厦门大学学位论文原创性声明 兹呈交的学位论文, 是本人在导师指导下独立完成的研究成果 本人在论文写作中参考的其他个人或集体的研究成果, 均在文中以明确方式标明 本人依法享有和承担 由此论文而产生的权利和责任 声明人 ( 签名 ): 年月日 1
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1 学校编码 :10384 分类号 密级 学号 :B UDC 厦门大学博士学位论文 南美白对虾 N- 乙酰 -β-d- 氨基葡萄糖苷酶的研究 Studies on the N-Acetyl-β-D-glucosaminidase from Prawn (Penaeus vannamei) 谢晓兰 指导教师姓名 : 陈清西 教授 厦门大学生命科学学院 专业名称 : 生物化学与分子生物学 论文提交日期 :2005 年 6 月 15 日 论文答辩时间 :2005 年 7 月 10 日 学位授予日期 :2005 年月 日 答辩委员会主席 : 周海梦 评 阅 人 : 2005 年 7 月
2 厦门大学学位论文原创性声明 兹呈交的学位论文, 是本人在导师指导下独立完成的研究成果 本人在论文写作中参考的其他个人或集体的研究成果, 均在文中以明确方式标明 本人依法享有和承担 由此论文而产生的权利和责任 声明人 ( 签名 ): 年月日 1
3 目 录 中文摘要...16 英文摘要...18 第一章引言 南美白对虾简况 形态特征 生活习性 养殖生物学特点 我国南美白对虾的养殖历史与现状 几丁质简介 N- 乙酰 -β-d- 氨基葡萄糖苷酶的研究概况 微生物 N- 乙酰 -β-d- 氨基葡萄糖苷酶 植物 N- 乙酰 -β-d- 氨基葡萄糖苷酶 动物 N- 乙酰 -β-d- 氨基葡萄糖苷酶 本课题的研究意义及研究内容...32 第二章实验材料 仪器与方法 材料与试剂 仪器 方法 蛋白质浓度的测定 酶活力和比活力的测定 酶的分离纯化 酶的纯度鉴定
4 2.3.5 酶的理化性质的测定 酶的分子量测定 酶分子的等电点测定 酶分子的亚基数及亚基分子量的测定 酶的氨基酸组成分析 酶分子的紫外吸收光谱测定 酶分子的内源荧光光谱测定 酶催化反应的动力学性质研究 酶催化 pnp-nag 水解的动力学参数测定 酶催化 pnp-nag 水解反应的活化能测定 酶催化反应最适 ph 的测定 酶催化反应最适温度的测定 酶的 ph 稳定性测定 酶的热稳定性测定 金属离子对酶活力的影响 有机污染物对酶活力的影响 效应物对酶活力的影响 效应物对酶的紫外吸收光谱的影响 效应物对酶的内源荧光光谱的影响 酶活性中心解离基团性质的研究 酶活性中心解离基团 pk e 的测定 酶活性中心解离基团的解离热焓 (ΔH 0 ) 的测定 酶活性中心必需基团的研究 酶的化学修饰 酶活性中心必需基团的定量分析
5 酶的抑制或失活作用动力学...44 第三章实验结果 南美白对虾 NAGase 的分离纯化与纯度鉴定 NAGase 的分离纯化 酶制剂的纯度鉴定 南美白对虾 NAGase 的基本性质 NAGase 的分子量测定 NAGase 的等电点测定 NAGase 的亚基数及亚基的分子量测定 NAGase 的氨基酸组成分析 NAGase 的紫外特征吸收光谱测定 NAGase 的内源荧光特征 NAGase 催化 pnp-nag 水解的动力学参数的测定 NAGase 催化 pnp-nag 水解反应的活化能测定 NAGase 催化反应最适 ph 及 ph 稳定性测定 NAGase 催化反应最适温度及温度稳定性的测定 金属离子对 NAGase 活力的影响 酶活性中心解离基团的动力学性质研究 酶活性中心解离基团解离常数的测定 温度对酶活性中心可解离基团解离常数的影响 酶催化活性中心功能基团的解离热焓 酶活性必需基团的研究 DTT 对酶的化学修饰 pcmb 对酶的化学修饰 组氨酸咪唑基的化学修饰
6 3.4.4 酶活性中心色氨酸残基的化学修饰 酶经 NBS 修饰后的光谱变化 酶活性中心必需色氨酸残基数的测定 羧基的化学修饰 氨基的化学修饰 精氨酸残基的化学修饰 修饰剂对酶的效应动力学 醋酸酐对酶活力影响及动力学研究 醋酸酐对酶活力的影响 醋酸酐对酶的抑制类型及抑制常数 醋酸酐对酶抑制作用的动力学模型 醋酸酐对酶的抑制动力学的微观速度常数测定 溴乙酸对酶活力影响及动力学研究 溴乙酸对酶活力的影响 溴乙酸对酶的抑制类型及抑制常数 溴乙酸对酶抑制作用的动力学模型 溴乙酸对酶的抑制动力学的微观速度常数测定 NBS 对酶的抑制作用动力学 NBS 对酶抑制作用的动力学过程 NBS 对酶的抑制动力学的微观速度常数测定 酶的催化机理研究 双底物双产物的反应机理判断依据 对虾 NAGase 催化 pnp-nag 水解反应的机理研究 几种有机污染物对酶活力影响及动力学研究 二氧六环对酶活力影响及动力学研究
7 二氧六环对酶活力影响 酶在二氧六环溶液中的失活动力学模型的建立 二氧六环对酶的失活动力学的微观速度常数测定 酶经不同浓度二氧六环变性的构象变化 甲醛对酶活力影响及动力学研究 甲醛对酶活力影响 酶在甲醛溶液中的失活机理 酶在甲醛溶液中的失活动力学模型的建立 甲醛对酶的失活动力学的微观速度常数测定 苯酚对酶活力影响及动力学研究 苯酚对酶的抑制类型及抑制常数 苯酚对酶抑制作用的动力学模型 苯酚对酶的抑制动力学的微观速度常数测定 效应物对酶活力影响及抑制作用机制 几种糖对酶活力的影响 几种糖对酶抑制作用类型及抑制速度常数的测定 几种氨基酸对酶活力的影响 酸性氨基酸对酶活力影响的浓度效应 酸性氨基酸对酶的抑制机理 酸性氨基酸对酶抑制作用类型及抑制速度常数的测定 几种饲料添加剂对酶活力的影响 盐酸甜菜碱及柠檬酸对酶的抑制机理 盐酸甜菜碱及柠檬酸对酶抑制作用类型及抑制速度常数 几种效应物对酶的抑制作用动力学研究 盐酸甜菜碱 (Bataine Hydrochloride) 对酶的抑制作用动力学
