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Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophile
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
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2013 (English)In: PeerJ, ISSN 2167-8359, E-ISSN 2167-8359, 46Article in journal (Refereed) Published
Abstract [en]

The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH) family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The recombinant enzyme (90 kDa) was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml) after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.

Place, publisher, year, edition, pages
2013. 46
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
URN: urn:nbn:se:ltu:diva-13703DOI: 10.7717/peerj.46Local ID: cfbe839d-e1bb-4f98-b32b-9f14b9ad48afOAI: oai:DiVA.org:ltu-13703DiVA: diva2:986656
Note
Validerad; 2013; Bibliografisk uppgift: Article numbere 46; 20130221 (ysko)Available from: 2016-09-29 Created: 2016-09-29 Last updated: 2017-11-24Bibliographically approved

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