Purification and characterization of two extracellular endochitinases from Massilia timonae |
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Authors: | Sina Adrangi Ahmad Reza Shahverdi Zargham Sepehrizadeh |
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Affiliation: | Department of Pharmaceutical Biotechnology, Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, PO Box 14155-6451, Tehran 14174, Iran Department of Medical Biotechnology, Faculty of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran 14174, Iran |
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Abstract: | Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, Km, and Vmax of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL−1, and 0.59 μmol μg−1 h−1, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL−1, and 1.36 μmol μg−1 h−1, respectively. Both enzymes were stimulated by Mn2+ and inhibited by Hg2+, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively. |
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Keywords: | Chitinase Massilia timonae Enzyme purification |
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