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Purification and Characterization of the Chitinase (ChiA) from Enterobacter sp. G-l

Author:
Jae Kweon Park, Kenji Morita, Ikuo Fukumoto, Yukikazu Yamasaki, Tsuyoshi Nakagawa, Makoto Kawamukai, Hideyuki Matsuda
Source:
Bioscience, biotechnology, and biochemistry 1997 v.61 no.4 pp. 684-689
ISSN:
1347-6947
Subject:
Enterobacter, Salvia hispanica, ammonium sulfate, biotechnology, calcium, carboxymethylcellulose, chitin, chitinase, chitosan, culture filtrates, fractionation, gel chromatography, isoelectric point, molecular weight, pH, polyacrylamide gel electrophoresis, temperature
Abstract:
Entevobacter sp. G-1 which produces chitinolytic and chitosanolytic enzymes, was previously isolated in our laboratory. One major chitinase, designated ChiA, was purified 42.9-fold from a culture filtrate of Entevobacter sp. G-L To purify the chitinase, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration on Sephadex G-100 column chromatography were used. The ChiA protein had a molecular weight of 60,000 estimated by SDS polyacrylamide gel electrophoresis and an isoelectric point of 6.6. The optimal pH and optimal temperature of ChiA against colloidal chitin were pH 7.0, and 40°C, respectively. The purified ChiA degraded colloidal chitin mainly to GlcNAc₂ with a small amount of GlcNAc₃ and GlcNAc₄. ChiA hydrolyzed flaked chitin, colloidal chitin, and ethylenglycol chitin, but did not hydrolyze carboxymethyl cellulose (CMC), nor >90% deacetylated flaked chitosan. The chitinase activity was 42% inhibited by 10mm EDTA, but was not inhibited by Ca²⁺ (<50 mm) or NaCl (<400 mm). The purified ChiA hydrolyzed colloidal chitin and chitin-related compounds in an endo splitting manner.
Agid:
6917331