Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

Biochemistry (Mosc). 2012 Nov;77(11):1303-11. doi: 10.1134/S0006297912110090.

Abstract

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cloning, Molecular
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Glycoside Hydrolases / chemistry
  • Glycoside Hydrolases / genetics
  • Glycoside Hydrolases / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Sordariales / enzymology*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity
  • Temperature

Substances

  • Fungal Proteins
  • Recombinant Proteins
  • Glycoside Hydrolases