Archives of Microbiology

, Volume 192, Issue 5, pp 383–393 | Cite as

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

  • Laura Mazzaferro
  • Lucrecia Piñuel
  • Marisol Minig
  • Javier D. Breccia
Original Paper


We screened for microorganisms able to use flavonoids as a carbon source; and one isolate, nominated Stilbella fimetaria SES201, was found to possess a disaccharide-specific hydrolase. It was a cell-bound ectoenzyme that was released to the medium during conidiogenesis. The enzyme was shown to cleave the flavonoid hesperidin (hesperetin 7-O-α-rhamnopyranosyl-β-glucopyranoside) into rutinose (α-rhamnopyranosyl-β-glucopyranose) and hesperetin. Since only intracellular traces of monoglycosidase activities (β-glucosidase, α-rhamnosidase) were produced, the disaccharidase α-rhamnosyl-β-glucosidase was the main system utilized by the microorganism for hesperidin hydrolysis. The enzyme was a glycoprotein with a molecular weight of 42224 Da and isoelectric point of 5.7. Even when maximum activity was found at 70°C, it was active at temperatures as low as 5°C, consistent with the psychrotolerant character of S. fimetaria. Substrate preference studies indicated that the enzyme exhibits high specificity toward 7-O-linked flavonoid β-rutinosides. It did not act on flavonoid 3-O-β-rutinoside and 7-O-β-neohesperidosides, neither monoglycosylated substrates. In an aqueous medium, the α-rhamnosyl-β-glucosidase was also able to transfer rutinose to other acceptors besides water, indicating its potential as biocatalyst for organic synthesis. The monoenzyme strategy of S. fimetaria SES201, as well as the enzyme substrate preference for 7-O-β-flavonoid rutinosides, is unique characteristics among the microbial flavonoid deglycosylation systems reported.


Glycoside hydrolase Diglycosidase Hesperidin α-Rhamnosyl-β-glucosidase 



This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Pampa (UNLPam) and Agencia Nacional de Promoción Científica y Técnica (ANPCyT) of Argentina. The authors gratefully thank the contributions of Eduardo Piontelli, Jorge Oyhenart and Alejandra Martínez in strain identification, Martin Hedström for mass spectrometry analysis and María Rita Martearena, Elsa Scaroni and Mirta Daz for the generous gift of flavonoids. Finally, we are indebted to Maria Andersson for helpful suggestions and critical reading of the manuscript.

Supplementary material

203_2010_567_MOESM1_ESM.pdf (40 kb)
Supplementary material 1 (PDF 40 kb)
203_2010_567_MOESM2_ESM.pdf (43 kb)
Supplementary material 2 (PDF 42 kb)
203_2010_567_MOESM3_ESM.pdf (33 kb)
Supplementary material 3 (PDF 32 kb)


