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Purification and Properties of Pullulanase from Bacillus halodurans

Author Affiliations

  • 1 Department of Biochemistry, Center for Post Graduate Studies, Jain University, Jayanagar 3rd Block, Bangalore-560011, INDIA

Int. Res. J. Biological Sci., Volume 2, Issue (3), Pages 35-43, March,10 (2013)


A pullulanase-producing bacterium was isolated from soil and identified as Bacillus halodurans based on the microscopic examination and biochemical tests. Maximum pullulanase production occurred in the presence of soluble pullulan at 1.5% concentration and in the presence of 0.5% peptone at pH 10.0 and 37°C. The purified alkaline pullulanase had a molecular mass of 37 ± 1 kDa, an optimum pH in the alkaline region (10.0) and optimum temperature of 50°C. The pullulanase activity was inhibited by Zn2+ and Cu2+ ions. Mg2+, Mn2+ and Fe2+ slightly inhibited the enzyme whereas Ca2+ had a stimulating effect on the enzyme activity. Ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), phenylmethylsulphonylfluoride (PMSF), tocylchloride methylketone (TLCK) and sodium azide (NaI) did not obviously inhibit the enzyme whereas N-ethylmalamide (NEM) and iodoacetic acid (IAA) had moderately inhibited the enzyme. N-bromosuccinimide (NBS) inhibited completely the enzyme activity suggesting that tryptophan is important for enzyme activity. The isolated enzyme was thermotolerant and alkolophilic which can thus be used in starch processing, detergent industry and other biotechnological applications.


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