Purification and Characterization of a Chitinolytic Enzyme Produced by Bacillus licheniformis GA9
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A bacterium producing a large amount of chitinolytic enzyme was isolated from the intestinal tract of earthworm. The isolate was identified as Bacillus licheniformis by 16S ribosomal RNA analysis and designated as B. licheniformis GA9. The enzyme was purified by 40−60% ammonium sulfate precipitation, diethyl-aminoethyl groups exchange chromatography, and gel filtration chromatography. The molecular weight was estimated to be 52.1 kDa and the N-terminal amino acid sequence was D-S-G-K-N-G-K-I-I-R-Y-YP- I-R. The optimum activity of the purified chitinolytic enzyme was shown at pH 5.0 and 40℃, and the enzyme was stable in the ranges of 20−50℃ and pH 5.0−6.0. Enzyme activity was increased by Co2+, while it was inhibited by Cu2+ and Fe2+. But it was recovered by chelating metals with ethylenediaminetetraacetic acid. The Km and Vmax values of the purified enzyme were 4.02 mg/ml and 0.52 mg/min, respectively. The chitinolytic enzyme characterized in this study has potential applications in areas such as biotechnology, biomedicine, agriculture, and nutrition.
Abstract
A bacterium producing a large amount of chitinolytic enzyme was isolated from the intestinal tract of earthworm. The isolate was identified as Bacillus licheniformis by 16S ribosomal RNA analysis and designated as B. licheniformis GA9. The enzyme was purified by 40−60% ammonium sulfate precipitation, diethyl-aminoethyl groups exchange chromatography, and gel filtration chromatography. The molecular weight was estimated to be 52.1 kDa and the N-terminal amino acid sequence was D-S-G-K-N-G-K-I-I-R-Y-YP- I-R. The optimum activity of the purified chitinolytic enzyme was shown at pH 5.0 and 40℃, and the enzyme was stable in the ranges of 20−50℃ and pH 5.0−6.0. Enzyme activity was increased by Co2+, while it was inhibited by Cu2+ and Fe2+. But it was recovered by chelating metals with ethylenediaminetetraacetic acid. The Km and Vmax values of the purified enzyme were 4.02 mg/ml and 0.52 mg/min, respectively. The chitinolytic enzyme characterized in this study has potential applications in areas such as biotechnology, biomedicine, agriculture, and nutrition.
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