  1. Ahn YO, Mizutani M, Saino H, Sakata K (2004) Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1. J Biol Chem 279:23405–23414PubMedCrossRefGoogle Scholar
  2. Barbagallo RN, Spagna G, Palmeri R, Restuccia C, Giudici P (2004) Selection, characterization and comparison of β-glucosidase from mould and yeasts employable for enological applications. Enz Microb Technol 35:58–66CrossRefGoogle Scholar
  3. Baumgertel A, Grimm R, Eisenbeiß W, Kreis W (2003) Purification and characterization of a flavonol 3-O-β-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench. Phytochem 64:411–418CrossRefGoogle Scholar
  4. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233–D238PubMedCrossRefGoogle Scholar
  5. Cassago A, Panepucci RA, Tortella Baião AM, Henrique-Silva F (2002) Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei. BMC Microbiol 2:14–18PubMedCrossRefGoogle Scholar
  6. Chuankhayan P, Hua Y, Svasti J, Sakdarat S, Sullivan PA, Ketudat Cairns JR (2005) Purification of an isoflavonoid 7-O-β-apiosyl-glucoside β-glycosidase and its substrates from Dalbergia nigrescens Kurz. Phytochem 66:1880–1889CrossRefGoogle Scholar
  7. Daiyasu H, Saino H, Tomoto H, Mizutani M, Sakata K, Toh H (2008) Computational and experimental analyses of furcatin hydrolase for substrate specificity studies of disaccharide-specific glycosidases. J Biochem 144:467–475PubMedCrossRefGoogle Scholar
  8. Genovés S, Gil JV, Vallés S, Casas JA, Manzanares P (2005) Assessment of the aromatic potential of palomino fino grape must using glycosidases. Am J Enol Vitic 56:188–191Google Scholar
  9. Gholipour Y, Nonami H, Erra-Balsells R (2008) In situ analysis of plant tissue underivatized carbohydrates and on-probe enzymatic degraded starch by UV-carbon nanotube-assisted laser desorption/ionization TOF mass spectrometry. Anal Biochem 383:159–167PubMedCrossRefGoogle Scholar
  10. Grimaldi A, McLean H, Jiranek V (2000) Identification and partial characterization of glycosidic activities of commercial strains of the lactic acid bacterium Oenococcus oeni. Am J Enol Viticul 51:362–369Google Scholar
  11. Hemingway KM, Alston MJ, Chappell CG, Taylor AJ (1999) Carbohydrate-flavour conjugates in wine. Carbohydr Polym 38:283–286CrossRefGoogle Scholar
  12. Henrissat B, Sulzenbacher G, Bourne Y (2008) Glycosyltransferases, glycoside hydrolases: surprise, surprise!. Curr Opin Struct Biol 18:527–533PubMedCrossRefGoogle Scholar
  13. Ibarz JM, Ferreira V, Hernández-Orte P, Loscos N, Cacho J (2006) Optimization and evaluation of a procedure for the gas chromatographic-mass spectrometric analysis of the aromas generated by fast acid hydrolysis of flavor precursors extracted from grapes. J Chromatogr A 1116:217–229CrossRefGoogle Scholar
  14. Jaworski A, Brückner H (2001) Sequences of polypeptide antibiotics stilboflavins, natural peptaibol libraries of the mold Stilbella flavipes. J Pept Scien 7:433–447CrossRefGoogle Scholar
  15. Kafanova TV, Busarova NG, Khudyakova YV, Isai SV (2008) Marine fungus Stilbella aciculosa as a potential producer of prostaglandins. Microbiology 77:451–454CrossRefGoogle Scholar
  16. Kato N, Suyama S, Shirokane M, Kato M, Kobayashi T, Tsukagoshi N (2002) Novel-glucosidase from Aspergillus nidulans with strong transglycosylation activity. Appl Environ Microbiol 68:1250–1256PubMedCrossRefGoogle Scholar
  17. Klose S, Tabatabai MA (2002) Response of glycosidases in soils to chloroform fumigation. Biol Fertil Soils 35:262–269CrossRefGoogle Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  19. Lai LB, Gopalan V, Glew RH (1992) Continuous spectrophotometric assays for β-glucosidases acting on the plant glucosides L-picein and prunasin. Anal Biochem 2:365–369CrossRefGoogle Scholar
