论文总字数：24926字

摘 要

1,2,4-丁三醇(简称BT)作为众多天然产物合成的中间体以及多种手性药物的合成前体，广泛应用于军工、烟草、化妆品、医药等领域。有研究证明，低效率的脱羧反应是BT合成的限速步骤，2-酮酸脱羧酶是BT生物合成中的关键酶。本课题旨在利用分子模拟技术发掘高效的催化3-脱氧-D-甘油-戊酮糖酸脱羧的酶，为提高BT生物合成效率提供优质的新酶源。

本实验利用相似基团反应在BRENDA、KEGG等数据库，筛选可能的脱羧酶。通过酶活检测筛选出目标酶，再通过分子克隆及表达优化，实现目标酶在大肠杆菌BL21中的高效异源表达。在重组酶分离纯化的基础上，分析其酶学性质及体外催化合成BT的效率，具体如下:

⑴分析3-脱氧-D-甘油-戊酮糖酸脱羧形成3,4-二羟基丁醛的反应机理，在KEGG等数据库中寻找类似的基团反应，借助分子模拟技术，并从底物和来源的角度，从Brenda数据库中虚拟筛选出了5个2-酮酸脱羧酶。利用分子对接软件将3-脱氧-D-甘油-戊酮糖酸作为对接的底物，以苯甲酰甲酸脱羧酶MDLC作为对照，按照对接自由能打分并排序分析，最终选择了在反应对接自由能上具有极大可行性的支链-α酮酸脱羧酶（KDCA）进行下一步研究。

⑵将所筛选出的支链-α酮酸脱羧酶（KDCA）以及对照酶苯甲酰甲酸脱羧酶（MDLC）构建到载体pGEX-6p-1上，并转化入大肠杆菌DH5α中进行表达。以MDLC作为对照，对所筛选出的脱羧酶进行酶活检测，结果表明，来源于乳酸乳球菌Lactococcus lactis的支链-α酮酸脱羧酶（KDCA）的比酶活明显高于MDLC。

⑶成功地纯化出支链-α-酮酸脱羧酶KDCA和木糖酸脱水酶XYLD，并完成了KDCA酶学性质研究，发现其最适pH为6.5，最适温度为30˚C，分析pH对KDCA酶稳定性的影响表明，KDCA在pH 6.0至7.0的范围内有良好的稳定性；热稳定性分析表明KDCA在30˚C孵育2h后仍能够保持90%以上的活性，但在60˚C孵育时几乎没有活性，表明KDCA是中温酶。KDCA在金属离子螯合剂下几乎无活性，表明该酶依赖于金属离子。1mM条件下Ca² 、Na ，对KDCA的酶活力没有产生很大影响。

⑷为了进一步提高BT的合成效率，本课题以木糖酸为底物，构建了1,2,4-丁三醇体外催化体系。经高效液相色谱检测分析，空质粒的催化液中均无法检测到1,2,4-丁三醇的产生，MDLC和KDCA催化液中均有明显的1,2,4-丁三醇的生成，其中MDLC体外催化24h的BT产量为0.175 g/L，KDCA体外催化24h的BT产量为0.337g/L，为MDLC的1.93倍，可见KDCA比MDLC在1,2,4-丁三醇生产方面的确更具优势。另外通过优化木糖酸脱水酶XYLD和2-酮酸脱羧酶KDCA添加比例，发现当木糖酸脱水酶XYLD与支链-α-酮酸脱羧酶 KDCA添加比例为1:4（mg/mg）时，体外催化24h后，催化液中剩余的底物木糖酸含量几乎被完全消耗，此时1,2,4-丁三醇的产量达到峰值，为1.78g/L。

关键词：1,2,4-丁三醇 2-酮酸脱羧酶 体外催化

Mining of 2-ketoacid decarboxylase, a key enzyme for the synthesis of butotriol by biological method

ABSTRACT

1,2,4-butyltriol (BT), as an intermediate of many natural products and a variety of chiral drugs, is widely used in military, tobacco, cosmetics, medicine and other fields. It has been proved that low efficiency decarboxylation is the rate limiting step of BT synthesis, and 2-ketoacid decarboxylase is the key enzyme in BT biosynthesis. The purpose of this study was to explore an efficient enzyme catalyzed by 3-deoxidization-D-glycerol-pentanolic acid decarboxylic acid by molecular simulation technique, and to provide a new enzyme source for improving the biosynthesis efficiency of BT.

