Cloning and expression of an endocellulase gene from a novel streptomyceteisolated from an East African soda lake

Alkaline cellulase-producing actinomycete strains were isolated from mud sa mples collected from East African soda lakes. The strains were identified a s novel Streptomyces spp. by 16S rDNA sequence analysis. A cellulase gene ( cel12A) from Streptomyces sp. strain 11AG8 was cloned by expression screeni ng of a genomic DNA library in Escherichia coli. From the nucleotide sequen ce of a 1.5-kb DNA fragment, an open reading frame of 1,113 nucleotides was identified encoding a protein of 371 amino acids. From computer analysis o f the sequence, it was deduced that the cel12A mature enzyme is a protein o f 340 amino acids. The protein contained a catalytic domain, a glycine-rich linker region, and a cellulose-binding domain of 221, 12, and 107 amino ac ids, respectively. FASTA analysis of the catalytic domain of cel12A classif ied the enzyme as a family 12 endoglucanase and the cellulose-binding domai n as a family Ha CBD. Streptomyces rochei EglS was determined as nearest ne ighbor with a similarity of 75.2% and 61.0% to the catalytic domain and the cellulose-binding domain, respectively. The cel12A gene was subcloned in a Bacillus high-expression vector carrying the Bacillus amyloliquefaciens am ylase regulatory sequences, and the construct was transformed to a Bacillus subtilis host strain. Crude enzyme preparations were obtained by ultrafilt ration of cultures of the Bacillus subtilis recombinant strain containing t he 11AG8 cel12A gene. The enzyme showed carboxymethylcellulase (CMCase) act ivities over a broad pH range (5-10) with an optimum activity at pH 8 and 5 0 degreesC. The enzyme retained more than 95% of its activity after incubat ion for 30 min under these conditions.