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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)
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | DoHyun Jo | - |
| dc.contributor.author | Moon, Jong KooK | - |
| dc.date.accessioned | 2018-11-08T07:55:53Z | - |
| dc.date.available | 2018-11-08T07:55:53Z | - |
| dc.date.issued | 2011-02 | - |
| dc.identifier.other | 11486 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/8516 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학 :분자과학기술학과,2011. 2 | - |
| dc.description.tableofcontents | List of Tables i List of Figures ii Abbreviations v Abstract vi I. General introduction 1 1. Chitin 1 1.1. Applications of chitin and chitosan 2 2. Chitinase 5 2.1. Classification and structure of chitinases 6 2.2. Substrates of chitinase 6 2.3. Chitinase functions and induction 9 2.4. PR-protein 10 3. N-acetyl-？-D-hexosaminidase 11 4. Ginseng 13 II. Isolation of chitinolytic enzymes from different parts of ginseng plants 15 1. Introduction 15 2. Materials and Methods 17 2.1. Materials 17 2.2. Methods 17 2.2.1. Extraction of crude enzymes from different parts of ginseng plants 17 2.2.2. Protein assay 17 2.2.3. Enzyme assay 18 2.2.4. Anion exchanger chromatography 18 2.2.5. Chromatofocusing 18 2.2.6. Electrophoresis and detection of enzyme activity 18 2.2.7. Chromatography of chitooligosaccharides 19 3. Results and Discussion 21 3.1. Anion exchanger chromatography of ginseng plants 21 3.2. Analysis of chitinolytic activity by SDS-PAGE 21 3.3. Separation of chitinases by chromatofocusing 27 3.4. Analysis of reaction products by the chitinase fractions separated by chromatofocusing 27 4. Summary 34 III. Purification and characterization of two chitinases from one-year-old Korean ginseng main roots and an induced chitinase by C. destructans 35 1. Introduction 35 2. Materials and Methods 36 2.1. Materials 36 2.2. Methods 36 2.2.1. Extraction of crude enzyme from ginseng roots 36 2.2.2. Anion exchanger chromatography 36 2.2.3. Cation exchanger chromatography 36 2.2.4. Hydrophobic interaction chromatography 37 2.2.5. Detection of purified protein and enzyme activity 37 2.2.6. Optimal pH and optimal temperature 37 2.2.7. Effects of incubation time with [3H]-chitin 37 2.2.8. Detection of Km and Vmax 37 2.2.9. Detection of substrate specificity 38 2.2.10. Chromatography of chitooligosaccharides 38 2.2.11. Determination of NH2-terminal amino acid sequence 39 2.2.12. Determination of amino acid sequence by LC-MS/MS 39 2.2.13. Fungal induction and characterization of induced chitinase from one-year-old Korean ginseng 39 3. Results and Discussion 40 3.1. Purification of two 31 kDa chitinase enzymes 40 3.1.1. Chitinolytic activity from different parts of one-year-old ginseng roots 40 3.1.2. Anion exchanger chromatography of crude enzyme from ginseng main root 44 3.1.3. Cation exchanger chromatography of unbound (QF1) fraction from anion exchanger chromatography 44 3.1.4. Hydrophobic interaction chromatography of bound (SBF1 and SBF2) fraction from cation exchanger chromatography 44 3.2. Enzymatic properties of the purified SBF1 and SBF2 52 3.2.1. Detection of optimal pH and temperature 52 3.2.2. Detection of Km and Vmax 52 3.2.4. Analysis of the reaction product by SBF1 and SBF2 52 3.2.3. Detection of substrate specificity 52 3.2.4. NH2-terminal amino sequence of SBF1 and SBF2 53 3.2.5. Determination of amino acid sequence by LC-MS/MS 64 3.3. Characterization of chitinase an induced by C.destructans from one-year-old Korean ginsneg lateral roots 68 3.3.1. Effect of the C. destructans challenge 68 3.3.2. Anion exchanger chromatogram of crude extract of lateral roots 68 3.3.3. Hydrophobic interaction chromatography of induced chitinase from anion exchanger (QB2) 68 3.4. Enzymatic properties of the purified HIC2 72 3.4.1. Effect on pH and temperature 72 3.4.2 Detection of Km and Vmax 72 4. Summary 75 IV. Purification and characterization of a N-acetyl-？-D-hexosaminidase from broiler serum 76 1. Introduction 76 2. Materials and Methods 77 2.1. Materials 77 2.2. Methods 77 2.2.1. Extraction of crude enzyme from broiler serum 77 2.2.2. Enzyme assay 77 2.2.3. Cation exchanger chromatography 78 2.2.4. Lectin Affinity chromatography 78 2.2.5. Hydrophobic interaction chromatography 78 2.2.6. Gel filtration chromatography 78 2.2.7. Protein assay 78 2.2.8. Detection of optimal pH and temperature 79 2.2.9. Detection of Km and Vmax 79 3. Results and Discussion 80 3.1. Purification of N-acetyl-？-D-hexosaminidase 80 3.1.1. Cation exchanger chromatography from broiler serum 80 3.1.2. Lectin affinity chromatography 80 3.1.3. Hydrophobic interaction chromatography 80 3.1.4. Gel filtration chromatography 84 3.2. Enzymatic properties of the purified BSH (broiler serum N-acetyl-？-D-hexosaminidase) 84 3.2.1. Substrate specificity 84 3.2.2. Optimal pH and optimal temperature 84 3.2.3. Detection of Km and Vmax 85 4. Summary 93 V. Conclusion 94 VI. References 96 국문요약 109 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 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) | - |
| dc.title.alternative | Jong Kook Moon | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Jong Kook Moon | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2011. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 569249 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011486 | - |
| dc.subject.keyword | Chitinase | - |
| dc.subject.keyword | ginseng | - |
| dc.subject.keyword | Broiler | - |
| dc.subject.keyword | serum | - |
| dc.description.alternativeAbstract | 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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