Purification and Comparison of Two Types of β-Galactosidases from Aspergillus oryzae

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Highly purified preparations of two types of acid β-galactosidases were obtained, respectively, from two crude enzyme preparations of Aspergillus oryzae and their properties were compared. Both the enzymes were purified by 2-propanol fractionation, and column chromatographies on DEAE-Sephadex A-50 and Sephadex G-200 or on hydroxyapatite. The purified enzymes, tentatively named β-galactosidase I and II of A. oryzae, appeared homogeneous on disc gel electrophoresis. They were most active at pH 4.7 with o-nitrophenyl β-_D-galactoside (ONPG) as a substrate and active at pH 4.5 toward lactose. β-Galactosidase I was stable over the pH range from 3 to 9,whereas β-galactosidase II showed a narrow range of pH stability between 4 and 7 as checked with ONPG. Optimum temperatures of β-galactosidase I and II were 53℃ and 45℃, respectively, in for 20 min of the enzyme reaction. The former enzyme was stable for more than 30 min at 60℃ and pH 5.0,while the latter one was rapidly inactivated at temperatures higher than 50℃ under the same conditions. Both the enzymes were markedly inactivated by 0.1 mM N-bromosuccinimide and 1 mM Hg.^<2+> THe K_m values of enzyme I and II were 1.8 and 0.88 mM for ONPG, and 38 and 35 mM for lactose substrate, respectively. The physicochemical properties of these two enzymes were very similar : their isoelectric points were 4.4-4.6,and their molecular weights were about 110,000. An appreciable difference found between them was the neutral sugar content : the mannose content of enzyme I was 7.0%, while that of enzyme II was 4.6%.
公益社団法人日本生物工学会の論文
1980-04-25

Purification and Comparison of Two Types of β-Galactosidases from Aspergillus oryzae

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