大骨鸡论文：碱性磷酸酶的提取及在线固定化研究[精选].docVIP

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；ALP粗酶液催化磷酸苯二钠的最适温度为500C,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