CLONING OF AN ENDO-(1-]4)-BETA-GLUCANASE GENE, CELA, FROM THE RUMEN BACTERIUM CLOSTRIDIUM SP (C-LONGISPORUM) AND CHARACTERIZATION OF ITS PRODUCT, CELA, IN ESCHERICHIA-COLI

A genomic library of Clostridium sp. ('C. longisporum') ATCC 49440 in the host Escherichia coli was screened for endo-beta-glucanases, and p lasmids pCM64 and pCM4 were isolated. The nucleotide sequence of a 362 0 bp fragment was found to contain a 1548 bp open reading frame (ORF), termed celA, which encodes an endo-(1-->4)-beta-glucanase, CelA, assi gned to family A4. N-terminal amino acid sequence determination reveal ed that pCM64 encoded the full-length celA gene, including a signal se quence, while pCM4 carried a 5'-truncated celA gene expressed as an N- terminal fusion protein, CelA Delta N', without a signal sequence. Cel A was secreted into the periplasm in E. coli. In this organism, proteo lytic 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 consis ted of the catalytic domain from which the cellulose-binding domain (C BD) had been removed (CelA Delta CBD). The intracellularly-located Cel A Delta N' was not subject to proteolytic degradation. The pH and temp erature optima of CelA were pH 4.8 and 43 degrees C, respectively. Cel A hydrolysed barley beta-glucan, lichenan, carboxymethylcellulose and xylan. It showed preferential activity against the larger cellooligosa ccharides (cellohexaose and cellopentaose); cellotetraose was the smal lest substrate degraded completely.