  20. Lehr NA, Meffert A, Antelo L, Sterner O, Anke H, Weber RWS (2006) Antiamoebins, myrocin B and the basis of antifungal antibiosis in the coprophilous fungus Stilbella erythrocephala (syn. S. fimetaria). FEMS Microbiol Ecol 55:105–112PubMedCrossRefGoogle Scholar
  21. Ma SJ, Mizutani M, Hiratake J, Hayashi K, Yagi K, Watanabe N, Sakata K (2001) Substrates specificity of β-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing. Biosci Biotechnol Biochem 65:2719–2729PubMedCrossRefGoogle Scholar
  22. Manthey JA, Grohmann K (1996) Concentrations of hesperidin and other orange peel flavonoids in citrus processing byproducts. J Agric Food Chem 44:811–814CrossRefGoogle Scholar
  23. Manzanares P, van den Broeck HC, de Graaff LH, Visser J (2001) Purification and characterization of two different α-l-rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus. Appl Environ Microbiol 67:2230–2234PubMedCrossRefGoogle Scholar
  24. Martearena M, Daz M, Ellenrieder G (2007) Synthesis of rutinosides and rutinose by reverse hydrolysis catalyzed by fungal α-l-rhamnosidases. Biocatal Biotransf 26:177–185CrossRefGoogle Scholar
  25. Martínez MA, Delgado OD, Breccia JD, Baigorí MD, Siñeriz F (2002) Revision of the taxonomic position of the xylanolytic Bacillus sp. MIR32 reidentified as Bacillus halodurans and plasmid-mediated transformation of B. halodurans. Extremophiles 6:391–395PubMedCrossRefGoogle Scholar
  26. Mauludin R, Müller RH (2008) Hesperidin smart crystals: redispersibility and improved solubility properties. Pharmacogenetics/Pharmacogenomics Virtual J ( 10: S2
  27. Miller GL (1959) Use of dinitrosalisylic acid (DNS) for determination of reducing sugars. Anal Chem 31:426–428CrossRefGoogle Scholar
  28. Mizutani M, Nakanishi H, Ema J, Ma SJ, Noguchi E, Inohara-Ochiai M, Fukuchi-Mizutani M, Nakao M, Sakata K (2002) Cloning of β-primeverosidase from tea leaves, a key enzyme in tea aroma formation. Plant Physiol 130:2164–2176PubMedCrossRefGoogle Scholar
  29. Møller HJ, Poulsen HJ (1996) Staining of Glycoproteins/Proteoglycans on SDS-Gels. In: Walker JM (ed) The protein protocols handbook. Humana Press, New York, pp 627–631CrossRefGoogle Scholar
  30. Monti D, Pišvejcová A, Křen V, Lama M, Riva S (2004) Generation of an α-l-rhamnosidase library and its application for the selective derhamnosylation of natural products. Biotechnol Bioeng 87:763–771PubMedCrossRefGoogle Scholar
  31. Monti D, Candido A, Cruz Silva MM, Křen V, Riva S, Danieli B (2005) Biocatalyzed generation of molecular diversity: selective modification of the saponin asiaticoside. Adv Synth Catal 347:1168–1174CrossRefGoogle Scholar
  32. Nakanishi F, Nagasawa Y, Kabaya Y, Sekimoto H, Shimomura K (2005) Characterization of lucidin formation in Rubia tinctorum L. Plant Physiol Biochem 43:921–928PubMedCrossRefGoogle Scholar
  33. Narikawa T, Shinoyama H, Fujii T (2000) A β-rutinosidase from Penicillium rugulosum IFO 7242 that is a peculiar flavonoid glycosidase. Biosci Biotechnol Biochem 64:1317–1319PubMedCrossRefGoogle Scholar
  34. Nonami H, Fukui S, Erra-Balsells R (1997) β-Carboline alkaloids as matrices for matrix-assisted ultraviolet laser desorption time-of flight mass spectrometry of proteins and sulfated oligosaccharides: a comparative study using phenylcarbonyl compounds, carbazoles and classical matrices. J Mass Spectrom 32:287–296CrossRefGoogle Scholar
  35. Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363PubMedCrossRefGoogle Scholar
  36. Orrillo AG, Ledesma P, Delgado OD, Spagna G, Breccia JD (2007) Cold-active α-l-rhamnosidase from psychrotolerant bacteria isolated from a sub-Antarctic ecosystem. Enz Microb Technol 40:236–241CrossRefGoogle Scholar
