生物催化法制备苯乙酮酸的研究

发布时间：2018-05-14 09:21

  本文选题：苯乙酮酸 + 生物催化　； 参考：《南京理工大学》2010年硕士论文

【摘要】： 苯乙酮酸(PGA)，，又称苯甲酰甲酸,是应用广泛的有机合成中间体。本实验室从铜绿假单胞菌(Pseudomonas aeruginosa)中克隆得到扁桃酸消旋酶的编码基因(mdlA)和扁桃酸脱氢酶基因(mdlB),己成功构建了可将扁桃酸转化为苯乙酮酸的重组表达菌株E.coli BL21 (DE3)/pEt30a (mdlA+mdlB),并且在IPTG诱导下有较高的表达量。本文在以上工作的基础上,从该工程菌株的性质出发,对菌体培养条件进行系统优化,旨在获得高菌体密度的同时保证有较高的蛋白表达量；除获得高密度、高活力菌体外,提高苯乙酮酸产率的另一个重要方面就是转化工艺条件的优化。菌体只有在最适宜的转化条件下,才能表现最佳的转化活力,保证苯乙酮酸的产率实现最大化。具体实验设计及结果如下： 对重组菌的基本性质和诱导培养条件进行了研究。选用乳糖代替IPTG作为诱导剂,采用摇瓶培养,相继确定了乳糖最适添加浓度为5g／L、添加时间4h、诱导培养时间8h、诱导前培养温度37℃,诱导培养温度30℃等一系列适合该工程菌高效表达重组蛋白的诱导条件。 为获得高密度、高活力菌体,采用统计学优化方法对该重组菌的培养基组成及培养条件进行优化研究。首先,利用Plackett-Burman试验设计筛确定三个主要影响因素,即碳、氮源浓度和pH值；在此基础上用最陡爬坡路径逼近最大响应区域；再以菌体转化比活力(即蛋白表达量)为考察指标进行CCD试验设计,并对实验数据进行回归分析,通过求解回归方程得到三个显著因素的最优取值,即碳源浓度6.34g／L,复合氮源浓度为13.56g／L,pH值为7.7。在此条件下,模型预测最高响应值为18.5276 U／g。经五批次培养验证实验,预测值与验证试验平均值非常接近,表明该模型能很好地预测实际的培养条件。 在对菌体培养条件进行优化,成功获得高密度、高活力菌体后,为进一步提高重组菌转化苯乙酮酸的能力,对重组菌转化条件进行了详细的研究。最终确定转化反应最适条件为：底物浓度为20g/L,细胞浓度为10g/L,磷酸体系浓度67mM, pH6.0,37℃,200rpm。在此条件下,转化比活力又有所提高,可达到20.5273U／g,转化反应进行8h即完成转化。 通过上述对培养条件和转化条件的优化实验,当底物扁桃酸浓度为2%时,重组菌在8h可完全转化,与未经优化的原始条件相比,在底物浓度提高了1倍的同时转化反应时间缩短了40h。
[Abstract]:Acetophenone acid PGA, also known as benzoyl formic acid, is a widely used intermediate in organic synthesis. From Pseudomonas aeruginosa), the coding gene of mandelic acid racemase was cloned from Pseudomonas aeruginosa) and the gene of mandelic dehydrogenase (MDLBN) was cloned from Pseudomonas aeruginosa. A recombinant expression strain, E.coli BL21 DE3 / pEt30a, was successfully constructed to transform mandelic acid into phenylketonic acid. And there was a high expression level induced by IPTG. On the basis of the above work and the properties of the engineering strain, the culture conditions of the strain were systematically optimized to obtain high bacterial density and ensure high protein expression, in addition to obtaining high density and high activity bacteria in vitro. Another important aspect of improving the yield of acetophenone acid is the optimization of conversion process conditions. Only under the most suitable transformation conditions can the bacteria exhibit the best transformation activity and ensure the maximum yield of acetophenone acid. The experimental design and results are as follows: The basic properties and induced culture conditions of recombinant bacteria were studied. Lactose instead of IPTG was used as inducer. The optimum concentration of lactose was 5 g / L, the addition time was 4 h, the induction time was 8 h, and the culture temperature before induction was 37 鈩

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