Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila
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 contr...
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PeerJ Inc.
2013-02-01
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| author | Anthi Karnaouri Evangelos Topakas Thomas Paschos Ioanna Taouki Paul Christakopoulos |
| author_facet | Anthi Karnaouri Evangelos Topakas Thomas Paschos Ioanna Taouki Paul Christakopoulos |
| author_sort | Anthi Karnaouri |
| collection | DOAJ |
| description | 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. |
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| spelling | doaj.art-8efeacddc14a422da23a21d69185a5022023-12-03T09:55:57ZengPeerJ Inc.PeerJ2167-83592013-02-011e4610.7717/peerj.4646Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophilaAnthi Karnaouri0Evangelos Topakas1Thomas Paschos2Ioanna Taouki3Paul Christakopoulos4Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, GreeceBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, GreeceBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, GreeceBiotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, GreeceBiochemical and Chemical Process Engineering, Division of Sustainable Process Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, Luleå, SwedenThe 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.https://peerj.com/articles/46.pdfGlycoside hydrolase family 3Myceliophthora thermophila Pichia pastoris Overexpressionβ-glucosidaseTransglycosylation |
| spellingShingle | Anthi Karnaouri Evangelos Topakas Thomas Paschos Ioanna Taouki Paul Christakopoulos Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila PeerJ Glycoside hydrolase family 3 Myceliophthora thermophila Pichia pastoris Overexpression β-glucosidase Transglycosylation |
| title | Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila |
| title_full | Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila |
| title_fullStr | Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila |
| title_full_unstemmed | Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila |
| title_short | Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila |
| title_sort | cloning expression and characterization of an ethanol tolerant gh3 β glucosidase from myceliophthora thermophila |
| topic | Glycoside hydrolase family 3 Myceliophthora thermophila Pichia pastoris Overexpression β-glucosidase Transglycosylation |
| url | https://peerj.com/articles/46.pdf |
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