Abstract
BackgroundIn the last years, the most outstanding trend for obtaining high added-value components and second-generation (2G) biofuels consisted on exploitation of plant biomass. But recently, 3G biofuels, based in algae biomass, have emerged as a great alternative for production of energy.ResultsIn this work, a versatile β-glucosidase from the ascomycete fungus Talaromyces amestolkiae has been purified, characterized, and heterologously expressed. The synthesis of this β-glucosidase (BGL-3) was not induced by cellulose, and the presence of a specific carbon source is not required for its production, which is uncommon for β-glucosidases. BGL-3, which was obtained from a basal medium with glucose as carbon source, was profusely secreted under carbon starvation conditions, which was corroborated by qRT-PCR assays. BGL-3 was purified from T. amestolkiae cultures in one step, and biochemically characterized. The enzyme showed high thermal stability, and very high efficiency on pNPG (Km of 0.14 mM and Vmax of 381.1 U/mg), cellobiose (Km of 0.48 mM and Vmax of 447.1 U/mg), and other cello-oligosaccharides. Surprisingly, it also showed remarkable ability to hydrolyze laminarin, a β-1,3-glucan present in algae. The recombinant enzyme, obtained in the yeast Pichia pastoris, exhibited kinetic and physicochemical properties similar to those found for the native protein. Enzyme efficiency was examined in wheat straw saccharification processes, in which BGL-3 worked better supplementing Celluclast 1.5L than the commercial cellulase cocktail N-50010. Besides, BGL-3 hydrolyzed laminarin more efficiently than a commercial laminarinase.ConclusionsA very efficient 1,4-β-glucosidase, which also showed activity over 1,3-β-glucose bonds, has been produced, purified, and characterized. This is the first report of such versatility in a 1,4-β-glucosidase. The application of this enzyme for saccharification of wheat straw and laminarin and its comparison with commercial enzymes suggest that it could be an interesting tool for the production of 2G and 3G biofuels.
Highlights
In the last years, the most outstanding trend for obtaining high added-value components and secondgeneration (2G) biofuels consisted on exploitation of plant biomass
One of them was induced exclusively by cellulosic substrates (g3821), while the other one (g377) was produced in the four carbon sources tested (Avicel, glucose, xylan, or pretreated wheat straw). This phenomenon is peculiar since, usually, cellulolytic enzymes require cellulose or its derivatives to be induced, and they are repressed by glucose or other metabolizable carbon sources [14]
Expression of bgl‐3 gene under carbon starvation In a previous work, we demonstrated that the fungus secreted detectable amounts of BGL when glucose was used as carbon source, this activity appeared upon glucose depletion
Summary
The most outstanding trend for obtaining high added-value components and secondgeneration (2G) biofuels consisted on exploitation of plant biomass. Laminarin is a predominantly linear β-(1,3)-glucan that can contain few branches of mannitol or glucose attached at O-6 positions of the main chain, and it is a potentially nice candidate to produce bioethanol 3G [6]. With this perspective, enzymes used in 2G bioethanol processes, like cellulase cocktails, including β-glucosidases, could represent a good alternative for efficient liberation of glucose from algae polysaccharides
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