论文总字数：20589字

摘 要

头孢菌素类抗生素是临床上广泛应用的重要抗生素，发展前景十分广阔。头孢孟多属于第二代半合成的头孢菌素类抗生素，具有抗菌活性强、抗菌谱广、毒性低、疗效高等特点。传统的合成头孢孟多工艺方法主要有活性酯法和酰氯法两种，但是传统制备方法工艺复杂，收率不高且对环境危害较大，我国目前抗生素生产工艺正处于传统的化学法全面向绿色酶法改变的过程。相比于化学法，目前正在大力发展的酶法合成半合成抗生素对环境危害较小，并且条件温和，操作简便。本研究通过对AxPGA进行分子改造，对AxPGA活性周围的残基进行丙氨酸扫描，筛选出对合成头孢孟多影响较大的位点αM263，并对αM263进行饱和突变以此筛选能获得高活性αM263G突变体。相比于野生型酶，αM263G突变体对头孢孟多的合成转化率由原来的36%提高到68.3%，反应时间从420min 降至240min，合成速率显著提高，并且达到最大转化率之后产物几乎没有二次水解。在此基础上，进一步探讨酶法合成头孢孟多催化条件的优化，结果表明，在25℃左右，pH为6.5时，头孢孟多的合成效率进一步提高。

关键词：青霉素G酰化酶 头孢孟多 丙氨酸扫描 饱和突变

The semi-rational modification of penicillin G acylase was used to improve the synthesis efficiency of Cefamandole

ABSTRACT

Cephalosporin antibiotics are important antibiotics that are widely used in clinical practice and have broad prospects for development. Cefamandole belongs to the second generation of semi-synthetic cephalosporin antibiotics with strong antibacterial activity, wide antibacterial spectrum, low toxicity and high curative effect. The traditional methods for the synthesis of Cefamandole sodium mainly include active ester method and acyl chloride method. However, the traditional methods for the preparation of Cefamandole sodium are complicated, with low yield and great harm to the environment. At present, the traditional chemical methods for the production of antibiotics in China are in the process of changing from traditional chemical methods to green enzymatic methods. Compared with chemical methods, enzymatic synthesis of semi-synthetic antibiotics is less harmful to the environment, and the conditions are mild and the operation is simple. In this study, through molecular modification of AxPGA, Alanine scanning was performed on the residues around the activity of AxPGA to screen out the site αM263, which had a great influence on the synthesis of Cefamandole, and saturation mutation was performed on the site, so as to screen out the mutant of high activity αM263G. Compared with the wild-type enzyme, αM263G mutant improved the synthesis efficiency of Cefamandole, and the conversion rate was increased from 36% to 68.3%, and the reaction time was reduced from 420 min to 240 min, The rate of synthesis increased significantly, and there is almost no secondary hydrolysis of the product after reaching the maximum conversion rate. On this basis, the catalytic conditions of enzymatic synthesis of Cefamandole were further optimized. The results showed that the synthesis efficiency of Cefamandole was further improved at about 25℃ and pH of 6.5.

Key words: Penicillin G acylase；Cefamandole ；Site-directed mutation；Alanine scanning

目录

摘要 I

ABSTRACT II

第一章 文献综述 1

1.1 PGA的简介 1

1.1.1青霉素酰化酶来源、分类 1

1.1.2青霉素G酰化酶结构组成 2

1.2 PGA催化合成头孢孟多反应机理 3

1.3 PGA的蛋白质工程 3

1.4 影响PGA催化合成的条件因素 4

1.4.1 温度影响 4

1.4.2 pH影响 5

1.5本课题研究内容与研究意义 5

1.5.1 研究内容 5

1.5.2 研究意义 5

第二章 AxPGA的分子改造以及在头孢孟多合成中的应用 7

2.1 前言 7

2.2 实验材料 7

2.3 实验方法 10

2.3.1 对AxPGA活性中心的残基进行丙氨酸扫描 10

2.3.2 选取AxPGA关键位点进行饱和突变 11

2.3.3 筛选高合成活性AxPGA突变体 11

2.3.4 AxPGA 野生型及其突变体在头孢孟多合成中的实际应用 12

2.3.5 HPLC 检测底物含量 12

2.3.6 酶法合成头孢孟多催化条件的优化 12

2.4 结果与讨论 12

2.4.1 丙氨酸扫描AxPGA活性中心残基结果 12

2.4.2筛选高合成活性AxPGA突变体 13

2.4.3 AxPGA 野生型和αM263G突变体在合成头孢孟多中的实际应用 14

2.4.4 酶法合成头孢孟多催化条件的优化 15

2.5 本章小结 17

第三章 结论与展望 18

3.1 结论 18

3.2 展望 18

参考文献 20

致谢 24

1. 文献综述

头孢菌素类抗生素对细菌的选择作用强，而对人体几乎没有毒性，具有抗菌谱广、抗菌作用强、耐青霉素酶、过敏反应比青霉素类抗生素少等优点，是一类高效、低毒、临床应用广泛的重要抗生素，发展前景十分广阔，占据了我国抗感染类药物的半壁江山[1，2，3]。随着头孢菌素原料药不断国产化，具有一定生产技术实力的企业向着简约化、规模化方向发展，从而带动了第二、三代头孢菌素产量的增长。在我国头孢菌素市场上，合资企业的产品销量占 35%左右，进口产品销量占25%左右，而国产药品销量不到 40%[4，5]。

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