title: Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-L-rhamnosyl-β-D-glucosidase active on flavonoids.
abstract: Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-β-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-β-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.
authors.affiliation: INCITAP - CONICET (National Scientific and Technical Research Council. Department of Chemistry, Faculty of Natural Sciences, National University of La Pampa (UNLPam), Av. Uruguay 151, (6300), Santa Rosa, La Pampa, Argentina.
authors.forename: Bárbara D
authors.initials: BD
authors.lastname: Neher
authors.affiliation: INCITAP - CONICET (National Scientific and Technical Research Council. Department of Chemistry, Faculty of Natural Sciences, National University of La Pampa (UNLPam), Av. Uruguay 151, (6300), Santa Rosa, La Pampa, Argentina.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Mazzaferro
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Michael
authors.initials: M
authors.lastname: Kotik
authors.affiliation: INCITAP - CONICET (National Scientific and Technical Research Council. Department of Chemistry, Faculty of Natural Sciences, National University of La Pampa (UNLPam), Av. Uruguay 151, (6300), Santa Rosa, La Pampa, Argentina.
authors.forename: Jorge
authors.initials: J
authors.lastname: Oyhenart
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Halada
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
authors.affiliation: INCITAP - CONICET (National Scientific and Technical Research Council. Department of Chemistry, Faculty of Natural Sciences, National University of La Pampa (UNLPam), Av. Uruguay 151, (6300), Santa Rosa, La Pampa, Argentina. javierbreccia@gmail.com.
authors.forename: Javier D
authors.initials: JD
authors.lastname: Breccia
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D047188:Chalcones / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006569:Hesperidin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008848:Micromonosporaceae / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012210:Rhamnose / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D047188:Chalcones; D004187:Disaccharides; D005419:Flavonoids; D006027:Glycosides; D011994:Recombinant Proteins; C539209:rutinose; C033022:hesperidin methylchalcone; D006569:Hesperidin; D001617:beta-Glucosidase; D012210:Rhamnose
keywords: Inverting glycosidase; Methylumbelliferyl-β-D-rutinoside; α-L-rhamnosidase; β-D-glucosidase