A novel, highly efficient ß-glucosidase with a cellulose-binding domain: characterization and properties of native and recombinant proteins.

BACKGROUND: Cellulose, the most abundant biopolymer on earth, is an alternative for fossil fuels as a renewable feedstock for the production of second-generation biofuels and other chemicals. The discovery of novel, highly efficient ß-glucosidases remains as one of the major bottlenecks for cellulose degradation. In this context, the ascomycete Talaromyces amestolkiae, isolated from cereal samples, has been studied as a promising source for these enzymes. RESULTS: BGL-2 is the major ß-glucosidase secreted by this fungus in the presence of cellulosic inductors. This enzyme possesses a CBD (Cellulose Binding Domain), an unusual feature among this type of proteins. Besides, when growing on cellulose, the fungus produced two different bgl-2 mRNAs that were cloned and expressed in Pichia pastoris. A complete recombinant protein (BGL-2*) and its truncated form, lacking CBD (BGL-2T*), have been purified, characterized and compared with the native enzyme (BGL-2). The three BGL-2 forms studied are highly stable in a wide pH range, but BGL-2T* showed an improved thermal stability at 50 °C after 72 h. Using p-nitrophenyl-ß-d-glucopyranoside as a substrate, the steady-state kinetic characterization of the three proteins showed a similar Km and kcat for BGL-2 and BGL-2*, while the truncated protein displayed a threefold higher value for kcat . All tested BGL-2 enzymes were as well highly efficient using cellobiose and other short oligosaccharides as a substrate. In view of biotechnological applications, the recombinant T. amestolkiae enzymes in saccharification of brewers' spent grain were studied, being comparable to commercial ß-glucosidase cocktails. CONCLUSION: A new ß-glucosidase from T. amestolkiae has been studied. The enzyme, containing a functional CBD, has been expressed in P. pastoris. The comparative analyses of the native protein and its recombinant forms, with and without CBD, suggest that they could be suitable tools for valorization of lignocellulosic biomass.