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

ABSTRACT: 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-?-1,4-glucanases, cellobiohydrolases, cellodextrinases, and ?-glucosidases. Converting cellobiose by ?-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production.In this study, a cDNA encoding ?-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.Our characterizations of the novel ?-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.

SUBMITTER: Chen HL

PROVIDER: S-EPMC3403894 | biostudies-other | 2012 Apr

REPOSITORIES: biostudies-other

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

Chen Hsin-Liang HL Chen Yo-Chia YC Lu Mei-Yeh Jade MJ Chang Jui-Jen JJ Wang Hiaow-Ting Christine HC Ke Huei-Mien HM Wang Tzi-Yuan TY Ruan Sz-Kai SK Wang Tao-Yuan TY Hung Kuo-Yen KY Cho Hsing-Yi HY Lin Wan-Ting WT Shih Ming-Che MC Li Wen-Hsiung WH Li Wen-Hsiung WH

Biotechnology for biofuels 20120419 1

<h4>Background</h4>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.<h4>Results</h4 ...[more]

PMID: 22515264

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Project description:Four types of ?-1,3-1,4 glucanase (?-glucanase, EC 3.2.1.73) genes, designated bglA13, bglA16, bglA51, and bglM2, were found in the cDNA library of Neocallimastix patriciarum J11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias to Streptococcus equinus. The presence of expansion and several predicted secondary structures in the 3' untranslated regions (3'UTRs) of bglA16 and bglM2 suggest that these two genes were duplicated recently, whereas bglA13 and bglA16, which contain very short 3'UTRs, were replicated earlier. These findings indicate that the ?-glucanase genes from N. patriciarum J11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. ?-Glucanase genes of Streptococcus equinus, Ruminococcus albus 7, and N. patriciarum J11 were cloned and expressed by Escherichia coli. The recombinant ?-glucanases cloned from S. equinus, R. albus 7, and N. patriciarum J11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial ?-glucanases were also significantly lower than those of the fungal ?-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal ?-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived ?-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated ?-glucanases, showed much higher k(cat) values than others. These results support the notion that duplicated ?-glucanase genes, namely, bglA16 and bglM2, increase the reaction efficiency of ?-glucanases and suggest that the catalytic efficiency of ?-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms in N. patriciarum J11.

Dataset Information

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

Publications

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

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