In this experiment, similar group reactions were used to screen possible decarboxylases in BRENDA,KEGG and other databases. The target enzyme was screened by enzyme activity detection, and then the target enzyme was expressed in E.coliBL21 by molecular cloning and expression optimization. On the basis of separation and purification of recombinant enzyme, the enzyme properties and the efficiency of catalytic synthesis of BT in vitro were analyzed as follows:

(1) The reaction mechanism of 3-deoxidization-D-glycerol-pentanolic acid decarboxylic acid to 3,4-dihydroxybutyraldehyde was analyzed. Similar group reactions were found in KEGG and other databases. With the help of molecular simulation technology, from the point of view of substrate and source, Five 2-ketoacid decarboxylases were virtual screened from Brenda database. Using molecular docking software, 3-deoxy-D-glycerol-pentanolic acid was used as the substrate of docking, and benzoylformic acid decarboxylase MDLC was used as control. According to the docking free energy score and sequencing analysis, Finally, the branch chain-α ketoacid decarboxylase (KDCA) , which has great feasibility in the free energy of reaction docking, is selected to carry out the next step of the study.

(2) The selected branch chain-α-ketoacid decarboxylase (KDCA) and control enzyme benzoylformic acid decarboxylase (MDLC) were constructed on vector pGEX-6p-1 and transformed into E.coliDH5α for expression. Compared with MDLC, the enzyme activity of the selected decarboxylase was detected. The results showed that the specific enzyme activity of chain-α-ketoacid decarboxylase (KDCA), which originated from Lactococcus lactis, was significantly higher than that of MDLC.

(3) The branch chain-α-ketoacid decarboxylase KDCA and xylose dehydrase XYLD, were successfully purified and the properties of KDCA enzyme were studied. It was found that the optimum pH was 6.5 and the optimum temperature was 30˚C. The effect of pH on the stability of KDCA enzyme was analyzed. KDCA has good stability in the range of pH 6.0 to 7.0. The thermal stability analysis showed that KDCA could maintain more than 90% activity after incubation with 30˚C for 2 h, but almost no activity at 60˚C, indicating that KDCA was a intermediate temperature enzyme. KDCA has little activity under metal ion chelating agent, indicating that the enzyme is dependent on metal ions. Under the condition of 1mM Ca² , Na , had no significant effect on the enzyme activity of KDCA.

(4) In order to further improve the synthesis efficiency of BT, the in vitro catalytic system of 1,2,4-butotriol was constructed with xylose acid as substrate. The production of 1,2,4-butotriol could not be detected in the catalytic solution of empty particles by high performance liquid chromatography (HPLC), and the formation of 1,2,4-butotriol was obvious in both MDLC and KDCA catalytic solutions. The yield of BT catalyzed by MDLC for 24 h was 0.175 g/L, and that of BT catalyzed by KDCA for 24 h was 0.337 g/L, which was 1.93 times higher than that of MDLC. It can be seen that KDCA does have an advantage over MDLC in the production of 1,2,4-butotriol.There are indeed more advantages in the production of 4-butotriol. In addition, by optimizing the addition ratio of xylose dehydrase XYLD and 2-ketoacid decarboxylase KDCA, it was found that when the ratio of xylose dehydrase XYLD to branch chain-α-ketoate decarboxylase KDCA was 1:4 (mg/mg), it was catalyzed in vitro for 24 hours. The remaining substrate xylose acid content in the catalytic solution was almost completely consumed. At this time, the yield of 4-butotriol reached the peak value of 1.78 g/L.

Keywords: 1,2,4-butanetriol；2-keto-acid decarboxylase；free-cell catalysis

目录

摘 要

ABSTRACT

目录

第一章 文献综述

1.1 研究背景 1

1.2 1,2,4-丁三醇的概况 1

1.2.1 1,2,4-丁三醇的性质 1

1.2.2 1,2,4-丁三醇的合成 2

1.3 研究目的和研究内容 3

第二章 实验材料与方法

2.1 材料与器材 4

2.1.1 实验试剂及仪器 4

2.1.2 培养基 4

2.1.3 十二烷基磺酸钠-聚丙烯酰胺凝胶电泳相关溶液的制备 4

2.1.4 菌株及质粒 4

2.2 实验方法 5

2.2.1 2-酮酸脱羧酶的筛选 5

2.2.2 2-酮酸脱羧酶在E.coliDH5α中表达体系的构建 5

2.2.2.1 质粒提取 5

2.2.2.2 目的基因的扩增 6

2.2.2.3 胶回收PCR产物 6

2.2.2.4 双酶切体系 6

2.2.2.5 连接载体pGEX-6P-1与目的基因片段 7

2.2.2.6 连接液转入感受态细胞 7

2.2.2.7 菌落PCR鉴定阳性重组子 7

2.2.3 木糖酸脱水酶、支链-α-酮酸脱羧酶在E.coliBL21中表达体系的构建 7

2.3 Ni柱亲和层析纯化 8

2.4 脱羧酶的酶活检测方法 8

2.5 蛋白质浓度检测 9

2.6 1,2,4-丁三醇的体外催化 9

第三章 结果与讨论

3.1 酮酸脱羧酶的虚拟筛选 10

3.2 酮酸脱羧酶的克隆表达及纯化 12

3.3 木糖酸脱水酶XYLD的克隆表达及纯化 15

3.4 支链-α-酮酸脱羧酶（KDCA）的酶学性质 15

3.5 1,2,4-丁三醇的体外催化 18

3.6 结果与讨论 19

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