title: Characterization of a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1.
abstract: BACKGROUND
  In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme(®) CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast(®) 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase.
  
  
  RESULTS
  In this study, for the first time, a β-glucosidase DT-Bgl gene (1359 bp) was identified in the genome of Anoxybacillus sp. DT3-1, and cloned and heterologously expressed in Escherichia coli BL21. Phylogenetic analysis indicated that DT-Bgl belonged to glycosyl hydrolase (GH) family 1. The recombinant DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-β-d-glucopyranoside (pNPG). The DT-Bgl was purified using an Ni-NTA column, with molecular mass of 53 kDa using an SDS-PAGE analysis. It exhibited optimum activity at 70 °C and pH 8.5, and did not require any tested co-factors for activation. The K m and V max values for DT-Bgl were 0.22 mM and 923.7 U/mg, respectively, with pNPG as substrate. The DT-Bgl displayed high glucose tolerance, and retained 93 % activity in the presence of 10 M glucose.
  
  
  CONCLUSIONS
  Anoxybacillus DT-Bgl is a novel thermostable β-glucosidase with low glucose inhibition, and converts long-chain cellodextrins to cellobiose, and further hydrolyse cellobiose to glucose. Results suggest that DT-Bgl could be useful in the development of a bioprocess for the efficient saccharification of lignocellulosic biomass.
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor Malaysia.
authors.forename: Chia Sing
authors.initials: CS
authors.lastname: Chan
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Lee Li
authors.initials: LL
authors.lastname: Sin
authors.affiliation: Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Kok-Gan
authors.initials: KG
authors.lastname: Chan
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor Malaysia.
authors.forename: Mohd Shahir
authors.initials: MS
authors.lastname: Shamsir
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor Malaysia.
authors.forename: Fazilah Abd
authors.initials: FA
authors.lastname: Manan
authors.affiliation: Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City, USA.
authors.forename: Rajesh Kumar
authors.initials: RK
authors.lastname: Sani
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81300 Skudai, Johor Malaysia.
authors.forename: Kian Mau
authors.identifier: 0000-0002-2839-8722
authors.initials: KM
authors.lastname: Goh
keywords: Anoxybacillus; Biofuel; Biomass conversion; Glucose tolerance; Glycosyl hydrolase