论文总字数：23442字

摘 要

过氧乙酸作为氧化剂、环氧化剂、杀菌消毒剂，在医疗卫生、化学工业及日常生活中用途广泛。传统的过氧乙酸生产工艺，是用过氧化氢和醋酸在硫酸的催化下反应而得，产品含量低，醋酸利用率低，且生产安全隐患大。而以酶法生产过氧乙酸已经成为了一种新的渠道。

本研究在已构建好的乙酰基木聚糖酯酶AXE的基础上，获得了一株催化效果较好的酶，具体内容及成果如下：

（1）确定反应底物。以不同短链乙酸酯为底物，在25 ℃ 下反应5 min后发现，以甘油三乙酸酯作为底物产生的过氧乙酸浓度最高，最终确定底物为甘油三乙酸酯。

（2）重组酯酶AXE用量的优化。1 ml反应体系中含有250 mmol/l 甘油三乙酸酯，1mol/l过氧化氢在25 ℃、pH:7.4条件下反应5 min后发现，该反应体系中重组酯酶的用量最优为0.30 mg/ml，可产生8259 ppm过氧乙酸。

（3）甘油三乙酸酯浓度的优化。1 ml反应体系中含有重组酯酶AXE 0.3 mg/ml，1 mol/l过氧化氢在25 ℃、pH:7.4条件下反应5 min，发现该反应体系中甘油三乙酸酯的浓度最优为300 mmol/l，可产生9613 ppm过氧乙酸。

（4）过氧化氢浓度优化。1 ml反应体系中含有300 mmol/l甘油三乙酸酯，重组酯酶AXE 0.3 mg/ml在25℃、pH:7.4条件下反应5 min，发现该反应体系中过氧化氢浓度最优为1 mol/l，可产生9613 ppm过氧乙酸。

（5）温度的优化。在不同温度下，1ml反应体系中含有300 mmol/l甘油三乙酸酯，重组酯酶AXE 0.3 mg/m，1 mol/l过氧化氢在pH:7.4条件下反应5 min，最终发现在20℃时，生产的过氧乙酸的量达到最高为10481 ppm, 具有显著的应用价值。

（6）pH的优化。在不同pH条件下，1 ml反应体系中含有300 mmol/l甘油三乙酸酯，重组酯酶AXE 0.3 mg/ml，1 mol/l过氧化氢在20℃条件下反应5 min，结果显示，当pH8.0时，生产的过氧乙酸的量达到最高为11385 ppm。

（7）固定化AXE酯酶生产过氧乙酸。1 ml反应体系中含有300 mmol/l甘油三乙酸酯，1 mol/l过氧化氢，1 g固定化酶，在pH 8.0、20℃条件下反应5 min，结果表明，1 g固定化后的重组酯酶AXE可产生10950 ppm的过氧乙酸。较之于游离酶具有更便捷、可回收使用等优点。

关键词：过氧乙酸 AXE 优化 固定化

Acetyl xylan esterase of Bacillus subtilis catalyzed the synthesis of

peracetic acid

ABSTRACT

Peracetic acid plays a unique role as a disinfectant, oxidizer and oxidizing agent. The traditional production process of peracetic acid is mixed by acetic acid and hydrogen peroxide, sulfuric acid as a catalyst. But the content of product and the utilization of acetic acid is low. By the way, the high concentration of hydrogen peroxide has a risk of explosive. It has become a new channel to produce preacetic acid catalyzed by Acetyl xylan esterases.

On the basis of the Acetyl xylan esterase of Bacillus subtilis, I carried out the following and access to a wealth of research results, the specific contents of this research is as follows:

(1) Determining the substrate. Reacting with different short chain acetate react at 25 ℃ for 5 minutes, the results show that the concentration of peracetic acid which reacts with glycerol triacetate is the highest, so I determined glycerol triacetate as the substrate.

