Glycosidase-catalysed synthesis of glycosides by an improved procedure for reverse hydrolysis: application to the chemoenzymatic synthesis of galactopyranosyl-(1→4)- O-α-galactopyranoside derivatives (original) (raw)
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Bioorganic & Medicinal Chemistry Letters, 2008
A b-galactosidase (from Aspergillus oryzae) preparation viz. EPRP (enzyme precipitated and rinsed with propanol), obtained by the removal of bulk water by precipitation with n-propanol, showed higher biological activity than the lyophilized powder. FT-IR study confirmed that EPRP had retained the a-helical content of the native structure better than the lyophilized form. Use of this formulation of b-galactosidase under low water conditions (temperature 55°C, reaction time of 4 h) gave enantioselectivity, E > 1000 for the stereoselective synthesis of (R)-(1-phenylethyl)-b-D D-galactopyranoside, starting from racemic 1-phenylethanol and D D-galactose. For racemic 2-octanol also, EPRP worked better. Under similar conditions, (R)-(2-octyl)-b-D Dgalactopyranoside was formed with an enantioselectivity, E = 38.
Enzymatic synthesis of galactosyl–xylose by Aspergillus oryzae β-galactosidase
Journal of Molecular Catalysis B-enzymatic, 2002
In aqueous medium, the reaction catalyzed by Aspergillus oryzae  -galactosidase with O-nitrophenyl--d-galactopyranoside (ONPG) in the presence of an acceptor leads to the synthesis of transglycosylation compounds in addition to the hydrolysis products (ONP and galactose). Our goal was to develop a simple system for the synthesis of galactosyl-xylose, a disaccharide of possible application to diagnostics. To maximize synthesis yields, we have studied the effect of several conditions: increase of acceptor concentration (0.05-2.7 M xylose), organic co-solvents (dimethylformamide, acetone) and reaction time.
Enzymatic synthesis of galactosyl�xylose by Aspergillus oryzae �-galactosidase
J Mol Catal B Enzym, 2002
In aqueous medium, the reaction catalyzed by Aspergillus oryzae -galactosidase with O-nitrophenyl--d-galactopyranoside (ONPG) in the presence of an acceptor leads to the synthesis of transglycosylation compounds in addition to the hydrolysis products (ONP and galactose). Our goal was to develop a simple system for the synthesis of galactosyl-xylose, a disaccharide of possible application to diagnostics. To maximize synthesis yields, we have studied the effect of several conditions: increase of acceptor concentration (0.05-2.7 M xylose), organic co-solvents (dimethylformamide, acetone) and reaction time. In the absence of co-solvents ONPG was completely consumed in 2 h; with 0.5 M xylose the maximum yield of galactosylxylose (16%) was attained at 60 min, while with 2.7 M xylose the yield reached 21%. Both co-solvents tested decreased the kinetics of ONPG convertion into products and 50% (v/v) dimethylformamide was deleterious to the synthesis. However, in 50% (v/v) acetone the synthesis yield was 12% and interestingly, the proportion of transglycosylation with respect to the reacted substrate was higher than in buffer. The synthesis of galactosyl-ethyleneglycol was also studied; it was achieved with extremely high yield and no detectable hydrolysis products. This proves that other acceptor alcohols can be preferred over water in some conditions.
Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool
Applied Microbiology and Biotechnology, 2014
Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-β-Dgalactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose β-1,4 linked to either N-acetylglucosamine or fucose, such as N -glycans derived from bovine fibrin and Caenorhabditis elegans . In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae , all enzymes also displayed various degrees of activity toward complex oligosaccharides containing β-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.
Journal of Biotechnology, 1999
Enzymatic synthesis of butylgalactoside via the transglycosylation reaction of lactose was carried out using b-galactosidase from Aspergillus oryzae. Reactions were performed in monophasic (butanol phase) and biphasic systems (emulsified aqueous in butanol phase). The results indicated that water content was an essential parameter of this reaction. In the case of the monophasic system, addition of water led to a significant increase of product concentration. The highest concentration was obtained at 17% (v/v) of water. Conversely, in the case of the biphasic system, increasing the aqueous/butanol phase ratio (v/v) provoked a decrease of the final butylgalactoside concentration. In this system, the reaction performance was lower than that in the monophasic system. Lactose favorably influenced the reaction synthesis. However, at high concentrations, it led to a drastic decrease of the product concentration.
Chemical and enzymatic synthesis of glycoconjugates 1. Enzymatic galactosylation of conduritol B
Tetrahedron Letters, 1995
&Galactosidmeactivitiesfromthe recombinanttbermophilicCLONEZYMEmglycosidase librarywers screenedat70"C for catalysisof atrarrsgalactosylation &omo-nitrophenyl$galactopyranosideto N-aeetylglucosamine.Threethermophilicglycosidases(GlyOO1-06, -07 and -09) were foundto producepredominantlythe~(1-4)-linkedisomer,Gal~(1-4)GlcNAcwith up to 610/0 yield and less than 10Yo of the hydrolysisside reactionproduct. Thus, commercialrecombinantthermophilic enzymelibrariesconstitutea novelclass of biocatalystsfor preparativeorganicsynthesis. 01997 Elsevier Science Ltd.
Bioorganic & Medicinal Chemistry Letters, 2005
The use of crude extract of the hepatopancreas of Aplysia fasciata, a large mollusc belonging to the order Anaspidea containing a b-galactosidase activity, was reported for the synthesis of different galactosides. Good yields with polar acceptors and the uncommon b-1-3 selectivity in the transgalactosylation reactions with most of the acceptors were observed. A b-1-2 selectivity in the hydrolytic conditions was also observed and discussed.
Synthesis of β-mercaptoethyl-glycosides by enzymatic reverse hydrolysis and transglycosylation
Biotechnology Letters, 1994
The synthetic potential of Almond eglucosidase and Jack bean a-mannosidase in the presence of high amounts of &mercaptoethanol as glycosyl acceptor for the synthesis of @mercaptoethyl-glycosides was studied. The regioselectivity, 0-glycosylation and/or Sglycosylation, and the stereoselectivity were analyzed with the reverse hydrolysis and the transglycosylation methods. With both enzymes, high yields of condensation are obtained without the use of chemical protective groups.