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KMID : 1160619970020010071
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Preventive Nutrition and Food Science
1997 Volume.2 No. 1 p.71 ~ p.76
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Purification and Characterization of Pullulanase from Klebsiella pnrumoniae NFB-320
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Yoo Seung-Seok
Yu Ju-Hyun
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Abstract
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Pullulanase was produced from the Klebsiella pneumoniae NFB-320 with the composition of 1.0% pullulan, 1.5% yeast extract, 0.2% K₂HPO₄ and 0.02% MgSO₄ ㆍ7H₂O(pH 5.5). The optimum temperature for activity of the pullulanase was 30℃ and the highest yield of the enzyme was obtained after cell growth at 30℃ for 18hr, and maintained until 24hr cultivation. The pullulanase was successively purified 52.6 folds with 7.8% yield by acetone precipitation, DEAE-cellulose column chromatography and gel filtrations. The purified enzyme hydrolyzed pullulan into maltotriose exclusively. Chemical and physical properties of purified pullulanase from Klebsiella pneumoniae NFB-320 were examined. The optimum pH and temperature for enzyme activity were 5.0 and 60℃, respectively. The enzyme was stable between pH 4 and 7, and up to 50℃. The effect of modification on the rate of enzyme reaction was studied with various chemicals and metal ions. The enzyme has been found to be inactivated by I₂ and N-bromosuccinimide(NBS), which probably indicated the involvement of tryptophan residues in the active center of the enzyme.
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KEYWORD
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Klebsiella pneumoniae, Pullulanase, purification, enzymatic properties
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