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

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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.

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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.

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  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

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  1. Gisela Weiz
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Correspondence to Javier D. Breccia.

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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

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