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Efficient Expression Of Transaminase ATA117in Escherichia Coli For The Production Of Sitagliptin

Posted on:2015-01-23Degree:MasterType:Thesis
Country:ChinaCandidate:L WangFull Text:PDF
GTID:2251330425484089Subject:Chemical Engineering
Abstract/Summary:PDF Full Text Request
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.
Keywords/Search Tags:aminotransferase, recombinant expression, response surface optimization, Sitagliptin
PDF Full Text Request
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