title: Alpha-L-fucosidase isoenzyme iso2 from Paenibacillus thiaminolyticus.
abstract: BACKGROUND
  α-L-Fucosidases are enzymes involved in metabolism of α-L-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which α-L-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed.
  
  
  METHODS
  Activity-based screening of a genomic library was used to isolate the gene encoding a novel α-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged α-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation.
  
  
  RESULTS
  Using a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5α cells, nucleotide sequence of a new α-L-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of α-L-fucosidase iso2 were tested using different acceptor molecules.
  
  
  CONCLUSIONS
  In this study, new enzyme α-L-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known α-L-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. Eva.Benesova@vscht.cz.
authors.forename: Eva
authors.initials: E
authors.lastname: Benešová
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. Petra.Lipovova@vscht.cz.
authors.forename: Petra
authors.initials: P
authors.lastname: Lipovová
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. hoficek@centrum.cz.
authors.forename: Jana
authors.initials: J
authors.lastname: Krejzová
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. terezia.kovalova@gmail.com.
authors.forename: Terezia
authors.initials: T
authors.lastname: Kovaľová
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. buchtovp@vscht.cz.
authors.forename: Patricie
authors.initials: P
authors.lastname: Buchtová
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. spiwokv@vscht.cz.
authors.forename: Vojtěch
authors.initials: V
authors.lastname: Spiwok
authors.affiliation: Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic. Blanka.Kralova@vscht.cz.
authors.forename: Blanka
authors.initials: B
authors.lastname: Králová
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002384:Catalysis; D006031:Glycosylation; D006868:Hydrolysis; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D056507:Paenibacillus / Q000201:enzymology*; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D005644:alpha-L-Fucosidase