Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes.
Recombinant Escherichia coli clones displaying thermostable beta-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-beta-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 beta-glucanase, which is now designated CelA. CelA, which has a calculated molecular mass of 29,732 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 beta-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 beta-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.
Liebl
W
W
Institut für Mikrobiologie, Technische Universität München, Germany.
Ruile
P
P
Bronnenmeier
K
K
Riedel
K
K
Lottspeich
F
F
Greif
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D001330:Electronic Data Processing; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D016364:Reading Frames; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012965:Sodium Chloride / Q000378:metabolism
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012965:Sodium Chloride; D002480:Cellulase