Purification and Characterization of Acidic Chitinases from Gizzards of Broiler ( Gallus gallus L. )
2000; Korean Society for Biochemistry and Molecular Biology; Volume: 33; Issue: 4 Linguagem: Inglês
ISSN
1976-670X
AutoresBeom Ku Han, Jong Kook Moon, Yeon Woo Ryu, Yun Hee Park, Do Hyun Jo,
Tópico(s)Enzyme Production and Characterization
ResumoAcidic chitinases from the gizzards of a broiler were purified to homogeneity, using precipitation with (NH₄)₂SO₄, ion exchanger chromatography, gel filtration, chromatofocusing and hydrophobic interaction chromatography The enzymes, GAC1 and GAC2, were purified 180- and 194folds with a recovery of 4.9% and 2.7%, respectively The molecular mass of GAC1 and GAC2 were 48.2 kDa and 57.8 kDa, respectively Chromatofocusing resulted in a pI of 3.1 for both enzymes. The purified enzymes were endochitinases that were devoid of β-N-acetylglucosaminidase and lysozyme activity. Kinetic studies using [³H]chitin indicate that GAC1 has a K_m and V_(max) of 1.97 ㎎/㎖ and 185 ㎎/㎎ protein/h, respectively The GAC2 has a K_m, and V_(max) of 0.42 ㎎/㎖ and 92.3 ㎎/㎎ protein/h, respectively at optimal pH and temperature (pH 5.0 and 60℃). When the pentamer and hexamer of Nacetylglucosamine (G1cNAc) were used as a substrate, the major product by GAC1 was the dieter of G1cNAc with a differential accumulation of the monomer and trimer, depending upon the substrate. However, the GAC2 produced the dieter and trimer in an equal quantity, regardless of the substrate used. The first 9 NH₂-terminal amino acid residues of the purified gizzard chitinase GAC1 and GAC2 shared a 100% homology. The first 25 NH₂-terminalamino acid residues of GAC1 also shared 55-60% homology with animal chitinasess and some animal proteins, such as whey protein and oviduct-specific proteins. However, little homology was found with either microbial and plant chitinases, or egg white lysozyme.
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