甘油脱氢酶和亮氨酸脱氢酶偶联合成1,3-二羟基丙酮和2-氨基丁酸
文献类型：学位论文
| 作者 | 苗维娟 |
| 学位类别 | 硕士 |
| 答辩日期 | 2007-06-08 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 王平 |
| 关键词 | 甘油脱氢酶 亮氨酸脱氢酶 辅酶再生 生物合成 |
| 其他题名 | Glycerol Dehydrogenase and Leucine Dehydrogenase Coupling |
| 学位专业 | 生物化工 |
| 中文摘要 | 甘油脱氢酶（glycerol dehydrogenase, GDH）以NAD+为辅酶，可将甘油转化为1,3-二羟基丙酮（1,3-dihydroxyacetone, DHA），DHA在化妆品、医药和化工领域都有着广泛的应用。 亮氨酸脱氢酶(leucine dehydrogease, LDH)以NADH为辅酶，可将2-丁酮酸还原胺化为手性医药中间体2-氨基丁酸。将甘油脱氢酶和亮氨酸脱氢酶相偶联，能够以甘油和2-丁酮酸为底物合成DHA和2-氨基丁酸，同时实现辅酶NAD+的循环再生。 以NADH为起始辅酶行偶联反应。结果表明，pH 8.5，1.0 mol/L的NH4OH-NH4Cl缓冲液适合偶联反应的进行，提高GDH的用量可以提高产物DHA和2-氨基丁酸的产量。 以NAD+为起始辅酶，当其初始浓度为0.1μmol/L时，TTN最高可以达到5110，但是产物浓度和底物的平衡转化率却仅为1.40％ （对甘油）和2.6％（对2-丁酮酸）。 通过分析各反应底物和产物对GDH和LDH活性的影响，表明产物DHA和2-氨基丁酸对GDH和LDH强烈的抑制效应是造成反应转化率低的主要原因。 对偶联反应体系中GDH和LDH的反应动力学模型进行了初步探索，为下一步系统建立完整的动力学模型提供了值得借鉴的方法和思路。 本文首次将GDH和LDH偶联，生产两种产物的同时实现辅酶循环再生，为酶法偶联实现辅酶再生提供了一种新的途径。此外，通过对偶联反应中的动力学常数的考察，为酶偶联法实现辅酶再生的多酶体系的设计和构建提供理论指导。 |
| 英文摘要 | With NAD+ as coenzyme, Glycerol dehydrogenase (GDH) can convert glycerol to 1,3-dihydroxyacetone (DHA), which is widely used in cosmetics, pharmacy and chemical engineering. Leucine dehydrogenase (LDH) can convert 2-oxobutyric acid to 2-aminobutyric acid with NADH as its coenzyme. 2-Aminobutyric acid is a chiral intermediate in pharmacy. In this article, GDH and LDH were coupled to produce DHA and 2-aminobutyric acid from glycerol and 2-oxobutyric acid, and coenzyme were regenerated in this multi-enzyme biocatalysis system. The effects of pH, buffer concentration, ratio of GDH to LDH, and NADH concentration on yields were investigated using NADH as starting coenzyme. The results indicated that for this coupling reaction, pH8.5, 1.0mol/L NH4OH-NH4Cl was a proper buffer, and the yields of DHA and 2-aminobutyric acid were improved by increasing the amount of GDH. When NAD+ was used as starting coenzyme, a high TTN of 5110 was obtained by lowing the NAD+ concentration to 0.1μmol/L, nevertheless, the substrate conversion at this condition were really low, which were only 1.4％ for glycerol and 2.6％ for 2-oxobutyric acid, respectively. By analyzing the effects of both substrates and products on the multi-enzymatic system, it was concluded that the strong inhibition of DHA and 2-aminobutyric acid on GDH and LDH was the main reason for such low conversion. In this thesis, it is the first time to synthesis DHA from glycerol by GDH, and the regeneration of coenzyme was achieved by coupling this reaction with LDH biocatalysis reaction which can produce 2-aminobutyric acid from 2-oxobutyric acid. The investigation on kinetics of this GDH-LDH coupling reaction provided important theoretical basis for a better design and construction of enzyme-coupled coenzyme regeneration multienzymatic system. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 85 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1137]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 苗维娟. 甘油脱氢酶和亮氨酸脱氢酶偶联合成1,3-二羟基丙酮和2-氨基丁酸[D]. 过程工程研究所. 中国科学院过程工程研究所. 2007. |
入库方式： OAI收割
来源：过程工程研究所
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