Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose

Abstract

Backgroundβ-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant β-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications.ResultsThe β-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of β-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel β-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2–7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-β-D-glucopyranoside (Km value of 0.62 mM) and cellobiose (Km value of 7.9 mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat for cellobiose was 67.7 s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290 mM. It was activated by glucose at concentrations lower that 200 mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600 mM glucose.ConclusionsThe article provides a useful novel β-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

Effect of Fermented Red Ginseng Extract Enriched in Ginsenoside Rg3 on the Differentiation and Mineralization of Preosteoblastic MC3T3-E1 Cells

“…7,8 b-Glucosidase (Bgp) hydrolyzes two molecules of glucose or glucose-substituted molecules by acting on b-1,4 bond of a compound. 9 As previously reported, b-galactosidase from Aspergillus oryzae and b-D-glucosidase from Bacteroides sp. have been used for the bioconversion of major ginsenosides Re and Rg1 into minor ginsenosides Rg2 and Rh1, respectively.…”

Cloning, Purification, and Characterization of a Thermostable β-Glucosidase from Thermotoga thermarum DSM 5069

“…2b), which is higher than the optimum for enzymes from Actinosynnema mirum (37 ºC), Azospirillum irakense (45 ºC), Flavobacterium johnsoniae (37 ºC), Martelella mediterranea (45 ºC), Myceliophthora thermophile (60 ºC), Aspergillus niger (70 ºC), and Thermoascus aurantiacus CBMAI-756 (75 ºC) (Rashid and Siddiqui 1997;Faure et al 2001;Leite et al 2008;Mao et al 2010;Hong et al 2012;Cui et al 2013;Zhao et al 2015). With good thermostability, the enzyme maintains its activity for a longer time, and the amount needed in the reaction is reduced (Pei et al 2012;Shi et al 2013). After pre-incubation at 80 ºC for 120 min, TthBgl retained 80% of the initial activity (Fig.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Moreover, β-glucosidases belonging to GH family 1 was found to be stimulated by lower glucose concentration and be tolerant to higher concentration. For examples, β-glucosidase from Microbispora bispora was activated in the presence of 300 mM glucose [135] and that reported in T. thermosaccharolyticum DSM 571 and T. aotearoense was activated by glucose concentration < 200 and 250 mM, respectively [85,136]. Similarly, recombinant GH 1 β-glucosidase from Exiguobacterium antarcticum B7 [84], Fervidobacterium islandicum [137], Thermotoga thermarum DSM 5069T [138] and Anoxybacillus DT3-1 [83] was tolerant to high glucose concentration e.g., Ki of 0.54-15 M. On the other hand, glucose tolerant β-glucosidase with Ki of 1.0-4.8 M has been identified from different environments using metagenomic approaches [45,53,106,111,139,140].…”

“…For instance, β-glucosidase from Anoxybacillus sp. DT3-1 [83], Exiguobacterium antarcticum B7 [84], and Thermoanaerobacterium thermosaccharolyticum DSM 571 [85] has been expressed in E. coli as fusion protein with six His tag at N-terminal and purified through Nickel-Nitrilotriacetic acid (Ni-NTA) chromatography [85,86,87]. Glutathione-S-Transferase (GST) affinity has been used for purification of recombinant β-glucosidase from Exiguobacterium oxidotolerans A011 expressed as fusion protein with GST which provides additional advantage by acting as a chaperone protein to facilitate protein folding, and provide a mean for their purification by immobilized glutathione column [88,89].…”