RESUMO
A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65⯰C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family ß-d-xylosidase. Using 4-nitrophenyl ß-d-xylopyranoside (pNPX) as a substrate, the KM, Vmax, kcat and kcat/KM kinetic parameters were determined as 0.25⯱â¯0.03â¯mM, 889.47⯱â¯28.54â¯U/mg, 39.20â¯s-1 and 156.8â¯mM-1â¯s-1, respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme's low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family ß-d-xylosidase.
Assuntos
Chloroflexi/enzimologia , Temperatura , Xilosidases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Xilosidases/química , Xilosidases/genéticaRESUMO
The phylum Chloroflexi is phylogenetically diverse and is a deeply branching lineage of bacteria that express a broad spectrum of physiological and metabolic capabilities. Members of the order Ktedonobacteriales, including the families Ktedonobacteriaceae, Thermosporotrichaceae, and Thermogemmatisporaceae, all have flexible aerobic metabolisms capable of utilizing a wide range of carbohydrates. A number of species within these families are considered cellulolytic and are capable of using cellulose as a sole carbon and energy source. In contrast, Ktedonobacter racemifer, the type strain of the order, does not appear to possess this cellulolytic phenotype. In this study, we confirmed the ability of Thermogemmatispora sp. strain T81 to hydrolyze cellulose, determined the whole-genome sequence of Thermogemmatispora sp. T81, and using comparative bioinformatics analyses, identified genes encoding putative carbohydrate-active enzymes (CAZymes) in the Thermogemmatispora sp. T81, Thermogemmatispora onikobensis, and Ktedonobacter racemifer genomes. Analyses of the Thermogemmatispora sp. T81 genome identified 64 CAZyme gene sequences belonging to 57 glycoside hydrolase families. The genome of Thermogemmatispora sp. T81 encodes 19 genes for putative extracellular CAZymes, similar to the number of putative extracellular CAZymes identified in T. onikobensis (17) and K. racemifer (17), despite K. racemifer not possessing a cellulolytic phenotype. These results suggest that these members of the order Ktedonobacteriales may use a broader range of carbohydrate polymers than currently described.