Production, purification and characterization of a constitutive intracellular alpha-galactosidase from the thermophilic fungus Humicola sp.
The thermophilic fungus Humicola sp constitutively produces intracellular alpha-galactosidase (1.33 U mg-1 protein) within 48 h at 45 degrees C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65 degrees C and an optimum pH of 5.0. Humicola alpha-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The KmS for p-nitrophenyl-alpha-D-galactopyranoside, O-nitrophenyl-alpha-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag+ and Hg2+. D-Galactose inhibited alpha-galactosidase competitively and the inhibition constant (Ki) for galactose was 11 mM.
Kotwal
S M
SM
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
Gote
M M
MM
Khan
M I
MI
Khire
J M
JM
D001203:Ascomycota / Q000737:chemistry / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D012834:Silver / Q000494:pharmacology; D014462:Ultrafiltration; D000519:alpha-Galactosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D012834:Silver; D000519:alpha-Galactosidase; D008628:Mercury