RESUMO
The nucleotide sequence of the celF gene of Clostridium thermocellum was determined. The open reading frame extended over 2217 bp. The encoded 739-aa polypeptide, CelF, with a Mw = 82,015, was an endoglucanase with activity against carboxymethylcellulose. The N terminus showed a typical signal peptide, and a cleavage site after Ala-27 was predicted. From residues 28 to 470, the sequence of CelF was related to the catalytic domains of type E2 endoglucanases, with a strong homology to the endoglucanases CelZ of Clostridium stercorarium and CenB of Cellulomonas fimi. The catalytic region was followed by a 134-aa segment also present in C. stercorarium CelZ and in C. fimi CenB, and belonging to the family of non-catalytic, presumably cellulose-binding domains first identified in Bacillus subtilis endoglucanase. A 21-aa segment rich in Pro/Thr/Ser residues separated the putative cellulose-binding region from the COOH-terminal region, which contained two conserved stretches of 24 amino acids closely similar to those previously described in endoglucanases CelA, CelB, CelD, CelE, CelH and CelX, and xylanase XynZ of C. thermocellum.
Assuntos
Celulase/genética , Clostridium/genética , DNA Bacteriano/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Sequência de Bases/genética , Clostridium/enzimologia , Técnicas In Vitro , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Plasmídeos/genéticaRESUMO
An endoxylanase encoded by the xynZ gene of Clostridium thermocellum was purified from Escherichia coli harbouring a fragment of the gene cloned in pUC8. The purified enzyme showed two active bands of Mr 41,000 and 39,000, the latter one presumably derived from the former through proteolysis. The enzyme was highly active on xylan and para-nitrophenyl-beta-D-xylobioside. The major end product of xylan hydrolysis was xylobiose. With an antiserum raised against the enzyme purified from E. coli, an immunoreactive polypeptide of Mr 90,000, corresponding to the entire xynZ gene product, was detected in a culture supernatant from C. thermocellum grown on cellulose. By immunological detection, xylanase Z was shown to be associated with a cellulose-binding, high-molecular-weight fraction whose properties coincided with those described previously for the cellulose-degrading complex of C. thermocellum known as the cellulosome.
Assuntos
Clostridium/enzimologia , Glicosídeo Hidrolases/isolamento & purificação , Xilosidases/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Clonagem Molecular , Clostridium/isolamento & purificação , Endo-1,4-beta-Xilanases , Escherichia coli , Peso Molecular , Frações Subcelulares/enzimologia , Xilosidases/genéticaRESUMO
The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined. The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids. A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z. This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C. thermocellum endoglucanases A, B, and D. Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis. Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19. An internal translational initiation site which was efficiently recognized in E. coli was tentatively identified 470 codons downstream from the actual start codon.
Assuntos
Clostridium/genética , Glicosídeo Hidrolases/genética , Xilosidases/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Deleção Cromossômica , Clonagem Molecular , Clostridium/enzimologia , Códon , Análise Mutacional de DNA , DNA Bacteriano/genética , Endo-1,4-beta-Xilanases , Genes Bacterianos , Dados de Sequência Molecular , Peso Molecular , Xilosidases/imunologiaRESUMO
The size and location of the 5' end of celA mRNA encoding endoglucanase A of Clostridium thermocellum were investigated in C. thermocellum and in an Escherichia coli clone that carries and expresses the celA gene. In E. coli, the 5' end of celA mRNA was located 134 bp upstream from the initiation codon and 10 bp downstream from a sequence homologous to the consensus sequence of E. coli sigma 70 and Bacillus subtilis sigma 43 (formerly sigma 55) vegetative promoters. In C. thermocellum, a minor transcript appeared to start from the same site, but a major species started 57 bp upstream from the coding sequence. The 5' end of this mRNA was preceded by a sequence reminiscent of B. subtilis sigma 28 vegetative promoters. In both organisms, the size of the transcript suggested that celA belongs to a monocistronic unit of transcription.
