title: A highly efficient β-glucosidase from the buffalo rumen fungus Neocallimastix patriciarum W5.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Hsin-Liang
authors.initials: HL
authors.lastname: Chen
authors.affiliation: Department of Biological Science & Technology, National Pingtung University of Science & Technology, Neipu Hsiang, Pingtung, 91201, Taiwan.
authors.forename: Yo-Chia
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Mei-Yeh Jade
authors.initials: MJ
authors.lastname: Lu
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.|Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Jui-Jen
authors.initials: JJ
authors.lastname: Chang
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Hiaow-Ting Christine
authors.initials: HC
authors.lastname: Wang
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.|Program in Microbial Genomics, National Chung-Hsing University, Taichung, 402, Taiwan.
authors.forename: Huei-Mien
authors.initials: HM
authors.lastname: Ke
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Tzi-Yuan
authors.initials: TY
authors.lastname: Wang
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Sz-Kai
authors.initials: SK
authors.lastname: Ruan
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Tao-Yuan
authors.initials: TY
authors.lastname: Wang
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Kuo-Yen
authors.initials: KY
authors.lastname: Hung
authors.affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei, 115, Taiwan.|Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, 402, Taiwan.|Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Hsing-Yi
authors.initials: HY
authors.lastname: Cho
authors.affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan.
authors.forename: Wan-Ting
authors.initials: WT
authors.lastname: Lin
authors.affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung-Hsing University - Academia Sinica, Taipei, 115, Taiwan.|Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan.|Biotechnology Center, National Chung-Hsing University, Taichung, 402, Taiwan.
authors.forename: Ming-Che
authors.initials: MC
authors.lastname: Shih
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, 115, Taiwan.|Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.|Biotechnology Center, National Chung-Hsing University, Taichung, 402, Taiwan.|Department of Ecology and Evolution, University of Chicago, Chicago, IL, 60637, USA.
authors.forename: Wen-Hsiung
authors.initials: WH
authors.lastname: Li