Alteration of the substrate specificity of Aspergillus oryzae beta-galactosidase by modification with polyethylene glycol.
beta-Galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae was modified with 2,4,6-trichloro-s-triazine derivatives of polyethylene glycol (activated BPEG) having molecular weights of 600, 1500, 2000, and 4000. Polyethylene glycol derivatives were attached to 6 of the 12 amino groups exposed on the surface of the enzyme. Upon modification, the enzymatic activity for a water-soluble substrate, o-nitrophenyl beta-D-galactopyranoside, was reduced with increasing molecular weight of the activated BPEG. On the contrary, the enzymatic activity for another substrate, 4-methylumbelliferyl beta-D-galactopyranoside, was increased upon modification. The Michaelis constants of native and modified enzymes for these two substrates were virtually the same. The effect of the modification was more marked in the enzymatic hydrolysis of the beta-galactosidic bond of amphipathic substrates. A fluorescent analog of naturally occurring galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, was hydrolyzed more rapidly by the modified enzyme than by the native one. The enzyme modified with activated BPEG of 1500 Da had the highest activity for this substrate. The beta-galactosidic bond of the terminal galactose of GM1-ganglioside (II3NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosyl -glucosylceramide) was cleaved by the modified but not by the native enzyme.
D001230:Aspergillus / Q000201:enzymology*; D001236:Aspergillus oryzae / Q000201:enzymology*; D005677:G(M1) Ganglioside; D005696:Galactosidases / Q000378:metabolism*; D007700:Kinetics; D011092:Polyethylene Glycols / Q000494:pharmacology; D011609:Psychosine / Q000031:analogs & derivatives; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*
C032551:galactosyl-N-dansyl-sphingosine; D011609:Psychosine; D005677:G(M1) Ganglioside; D011092:Polyethylene Glycols; D005696:Galactosidases; D001616:beta-Galactosidase