论文总字数：20964字

摘 要

本论文研究了葡萄糖脱氢酶（GDH）和NADH氧化酶（NOX）交联复合聚集体（combi-CLEAs）的制备方法，并将固定化酶用于葡萄糖酸钠的合成。通过单因素实验法，研究了沉淀剂种类和浓度、复合酶浓度、交联剂浓度和交联时间等因子对combi-CLEAs回收率的影响。其中四因素在一定范围内对酶活回收率影响较大，该范围分别为：戊二醛浓度8.5~12.5 mM、酶浓度9~15 mg/ml、交联时间0.5~1.5 h和交联温度20~30 ℃。采用正交实验优化后的最优组合，固定化酶回收率可达55%。对固定化酶催化条件进行了研究，对一定量的酶和辅酶，当底物浓度高于30 mM，催化速率不在随底物浓度增加增加，而是基本维持稳定，30 mM的底物已达饱和；当维持酶量300 μl，底物浓度30 mM，总体系2 mL时，发现当辅酶浓度高于0.2 mM时，再增加辅酶浓度，酶催化速率基本不变，而辅酶浓度低于0.1 mM时，辅酶浓度对酶催化速率影响较大，因此在该条件下最适辅酶浓度度为0.1~0.2mM。最后对固定化酶和游离酶催化进行了比较，底物浓度50 g/l，总体系20 ml，反应时间12 h，固定化酶在前10 h催化速率维持稳定，而游离酶6 h后催化速率开始下降。最终固定化酶催化底物转化率达到100%，游离酶只有71%。

关键词：葡萄糖脱氢酶 NADH氧化酶 交联酶聚集体 酶活回收率

ABSTRACT

This paper studied the glucose dehydrogenase and aggregate NADH oxidase efficient crosslinking production methods of sodium gluconate.Immobilized enzyme, which was prepared by crosslinking the precipitating agent is 80% ammonium sulphate, for glutaraldehyde crosslinking agent.After respectively, enzyme concentration, concentration of glutaraldehyde crosslinking temperature and the crosslinking time on enzyme activity recovery after crosslinking.Found that four factors impact on the enzyme activity recovery within a certain scope is bigger, the scope are: 8.5 ~ 12.5 mM, glutaraldehyde concentration enzyme concentration 9 ~ 15 mg/ml, crosslinking time 0.5 ~ 1.5 h and crosslinking temperature 20 ~ 30 ℃.After using the orthogonal experimental points to optimize the production process, the optimal combination of production with immobilized enzyme recovery can reach 55%. Catalytic conditions of immobilized enzyme was studied, to a certain amount of enzyme and coenzyme, when the substrate concentration higher than 30 mm, catalytic rate is not increased with the increase of the substrate concentration, but the basic stability of 30 mm substrate has reached its saturation;While maintaining enzyme amount 300 ul, 30 mM, initial concentration of total system 20 ml, found that when the coenzyme concentration is higher than 0.2 mM, to increase the concentration of coenzyme, enzyme catalytic rate basically remain unchanged, and coenzyme concentration is lower than 0.1 mM, coenzyme concentration had a greater influence on the rate of enzyme, so under the condition of the optimal concentration of coenzyme degree is 0.1 ~ 0.2 mM.Finally compares the immobilized enzyme and free enzyme catalysis, 50 g/l, initial concentration of total system 20 ml, reaction practice 12 h, stability of the immobilized enzyme catalytic rate in the first 10 h, and the free enzyme catalytic rate began to decline after 6 h.Eventually immobilized enzyme catalytic substrate conversion rate reached 100%, free enzyme was only 71%.

Key words: glucose dehydrogenase; NADH oxidase; Cross-linked enzyme aggretates; recovery activity

目 录

摘 要 I

ABSTRACT II

第一章 文献综述 1

1.1 葡萄糖脱氢酶的基本性质 1

1.1.1 葡萄糖脱氢酶的结构 1

1.1.2 葡萄糖脱氢酶的基本性质 1

1.2 NADH氧化酶的概述 1

1.3交联酶聚集体技术的研究进展 1

1.4葡萄糖酸钠的性质的性质、应用及制备方法 4

1.4.1 葡萄糖酸钠的性质 4

1.4.2葡萄糖酸钠的应用 4

1.4.3葡萄酸钠的制备方法 5

1.4.4 酶法制备葡萄酸钠 5

1.5 本论文的主要研究内容 5

第二章 实验材料与方法 6

2.1引言 6

2.2 实验材料和设备 6

2.2.1 主要试剂 6

2.2.2 主要仪器 6

2.3 实验方法 7

2.3.1 Combi-CLEAs聚集体的制备方法 7

2.3.2 双酶酶活定义及活力测定方法 7

2.3.3 双酶共固定化条件选择 8

第三章 实验结果与讨论 10

3.1 不同酶浓度对酶活回收率影响 10

3.2 不同交联温度对酶活回收率影响 10

3.3 不同戊二醛浓度对酶活回收率影响 11

3.4 不同交联时间对酶活回收率影响 11

3.5 正交实验优化combi-CLEAs生产制备工艺 12

3.6 固定化酶在不同温度下的稳定性 13

3.7 底物浓度对酶催化效率影响 14

3.7.1 不同底物浓度对葡萄糖消耗速率影响 14

3.7.2不同底物浓度对酶催化反应速率影响 14

3.8 辅酶量对酶催化效率影响 15

3.8.1 不同辅酶量对葡萄糖消耗速率影响 15

3.8.2 不同辅酶量对酶催化反应速率影响 16

3.9 combi-CLEAs重复利用效果 16

3.10 comni-CLEAs和游离酶在催化葡萄糖生成葡萄糖酸反应中应用比较 17

第四章 结论与展望 18

4.1 结论 18

4.2展望 19

参考文献 20

致谢 25

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