Skip to main content
SpringerLink
Log in

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Diglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-β-glucosidase I and II (αRβG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-β-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRβG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRβG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRβG I. On the other hand, αRβG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRβG I showed exclusive specificity toward 7-O-β-rutinosyl flavonoids, whereas αRβG II hydrolyzed both 7-O-β-rutinosyl- and 3-O-β-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-β-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRβG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Aspeborg H, Coutinho PM, Wang Y, Brumer H, Henrissat B (2012) Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 12:186

    Article  CAS  Google Scholar 

  • Bassanini I, Krejzová J, Panzeri W, Křen V, Monti D, Riva S (2017) A sustainable one-pot two-enzymes synthesis of naturally occurring arylalkyl glucosides. ChemSusChem 10:2040–2045

    Article  CAS  Google Scholar 

  • Bojarová P, Křen V (2009) Glycosidases: a key to tailored carbohydrates. Trends Biotechnol 27:199–209

    Article  Google Scholar 

  • Cutfield SM, Davies GJ, Murshudov G, Anderson BF, Moody PCE, Sullivan PA, Cutfield JF (1999) The structure of the exo-β-(1,3)-glucanase from Candida albicans in native and bound forms: relationship between a pocket and groove in family 5 glycosyl hydrolases. J Mol Biol 294:771–783

    Article  CAS  Google Scholar 

  • Fries A, Mazzaferro LS, Grüning B, Bisel P, Stibal K, Buchholz PCF, Pleiss J, Sprenger GA, Müller M (2019) Alteration of the route to menaquinone towards isochorismate-derived metabolites. ChemBioChem. https://doi.org/10.1002/cbic.201900050

    Article  CAS  Google Scholar 

  • Gerlt JA (2017) Genomic enzymology: web tools for leveraging protein family sequence-function space and genome context to discover novel functions. Biochem 56:4293–4308

    Article  CAS  Google Scholar 

  • Goudot A, Pourceau G, Meyer A, Gehin T, Vidal S, Vasseur JJ, Morvan F, Souteyrand E, Chevolot Y (2013) Quantitative analysis (Kd and IC50) of glycoconjugates interactions with a bacterial lectin on a carbohydrate microarray with DNA Direct Immobilization (DDI). Biosens Bioelectron 40:153–160

    Article  CAS  Google Scholar 

  • Gudmundsson M, Hansson H, Karkehabadi S, Larsson A, Stals I, Kim S, Sunux S, Fujdala M, Larenas E, Kaper T, Sandgren M (2016) Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii. Acta Cryst 72:860–870

    CAS  Google Scholar 

  • Ishikawa M, Kawasaki M, Shiono Y, Koseki T (2018) A novel Aspergillus oryzae diglycosidase that hydrolyzes 6-O-α-L-rhamnosyl-β-D-glucoside from flavonoids. Appl Microbiol Biotechnol 102:3193–3201

    Article  CAS  Google Scholar 

  • Katayama S, Ohno F, Yamauchi Y, Kato M, Makabe H, Nakamura S (2013) Enzymatic synthesis of novel phenol acid rutinosides using rutinase and their antiviral activity in vitro. J Agric Food Chem 61:6917–6962

    Article  Google Scholar 

  • Karkehabadi S, Helmich KE, Kaper T, Hansson H, Mikkelsen N, Gudmundsson M, Piens G, Fujdala M, Banerjee G, Scott-Craig J, Walton JD, Phillips GN, Sandgren M (2014) Biochemical characterization and crystal structures of a fungal family 3 glucosidase, Cel3A from Hypocrea jecorina. J Biol Chem 289:31624–31637

    Article  CAS  Google Scholar 

  • Kato Y, Matsushita J, Kubodera T, Matsuda K (1985) A novel enzyme producing isoprimeverose from oligoxyloglucans of Aspergillus oryzae. J Biochem 97:801–810

    Article  CAS  Google Scholar 

  • Křen V, Martínková L (2001) Glycosides in medicine: “The role of glycosidic residue in biological activity”. Curr Med Chem 8:1303–1328

    Article  Google Scholar 

  • Křen V, Řezanka T (2008) Sweet antibiotics – the role of glycosidic residues in antibiotic and antitumor activity and their randomization. FEMS Microbiol Rev 32:858–889

    Article  Google Scholar 

  • Ma SJ, Mizutani M, Hiratake J, Hayashi K, Yagi K, Watanabe N, Sakata K (2001) Substrate specificity of β-primeverosidase, a key enzyme in aroma formation during oolong tea and black tea manufacturing. Biosci Biotechnol Biochem 65:2719–2729

    Article  CAS  Google Scholar 

  • Matsuzawa T, Mitsuishi Y, Kameyama A, Yaoi K (2016) Identification of the gene encoding isoprimeverose-producing oligoxyloglucan hy- drolase in Aspergillus oryzae. J Biol Chem 291:5080–5087

    Article  CAS  Google Scholar 

  • Mazzaferro L, Breccia JD (2011) Functional and biotechnological insights into diglycosidases. Biocat Biotransf 29:103–112

    Article  CAS  Google Scholar 

  • Mazzaferro LS,  Weiz G,  Braun L,  Kotik M,  Pelantová H,  Křen V,  Breccia JD (2019) Enzyme-mediated transglycosylation of rutinose (6- -α- -rhamnosyl- -glucose) to phenolic compounds by a diglycosidase from sp. DSM 24697 . Biotech Appl Biochem 66 :53–59

  • Mazzaferro L, Piñuel L, Minig M, Breccia JD (2010) Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria. Arch Microbiol 192:383–393 Erratum in: Arch Microbiol 2011 193:461

    Article  CAS  Google Scholar 

  • Mazzaferro LS, Piñuel L, Erra-Balsells R, Giudicessi SL, Breccia JD (2012) 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 347:69–75

