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

ABSTRACT: 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 ?-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 ?-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-?-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 ?-glucosidase cocktails. Conclusion:A new ?-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.

SUBMITTER: Mendez-Liter JA

PROVIDER: S-EPMC5674860 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Méndez-Líter J A JA Gil-Muñoz J J Nieto-Domínguez M M Barriuso J J de Eugenio L I LI Martínez M J MJ

Biotechnology for biofuels 20171106

<h4>Background</h4>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 β-glucosidases remains as one of the major bottlenecks for cellulose degradation. In this context, the ascomycete <i>Talaromyces amestolkiae,</i> isolated from cereal samples, has been studied as a promising source for these enzymes.<h4>Results</h4>BGL-2 is th ...[more]

PMID: 29142591

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Project description:The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively. The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD(CelK) contains 1 mol of calcium per mol. The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N(CelK). The CBD-N(CelK) and D192A retained binding parameters close to that of the intact CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded.

Dataset Information

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

Publications

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

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