RESUMO
An artificial, bifunctional, thermostable cellulase-xylanase enzyme from Thermotoga maritima by gene fusion. The fusion protein exhibited both cellulase and xylanase activity when xynA was fused downstream of cel5C but no activities were shown when xynA was fused upstream of cel5C. The enzyme was optimally active at pH 5.0 and 80 degrees C over 30 min. E. coli expressed the fusion enzyme, with an apparent molecular mass of approximately 152 kDa by carboxymethyl cellulose- and xylan-SDS-PAGE.
Assuntos
Biotecnologia/métodos , Celulase/química , Thermotoga maritima/metabolismo , Xilosidases/química , Celulose/química , DNA/química , Eletroforese em Gel de Poliacrilamida , Enzimas/química , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes de Fusão/química , Temperatura , Xilosidases/metabolismoRESUMO
The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.
Assuntos
Celulase/genética , Dickeya chrysanthemi/enzimologia , Dickeya chrysanthemi/genética , Genes Bacterianos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Celulase/química , Celulase/metabolismo , Clonagem Molecular , Sequência Conservada , DNA Bacteriano/genética , Evolução Molecular , Glicosídeo Hidrolases/genética , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
An artificial bifunctional enzyme, cellulase-beta-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and beta-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or beta-glucosidase. The fusion enzyme was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70 degrees C over 30 min, and optimal beta-glucosidase activity at pH 7.0 and 80 degrees C over 30 min.
Assuntos
Celulase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Thermotoga maritima/enzimologia , beta-Glucosidase/metabolismo , Celulase/genética , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Modelos Genéticos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Temperatura , Thermotoga maritima/genética , beta-Glucosidase/genéticaRESUMO
A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.