The Extraction Of Big-bone Chicken Liver Alkaline Phosphatase And Its On-line Immobilization

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.