title: Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4.
abstract: Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K(m)) than BxlB towards para-nitrophenyl β-D-xylopyranoside (pNPX) and β-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K(m) decreased with increasing β-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different β-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved β-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, β-D-Xylp-(1→4)-D-Lyxp, β-D-Xylp-(1→6)-D-Galp, β-D-Xylp-(1→4)-α-D-Glcp-(1→3)-β-D-Fruf, β-D-Xylp-(1→4)-α-D-Glcp-(1→2)-β-D-Fruf, and β-D-Xylp-(1→6)-β-D-Fruf-(2→1)-α-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 β-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined.
authors.affiliation: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Adiphol
authors.initials: A
authors.lastname: Dilokpimol
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Gotfredsen
authors.forename: Maaike
authors.initials: M
authors.lastname: Appeldoorn
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Baumann
authors.forename: Natsuko
authors.initials: N
authors.lastname: Nakai
authors.forename: Henk A
authors.initials: HA
authors.lastname: Schols
authors.forename: Maher Abou
authors.initials: MA
authors.lastname: Hachem
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001233:Aspergillus nidulans; D002384:Catalysis; D004187:Disaccharides / Q000138:chemical synthesis; D008968:Molecular Conformation; D015394:Molecular Structure; D008970:Molecular Weight; D009844:Oligosaccharides / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013237:Stereoisomerism; D014312:Trisaccharides / Q000138:chemical synthesis; D014994:Xylose / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D014995:Xylosidases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D004187:Disaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; D014994:Xylose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase