RESUMEN
Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box-Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.
Asunto(s)
Deshidrogenasas de Carbohidratos/metabolismo , Proteínas Fúngicas/metabolismo , Termitomyces/enzimología , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/genética , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Especificidad por Sustrato , Termitomyces/química , Termitomyces/genética , Termitomyces/aislamiento & purificaciónRESUMEN
BACKGROUND: Fungus-cultivating termites make use of an obligate mutualism with fungi from the genus Termitomyces, which are acquired through either vertical transmission via reproductive alates or horizontally transmitted during the formation of new mounds. Termitomyces taxonomy, and thus estimating diversity and host specificity of these fungi, is challenging because fruiting bodies are rarely found. Molecular techniques can be applied but need not necessarily yield the same outcome than morphological identification. METHODOLOGY: Culture-dependent and culture-independent methods were used to comprehensively assess host specificity and gut fungal diversity. Termites were identified using mitochondrial cytochrome oxidase II (COII) genes. Twenty-three Termitomyces cultures were isolated from fungal combs. Internal transcribed spacer (ITS) clone libraries were constructed from termite guts. Presence of Termitomyces was confirmed using specific and universal primers. Termitomyces species boundaries were estimated by cross-comparison of macromorphological and sequence features, and ITS clustering parameters accordingly optimized. The overall trends in coverage of Termitomyces diversity and host associations were estimated using Genbank data. RESULTS AND CONCLUSION: Results indicate a monoculture of Termitomyces in the guts as well as the isolation sources (fungal combs). However, cases of more than one Termitomyces strains per mound were observed since mounds can contain different termite colonies. The newly found cultures, as well as the clustering analysis of GenBank data indicate that there are on average between one and two host genera per Termitomyces species. Saturation does not appear to have been reached, neither for the total number of known Termitomyces species nor for the number of Termitomyces species per host taxon, nor for the number of known hosts per Termitomyces species. Considering the rarity of Termitomyces fruiting bodies, it is suggested to base the future taxonomy of the group mainly on well-characterized and publicly accessible cultures.
Asunto(s)
ADN Espaciador Ribosómico/genética , Isópteros/genética , Simbiosis/genética , Termitomyces/genética , Termitomyces/aislamiento & purificación , Animales , ADN de Hongos/genética , Variación Genética , Isópteros/microbiología , Isópteros/fisiología , Filogenia , Termitomyces/clasificaciónRESUMEN
The microbial communities harbored in the gut and fungus comb of the fungus-growing termite Odontotermes formosanus were analyzed by both culture-dependent and culture-independent methods to better understand the community structure of their microflora. The microorganisms detected by denaturing gradient gel electrophoresis (DGGE), clonal selection, and culture-dependent methods were hypothesized to contribute to cellulose-hemicellulose hydrolysis, gut fermentation, nutrient production, the breakdown of the fungus comb and the initiation of the growth of the symbiotic fungus Termitomyces. The predominant bacterial cultivars isolated by the cultural approach belonged to the genus Bacillus (Phylum Firmicutes). Apart from their function in lignocellulosic degradation, the Bacillus isolates suppressed the growth of the microfungus Trichoderma harzianum (genus Hypocrea), which grew voraciously on the fungus comb in the absence of termites but grew in harmony with the symbiotic fungus Termitomyces. The in vitro studies suggested that the Bacillus sp. may function as mutualists in the termite-gut-fungus-comb microbial ecosystem.