8 盐酸甜菜碱对酶抑制作用的动力学过程 在盐酸甜菜碱存在下酶催化不同浓度底物水解的动力学 过氧化氢对酶活力影响及动力学研究 过氧化氢对酶活力的影响 过氧化氢对酶的抑制类型 过氧化氢对酶抑制作用的动力学模型 H 2 O 2 对酶的抑制动力学的微观速度常数测定 酶经 H 2 O 2 修饰后的荧光光谱变化 柠檬酸 (Citric Acid) 对酶的抑制作用动力学 柠檬酸对酶抑制作用的动力学过程 在柠檬酸存在下酶催化不同浓度底物水解的动力学 NAG 对酶的抑制作用动力学 NAG 对酶抑制作用的动力学模型 在 NAG 存在下酶催化不同浓度底物水解的动力学 酶在盐酸胍变性作用下的失活与去折叠 酶经不同浓度盐酸胍变性后活力与构象变化的比较 酶经不同浓度盐酸胍变性的活力变化 酶经不同浓度盐酸胍变性的构象变化 在盐酸胍变性后酶的活力变化与构象变化的比较 酶在盐酸胍溶液中的失活作用动力学 酶在盐酸胍溶液中的失活机理判断 酶在盐酸胍溶液中的失活类型判断 酶在盐酸胍溶液中的失活动力学模型的建立 酶在盐酸胍溶液中的失活动力学常数求法 酶在盐酸胍溶液中的失活动力学的微观速度常数测定
9 第四章讨论 南美白对虾内脏 NAGase 分离纯化与酶学性质 酶活性中心解离常数的研究 酶活性必需基团的修饰及动力学研究 酶催化机理的研究 有机污染物对酶活力的影响 效应物对酶活力的影响 酶在盐酸胍变性作用下的失活与去折叠研究 结论 参考文献 已发表的学术论文 已接受待发表的研究论文 致谢
10 Contents Chinese Abstract...16 English Abstract...18 Chapter 1. Introduction Review of Penaeus vannamei Shape Character Life Behaviour Biological Characteristic of Breeding The Breeding History and Status of Penaeus vannamei Review of Chitin Survey of Research on N-Acetyl-β-D- glucosaminidase N-Acetyl-β-D- glucosaminidase from Microorganisms N-Acetyl-β-D- glucosaminidase from Plants N-Acetyl-β-D- glucosaminidase from Animals Significance and Contents of the Research...32 Chapter 2. Materials and Methods Materials and Reagents Instruments Methods Assay of Protein Concentration Assay of the NAGase Activity Purification of the NAGase
11 2.3.4 Assay of the NAGase Homogenity Determination of the NAGase Properties Determination of the Molecular Weight Determination of the Isoelectric Point Determination of the number and Molecular Weight of Subunit Assay of Amino Acids Contents Determination of Uv-absorption Spectra Determination of Intrinsic Fluorescence Spectra Assay of Kinetics Properties Determination of K m for hydrolysis of pnp-nag Determination of the Activation Energy Determination of the Optimum ph Determination of the Optimum Temperature Determination of the ph Stability Determination of the Thermal Stability Effects of Metal Ions on the NAGase Effects of Organic Pollutants on the NAGase Effects of Some Effectors on the NAGase Effects of Some Effectors on the Uv-absorption Spectra Effects of Some Effectors on the Intrinsic Fluorescence Spectra Study on the Characters of Ionization Groups of the Active Site Determination of pk e of Enzyme Active Site Determination of ΔH 0 of Enzyme Active Site Study on the Essential Groups of Enzyme Active Site Chemical Modification of NAGase
12 Quantitative Assay of the Essential Groups Assay of the Inhibition or Inactivation Kinetics...44 Chapter 3. Results Purification and Homogenity of the NAGase Isolation and Purification of the NAGase Homogenity of the Purified NAGase Basic Properties of the NAGase Molecular Weight of the NAGase Isoelectric Point of the NAGase Subunit Numbers and Subunit Molecular Weight Amino Acids Contents of the NAGase Uv-absorption Spectra of the NAGase Intrinsic Fluorescence Spectra of the NAGase The Value of K m for pnp-nag Hydrolysis The Value of Activation energy for pnp-nag Hydrolysis The Optimum ph and ph Stability The Optimum Temperature and Thermal Stability Effects of Metal Ions on the NAGase Characters of Ionization Groups from Enzyme Active Site The Value of pk e of Enzyme Active Site Effects of Temperature on pk e of Enzyme Active Site The Value of ΔH 0 of Enzyme Active Site Essential Groups of Enzyme Active Site Chemical Modification of NAGase by DTT Chemical Modification of NAGase by pcmb
