RESUMEN
An intracellular ß-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140â¯kDa) was optimally active at 65⯰C and pHâ¯5.5, exhibited half life of 10â¯h at 60⯰C, 78 and 88% residual activity after 24â¯h, at pHâ¯4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by ß-mercaptoethanol. In the presence of p-nitrophenyl-ß-d-xylopyranoside, AbXyl exhibited Km of 0.19â¯mM, Kcat of 453.29â¯s-1, Kcatâ¯Km-1 of 2322â¯s-1â¯mM and was moderately influenced by xylose (Ki 21.25â¯mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.
Asunto(s)
Agricultura , Anoxybacillus/enzimología , Carbohidratos/química , Residuos Industriales , Xilosidasas/metabolismo , Anoxybacillus/citología , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Hidrólisis , Espacio Intracelular/enzimología , Especificidad por Sustrato , Temperatura , Xilosidasas/antagonistas & inhibidoresRESUMEN
Four closely related facultative anaerobe, moderately thermophilic, Gram positive rods (JS1(T), JS5, JS11, and JS15) were isolated from sediment samples from a hot spring at Suryakund, Jharkhand, India. Colonies were pale yellow, rough surface with uneven edges on TSA after 72 h incubation. Heterotrophic growth was observed at 40-60°C and pH 5.5-11.5; optimum growth occurred at 55°C and pH 7.5. 16S rRNA gene sequence analysis revealed the strains belong to genus Anoxybacillus. DNA-DNA homology values among strains were above 70% and showed distinct ERIC and REP PCR profile. On the basis of morphology and biochemical characteristics, strain JS1(T) was studied further. Strain JS1(T) showed 99.30% sequence similarity with A. flavithermus subsp. yunnanensis, 99.23% with A. mongoliensis, 99.16% with A. eryuanensis, 98.74% with A. flavithermus subsp. flavithermus, 98.54% with A. tengchongensis, 98.51% with A. pushchinoensis, 97.91% with A. thermarum, 97.82% with A. kaynarcensis, 97.77% with A. ayderensis and A. kamchatkensis, 97.63% with A. salavatliensis, 97.55% with A. kestanbolensis, 97.48% with A. contaminans, 97.27% with A. gonensis and 97.17% with A. voinovskiensis. In 16S rRNA secondary structure based phylogenetic comparison, strain JS1(T) was clustered with Anoxybacillus eryuanensis, A. mongoliensis, and A. flavithermus subsp. yunnanensis and showed 15 species specific base substitutions with maximum variability in helix 6. Moreover, DNA-DNA relatedness between JS1(T) and the closely related type strains were well below 70%. The DNA G+C content was 42.1 mol%. The major fatty acids were C(15:0 iso), C(16:0 iso) and C(17:0iso). The polar lipids were a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethnolamine, a phosphatidylcholine, a phosphatidyl monomethylethanolamine and four unknown lipids. Based on polyphasic approach, strain JS1(T) represent a novel species of the genus Anoxybacillus for which Anoxybacillus suryakundensis sp. nov. is proposed. The type strain is JS1(T) (= DSM 27374(T) = LMG 27616(T) =JCM19211(T)).