Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus.
An extracellular alpha-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha-D-galactosides but efficiently hydrolyzed alpha-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl alpha-galactopyranoside and galactosyl(1)mannotriose, followed by (1)H NMR spectroscopy, pointed out the alpha-anomer of D-galactose was the primary product of hydrolysis from which the beta-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.
Puchart
V
V
Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.
Vrsanská
M
M
Bhat
M K
MK
Biely
P
P
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D005658:Fungi / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000031:analogs & derivatives; D016695:Glycosyltransferases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013237:Stereoisomerism; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008351:Mannans; C012990:galactomannan; D016695:Glycosyltransferases; D000519:alpha-Galactosidase; D005690:Galactose