13 3.4.3 Chemical Modification of Histidine Residue of NAGase Modification of Tryptophan Residue of NAGase Uv-absorption Spectra of NAGase in the NBS Solution Essential Tryptophan Residues Number of NAGase Active Site Modification of Carboxyl Groups Modification of Amino Groups Modification of Arginine Residue Inhibition Kinetics of Chemical Modificators on NAGase Effect of Acetic Anhydride on NAGase and Kinetics Effect of Acetic Anhydride on NAGase Inhibitory Type and Inhibition Constants of Acetic Anhydride Inhibition Kinetics Model of Acetic Anhydride Microscopic Inhibition Rate Constants of Acetic Anhydride Effect of Bromacetic Acid on NAGase and Kinetics Effect of Bromacetic Acid on NAGase Inhibitory Type and Inhibition Constants of Bromacetic Acid Inhibition Kinetics Model of Bromacetic Acid Microscopic Inhibition Rate Constants of Bromacetic Acid Inhibition Kinetics of NBS Inhibition Kinetics Course of NBS Hydrolysis Course of pnp-nag at NBS Catalyze Mechanism of NAGase for pnp-nag Hydrolysis Judge Rule of Bi-substrates and Bi-products Mechanism Catalytic Mechanism of NAGase for pnp-nag Hydrolysis Effects of Some Organic Pollutants on NAGase and Kinetics
14 3.7.1 Effect of Dioxane on NAGase and Kinetics Effect of Dioxane on NAGase Inactivation Kinetics Model of Dioxane Microscopic Inactivation Rate Constants of Dioxane Conformation Change of NAGase in Dioxane Solution Effect of Formaldehyde on NAGase and Kinetics Effect of Formaldehyde on NAGase Inactivation Mechanism of Formaldehyde Inactivation Kinetics Model of Formaldehyde Microscopic Inactivation Rate Constants of Formaldehyde Effect of Phenol on NAGase and Kinetics Inhibitory Type and Inhibition Constants of Phenol Inhibition Kinetics Model of Phenol Microscopic Inhibition Rate Constants of Phenol Effects of Some Effectors on NAGase and Inhibitory Mechanism Effects of Some Saccharide Substance on NAGase Inhibitory Type and Inhibition Constants of Saccharide Substance Effects of Some Amino Acids on NAGase Effects of Acidic Amino Acids on NAGase Inhibitory Mechanism of Acidic Amino Acids Inhibitory Type and Inhibition Constants of Acidic Amino Acids Effects of Some Additives on NAGase Inhibitory Mechanism of Bataine Hydrochloride and Citric Acid Inhibitory Type and Inhibition Constants of Bataine Hydrochloride and Citric Acid