    Article  CAS  Google Scholar 

  • Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Minig M,  Mazzaferro LS,  Erra-Balsells R,  Petroselli G,  Breccia JD (2011) α-Rhamnosyl-β-glucosidase-Catalyzed Reactions for Analysis and Biotransformations of Plant-Based Foods. J Agric Food Chem 59 :11238–11243

    Article  CAS  Google Scholar 

  • 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 130:2164–2176

    Article  CAS  Google Scholar 

  • Neher BD, Mazzaferro LS, Kotik M, Oyhenart J, Halada P, Křen V, Breccia JD (2016) Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-β-D-glucosidase active on flavonoids. Appl Microbiol Biotechnol 100:3061–3070

    Article  CAS  Google Scholar 

  • Patrick WM, Nakatani Y, Cutfield SM, Sharpe ML, Ramsay RJ, Cutfield JF (2010) Carbohydrate binding sites in Candida albicans exo-β-1,3-glucanase and the role of the Phe-Phe ‘clamp’ at the active site entrance. FEBS J 277:4549–4561

    Article  CAS  Google Scholar 

  • Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786

    Article  CAS  Google Scholar 

  • Peterson JJ, Beecher GR, Bhagwat SA, Dwyer JT, Gebhardt SE, Haytowitz DB, Holden JM (2006) Flavanones in grapefruit, lemons, and limes: a compilation and review of the data from the analytical literature. J Food Compos Anal 19:S74–S80

    Article  CAS  Google Scholar 

  • Pozzo T, Pasten JL, Karlsson EN, Logan DT (2010) Structural and functional analyses of glucosidase 3B from Thermotoga neapolitana: A thermostable three-domain representative of glycoside hydrolase 3. J Mol Biol 397:724–739

    Article  CAS  Google Scholar 

  • Robinson MA, Charlton ST, Garnier P, Wang X, Davis SS, Perkins AC, Frier M, Duncan R, Savage TJ, Wyatt DA, Watson SA, Davis BG (2004) LEAPT: lectin-directed enzyme-activated prodrug therapy. Proc Natl Acad Sci U S A 101:14527–14532

    Article  CAS  Google Scholar 

  • Šimčíková D, Kotik M, Weignerová L, Halada P, Pelantová H, Adamcová K, Křen V (2015) α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) from Aspergillus niger: characterization and synthetic potential of a novel diglycosidase. Adv Synth Catal 356:1–12

    Google Scholar 

  • Stanke M, Morgenstern B (2005) AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res 33(suppl 2):W465–W467

    Article  CAS  Google Scholar 

  • Suzuki K, Sumitani JI, Nam YW, Nishimaki T, Tani S, Wakagi T, Kawaguchi T, Fushinobu S (2013) Crystal structures of glycoside hydrolase family 3 glucosidase 1 from Aspergillus aculeatus. Biochem J 452:211–221

    Article  CAS  Google Scholar 

  • Takahashi M, Konishi T, Takeda T (2011) Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis. Appl Microbiol Biotechnol 91:1073–1082

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  Google Scholar 

  • Tsuruhami K, Mori S, Amarume S, Saruwatari 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 70:691–698

    Article  CAS  Google Scholar 

  • Tsuruhami K, Mori S, Koide Y (2011) Diglycosidase and gene encoding the same. Patent US 7998721 B2

  • Weiz G,  Breccia JD,  Mazzaferro LS (2017) Screening and quantification of the enzymatic deglycosylation of the plant flavonoid rutin by UV–visible spectrometry. Food Chem 229:44–49

    Article  CAS  Google Scholar 

  • 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 66:801–807

    Article  CAS  Google Scholar 

  • Yoshida E, Hidaka M, Fushinobu S, Koyanagi T, Minami H, Tamaki H, Kitaoka M, Katayama T, Kumagai H (2010) Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 glucosidase from Kluyveromyces marxianus. Biochem J 431:39–49

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the National University of La Pampa (UNLPam), the National Council of Scientific and Technical Research (CONICET), and The National Agency for Science and Technology Promotion (ANPCyT) of Argentina. The return fellowship to L.S.M. (grant number 1147746) from the Alexander von Humboldt Foundation is sincerely acknowledged. We acknowledge support from the Czech Science Foundation (grant no. 19-00091S). Funding of the bilateral cooperation project 7AMB13AR005 by the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic and the Ministry of Science, Technology and Innovation (MINCYT) of Argentina is acknowledged.

Author information

Author notes

  1. Laura S. Mazzaferro and Michael Kotik contributed equally to this work.

Authors and Affiliations

  1. Facultad de Ciencias Exactas y Naturales, Instituto de Ciencias de la Tierra y Ambientales de La Pampa (INCITAP), Universidad Nacional de La Pampa – Consejo Nacional de Investigaciones Científicas y Técnicas (UNLPam-CONICET), Av. Uruguay 151, 6300, Santa Rosa, La Pampa, Argentina

    Gisela Weiz, Laura S. Mazzaferro, Bárbara D. Neher & Javier D. Breccia

  2. Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20, Prague, Czech Republic

    Michael Kotik & Vladimír Křen

  3. Laboratory of Molecular Structure Characterization, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20, Prague, Czech Republic

    Petr Halada

Authors
  1. Gisela Weiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laura S. Mazzaferro
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Kotik
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bárbara D. Neher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Petr Halada
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vladimír Křen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Javier D. Breccia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Javier D. Breccia.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 941 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Weiz, G., Mazzaferro, L.S., Kotik, M. et al. The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities. Appl Microbiol Biotechnol 103, 9493–9504 (2019). https://doi.org/10.1007/s00253-019-10180-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-019-10180-y

Keywords

Search

Navigation