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Purification and Properties of a Thermostable Inulinase (β‐ D ‐Fructan Fructohydrolase) from Bacillus stearothermophilus KP1289
Author(s) -
Keto Kumiko,
Araki Toshihiro,
Kitamura Tae,
Morita Naoko,
Moori Mika,
Suzuki Yuzuru
Publication year - 1999
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/(sici)1521-379x(199907)51:7<253::aid-star253>3.0.co;2-7
Subject(s) - inulinase , inulin , fructan , chemistry , raffinose , fructose , michaelis–menten kinetics , sucrose , enzyme , isoelectric point , thermophile , chromatography , thermostability , maltose , enzyme assay , biochemistry , food science
A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41 °C to 69 °C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight ( M r ) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively. The enzyme was active between 30 and 75 °C and at pH 4.5—8.6 with an optimum at 60 °C and pH 6.1. At 69 °C and pH 7.0 the half‐life of the enzyme was 10 min. The enzyme released fructose exo‐wise from the non‐reducing end of inulin ( M r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60 °C and pH 5.0 were 80 mM (360 mg/mL), 460 s —1 , and 5.8 s —1 mM —1 , respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0.50:0.16. The enzyme was classified as a thermophilic thermostable β‐ D ‐fructan fructohydrolase (EC 3.2.1.80).