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Volume 142, Issue 9

Analysis of a DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes Free

Abstract

Recombinant clones displaying thermostable -glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium MSB8 (DSM 3109), and the nucleotide sequence of a 1,4--glucanase gene designated was determined. Amino-terminal sequencing of cellulase I previously detected in cells indicated that the gene encodes this -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 -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 -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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Keyword(s): cellulase , nucleotide sequence , purification , thermostability and β-glucanase
© Society for General Microbiology 1996
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1996-09-01
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Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes
Microbiology 142, 2533 (1996); https://doi.org/10.1099/00221287-142-9-2533
/content/journal/micro/10.1099/00221287-142-9-2533
/content/journal/micro/10.1099/00221287-142-9-2533
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