Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria

Mazzaferro, Laura S.; Piñuel, Lucrecia; Minig, Marisol; Breccia, Javier D.

doi:10.1007/s00203-010-0567-7

Cited by 43 publications

(61 citation statements)

References 49 publications

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“…This implies that this enzyme, as well as several other enzymes reported, was highly stabilized by multipoint covalent immobilization with a half-life approximately three times higher in comparison with the soluble protein [Mateo et al, 2006]. Although knowledge of the primary and secondary structure of αRβGl is limited, the stabilization achieved by immobilization on Ag-G would suggest an important number of lysine residues fairly exposed in the protein surface and ready to react with the glyoxyl groups of the support [Mazzaferro et al, 2010]. The immobilization procedure onto Ag-G can be optimized by adjusting different variables, such as the time under alkaline conditions and the density of reactive groups on the support [Mateo et al, 2006].…”

Section: Stability Of αRβgl Preparationsmentioning

confidence: 97%

“…1 ; table 2 ). The biotransformation was carried out in the presence of different concentrations of DMSO to overcome the low solubility of hesperidin in water (0.32 m M ) [Mazzaferro et al, 2010]. To increase the soluble substrate concentration, 50% v/v DMSO was used, dissolving up to 5 m M of hesperidin.…”

Section: Engineering the Reaction Media To Improve Hesperetin Yieldmentioning

confidence: 99%

“…DSM24697 was cultured using hesperidin as the carbon source and the enzyme was purified following the procedure previously described by Mazzaferro et al [2010].…”

Section: Strain Growth Conditions and Protein Purificationmentioning

confidence: 99%

“…This enzyme, α-rhamnosyl-β-glucosidase (αRβGl; EC 3.2.1.168), is a distinctive endoglycosidase specific for 7-O -rutinosilate flavonoids ( fig. 1 ) [Mazzaferro et al, 2010]. It also enables the transglycosylation of rutinosyl units using the flavonoids hesperidin or hesperidin methyl chalcone as rutinose donors, which are abundant and inexpensive by-products of the citrus industry .…”

Section: Introductionmentioning

confidence: 99%

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Production of Hesperetin Using a Covalently Multipoint Immobilized Diglycosidase from Acremonium sp. DSM24697

et al. 2013

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The diglycosidase α-rhamnosyl-β-glucosidase (EC 3.2.1.168) from the fungus Acremonium sp. DSM24697 was immobilized on several agarose-based supports. Covalent multipoint immobilization onto glyoxyl-activated agarose was selected as the more stable preparation at high concentration of dimethyl sulfoxide (DMSO) and high temperature. The optimal conditions for the immobilization process involved an incubation of the enzyme with agarose beads containing 220 μmol of glyoxyl groups per gram at pH 10 and 25°C for 24 h. The hydrolysis of hesperidin carried out in 10% v/v DMSO at 60°C for 2 h reached 64.6% substrate conversion and a specific productivity of 2.40 mmol h^-1 g^-1. Under these conditions, the process was performed reutilizing the catalyst for up to 18 cycles, maintaining >80% of the initial activity and a constant productivity 2.96 ± 0.42 µmol^-1 h^-1 g^-1. To the best of our knowledge, such productivity is the highest achieved for hesperetin production through an enzymatic approach.

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Section: Stability Of αRβgl Preparationsmentioning

confidence: 97%

Section: Engineering the Reaction Media To Improve Hesperetin Yieldmentioning

confidence: 99%

“…DSM24697 was cultured using hesperidin as the carbon source and the enzyme was purified following the procedure previously described by Mazzaferro et al [2010].…”

Section: Strain Growth Conditions and Protein Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production of Hesperetin Using a Covalently Multipoint Immobilized Diglycosidase from Acremonium sp. DSM24697

et al. 2013

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“…-L-Rhamnosidase (EC 3.2.1.40) cleaves terminal ␣-L-rhamnose from a large number of natural products, such as flavonoids, saponins, and many other natural glycosides, and this enzyme has been used in the food industry for eliminating the naringin bitterness of citrus juices (4,20,25,26), removing hesperidin crystals from orange juices and releasing 7-O-␤-D-glucoside-hesperetin, an important precursor in sweetener production (17,24), and enhancing wine aromas (7,6,22). In addition, the deglycosylation of flavonoids in vitro by ␣-L-rhamnosidase has ushered in a new era of drug development (5,8,19,27).…”

