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KMID : 0613820020120010077
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Journal of Life Science
2002 Volume.12 No. 1 p.77 ~ p.86
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Purification and characterization of the chitinase from Bacillus subtilis JK-56
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Kim Nak-Won
Jeong Yong-Kee
Jun Hong-Ki
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Abstract
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Chitin, a β-1,4 polymer of N-acetyl-D-glucosamine, is one of the most abundant organic compounds in nature. Chitinase (EC 3.2.1.14) is an enzyme that degrades chitin to chito-oligosaccharides, diacetyl chitobiose and N-acetyl-Dglucosamine. An extracellular chitinase-producing bacterial strain was isolated from soil and named to as Bacillus subtilis JK-56. Optimum culture condition of B. subtilis JK-56 for the production of chitinase was 1% chitin, 0.5% polypepton, 0.1% KCl, 0.05% MnSO4；4H2O, 37줛, initial pH 7.0 and 40 hour culture time. When B. subtilis JK-56 was grown in the optimum medium, one major active band and two minor active bands were detected by native-PAGE and active staining of the gel. Among them, the major band was purified from the culture supernatant by 70% ammonium sulfate precipitation and native-PAGE with BIO-RAD Model 491 Prep-Cell and named as Chi-56A. Its molecular weight was estimated to be 53kDa monomer and the isoelectric point (pI) was pH 4.3. The pH and temperature for the optimum activity of Chi-56A were pH 6.0 and 65℃, respectively. Chi-56A was stable up to 65℃ and in alkaline region. Its Km value for colloidal chitin was 17.33g/L. HPLC analysis of the reaction products confirmed that Chi-56A was an exo type chitinase.
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KEYWORD
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chitinase, chitin, Bacillus subtilis
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