  37. Polaina J, MacCabe AP (2007) Industrial enzymes. Function and applications. Springer, NetherlandsCrossRefGoogle Scholar
  38. Promega (2008) Protocols and application guide. Promega Corporation, Madison, WisconsinGoogle Scholar
  39. Puder C, Wagner K, Vettermann R, Hauptmann R, Potterat O (2005) Terphenylquinone inhibitors of the Src protein tyrosine kinase from Stilbella fimetaria. J Nat Produc 68:323–326CrossRefGoogle Scholar
  40. Russell NJ (2000) Toward a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles 4:83–90PubMedCrossRefGoogle Scholar
  41. Sang-Joon L, Omori T, Kodama T (1990) Purification and some properties of rutinosidase from Arthrobacter sp. Kor J Appl Microbiol Biotech 18:360–367Google Scholar
  42. Sarry JE, Gunata Z (2004) Plant and microbial glycoside hydrolases: volatile release from glycosidic aroma precursors. Food Chem 87:509–521CrossRefGoogle Scholar
  43. Schimel JP, Fahnestock J, Michaelson G, Mikan C, Ping C-L, Romanovsky VE, Welker J (2006) Cold-season production of CO2 in Arctic soils: can laboratory and field estimates be reconciled through a simple modeling approach? Arct Antarc Alp Res 38:249–256CrossRefGoogle Scholar
  44. Seifert KA (1985) A monograph of Stilbella and some allied Hyphomycetes. Stud Mycol 27:1–235Google Scholar
  45. Shaw LJ, Morris P, Hooker JE (2006) Perception and modification of plant flavonoid signals by rhizosphere microorganisms. Environ Microbiol 8:1867–1880PubMedCrossRefGoogle Scholar
  46. Slapeta J, Moreira D, López-García P (2005) The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes. Proc R Soc B 272:2073–2081PubMedCrossRefGoogle Scholar
  47. Spagna G, Barbagallo RN, Greco E, Manenti I, Pifferi P (2002) A mixture of purified glycosidases from Aspergillus niger for oenological application immobilized by inclusion in chitosan gels. Enz Microb Technol 30:80–89CrossRefGoogle Scholar
  48. Tsuruhami K, Mori S, Amarume S, Sarawatari S, Murata T, Hirakake J, Sakata K, Usui T (2006) Isolation and characterization of a β-primeverosidase-like enzyme from Penicillium multicolor. Biosci Biotechnol Biochem 70:691–698PubMedCrossRefGoogle Scholar
  49. Wang D, Kurasawa E, Yamaguchi Y, Kubota K, Kobayashi A (2001) Analysis of glycosidically bound aroma precursors in tea leaves. 2. Changes in glycoside contents and glycosidase activities in tea leaves during the black tea manufacturing process. J Agric Food Chem 49:1900–1903PubMedCrossRefGoogle Scholar
  50. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR protocols: a guide to methods and applications. Academic Press, Orlando, pp 315–322Google Scholar
  51. Williams PJ, Strauss CR, Wilson B, Massy-Westropp RA (1982) Use of C18 reversed-phase liquid chromatography for the isolation of monoterpene glycosides and nor-isoprenoid precursors from grape juice and wines. J Chromatogr 235:471–480CrossRefGoogle Scholar
  52. Yamamoto S, Okada M, Usui T, Sakata K (2002) Isolation and characterization of a β-primeverosidase-like endo-manner β-glycosidase from Aspergillus fumigatus AP-20. Biosci Biotechnol Biochem 66:801–807PubMedCrossRefGoogle Scholar
  53. Yamamoto S, Okada M, Usui T, Sakata K, Toumoto A, Tsuruhami K (2006) Diglycosidase isolated from microorganisms. US Patent 7109014Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Laura Mazzaferro
    • 1
  • Lucrecia Piñuel
    • 1
  • Marisol Minig
    • 1
  • Javier D. Breccia
    • 1
  1. 1.CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), Departamento de Química, Facultad de Ciencias Exactas y NaturalesUniversidad Nacional de La Pampa (UNLPam)Santa Rosa, La PampaArgentina

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