Studies on the chitinolytic enzymes from Korean Ginseng (panax ginseng C.A. Meyer) and Broiler (Gallus gallus L.) Serum (고려인삼과 닭 혈액에서의 키틴 분해 효소에 대한 연구)

Alternative Title
Jong Kook Moon
Moon, Jong KooK
Alternative Author(s)
Jong Kook Moon
DoHyun Jo
일반대학원 분자과학기술학과
The Graduate School, Ajou University
Publication Year
Alternative Abstract
In this study, chitinase activity profiles were determined using the leaves, stems, and roots of Korean ginseng by performing chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) activity staining. Panax ginseng has chitinases of different molecular weights, which are specific to tissues, and a 31 kDa chitinase common in all tissues. In the roots, 2 chitinases of molecular weights 43 kDa and 34 kDa were specifically detected. We also found the major pI fraction by chromatofocusing different ginseng tissues the values were 4.1 for stems, 7.2 for leaves, and 9.3 and 10.0 for roots. None of these chitinases could hydrolyze the natural trimer of N-acetylglucosamine (GlcNAc) or N-acetyl glucosamine oligosaccharides [4MU-(GlcNAc)1?3], although they were active with [3H]-chitin. We did not obtain any reaction product with (GlcNAc)3-6 in the stem pI 4.1 sample. Because the leaf pI 7.2 sample hydrolyzed (GlcNAc)6 to produce 3 fragments, we concluded that this enzyme had 1 cleavage site on (GlcNAc)6. On the same lines, we also found that the enzyme in the root pI 10 sample had 2 cleavage sites on (GlcNAc)6. To characterize the major chitinase we purified two 31 kDa proteins from one-year-old Korean ginseng. These two enzymes (SBF1 and SBF2) were purified 70- and 81-fold with yields of 0.75 and 1.25%, respectively, and exhibited optimal pH and temperature ranges of 5.0-5.5 and 40-50oC. With [3H]-chitin as a substrate, Km and Vmax values of SBF1 were 4.6 mM and 220 mmol/mg-protein/h, respectively, while those of SBF2 were 7.14 mM and 287 mmol/mg-protein/h. The purified enzymes showed markedly less activity with p-nitrophenyl-N-acetylglucosaminide and fluorescent 4-methylumbelliferyl glycosides of N-acetylglucosamine oligomers than with [3H]-chitin. End-product inhibition of both enzymes demonstrated that both are endochitinases with different N-acetylglucosaminidase activity. Furthermore, the NH2-terminal sequence of SBF1 showed a high degree of homology with other plant chitinases where as the NH2-terminal amino acid of SBF2 was blocked. Internal sequence was also determined by LC-MS/MS. Infection with Cylindrocarpon destructans, a fungus, is a major disease of ginseng roots that cause rotting. When ginseng roots were challenged with C. destructans, the specific activity of the lateral roots increased the most compared to any other part. Anion-exchange chromatography revealed that the relative activity of the fractions eluted at pH 7.0 and with 1 M NaCl had increased by 240% and 350%, respectively, while the relative activity of the unbound fraction decreased by 43%. The pH 7.0-eluted fraction showed a single activity band of 29 kDa. HIC2 had a dual optimum pH of 4.0 and 6.0. The Km and Vmax values of HIC2 were 8.4 mM and 7.3 mmol/mg-protein/h, respectively. An N-acetyl-?-D-hexosamindase (HEX) was purified and characterized from broiler serum. This enzyme was a glycoprotein with a molecular weight of 69 kDa, as determined by gel filtration. The pH and temperature of the enzyme were 4.0 and 60oC, respectively. The enzyme had a higher specificity for 4MU-GlcNAc than the other 4MU derivatives of GlcNAc oligomer. The Km and Vmax against 4MU-GlcNAc were 27 ?M and 14.9 ?mol/mg-protein/h, respectively.

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