- α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) from Aspergillus niger: Characterization and synthetic potential of a novel diglycosidase
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We report the first heterologous production of a fungal rutinosidase (6-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α-L-rhamnosidase-catalyzed synthesis of a chromogenic substrate for a rutinosidase assay - para-nitrophenyl β-rutinoside - is described.
- imkov, Daniela,Kotik, Michael,Weignerov, Lenka,Halada, Petr,Pelantov, Helena,Adamcov, Kateina,Ken, Vladimr
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p. 107 - 117
(2015/01/30)
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- Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β- glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside
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Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-β-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na]+ ions correlated with the theoretical monoisotopic mass (calcd [M+Na]+: 507.44 m/z; obs. [M+Na]+: 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-β-glucosidases by zymographic analysis.
- Mazzaferro, Laura S.,Pi?uel, Lucrecia,Erra-Balsells, Rosa,Giudicessi, Silvana L.,Breccia, Javier D.
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scheme or table
p. 69 - 75
(2012/02/05)
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