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confidence: 99%

Aspergillus niger DLFCC-90 Rhamnoside Hydrolase, a New Type of Flavonoid Glycoside Hydrolase

Liu

Zhang

et al. 2012

Appl Environ Microbiol

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A novel rutin-␣-L-rhamnosidase hydrolyzing ␣-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the ␣-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13. ␣ -L-Rhamnosidase (EC 3.2.1.40) cleaves terminal ␣-L-rhamnose from a large number of natural products, such as flavonoids, saponins, and many other natural glycosides, and this enzyme has been used in the food industry for eliminating the naringin bitterness of citrus juices (4,20,25,26), removing hesperidin crystals from orange juices and releasing 7-O-␤-D-glucoside-hesperetin, an important precursor in sweetener production (17,24), and enhancing wine aromas (7,6,22). In addition, the deglycosylation of flavonoids in vitro by ␣-L-rhamnosidase has ushered in a new era of drug development (5,8,19,27). Hitherto, ␣-L-rhamnosidases from fungi, bacteria, and animals have been purified and characterized (29), and several genes encoding ␣-Lrhamnosidases have also been cloned (1, 2, 3, 9, 16). Thus far, the ␣-L-rhamnosidases are categorized into four glycoside-hydrolase (GH) families, 28, 78, 106, and NC (nonclassified), in the CAZy (carbohydrate-active enzymes) database based on amino acid sequence similarities (10, 11).In this paper, a novel rutin-␣-L-rhamnosidase was purified and characterized from Aspergillus niger DLFCC-90, and the gene (rhaR) encoding this enzyme was also cloned and expressed in Pichia pastoris GS115.The strain A. niger DLFCC-90, obtained from the Culture Collection of Biotechnology Engineering of Dalian Polytechnic University (Dalian, China), was cultured with shaking at 28 to 30°C for 72 to 96 h in a wort medium of 5.0 Baume degrees containing 2% extract from flowers of Sophora japonica (Huai Hua in Chinese). To obtain pure enzyme, the cell-free culture was treated by a 3-step method, i.e., ammonium sulfate precipitation (75% saturation), a Sephadex G-75 column (Amersham Pharmacia) eluted with 0.02 M acetate buffer (pH 5.0), and a DEAE 52-cellulose column (Amersham Pharmacia) eluted with a linear gradient of KCl (0.0 to 0.5 M) in 0.02 M acetate buffer (pH 5.0). The determination of the protein concentration was performed as described in reference 13. The enzymatic activity was measured using 2.0 mg/ml rutin (Sigma) in 0.02 M acetate buffer (pH 5.0) as a substrate after a reaction at 50°C for 18 h, and products of rhamnose and isoquercitrin from the enzyme reaction were detected to calculate the enzyme activity (18, 28). After the above-described 3 steps of purification, the enzyme's specific activity was increased 5.3-fold. The purified enzyme migrated as a single band on the 12% SDS-PAGE gel (12) with an apparent molecular mass of about 66 kDa (Fig. 1A), which was similar to those for the previously reported ␣-L-rhamnosidase from Curvularia lunata (6) and naringinase from Aspergillus sojae (5). The 3-step-purified enzyme gave one major p...

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α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

et al. 2014

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Abstract:We report the first heterologous production of a fungal rutinosidase (6-O-a-l-rhamnopyranosyl-b-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 a-l-rhamnosidase-catalyzed synthesis of a chromogenic substrate for a rutinosidase assay -para-nitrophenyl b-rutinoside -is described.

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Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria

Cited by 43 publications

References 49 publications

Production of Hesperetin Using a Covalently Multipoint Immobilized Diglycosidase from <b><i>Acremonium</i></b> sp. DSM24697

Production of Hesperetin Using a Covalently Multipoint Immobilized Diglycosidase from <b><i>Acremonium</i></b> sp. DSM24697

Aspergillus niger DLFCC-90 Rhamnoside Hydrolase, a New Type of Flavonoid Glycoside Hydrolase

α‐L‐Rhamnosyl‐β‐D‐glucosidase (Rutinosidase) from Aspergillus niger: Characterization and Synthetic Potential of a Novel Diglycosidase

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