Abstract
An extracellular β-galactosidase from a thermophilic fungus
Rhizomucor sp. has been purified to homogeneity by successive DEAE cellulose chromatography followed by gel filtration on Sephacryl S-300. The native molecular mass of the enzyme is 250 000 and it is composed of two identical subunits with molecular mass of 120 000. It is an acidic protein with a p
I of 4.2. Purified β-galactosidase is a glycoprotein and contains 8% neutral sugar. The optimum pH and temperature for enzyme activity are 4.5 and 60°C, respectively. The enzyme is stable at 60°C for 4 h, and has a
t
1/2 of 150 min at 70°C which is one of the highest reported for fungal β-galactosidases. Substrate specificity studies indicated that the enzyme is specific for β-linked galactose residues with a preference for
p-nitrophenyl-β-
D-galactopyranoside (
pNPG). The
K
m and
V
max values for the synthetic substrates
pNPG and
o-nitrophenyl-β-
D-galactopyranoside (
oNPG) were 0.66 mM and 1.32 mM; and 22.4 mmol min
−1 mg
−1 and 4.45 mmol min
−1 mg
−1, respectively, while that for the natural substrate, lactose, was 50.0 mM and 12 mmol min
−1 mg
−1. The end product galactose and the substrate analogue isopropyl thiogalactopyranoside (ITPG) inhibited the enzyme with
K
i of 2.6 mM and 12.0 mM, respectively. The energy of activation for the enzyme using
pNPG and
oNPG were 27.04 kCal and 9.04 kCal, respectively. The active site characterization studies using group-specific reagents revealed that a tryptophan and lysine residue play an important role in the catalytic activity of the enzyme.