Cloning, sequence analysis, and characterization of a novel β-glucosidase-like activity from Pichia etchellsii

Roy, Priyanka; Mishra, Saroj; Chaudhuri, Tapan K.

doi:10.1016/j.bbrc.2005.08.067

Cited by 17 publications

(6 citation statements)

References 30 publications

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“…GH3 β-glucosidases have been purified and characterized from many organisms, including bacteria, eukaryotes, and archaea (http://www.cazy.org/fam/acc_GH.html; Roy et al 2005;Kudou et al 2014). Thermotoga thermarum 5069 is a hyperthermophilic bacterium that grows at 80 ºC; it was isolated from continental solfataric springs at Lac Abbe in Djibouti, Somalia, Africa (Windberger et al 1989).…”

Section: Introductionmentioning

confidence: 99%

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

Long¹,

Shi²,

Li³

et al. 2016

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A 56-kDa β-glucosidase (TthBgl) derived from Thermotoga thermarum DSM 5069 was expressed and purified from Escherichia coli BL21 (DE3). The purified enzyme showed hydrolytic activity towards only pnitrophenyl-β-D-glucopyranoside among the synthetic glycosides tested. The pH maximum was 5.0, and under the conditions tested, maximal activity was at 85 ºC, and pH stability occurred from 5.0 to 6.0. After being incubated at 80 ºC for 120 min, TthBgl retained 80% of its original activity. The β-glucosidase had no apparent requirement for metal ions or other co-factors, but its activity was significantly inhibited by 0.1% SDS and 1mM Cu 2+ , in which only 3% and 10% residual activity was maintained, respectively. The Vmax of TthBgl was 8.79 U mg -1 for pnitrophenyl-β-D-glucopyranoside, while the Km was 2.41 mM. The Enzyme activity was gradually inhibited by the addition of glucose, but remained approximately 50% of its original value in 500 mM glucose. 789.25 mg/L glucose was released from cellobiose by the incubation of 0.2 U/mL TthBgl for 9 h at 75 ºC. According to a phylogenetic analysis, TthBgl belongs to the glycosyl hydrolase family 3 (GH3).

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Section: Introductionmentioning

confidence: 99%

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

Long¹,

Shi²,

Li³

et al. 2016

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“…This may not be entirely due to the system being inefficient as this yeast produces multiple β-glucosidases and the initial activity in the cell-free extract is likely to be due to these "minor" enzymes. Three additional β-glucosidases (and β-glucosidase-like enzymes) have also been identified in this yeast by expression in Escherichia coli [26][27][28]. All the chromatographic steps were reproducible with consistent separation pattern and purity as observed by SDS-PAGE.…”

Section: Binding and Elutionmentioning

confidence: 84%

Purification of ß-glucosidases from Pichia etchellsii Using CIM Monolith Columns

Gaonkar

Mishra

Vijayalakshmi

2010

Appl Biochem Biotechnol

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β-Glucosidases (EC 3.2.1.21) are industrially important glycosyl hydrolases used for cellulose saccharification as well as for synthesis of glyco-conjugates. Crystal structure of only one β-glucosidase of family 3 of the glycosyl hydrolase families is available due to difficulty in purification of these closely related enzymes from a given source. Multiple steps used during purification result in low yield, making it difficult to study their properties. Conditions for purification of two closely related β-glucosidases (BGL I and BGL II) of family 3 from Pichia etchellsii were investigated in this study. Two weak anion exchange columns convective interaction media-diethyl amino ethyl (CIM-DEAE) and CIM-ethylenediamine (CIM-EDA) were used for this purpose. The results obtained at 0.34 ml disk (CIM-DEAE) level were scaled up to 8 ml CIM-DEAE tube column wherein BGL I and BGL II were separated from the major contaminants in the cell-free extract. The recovered enzymes were completely resolved in the second step using CIM-EDA. A final specific activity of 9,180 IU/mg and 2,345.3 IU/mg was achieved for BGL I and BGL II respectively with an overall yield of 33%. The system should be applicable to resolution of other closely related enzymes from this family.

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“…In 2005, only one putative β-glucosidase from Pichia etchellsii was identified and showed no homology with known β-glucosidases. In contrast, this protein shares a high degree of identity with several cell-surface-associated proteins (Roy et al, 2005).…”

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confidence: 92%

Characterization of a novel β-glucosidase-like activity from a soil metagenome

Jiang

et al. 2009

J Microbiol.

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We report the cloning of a novel beta-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known beta-glucosidases. The putative beta-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed D-glucosyl-beta-(l-4)-D-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42 degrees C using p-nitrophenyl-beta-D-glucoside as the substrate. A CaCl(2) concentration of 1 mM was required for optimal activity. The putative beta-glucosidase had an apparent K(m) value of 0.19 mM, a V(max) value of 4.75 U/mg and a k (cat) value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.

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Cloning, sequence analysis, and characterization of a novel β-glucosidase-like activity from Pichia etchellsii

Cited by 17 publications

References 30 publications

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

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

Purification of ß-glucosidases from Pichia etchellsii Using CIM Monolith Columns

Characterization of a novel β-glucosidase-like activity from a soil metagenome

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