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Gene cloning and characterization of a novel cellulose-binding beta-glucosidase from Phanerochaete chrysosporium.

ABSTRACT: Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular beta-glucosidase (CBGL) from Phanerochaete chrysosporium suggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that cbgl is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles, cbgl-1 and cbgl-2, of a single gene.

SUBMITTER: Li B

PROVIDER: S-EPMC106459 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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Gene cloning and characterization of a novel cellulose-binding beta-glucosidase from Phanerochaete chrysosporium.

Li B B Renganathan V V

Applied and environmental microbiology 19980701 7

Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular beta-glucosidase (CBGL) from Phanerochaete chrysosporium suggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that cbgl is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles, cbgl-1 and cbgl-2, of a single gene. ...[more]

PMID: 9647863

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Project description:BackgroundCellulose, 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 Talaromyces amestolkiae, isolated from cereal samples, has been studied as a promising source for these enzymes.ResultsBGL-2 is the major β-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-β-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 β-glucosidase cocktails.ConclusionA new β-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.

Dataset Information

Gene cloning and characterization of a novel cellulose-binding beta-glucosidase from Phanerochaete chrysosporium.

Publications

Gene cloning and characterization of a novel cellulose-binding beta-glucosidase from Phanerochaete chrysosporium.

Similar Datasets

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