyl cellulose but not from cells grown on glycerol or glucose. Transcription started at a cluster of sites 53 to 59 nucleotides upstream of a GUG translation initiation codon and terminated at either of two closely spaced C residues immediately downstream of a region of potential secondary structure. The size of the transcript was approximately 3.5 kilobases, sufficient to encode a polypeptide of 130 kilodaltons. The 130-kilodalton polypeptide is designated endoglucanase C (CenC), and the gene encoding it is designated cenC.