15 3.9 Inhibition Kinetics of Some Effectors for NAGase Inhibition Kinetics of Bataine Hydrochloride Inhibition Kinetics Course of Bataine Hydrochloride Hydrolysis Course of pnp-nag at Bataine Hydrochloride Effect of H 2 O 2 on NAGase and Kinetics Effect of H 2 O 2 on NAGase Inhibitory Type of H 2 O Inhibition Kinetics Model of H 2 O Microscopic Inhibition Rate Constants of H 2 O Fluorescence Spectra of NAGase in the H 2 O 2 Solution Inhibition Kinetics of Citric Acid Inhibition Kinetics Course of Citric Acid Hydrolysis Course of pnp-nag at Citric Acid Inhibition Kinetics of NAG Inhibitory Kinetics Model of NAG Hydrolysis Course of pnp-nag at NAG Inactivation and Unfolding of NAGase in Gu HCl Solution Comparison of Changes of NAGase Activity and Conformation Changes of NAGase Activity Changes of NAGase Conformation Comparison of Changes of Activity and Conformation Inactivation Kinetics of Gu HCl Inactivation Mechanism of Gu HCl Inactivation Type of Gu HCl Inactivation Kinetics Model of Gu HCl
16 Method of Determination of Inactivation Rate Constants of Gu HCl Determination of Inactivation Rate Constants of Gu HCl Chapter 4. Discussion Purification and Enzymatic Properties of NAGase Ionization Constants of Enzyme Active Site Modification and Kinetics Essential Groups of Enzyme Catalyze Mechanism of NAGase Effects of Organic Pollutants on NAGase Effects of Effectors on NAGase Inactivation and Unfolding of NAGase in Gu HCl Solution Conclusions References Papers Acknowledgements
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学校编码 :10384 分类号密级 学号 : UDC 硕士 学位论文 棉铃虫 N- 乙酰 -β-d- 氨基葡萄糖苷酶活力调控的研究 Studies on regulation of the N-Acetyl-β-D-glucosaminidase from Helicoverpa
学校编码 :10384 分类号密级 学号 :200326120 UDC 硕士 学位论文 Studies on regulation of the N-Acetyl-β-D-glucosaminidase from Helicoverpa armigera 王 君 指导教师姓名 : 陈清西教授 专业名称 : 生物化学与分子生物学 论文提交日期 :2006 年 4 月 15 日 论文答辩时间 :2006
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厦 门 大 学 学 位 论 文 原 创 性 声 明 本 人 呈 交 的 学 位 论 文 是 本 人 在 导 师 指 导 下, 独 立 完 成 的 研 究 成 果 本 人 在 论 文 写 作 中 参 考 其 他 个 人 或 集 体 已 经 发 表 的 研 究 成 果, 均 在 文 中 以 适 当 方
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The Discussion on Manchu-City of Foochow in Qing Dynasty I Abstract II Abstract This thesis aims to, from the research on the Eight-Banner garrison in Foochow, explore the living situation of the banner-people
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1 20 60 < > < > 2. 3 1 1.Davis,John Francis,"The Poetry of Chinese" Journal of The Royal Asiatic Society of Great Britian and Ireland 11(1830),(Translation of chapter 3 of the novel) 1830 1792 2.Tom Rorbert,"The
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厦门大学博士学位论文东南亚华侨民族主义发展研究 (1912-1928) 姓名 : 张坚申请学位级别 : 博士专业 : 专门史指导教师 : 李国樑 20020101 东南亚华侨民族主义发展研究