(2) Optimization of the amount of AXE. 250 mmol/l glycerol triacetate, 1mol/l hydrogen peroxide in the reaction system of 1ml, react at 25 ℃, pH: 7.4 for 5 minute. The results show that the best amount of AXE in this system is 0.30mg/ml, which produced 8259 ppm peracetic acid.

(3) Optimization of the concentration of glycerol triacetate. 0.3mg/ml AXE and 1mol/l hydrogen peroxide in the reaction system of 1ml, react at 25 ℃, pH: 7.4 for 5 minute. The results show that the best concentration of glycerol triacetate is 300mmol/l, which produced 9613 ppm peracetic acid.

(4) Optimization of the concentration of hydrogen peroxide. 300 mmol/l glycerol triacetate and 0.3 mg/ml AXE in the reaction system of 1 ml, react at 25 ℃, pH: 7.4 for 5 minute. I found that the best concentration of hydrogen peroxide is 1 mol/l, which produced 9613ppm peracetic acid.

(5) Optimization of temperature. 300mmol/l glycerol triacetate, 0.3 mg/ml AXE and 1 mol/l hydrogen peroxide in the reaction system of 1 ml, react at pH7.4 for 5 minute. I found that when the temperature is 20 ℃, the system produced 10481 ppm peracetic acid, which is the highest concentration.

(6) Optimization of pH. At different pH conditions, 300 mmol/l glycerol triacetate, 0.3 mg/ml AXE and 1mol/l hydrogen peroxide in the reaction system of 1ml, react at 20 ℃ for 5 minute. The results show that when pH is 8.0, the amount of peracetic acid production reaches up to 11385 ppm.
(7) Producing peracetic acid by immobilized AXE. 300mmol/l glycerol triacetate, 0.3 mg/ml immobilized AXE and 1mol/l hydrogen peroxide in the reaction system of 1ml, react at 20 ℃, pH 8.0 for 5 minute. The results show that 1g immobilized AXE can produce 10950 ppm peracetic acid. Compared to the free enzyme, immobilized enzyme is more convenient and can be used many times.

Key words: Peracetic acid; AXE; Optimization; immobilized

目录

摘 要 I

ABSTRACT III

第一章 文献综述 1

1.1 引言 1

1.2 AXE概述 1

1.2.1乙酰木聚糖酯酶（AXE） 2

1.2.2 AXE的酶学性质 2

1.3过氧乙酸 3

1.3.1过氧乙酸简介 3

1.3.2化学法生产过氧乙酸 3

1.3.3酶法生产过氧乙酸 5

1.4过氧乙酸的分析方法 7

1.4.1液相色谱法 7

1.4.2 ABTS分光光度法 7

1.5本研究的目的意义及研究内容 8

第二章 材料与方法 9

2.1实验材料 9

2.1.1 实验仪器 9

2.1.2实验试剂 9

2.1.3溶液的配置 10

2.1.4 培养基 10

2.2方法 11

2.2.1酯酶的诱导表达方法 11

2.2.2 构建新基因工程菌E. coli BL21pET28a-Cah并对该酯酶进行5 L 罐规模诱导表达的方法 13

2.2.4 重组酯酶催化生产过氧乙酸的条件优化 16

2.2.5固定化AXE酯酶生产过氧乙酸 16

第三章 实验结果与讨论 17

3.1 5 L罐规模诱导表达AXE的方法 17

3.2 不同短链乙酸酯为底物，重组酯酶催化生产过氧乙酸及过氧乙酸的检测。 18

3.3重组酯酶催化生产过氧乙酸的条件优化 18

3.3.1重组酯酶AXE用量 18

3.3.2甘油三乙酸酯浓度 19

3.3.3过氧化氢浓度浓度 20

3.3.4温度的优化 20

3.3.5 pH的优化 21

3.4固定化AXE酯酶生产过氧乙酸 21

第四章 结论与展望 23

4.1结论 23

参考文献 25

致谢 28

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