title: Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus.
abstract: A new inducible intracellular beta-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native beta-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The beta-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47 degrees C for 2 h. After 191 h at 50 degrees C, the remaining beta-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The beta-galactosidase hydrolyzed p- and o-NO2-phenyl-beta-D-galactopyranoside, lactose, lactulose, MeOH-beta-D-galactopyranoside, phenyl-beta-D-galactopyranoside, and p-NO2-phenyl-alpha-L-arabinopyranoside. The kinetic constants (Km) measured for p- and o-NO2-phenyl-beta-D-galactopyranoside and beta-lactose were 4.8, 11.3, and 18.2 mM, respectively.
authors.affiliation: Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Fischer
authors.forename: C
authors.initials: C
authors.lastname: Scheckermann
authors.forename: F
authors.initials: F
authors.lastname: Wagner
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase