Abstract
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.
Highlights
Cellulose that is the most abundant biopolymer on earth, is formed by β-(1,4)-linked D-glucose units, where adjacent D-glucoses are flipped making cellobiose the fundamentalHow to cite this article Karnaouri et al (2013), Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila
Cellulose is mainly present as crystalline fibers that are highly resistant to hydrolysis, its content in biomass is typically larger compared to hemicellulose, and as a result, cellulases are the key enzymes for bioethanol production (Ragauskas et al, 2006)
Enzymes that modify complex carbohydrates, such as cellulases, together with their accessory noncatalytic carbohydrate binding modules (CBMs), have been grouped into sequence-based families on the continuously updated Carbohydrate-Active EnZymes (CAZy) database (Cantarel et al, 2009). β-Glucosidases (EC 3.2.1.21) which act on soluble cello-oligosaccharides produced by the action of β-1,4-endoglucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), including cellobiose, towards the release of D-glucose, belong to glycosyl hydrolase (GH) families 1, 3, 5, 9, 30 and 116. β-Glucosidases hydrolyze soluble cellodextrins and cellobiose to D-glucose and relieve the system from end product inhibition (Himmel et al, 2007)
Summary
Cellulose that is the most abundant biopolymer on earth, is formed by β-(1,4)-linked D-glucose units, where adjacent D-glucoses are flipped making cellobiose the fundamentalHow to cite this article Karnaouri et al (2013), Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila. Cellulose is mainly present as crystalline fibers that are highly resistant to hydrolysis, its content in biomass is typically larger compared to hemicellulose, and as a result, cellulases are the key enzymes for bioethanol production (Ragauskas et al, 2006). As shown in various studies (Bezerra & Dias, 2005; Gruno et al, 2004; Zhao et al, 2004) the cellobiose released during the hydrolysis of lignocellulose for ethanol production has a high inhibitory effect on cellulases. The presence of sufficient β-glucosidase activity in the enzyme mixture is shown to increase the hydrolysis performance by more than 20% reaching even 40% increase in the total ethanol production of the processes (Xin, Yinbo & Peiji, 1993; Han & Chen, 2008). Lies the need of β-glucosidase activity in the lignocellulosic multi-enzyme systems
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