title: Facile production of Aspergillus niger α-N-acetylgalactosaminidase in yeast.
abstract: α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI4.4, K(M) 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-α-d-galactopyranoside, and optimum enzyme activity was achieved at pH2.0-2.4 and 50-55°C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant α-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant α-GalNAc-ase.
authors: Laboratory of Protein Architecture, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague, Czech Republic.
authors: Hynek
authors: H
authors: Mrázek
authors: Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
authors: Oldřich
authors: O
authors: Benada
authors: Laboratory of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.
authors: Petr
authors: P
authors: Man
authors: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague, Czech Republic.
authors: Ondřej
authors: O
authors: Vaněk
authors: Center for Biocatalysis and Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors: Vladimír
authors: V
authors: Křen
authors: Laboratory of Protein Architecture, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague, Czech Republic.
authors: Karel
authors: K
authors: Bezouška
authors: Center for Biocatalysis and Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 40 Prague, Czech Republic. Electronic address: lenka@wcontact.cz.
authors: Lenka
authors: L
authors: Weignerová
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D018929:Cell Culture Techniques; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D048809:alpha-N-Acetylgalactosaminidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D048809:alpha-N-Acetylgalactosaminidase