Recombinant clones displaying thermostable β-glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium MSB8 (DSM 3109), and the nucleotide sequence of a 1,4-β-glucanase gene designated was determined. Amino-terminal sequencing of cellulase I previously detected in cells indicated that the gene encodes this β-glucanase, which is now designated CelA. CelA, which has a calculated molecular mass of 29732 Da, was purified from a recombinant strain to apparent homogeneity as judged by SDS-PAGE with a 44% yield. The enzyme was most active against soluble substrates such as mixed-linkage β-glucan and CM-cellulose. CelA displayed remarkable thermostability, which was enhanced in the presence of high concentrations of salt. Downstream of the gene we found a second open reading frame, whose nucleotide sequence was 58% identical to Experimental proof that also encodes a β-glucanase was obtained by separation from and expression in under the control of an efficient host promoter. According to the deduced amino acid sequences, CelB, in contrast to CelA, contains a signal peptide at the amino terminus. CelB and CelA had similar substrate specificities and temperature optima, but differed in their pH optima. Also, the addition of salt had a less stabilizing effect on CelB than on CelA. Nine 30 bp direct repeats, each itself representing a sequence with imperfect dyad symmetry, were detected upstream of the cellulase gene cluster.


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