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Purification and Characterization of Exoinulinase Enzyme from Streptomyces grisenus

Author(s): Eman Y. Tohamy

Journal: Pakistan Journal of Biological Sciences
ISSN 1028-8880

Volume: 9;
Issue: 5;
Start page: 911;
Date: 2006;
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Keywords: Streptomyces grisenius | purification | extracellular inulinase | N-Terminal sequence analysis

Streptomyces grisenus isolated from soil, was found to produce a very active inulinase enzyme. The optimum growth and inulinase activity occurred in the presence of 0.5% inuline, 0.3% NaNO3 and 0.5% CaCl2 in the production broth medium. Maximum growth was observed after 2 days at 30°C and pH 7 under shaking condition. Ammonium sulphate (70%) precipitation of cell free filtrate of Streptomyces grisenus increase the specific enzyme activity by 2.3 folds, further purification by gel filtration using Sephadex G100 increased the specific enzyme activity to 6.2 folds, respectively. Analysis of purified enzyme using gel electrophoresis revealed an apparent molecular weight of 140 KDa. Characterization of purified inulinase enzyme showed the maximum activity at pH 7, increased activity up to 40°C and incubated for 24 h. It was showed that inulinase activity was inhibited by all compounds (ZnSo4, MgSo4, MnSo4, FeSo4, CuSo4 and MnCl2) and the loss of activity reached to about 100, 93, 80, 76, 42 and 20%, respectively. However pure enzyme was stable in the presence of CaCl2. The purified enzyme was immobilized on (DEAE cellulose with 6% glutaraldehyde) gave better yield of activity, conc. of enzyme constant between 1.4 μM of inuline. The purified inulinase enzyme was subjected to N-terminal sequence analysis.

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