Cloning of an endo-(1→4)-β-glucanase gene, celA, from the rumen bacterium Clostridium sp. (‘C. longisporum’) and characterization of its product, CelA, in Escherichia coli

SUMMARY: A genomic library of Clostridium sp. (‘C. longisporum’) ATCC 49440 in the host Escherichia coli was screened for endo-β-glucanases, and plasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 3620 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1→4)-β-glucanase, CelA, assigned to family A4. N-terminal amino acid sequence determination revealed that pCM64 encoded the full-length celA gene, including a signal sequence, while pCM4 carried a 5′-truncated celA gene expressed as an N-terminal fusion protein, CelAΔN’, without a signal sequence. CelA was secreted into the periplasm in E. coli. In this organism, proteolytic cleavage of CelA at or near a putative linker region resulted in the appearance of two active polypeptides of molecular masses 57 and 47 kDa. The former was the full-length enzyme, while the latter consisted of the catalytic domain from which the cellulose-binding domain (CBD) had been removed (CelAΔCBD). The intracellularly-located CelAΔN’ was not subject to proteolytic degradation. The pH and temperature optima of CelA were pH 4.8 and 43 °C, respectively. CelA hydrolysed barley β-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cello-oligosaccharides (cellohexaose and cellopentaose); cellotetraose was the smallest substrate degraded completely.