Dataset Information

A Novel β-Glucosidase From Chryseobacterium scophthalmum 1433 for Efficient Rubusoside Production From Stevioside.

ABSTRACT: As a natural sweetening and solubilizing agent, rubusoside has great potential in the application of healthy beverages and pharmaceuticals. However, the direct extraction and purification of rubusoside from raw materials is inefficient. In this work, a novel β-glucosidase (CsBGL) was obtained from Chryseobacterium scophthalmum 1433 through screening of the environmental microorganisms. CsBGL markedly hydrolyzed sophorese (Glcβ1-2Glc) and laminaribiose (Glcβ1-3Glc), but for steviol glycosides, it only hydrolyzed the C-13/C-19-linked sophorese, instead of the C-13/C-19-linked Glcβ1-2[Glcβ1-3]Glc trisaccharide and Glcβ1-monosaccharide. It efficiently hydrolyzed stevioside (240 g/L) to produce rubusoside (99% yield) at 47.5°C for 70 min. Even when using a crude steviol glycosides extract (500 g/L) containing ∼226 g/L stevioside as the substrate, CsBGL could also convert stevioside to rubusoside (99% yield) at 47.5°C for 2 h, in which the rubusoside concentration increased from the initial 42 g/L to the final 222 g/L. These results reveal that CsBGL would be a promising biocatalyst for the industry-scale production of rubusoside from stevioside or/and the crude steviol glycosides extract.

SUBMITTER: Yan Z

PROVIDER: S-EPMC8546341 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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A Novel β-Glucosidase From Chryseobacterium scophthalmum 1433 for Efficient Rubusoside Production From Stevioside.

Yan Zhenxin Z Cao Xueting X Yang Xiao X Yang Shida S Xu Li L Jiang Xukai X Xiao Min M

Frontiers in microbiology 20211012

As a natural sweetening and solubilizing agent, rubusoside has great potential in the application of healthy beverages and pharmaceuticals. However, the direct extraction and purification of rubusoside from raw materials is inefficient. In this work, a novel β-glucosidase (CsBGL) was obtained from Chryseobacterium scophthalmum 1433 through screening of the environmental microorganisms. CsBGL markedly hydrolyzed sophorese (Glcβ1-2Glc) and laminaribiose (Glcβ1-3Glc), but for s ...[more]

PMID: 34712213

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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.

Project description:BackgroundCellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production.ResultsIn this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system.ConclusionOur characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Dataset Information

A Novel β-Glucosidase From Chryseobacterium scophthalmum 1433 for Efficient Rubusoside Production From Stevioside.

Publications

A Novel β-Glucosidase From <i>Chryseobacterium scophthalmum</i> 1433 for Efficient Rubusoside Production From Stevioside.

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