摘 要

苯乳酸（PLA）的化学结构为2-羟基-3-苯基乳酸，同时也称为3-苯基乳酸或者β-苯乳酸。已知PLA是由乳酸菌发酵产生的代谢物，属于芳香族有机酸类。苯乳酸的热稳定性高、安全性高，并且没有毒副作用，而且它的抑菌图谱广、溶解性能也比较好，同时又兼具较为宽泛的pH作用范围，故而容易在食品体系中得到扩散，所以在食品应用工业中将其演变成为一种新型的生物防腐剂具有广阔的发展前景。

课题组前期从食用酸奶中筛选出了一株高产苯乳酸的植物乳杆菌，对该菌株基因组进行分析并设计特异性引物，利用PCR扩增其D-乳酸脱氢酶编码基因d-ldh，该基因大小为1038bp，编码345个氨基酸，属于NADH依赖性氧化还原酶，命名为D-Ldh。用限制性内切酶EcoR I和Xho I对pET-28a( )载体进行过双酶切，将目的片段经相同酶切后构建重组表达载体，将带有目的基因的重组质粒转化至表达宿主E. coli BL21，测序验证无误后将重组菌株在IPTG诱导下，25^oC，18 h诱导表达D-乳酸脱氢酶，SDS-PAGE结果表明重组蛋白表达成功。

将重组表达D-乳酸脱氢酶利用Ni -NTA亲和层析进行纯化，获得电泳纯D-乳酸脱氢酶D-Ldh用于后续苯丙酮酸催化转化试验。在pH 6.2，底物浓度为50 mM，NADH辅酶因子浓度30 mM，37^oC条件下进行催化转化。HPLC结果表明重组菌株可高效转化苯丙酮酸至苯乳酸，转化率达到72%，产物浓度达到0.6 g/L，进一步酮催化产物进行抑菌测试，对金黄色葡萄球菌和枯草芽孢杆菌均有较好的抑制效果。

关键词：苯丙酮酸 苯乳酸 乳酸脱氢酶 重组质粒 转化 催化

Construction and application of engineering strain for D- lactic acid biosynthesis

ABSTRACT

Benzene lactic acid (PLA), also known as 3-phenyl lactic acid or beta-phenyl lactic acid, is known to be a metabolite produced by lactic acid bacteria and belongs to aromatic organic acids. PLA is high stability, non-toxic, antibacterial spectrum, excellent solubility, but also has the function of pH range is more broad and thermal stability, it is easy to spread in the food system, food industry in the application will be developed as a new biological preservative has broad prospects for development.

In ourprevious from eating yogurt were screened from Lactobacillus plantarum strain of high yield of phenyllactic acid, through the analysis of specific primers designed to amplify the genome of this genus, D- lactic acid dehydrogenase encoding gene by PCR gene, the size is 1038bp, encoding 345 amino acids, which belongs to the NADH dependent oxidoreductase, named D-Ldh. PET-28a on Restriction endonuclease EcoR I and Xho I ( ) vector after double enzyme digestion, the fragment with the same restriction enzyme to construct the recombinant expression vector, the recombinant plasmid with target gene was transformed into expression host E. coli BL21, Sequencing proved correct, the recombinant strain induced by IPTG, 25oC, H 18 D- expression induced by lactate dehydrogenase SDS-PAGE, the results showed that the recombinant protein was successfully expressed.

The recombinant expression of D- lactate dehydrogenase was purified by Ni -NTA affinity chromatography, and the electrophoretic pure D- lactate dehydrogenase D-Ldh was obtained. Under the condition of pH 6.2, substrate concentration was 50 mM, NADH cofactor concentration was 30 mM, under 37oC conditions for catalytic conversion. The results showed that the recombinant strain HPLC can efficiently converting phenylpyruvic acid to lactic acid benzene, the conversion rate of 72%, the product concentration reached 0.6 g/L, further ketone catalyst products were bacteriostatic test of Staphylococcus aureus and Bacillus subtilis had good inhibitory effect.

Key Words: benzene pyruvic acid;benzene lactic acid;lactate dehydrogenase;recombinant plasmid, conversion;catalyze

目 录

摘要 I

ABSTRACT II

第一章 前言 1

第二章 文献综述 2

2.1 苯乳酸的简介 2

2.1.1 苯乳酸的理化特性 2

2.1.2 苯乳酸的抑菌作用机理 2

2.1.3 苯乳酸的发展前景 2

2.1.4 产苯乳酸微生物的分布 2

2.2 乳酸脱氢酶简介 3

2.2.1 乳酸脱氢酶来源 3

2.2.2 乳酸脱氢酶特性 3

2.3 课题相关信息 3

2.3.1 课题来源 3

2.3.2 研究的主要内容 4

第三章 材料与方法 5

3.1 实验材料 5

3.1.1 菌株与载体 5

3.1.2 目的基因 5

3.1.3 抑菌实验菌株 5

3.2主要实验试剂 5

3.3主要实验仪器 6

3.4培养基及部分溶液的配置 6

3.5 试验方法 7

3.5.1 普通操作 8

3.5.2 实验流程操作 10

3.6 利用抑菌实验检测苯乳酸 12

3.7 苯乳酸的测定 13

第四章 结果与讨论 14

4.1 乳酸脱氢酶基因的克隆和重组质粒的构建 14

4.1.1 乳酸脱氢酶基因的获取 14

4.1.2 双酶切质粒 14

4.1.3 重组质粒的构建 15

4.1.4 小结 16

4.2 重组质粒的转化 16

4.2.1转化 16

4.2.2 诱导 16

4.2.3 验证 16

4.3 产物验证 17

4.3.1 发酵培养 17

4.3.2 液相检测 17

4.3.3 抑菌检测 18

4.4 结果讨论 18

第五章 总结 20

参考文献 21

致谢 24

第一章 前言

苯乳酸是真菌，乳酸菌，丙酸菌等多种微生物的次级代谢产物，其作为添加剂可以应用到动物饲料生产，医药、化妆品行业等多个领域。鉴于苯乳酸对人和动物的天然无毒性以及自身广泛的抗菌作用，因而被作为生物防腐剂应用于食品行业符合食品安全卫生的要求，在另一方面也更加经济环保。

乳酸脱氢酶是苯乳酸生物合成的关键酶，其属于胞内酶且微生物体内含量低，所以乳酸脱氢酶的编码基因要想成功地在微生物体内得到表达，就得受到多种因素的严格调控，因而造成胞内乳酸脱氢酶含量较低，使得苯基乳酸产量受到了多方面的限制。在本次试验中为了提高苯乳酸的产量，我们利用基因工程的技术手段，通过构建含有乳酸脱氢酶基因的大肠杆菌工程菌株以苯丙酮酸作为底物转化合成苯乳酸。