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A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5.

Chen HL, Chen YC, Lu MY, Chang JJ, Wang HT, Ke HM, Wang TY, Ruan SK, Wang TY, Hung KY, Cho HY, Lin WT, Shih MC, Li WH - Biotechnol Biofuels (2012)

Bottom Line: Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan. mcshih@gate.sinica.edu.tw.

ABSTRACT

Background: Cellulose, 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.

Results: In 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.

Conclusion: Our 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.

No MeSH data available.


Related in: MedlinePlus

The effects of Mg2+and Mn2+cations, to a final concentration of 1 and 10 mM, on the activity of purified NpaBGS at pH 6.0. The activity of NpaBGS (open square), NpaBGS with Mg2+ (gray square) and NpaBGS with Mn2+ (black square) were compared.
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Figure 3: The effects of Mg2+and Mn2+cations, to a final concentration of 1 and 10 mM, on the activity of purified NpaBGS at pH 6.0. The activity of NpaBGS (open square), NpaBGS with Mg2+ (gray square) and NpaBGS with Mn2+ (black square) were compared.

Mentions: We examined whether Mg2+ and Mn2+ could enhance pH or temperature tolerance of NpaBGS. We found no effect of these ions on pH tolerance (data not shown), but addition of either cation increased NpaBGS’s activity at higher temperatures (Figure 3). Interestingly, significant enhancement of the activity by the supplement of Mn2+ was found at 50°C over those assayed at 40°C, suggesting the potential of in vitro application in digesting cellulose at elevated temperatures by supplementing enhancing cations.


A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5.

Chen HL, Chen YC, Lu MY, Chang JJ, Wang HT, Ke HM, Wang TY, Ruan SK, Wang TY, Hung KY, Cho HY, Lin WT, Shih MC, Li WH - Biotechnol Biofuels (2012)

The effects of Mg2+and Mn2+cations, to a final concentration of 1 and 10 mM, on the activity of purified NpaBGS at pH 6.0. The activity of NpaBGS (open square), NpaBGS with Mg2+ (gray square) and NpaBGS with Mn2+ (black square) were compared.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3403894&req=5

Figure 3: The effects of Mg2+and Mn2+cations, to a final concentration of 1 and 10 mM, on the activity of purified NpaBGS at pH 6.0. The activity of NpaBGS (open square), NpaBGS with Mg2+ (gray square) and NpaBGS with Mn2+ (black square) were compared.
Mentions: We examined whether Mg2+ and Mn2+ could enhance pH or temperature tolerance of NpaBGS. We found no effect of these ions on pH tolerance (data not shown), but addition of either cation increased NpaBGS’s activity at higher temperatures (Figure 3). Interestingly, significant enhancement of the activity by the supplement of Mn2+ was found at 50°C over those assayed at 40°C, suggesting the potential of in vitro application in digesting cellulose at elevated temperatures by supplementing enhancing cations.

Bottom Line: Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan. mcshih@gate.sinica.edu.tw.

ABSTRACT

Background: Cellulose, 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.

Results: In 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.

Conclusion: Our 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.

No MeSH data available.


Related in: MedlinePlus