【作者】 王璐；

【导师】 徐刚； 杨立荣； 吴坚平；

【作者基本信息】 浙江大学 ， 化学工程， 2014， 硕士

【摘要】 转氨酶ATA117在合成手性胺类药物西他列汀中具有重要用途,本论文构建了不同的表达体系,优化表达条件,以实现ATAT117的高效表达,并对重组转氨酶的发酵工艺和产品西他列汀的制备工艺进行研究。我们首先以E.coli BL21(DE3)作为宿主,构建了PBV220-ATA117, pCDFDuet-ATA117, pETDuet-ATA117和pRSFDuet-ATA117四种重组载体,从蛋白表达水平和催化活力上进行了考察,选出表达能力较高的载体pETDuet-ATA117,其活力为137U/L,比活为0.24U/mg粗蛋白。对重组转氨酶的酶学性质进行了表征,其最适pH为9.0,在pH7.0-10.0的范围内都表现出很高的稳定性,该酶同时也具有很好的热稳定性,在反应温度45℃时,酶活力半衰期达到47.8h,在常温下,半衰期超过100h。为了提高Eco(pETDuet-ATA117)的发酵酶活,我们先使用单因素法和响应面法结合的方式对培养基成分进行了优化,得到最佳培养基配方：甘油14.45g/L,酵母粉7.27g/L,胰蛋白胨5.72g/L, NaC110g/L, K2HP044g/L。在优化培养基的基础上又对诱导条件进行了研究,最适诱导条件下酶活为713.7U/L。随后进行了lOL发酵罐中的分批和补料发酵工艺研究,最终菌体密度达到29.78g/L,酶活力达到3971U/L,比LB培养基发酵提高了30多倍。通过制备工艺的优化,50g/L底物浓度时,最佳的助溶剂为40%的DMSO,最佳的底物：细胞：辅酶比例为50：12.5：0.37,在此比例下,采用全细胞催化,8h可将底物完全转化为西他列汀,ee>99.95%。随后对产物西他列汀的分离纯化工艺进行了初步探索,最终收率达到80%。更多还原

【Abstract】 Aminotransferase ATA117has an important application in the synthesis of chiral amine drugs-Sitagliptin. We construct different expression systems to make the ATA117gene expressed better. The best engineered bacteria was selected for the study of fermentation and the preparation of sitagliptin.Fristly, four engineered plasmids PBV220-ATA117, pCDFDuet-ATA117, pETDuet-ATA117and pRSFDuet-ATA117were constructed, the protein expression and catalytic activity were the two examined factors. The results suggested that pETDuet-ATA117is our best choice.The enzymatic properties of recombinant aminotransferase ATA117were characterized. The optimum pH is9.0. The ATA117shows better stability in the range of pH7.0-10.0. The enzyme also has a good thermal stability. In the reaction temperature of45℃, the half-life of the enzyme activity have achieved47.8h. At room temperature, the half-life is more than100h.In order to achieve higher fermentation activity of aminotransferase ATA117, the single-factor method and the response surface method were used to optimize the medium components. The optimized concentration of each medium component is(g/L):Glycerol14.45, Yeast extract7.27, Tryptone5.72, NaC110, K2HPO44. On the basis of the medium optimization, we study the induction conditions of recombinant transaminase ATA117. After that, the batch and the fed-batch fermentation process have been investigated in a10L fermentor. We finally get the cell density(dry weight) of29.78g/L and enzymatic activity of3971U/L, which were more than30times higher than that of LB medium.Under the40%DMSO concentration,50:12.5:0.37substrate, cell, coenzyme ratio, it takes8hours to transfer almost all of the substrates to Sitagliptin. The product separation and purification technology also have been explored and a final yield of80%was achieved.更多还原