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Journal ArticleDOI

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

TL;DR: In this paper, the stability of almond β-glucosidase in five different organic media was evaluated, and the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively.
Abstract: The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions...
Citations
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Journal ArticleDOI
TL;DR: The crystal structure of the thermostable beta-glucosidase from Thermotoga neapolitana is determined, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 A.

133 citations

Journal ArticleDOI
TL;DR: A novel thermostable variant of a family 3 beta-glucosidase to be an efficient catalyst in alkyl-gl glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule.

72 citations

Journal ArticleDOI
TL;DR: β-Glucosidase II, encoded by the βglu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109 and placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data.
Abstract: β-Glucosidase II (Bgl II), encoded by the βglu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109. The enzyme had a molecular mass of 176 kDa and was a dimer with an apparent subunit mass of 83 kDa. It exhibited broad substrate specificity and hydrolyzed β-linked gluco-disaccharides and oligosaccharides, salicin, and cyanogenic glucoside amygladin. The unusually high hydrolytic activity of 7,680 units min−1 g−1 protein was obtained on sophorose. Competition experiments performed using differently linked β-disaccharides indicated these to be hydrolyzed at the same active site. Transglycosylation activity leading to the biosynthesis of several disaccharides and oligosaccharides was observed. The enzyme was placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data. The involvement of His as a catalytically important residue was confirmed by diethylpyrocarbonate modification. Pre-incubation of the purified enzyme with 5 mM p-nitrophenyl-β-d-glucoside offered 2.5-fold higher residual activity compared with unbound enzyme, indicating protection at the active site. The feasibility of this enzyme as a biocatalyst of choice for the synthesis of glyco-conjugates is discussed.

24 citations


Cites background from "Transglycosylation Catalyzed by Alm..."

  • ...Bgl II has been extensively studied for its ability to synthesize cellooligosaccharides (Bhatia et al. 2002b), alkyl and terpenyl glucosides (Bacchawat et al. 2004), and the glycosylasparagine mimic β-1-N-acetamido-D-glucopyranose as β(1→6) and β(1→3) isomers(Kannan et al. 2004)....

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  • ...2004), and a series of disaccharides that mimic the structure of cell surface receptors (Kannan et al. 2004)....

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  • ...…(Ajisaka et al. 1987; Bhatia et al. 2002b), alkyl (Vic et al. 1997; Wallecha and Mishra 2003), amino (Wang and Bertozzi 2001), and terpenyl glucosides (Gunata et al. 1994; Bacchawat et al. 2004), and a series of disaccharides that mimic the structure of cell surface receptors (Kannan et al. 2004)....

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  • ...glycosylasparagine mimic β-1-N-acetamido-D-glucopyranose as β(1→6) and β(1→3) isomers(Kannan et al. 2004)....

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Journal ArticleDOI
TL;DR: A genomic DNA fragment encoding a novel beta-glucosidase-like activity of the yeast Pichia etchellsii was cloned and expressed in Escherichia coli, and the encoded protein, MUGA, was purified by about 53-fold and characterized as a monomer of 52.1kDa by SDS-PAGE and MALDI-TOF.

18 citations


Cites background from "Transglycosylation Catalyzed by Alm..."

  • ...Both the enzymes have been well characterized [11,12] and BglII has been extensively used for synthesis of oligosaccharides [13], alkyl and terpene glucosides [14], and asparagine mimetics [15]....

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Journal ArticleDOI
TL;DR: The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides, among the very few that could synthesize a wide variety of glycoconjugates.
Abstract: The biosynthetic activity of yeast Pichia etchellsii beta-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

11 citations

References
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Journal ArticleDOI
TL;DR: The aim of the present article is to describe the sources and properties of recombinant β-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.
Abstract: β-Glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the s...

476 citations


"Transglycosylation Catalyzed by Alm..." refers background in this paper

  • ...The stability of cloned P. etchellsii b-glucosidase II in different aqueous organic media has been reported (Bhatia et al., 2002b)....

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  • ...Among glycosidases, b-galactosidases have been extensively used for synthesis of oligosaccharides. bGlucosidases, on the other hand, have more often been employed for glycoside synthesis than to synthesize disaccharides (Bhatia et al. , 2002a)....

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Journal ArticleDOI
TL;DR: Telltale surface sugars enable cells to identify and interact with one another and new drugs aimed at those carbohydrates could stop infection and inflammation.
Abstract: Telltale surface sugars enable cells to identify and interact with one another. New drugs aimed at those carbohydrates could stop infection and inflammation.

409 citations


"Transglycosylation Catalyzed by Alm..." refers background in this paper

  • ...Oligosaccharides and polysaccharides of cell surface glycoproteins and glycolipids play crucial roles in cellular communication phenomena and also as receptor sites for bacteria and viruses (Sharon and Lis, 1993)....

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Journal ArticleDOI
TL;DR: Evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for thecell wall of the Ascomycetes, pointing to the existence of cell wall repair mechanisms that compensate for cell damage.

378 citations


"Transglycosylation Catalyzed by Alm..." refers background in this paper

  • ...Nearly half of the cell wall is made up of b-D-(10/3)glucan only (Kapteyn et al. , 1999)....

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Journal ArticleDOI

111 citations


"Transglycosylation Catalyzed by Alm..." refers background in this paper

  • ...Specifically mutated glycosidases, also called glycosynthases, have been reported to synthesize oligosaccharides in very high yield (Mackenzie et al. , 1998)....

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