当前位置:首页 >> 农林牧渔 >>

大骨鸡论文:碱性磷酸酶的提取及在线固定化研究


大骨鸡论文: 大骨鸡论文:碱性磷酸酶的提取及在线固定化研究

【中文摘要】碱性磷酸酶广泛存在于动物、植物、微生物中,直 接参与磷酸基团的转移和代谢过程。 固定化碱性磷酸酶可以作为分子 识别元件,进行多种农药、重金属离子的检测。顺序注射系统在固定 化酶在线制备方面具有巨大潜能。在此基础上,本论文进行了以下四 方面的研究:1.庄河大骨鸡骨骼粗壮,其生长发育与成骨细

胞的快速 分化关系密切,而成骨细胞的分化又是在 ALP 作用下进行。本论文以 大骨鸡肝作为酶源,分别采用有机溶剂沉淀法和盐析法提取 ALP 粗酶 液,考察了 ALP 的催化动力学性质。结果表明:两种方法提取的 ALP 单位质量比活力相近; 粗酶液催化磷酸苯二钠的最适温度为 500C, ALP 最适酸度为 10.8,米氏常数为 Km=1.59mmol/L。Pb2+、Ba2+对 ALP 活 力有抑制作用,Ca2+对 ALP 活力有激活作用。2.以 XC-72/PVC 复合材 料作为载体,顺序注射系统-固相微柱-紫外可见吸收光谱系统为操作 平台,对大骨鸡肝 ALP 进行在线固定化研究,并比较固定化 ALP 与游离 ALP 的理化性质。结果表明:与游离 ALP 相比,固定化 ALP 适宜温度 和酸度范围范围均扩大,分别为 50℃~60℃及 pH 9.0~10;对底物的 亲和性增强,米氏常数为 Km= 0.125 mmol/L; Ca2+浓度在 0.9 mmol/L 以下对固定化 ALP 活力有激活作用,1.0 mmol/L 以上为抑制作用。3. 分别采用戊二醛交联的壳聚糖和复合材料 XC-72/PVC 对大肠杆菌 ALP 进行了固定化,以生色底物磷酸苯二钠和生荧底物 4-甲基伞形酮磷酸 酯考察了固定化 ALP 对底物的亲和力,同时考察了载体材料对两种底

物的吸附情况。结果表明:固定化 ALP 对磷酸苯二钠的亲和力强于对 4-MUP; 戊二醛修饰的壳聚糖对生荧底物和对应产物的吸附作用显著, 而 XC-72/PVC 仅对生荧产物吸附严重; 两种材料对生色底物均无吸附 作用,对产物略有吸附。 4.采用戊二醛交联的壳聚糖作为载体,考察了 ALP 在壳聚糖表面的在线共价偶联性能,结果表明:利用共价交联法 在线制备的固定化 ALP 活力明显低于物理吸附法制备的固定化 ALP 活 力,且固定化 ALP 的稳定性良好,说明在顺序注射系统中利用共价偶 联法在线固定化 ALP 有待进一步探讨。 【英文摘要】Alkaline phosphatase exists widely in animals, plants and microbes, it directly participate in phosphoric acid groups and metabolic processes. Immobilized-ALP is served as molecular recognition components to detecting pesticides and heavy metal ions. There is tremendous potential of this study on employing sequential injection system to online-immobilized enzyme. On the basis, this thesis has completed the four sorts of work as the following:Part I The big-bone chicken bone is brawny, its growth relate closed to the rapid differentiation of osteoblasts, however the ALP active in the differentiation of osteoblasts. In this thesis, the organic solvent precipitation and salting-out methods were used to extract ALP from big-bone chicken liver, and the kinetic properties of the extracted ALP were also studied. The results indicated that the

