Abstract
An exo-inulinase from strain Bacillus sp. SG7 was isolated and purified. A two-phase system PEG/Dextran, size-exclusion chromatography and ion-exchange chromatography were used in the purification process. The enzyme was purified to homogeneity with specific inulinase activity 18.47 U/mg protein and specific invertase activity 196.5 U/mg protein, purification fold of 10.44 and 27.4% yield. The molecular mass of the purified enzyme was estimated to be 56 000 Da. Strong inhibitors of enzyme activity are Pb, Hg, Zn and Cu ions with inhibition levels rising up to 55% for Cu and 95% for Pb. SDS totally inhibited the purified inulinase. The kinetic constants Km and Vmax for inulin as substrate were determined to be 1.0 mg/mL and 6.25 mg/mL.h, respectively. The pH optimum is at pH 7.0 and the enzyme is stable between pH 6.0 and pH 7.5, while retaining 100% of its initial activity between pH 6.5 and pH 7.0. The temperature optimum for the purified inulinase from strain Bacillus sp. SG7 was at 60°С. In the presence of inulin the purified inulinase sustains its activity at 100% for 55 minutes at 65°С. After the 70th minute the residual activity is 63% of the initial. The enzyme showed capacity to hydrolyse sucrose, raffinose and inulin from which it liberated only fructose units showing, therefore, an exo-action mechanism. The inulins from chicory (Cichorium intibus), from dahlia (Dahlia pinnata) and Jerusalem artichoke (Helianthus tuberosus) roots were hydrolysed by the purified enzyme.
Highlights
Inulin belongs to a class of carbohydrates known as fructans – polymers composed mainly of fructose units, and typically with a terminal glucose molecule
Bacterial cells were separated by centrifuging at 4000 rpm for 20 min and the clear supernatant was used in the purification steps
The results from the two-phase separation in system of PEG 6000 and dextran 500 used for the purification of an inulinase from strain Bacillus sp
Summary
Inulin belongs to a class of carbohydrates known as fructans – polymers composed mainly of fructose units, and typically with a terminal glucose molecule. Chemical hydrolysis of inulin with organic or mineral acids or through heterogeneous catalysis with solid acid catalysts, such as acid cation exchange resins or oxidized activated carbon, is disadvantageous because it results in unwanted side products and coloring compounds This requires special treatment, rendering the process inefficient. Inulinases are fructofuranosyl hydrolases produced by a wide range of microorganisms (Singh and Gill, 2006) These enzymes possess an invertase activity (hydrolysis of saccharose). Allais et al (1987a) isolated a thermophillic strain from genus Bacillus that produces an inulin inductive inulinase. The thermophillic soil isolate Bacillus stearothermophilus KP1289, which develops at temperatures ranging from 41 to 69°C, produces an inulin inductive extracellular inulinase with molecular mass of
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