Dataset Information

Novel pH-Stable Glycoside Hydrolase Family 3 ?-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

ABSTRACT: This paper reports on a novel ?-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu(2+) tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-?-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

SUBMITTER: Nieto-Dominguez M

PROVIDER: S-EPMC4542250 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

Nieto-Domínguez Manuel M de Eugenio Laura I LI Barriuso Jorge J Prieto Alicia A Fernández de Toro Beatriz B Canales-Mayordomo Ángeles Á Martínez María Jesús MJ

Applied and environmental microbiology 20150706 18

This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or ...[more]

PMID: 26150469

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Project description:BackgroundGlycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal β-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated.ResultsThe β-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of β-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-β-D-xylopyranoside of 0.20 mM and 69.3 s-1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) β-D-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR.ConclusionsA β-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) β-D-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.

Dataset Information

Novel pH-Stable Glycoside Hydrolase Family 3 ?-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

Publications

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation.

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