埃切毕赤酵母的发酵优化及其催化 摘 要：手性药物在新药开发中越来越受到重视。生物催化具有反应条件温和、高度选择性、产物纯度高等优点。手性醇是许多手性药物合成的关键手性砌块，利用微生物醇脱氢酶作为生物催化剂可以在温和的条件下合成手性醇。本文对一株产醇脱氢酶的埃切毕赤酵母的发酵优化进行了研究。系统研究了碳源、氮源、pH值、金属离子对发酵产酶的影响。发现最适培养条件为：葡萄糖15g/L、蛋白胨5g/L、硫酸銨5g/L、硫酸鋅0.01g/L、pH值7。经发酵优化后，酶活由初始的8.27U/L提高到21.75U/L。
关键词：生物催化；埃切毕赤酵母；发酵优化；羰基还原酶；手性醇
Optimizing the fermentation of Pichia etchellsii and the study of its catalytic performance
Abstract: Chiral drugs draw more and more attention in the development of new drugs. Biocatalysis has several advantages such as mild reaction conditions, high selectivity and high product purity. Chiral alcohols are key chiral building blocks in the synthesis of many chiral drugs. They can be synthesized under mind conditions by using microbial alcohol dehydrogenase as biocatalyst. In this paper, the fermentation of Pichia etchellsii, an alcohol dehydrogenase producing strain, was optimized and its catalytic performance was evaluated. The effect of carbon sources, nitrogen source, pH value and metal ions on enzyme activity during fermentation was systematically investigated. It was found the optimal conditions for the fermentation are the following: Glucose 15g/L, Protein 5g/L, (NH4)2SO4 5g/L, ZnSO4 0.01g/L, pH 7. After fermentation optimization, the enzyme activity was increased from 8.27U/L to 21.75U/L.
Key words: biocatalysis; Pichia etchellsii; fermentation optimization; alcohol dehydrogenase; chiral alcohol;
目 录
1 研究背景 1
1.1 手性化合物的意义和应用 1
1.2 生物催化法制备不对称手性化合物 1
1.3 生物催化羰基还原反应原理 2
1.4 生物催化法合成手性醇类化合物 3
1.5 羰基还原酶的发酵优化 4
1.6 研究背景 4
1.7 研究内容 5
1.7.1 碳源的影响 5
1.7.2 初始pH的影响 5
1.7.3 氮源的影响 5
1.7.4 金属离子的影响 6
2 实验的技术路线和流程 7
2.1产醇脱氢酶埃切毕赤酵母发酵条件的优化的技术路线 7
3 试验方法与材料 7
3.1 实验材料 7
3.1.1 主要试剂 7
3.1.2 主要仪器 8
3.2 试验方法 8
3.2.1 优化前培养条件 8
3.2.2 优化培养条件 8
3.2.3 OD值的测定 8
3.2.4 细胞干重的测定 9
3.2.5 酶活测定方法 9
3.2.6标准曲线的绘制 10
4 结果与讨论 10
4.1 碳源优化 10
4.1.1 碳源种类对生长及产酶的影响 10
4.1.2 碳源浓度对生长及产酶的影响 11
4.2 氮源优化 12
4.2.1 氮源种类对生长及产酶的影响 12
4.2.2 复合氮源的对生长及产酶的影响 13
4.2.3 复合氮源浓度对生长及产酶的影响 14
4.3 金属离子优化 15
4.3.1 金属离子种类对生长及产酶的影响 15
4.3.2 锌离子浓度对生长及产酶的影响 16
4.4 初始pH值优化 17
4.4.1 最佳初始发酵pH值优化 17
5 总结与展望 19
致谢 20
参考文献 21，3458
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