The diglycosidase α-rhamnosyl-β-glucosidase (EC 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.

Arakawa T, Kita Y, Timasheff SN: Protein precipitation and denaturation by dimethyl sulfoxide. Biophys Chem 2007;131:62-70.
Benavente-García O, Castillo J, Marin FR, Ortuño A, Del Río JA: Uses and properties of citrus flavonoids. J Agric Food Chem 1997;45:4505-4515.
Chang HY, Lee YB, Bae HA, Huh IY, Nam SH, Sohn HS, Lee HJ, Lee SB: Purification and characterization of Aspergillus sojae naringinase: the production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG-CoA reductase. J Agric Food Chem 2011;124:234-241.
Di Majo D, Giammanco M, La Guardia M, Tripoli E, Giammanco S, Finotti E: Flavanones in citrus fruit: structure-antioxidant activity relationships. Food Res Int 2005;38:1161-1166.
Ellenrieder G, Blanco S, Daz M: Hydrolysis of supersaturated naringin solutions by free and immobilized naringinase. Biotechnol Tech 1998;12:63-65.
Fernandez-Lafuente R, Rosell CM, Rodriguez V, Santana C, Soler G, Bastida A, Guisan JM: Preparation of activated supports containing low pK amino groups: a new tool for protein immobilization via the carboxyl coupling method. Enzyme Microb Technol 1993;15:546-550.
Genovés S, Gil JV, Vallés S, Casas JA, Manzanares P: Assessment of the aromatic potential of palomino fino grape must using glycosidases. Am J Enol Viticult 2005;56:188-191.
Guisán J: Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb Technol 1988;10:375-382.
Hemingway KM, Alston MJ, Chappell CG, Taylor AJ: Carbohydrate-flavour conjugates in wine. Carbohydr Polym 1999;38:283-286.
Henley JP, Sadana A: Deactivation theory. Biotechnol Bioeng 1986;28:1277-1285.
Mandalari G, Bennett RN, Kirby AR, Lo Curto RB, Bisignano G, Waldron KW, Faulds CB: Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from Bergamot (Citrus bergamia Risso) peel. J Agric Food Chem 2006;54:8307-8313.
Manthey JA, Grohmann K: Concentrations of hesperidin and other orange peel flavonoids in citrus processing byproducts. J Agric Food Chem 1996;44:811-814.
Mateo C, Abian O, Bernedo M, Cuenca E, Fuentes M, Fernandez-Lorente G, Palomo JM, Grazu V, Pessela BCC, Giacomini C, Irazoqui G, Villarino A, Ovsejevi K, Batista-Viera F, Fernandez-Lafuente R, Guisan JM: Some special features of glyoxyl supports to immobilize proteins. Enzyme Microb Technol 2005;37:456-462.
Mateo C, Bolivar JM, Godoy CA, Rocha-Martin J, Pessela BC, Curiel JA, Muñoz R, Guisan JM, Fernandez-Lorente G: Improvement of enzyme properties with a two-step immobilizaton process on novel heterofunctional supports. Biomacromolecules 2010;11:3112-3117.
Mateo C, Palomo JM, Fuentes M, Betancor L, Grazu V, López-Gallego F, et al: Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb Technol 2006;39:274-280.
Mazzaferro LS, Breccia JD: Functional and biotechnological insights into diglycosidases. Biocatalysis Biotransform 2011;29:103-112.
Mazzaferro LS, Breccia JD: Quantification of hesperidin in citrus-based foods using a fungal diglycosidase. Food Chem 2012;134:2338-2344.
Mazzaferro LS, Piñuel L, Erra-Balsells R, Giudicessi SL, Breccia JD: Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside. Carbohydr Res 2012;347:69-75.
Mazzaferro LS, Piñuel L, Minig M, Breccia JD: Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria. Arch Microbiol 2010;192:383-393, erratum in Arch Microbiol 2011;193:461.
Nam HK, Hong SH, Shin KC, Oh DK: Quercetin production from rutin by a thermostable β-rutinosidase from Pyrococcus furiosus. Biotechnol Lett 2012;34:483-489.
Orrillo AG, Ledesma P, Delgado OD, Spagna G, Breccia JD: Cold-active α-L-rhamnosidase from psychrotolerant bacteria isolated from a sub-Antarctic ecosystem. Enzyme Microb Technol 2007;40:236-241.
Pedroche J, Yust MM, Mateo C, Fernández-Lafuente R, Girón-Calle J, Alaiz M, Vioque J, Guisán JM, Millán F. Effect of the support and experimental conditions in the intensity of the multipoint covalent attachment of proteins on glyoxyl-agarose supports: correlation between enzyme-support linkages and thermal stability. Enzyme Microb Technol 2007;40:1160-1166.
Piñuel L, Mazzaferro LS, Breccia JD: Operational stabilization of fungal α-rhamnosyl-β-glucosidase by immobilization on chitosan composites. Process Biochem 2011;46:2330-2335.
Reshmi R, Sugunan S: Improved biochemical characteristics of cross-linked β-glucosidase on nanoporous silica foams. J Mol Catal B: Enzym 2012;85-86:111-118.
Ribeiro MH: Naringinases: occurrence, characteristics, and applications. Appl Microbiol Biotechnol 2011;90:1883-1895.
Scaroni E, Cuevas C, Carrillo L, Ellenrieder G: Hydrolytic properties of crude α-L-rhamnosidases produced by several wild strains of mesophilic fungi. Lett Appl Microbiol 2002;34:461-465.
Sheldon RA: Enzyme immobilization: the quest for optimum performance. Adv Synth Catal 2007;349:1289-1307.
Spagna G, Barbagallo RN, Greco E, Manenti I, Pifferi PG: A mixture of purified glycosidases from Aspergillus niger for oenological application immobilized by inclusion in chitosan gels. Enzyme Microb Technol 2002;30:80-89.
Wang D, Kurasawa E, Yamaguchi Y, Kubota K, Kobayashi A: Analysis of glycosidically bound aroma precursors in tea leaves. II. Changes in glycoside contents and glycosidase activities in tea leaves during the black tea manufacturing process. J Agric Food Chem 2001;49:1900-1903.
Yoon JH, McKenzie D: A comparison of the activities of three β-galactosidases in aqueous-organic solvent mixtures. Enzyme Microb Technol 2005;36:439-446.
You do not currently have access to this content.