A simple method for purifying glycosidases: α-l-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine

Spagna, G.; Barbagallo, Riccardo N.; Martino, Ascanio; Pifferi, Pier Giorgio

doi:10.1016/s0141-0229(00)00236-2

Cited by 79 publications

(68 citation statements)

References 39 publications

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“…Several grapevine fungal pathogens, such as Aspergillus and Botrytis, produce large amounts of glycosidase activities that also have high levels of specificity for purified wine glycosides (177). Accordingly, Aspergillus is a common source of commercial enzyme preparations that have glycosidic activities; however, these preparations are often impure, requiring resolution before characterization in the laboratory (258,259,261), and they have undesirable effects on the wine (1, 115, 296). More importantly, the enzymes of fungi are frequently ineffective in 5720 MINIREVIEW APPL.…”

Section: Glycosidasesmentioning

confidence: 99%

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Matthews

Grimaldi

Walker

et al. 2004

Appl Environ Microbiol

158

101

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Section: Glycosidasesmentioning

confidence: 99%

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Matthews

Grimaldi

Walker

et al. 2004

Appl Environ Microbiol

158

101

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“…The combination of ␤-D-glucosidase and ␣-L-rhamnosidase allows the sequential release of volatile compounds from these precursors (18). Several authors (9,32,46) have demonstrated the enological value of fungal (Aspergillus sp.) rhamnosidases.…”

mentioning

confidence: 99%

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

Michlmayr

Brandes

Eder

et al. 2011

Appl Environ Microbiol

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␣-L-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ram and ram2) from Pediococcus acidilactici DSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial ␤-glucosidase, both enzymes released the monoterpenes linalool and cis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/ nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substrate p-nitrophenyl-␣-L-rhamnopyranoside (V max ‫؍‬ 243 U mg ؊1 ). In contrast, Ram2 displayed considerable specificity toward hesperidin (V max ‫؍‬ 34 U mg ؊1 ) and, especially, rutinose (V max ‫؍‬ 1,200 U mg ؊1 ), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.␣-L-Rhamnosidases (EC 3.2.1.40) catalyze the hydrolysis of a wide spectrum of natural glycosides containing terminal Lrhamnose. The biotechnological interest in rhamnosidases includes the debittering of citrus fruit juices (naringinase and hesperidinase), the release of flavonoids from rhamnosylated precursors, and the production of L-rhamnose as starting material for chemical synthesis (52). Further, rhamnosidases may play a crucial role in wine making due to their contributions to releasing grape-derived aroma compounds (mainly terpenes and terpenic alcohols). Depending on the grape variety, rutinosides (␣-L-rhamnopyranosyl-␤-D-glucopyranoside) constitute 6 to 13% of the terpenic glycosides found in wine (29). The combination of ␤-D-glucosidase and ␣-L-rhamnosidase allows the sequential release of volatile compounds from these precursors (18). Several authors (9, 32, 46) have demonstrated the enological value of fungal (Aspergillus sp.) rhamnosidases. Based on their adaptation to the harsh wine milieu and their general hydrolytic abilities toward different glycosides (16,17,50), wine-related lactic acid bacteria (LAB) have attracted much interest as a potential source of novel glycosidases (34).Consequently, several (intracellular) glycosid...

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“…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).…”

mentioning

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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A simple method for purifying glycosidases: α-l-rhamnopyranosidase from Aspergillus niger to increase the aroma of Moscato wine

Cited by 79 publications

References 39 publications

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

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

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