本文關(guān)鍵詞：雞論文，由筆耕文化傳播整理發(fā)布。

大骨雞論文：堿性磷酸酶的提取及在線固定化研究

【中文摘要】堿性磷酸酶廣泛存在于動物、植物、微生物中,直

接參與磷酸基團的轉(zhuǎn)移和代謝過程。固定化堿性磷酸酶可以作為分子識別元件,進行多種農(nóng)藥、重金屬離子的檢測。順序注射系統(tǒng)在固定化酶在線制備方面具有巨大潛能。在此基礎(chǔ)上,本論文進行了以下四方面的研究：1.莊河大骨雞骨骼粗壯,其生長發(fā)育與成骨細胞的快速分化關(guān)系密切,而成骨細胞的分化又是在ALP作用下進行。本論文以大骨雞肝作為酶源,分別采用有機溶劑沉淀法和鹽析法提取ALP粗酶液,考察了ALP的催化動力學(xué)性質(zhì)。結(jié)果表明：兩種方法提取的ALP單位質(zhì)量比活力相近；ALP粗酶液催化磷酸苯二鈉的最適溫度為500C,最適酸度為10.8,米氏常數(shù)為Km=1.59mmol/L。Pb2+、Ba2+對ALP活力有抑制作用,Ca2+對ALP活力有激活作用。2.以XC-72/PVC復(fù)合材料作為載體,順序注射系統(tǒng)-固相微柱-紫外可見吸收光譜系統(tǒng)為操作平臺,對大骨雞肝ALP進行在線固定化研究,并比較固定化ALP與游離ALP的理化性質(zhì)。結(jié)果表明：與游離ALP相比,固定化ALP適宜溫度和酸度范圍范圍均擴大,分別為50℃～60℃及pH 9.0～10；對底物的親和性增強,米氏常數(shù)為Km= 0.125 mmol/L; Ca2+濃度在0.9 mmol/L以下對固定化ALP活力有激活作用,1.0 mmol/L以上為抑制作用。3.分別采用戊二醛交聯(lián)的殼聚糖和復(fù)合材料XC-72/PVC對大腸桿菌ALP進行了固定化,以生色底物磷酸苯二鈉和生熒底物4-甲基傘形酮磷酸酯考察了固定化ALP對底物的親和力,同時考察了載體材料對兩種底

物的吸附情況。結(jié)果表明：固定化ALP對磷酸苯二鈉的親和力強于對4-MUP；戊二醛修飾的殼聚糖對生熒底物和對應(yīng)產(chǎn)物的吸附作用顯著,而XC-72/PVC僅對生熒產(chǎn)物吸附嚴重；兩種材料對生色底物均無吸附作用,對產(chǎn)物略有吸附。4.采用戊二醛交聯(lián)的殼聚糖作為載體,考察了ALP在殼聚糖表面的在線共價偶聯(lián)性能,結(jié)果表明：利用共價交聯(lián)法在線制備的固定化ALP活力明顯低于物理吸附法制備的固定化ALP活力,且固定化ALP的穩(wěn)定性良好,說明在順序注射系統(tǒng)中利用共價偶聯(lián)法在線固定化ALP有待進一步探討。

【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.

【關(guān)鍵詞】大骨雞 堿性磷酸酶 分離純化 順序注射 在線固定化

【英文關(guān)鍵詞】big-bone chicken liver alkaline

phosphatase extraction sequential injection on-line immobilization

【目錄】堿性磷酸酶的提取及在線固定化研究

4-5Abstract5-61 緒論9-28摘要1.1 堿性磷酸酶

簡介9-181.1.1 ALP的結(jié)構(gòu)及理化性質(zhì)10-11

1.1.3 ALP的分離純化16-18

1.2.1 固定化酶方法

1.2.3 固定化酶性質(zhì)19-2223-251.1.2 1.2 固1.2.2 固定1.3 順序注ALP的檢測11-16定化酶18-25化載體22-23

射-固相生物反應(yīng)器系統(tǒng)25-26

目標(biāo)26-28

28-45

29-321.4 本論文的工作思路和研究2 大骨雞肝ALP提取及在線固定化2.1 引言28-292.2 實驗部分2.2.2 有機溶劑沉淀

2.2.4 ALP的

2.3 結(jié)

2.3.2 大骨2.2.1 儀器與試劑29-30法提取ALP30活性測定30-31果與討論32-442.2.3 鹽析法提取ALP302.2.5 ALP的在線固定化31-322.3.1 ALP比活力測定32

雞肝ALP的酶促反應(yīng)動力學(xué)性質(zhì)32-39

面在線固定化條件優(yōu)化39

學(xué)性質(zhì)39-43

43-442.3.3 ALP在XC-72表2.3.4 固定化ALP的酶促反應(yīng)動力2.3.5 固定化ALP與游離ALP活力比較3 大腸桿菌堿性磷酸酶的固定

3.2 實驗部分

3.2.2 大腸桿菌ALP

3.3 2.4 結(jié)論44-45化研究45-5846-473.1 引言45-463.2.1 儀器與試劑46-47的在線固定化47

結(jié)果與討論47-573.2.3 固定化ALP的活性檢測473.3.1 固定化方法47-49

49-523.3.2 固3.3.3 固定化定化載體材料與底物和產(chǎn)物的相互作用

ALP的性質(zhì)52-57

考文獻59-643.4 結(jié)論57-58結(jié)論58-59參攻讀碩士學(xué)位期間發(fā)表學(xué)術(shù)論文情況

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  本文關(guān)鍵詞：雞論文，由筆耕文化傳播整理發(fā)布。