Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria
- 374 Downloads
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.
KeywordsGlycoside 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.
- 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
- 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
- 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
- Mauludin R, Müller RH (2008) Hesperidin smart crystals: redispersibility and improved solubility properties. Pharmacogenetics/Pharmacogenomics Virtual J (http://www.aapsj.org) 10: S2
- 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
- Promega (2008) Protocols and application guide. Promega Corporation, Madison, WisconsinGoogle Scholar
- 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
- Seifert KA (1985) A monograph of Stilbella and some allied Hyphomycetes. Stud Mycol 27:1–235Google Scholar
- 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
- 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
- Yamamoto S, Okada M, Usui T, Sakata K, Toumoto A, Tsuruhami K (2006) Diglycosidase isolated from microorganisms. US Patent 7109014Google Scholar