Purification and characterization of Aspergillus ficuum endoinulinase.
Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000 +/- 500 and 66,000 +/- 1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800 +/- 1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1 +/- 1.0 mM and 773 +/- 60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.
Faculty of Biological Sciences, Chonbuk National University, Chonju, Korea. tbuhm@moak.chonbuk.ac.kr
T B
TB
Uhm
M S
MS
Chung
S H
SH
Lee
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F
Gourronc
I
I
Housen
J H
JH
Kim
J
J
Van Beeumen
B
B
Haye
J
J
Vandenhaute
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007444:Inulin; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D010446:Peptide Fragments; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase