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Microbiology 142 (1996), 2533-2542; DOI  10.1099/00221287-142-9-2533
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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

Wolfgang Liebl1,3, Peter Ruile1, Karin Bronnenmeier1, Katrin Riedel1, Friedrich Lottspeich2 and Ingrid Greif1

Institut für Mikrobiologie, Technische Universität München, Arcisstraße 21, D-80290 München, Germany
Max-Planck-Institut für Biochemie, Martinsried, Germany

3Author for correspondence: Wolfgang Liebl. Tel: +49 89 28922378. Fax: +49 89 28922360.

ABSTRACT

Recombinant Escherichia coli clones displaying thermostable β-glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium Thermotoga maritima MSB8 (DSM 3109), and the nucleotide sequence of a 1,4-β-glucanase gene designated celA was determined. Amino-terminal sequencing of cellulase I previously detected in T. maritima cells indicated that the celA 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 E. coli 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 celA gene we found a second open reading frame, celB, whose nucleotide sequence was 58% identical to celA. Experimental proof that celB also encodes a β-glucanase was obtained by separation from celA and expression in E. coli 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 celA-celB cellulase gene cluster.


Keywords: cellulase, β-glucanase, nucleotide sequence, purification, thermostability




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