optimum pH and temperature for the extracted ALP to catalyze the phenylphosphoric acid disodium is 10.8 and 50℃ respectively, the Michaelis-Menten constant (Km) is 1.59 mmol/L. Pb+ is an inhibitor to ALP, Ba2+ is an activator, and Ca2+ is an activator.Part II Employing a sequential injection system to online-immobilized ALP by the composite material XC-72/PVC as carrier, then the properties of the free ALP and immobilized-ALP were compared to study. The results indicated that the range of optimum pH and temperature for the immobilized-ALP are all expand, the appropriate temperature range of the immobilized-ALP is 50~60℃, and the suitable acidity range is 9.0-10, the Michaelis-Menten constant is 0.125 mmol/L. Ca2+ have activated function to the immobilized-ALP when the concentration is following 0.9 mmol/L, but as an inhibition when the concentration more than 1.0 mmol/L.Part III The glutaraldehyde crosslinking chitosan and the composite materials XC-72/PVC were used to immobilizted the ALP respectively. The affinity of substrate and immobilized-ALP were studied respectively, using the disodium phenyl phosphate hydrate as the phenacylmethylene substrate and 4-methylumbelliferyl phosphate as the fluorescence substrate. The adsorption was investigated between the carriers and the

substrates. The results indicated that the affinity of immobilized-ALP to phosphoric acid sodium benzodiazepines is better than the 4-MUP, the chitosan modified by glutaraldehyde has a significantly adsorption to the 4-MUP and the catalyze product, while XC-72/PVC only has a serious adsorption to the fluorescence product. Two kinds of material have no attraction to the disodium phenyl phosphate hydrate and a slightly adsorption to the product.PartⅣThe cross-linking modificated chitosan by glutaraldehyde was used as a carrier to investigated the covalent coupling performance about the ALP on the surface of chitosan. The results indicated that the activity of immobilized-ALP is lower than the physical adsorption immobilized-ALP significantly, the stability of immobilized-ALP is good. The study on covalent coupling method and employing sequential injection system to online immobilization ALP need to be further explored. 【关键词】 大骨鸡 碱性磷酸酶 分离纯化 顺序注射 在线固定化 【英文关键词】big-bone chicken liver phosphatase extraction alkaline

sequential injection

on-line immobilization 【目录】碱性磷酸酶的提取及在线固定化研究 4-5 Abstract 5-6 1 绪论 9-28 摘要 1.1 碱性磷酸酶

简介 9-18

1.1.1 ALP 的结构及理化性质 10-11 1.1.3 ALP 的分离纯化 16-18 1.2.1 固定化酶方法 1.2.3 固定化酶性质 19-22 23-25

1.1.2 1.2 固 1.2.2 固定 1.3 顺序注

ALP 的检测 11-16 定化酶 18-25 化载体 22-23

射-固相生物反应器系统 25-26 目标 26-28 28-45 29-32

1.4 本论文的工作思路和研究

2 大骨鸡肝 ALP 提取及在线固定化 2.1 引言 28-29 2.2 实验部分 2.2.2 有机溶剂沉淀 2.2.4 ALP 的 2.3 结 2.3.2 大骨

2.2.1 仪器与试剂 29-30

法提取 ALP 30 活性测定 30-31 果与讨论 32-44

2.2.3 盐析法提取 ALP 30

2.2.5 ALP 的在线固定化 31-32 2.3.1 ALP 比活力测定 32

鸡肝 ALP 的酶促反应动力学性质 32-39 面在线固定化条件优化 39 学性质 39-43 43-44

2.3.3 ALP 在 XC-72 表

2.3.4 固定化 ALP 的酶促反应动力

2.3.5 固定化 ALP 与游离 ALP 活力比较 3 大肠杆菌碱性磷酸酶的固定 3.2 实验部分 3.2.2 大肠杆菌 ALP 3.3

2.4 结论 44-45

化研究 45-58 46-47

3.1 引言 45-46

3.2.1 仪器与试剂 46-47

的在线固定化 47 结果与讨论 47-57

3.2.3 固定化 ALP 的活性检测 47 3.3.1 固定化方法 47-49 49-52

3.3.2 固 3.3.3 固定化

定化载体材料与底物和产物的相互作用 ALP 的性质 52-57 考文献 59-64

3.4 结论 57-58

结论 58-59



攻读硕士学位期间发表学术论文情况

64-65

致谢

65


相关文章:
大骨鸡论文:碱性磷酸酶的提取及在线固定化研究
大骨鸡论文: 大骨鸡论文:碱性磷酸酶的提取及在线固定化研究 【中文摘要】碱性磷酸酶广泛存在于动物、植物、微生物中,直 接参与磷酸基团的转移和代谢过程。 固定化...
更多相关标签: