重组大肠杆菌全细胞用于苯乙酮酸的绿色生物合成
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摘要
苯乙酮酸是化学合成中重要的合成砌块,可用于合成多种药物中间体,探索苯乙酮酸的绿色合成工艺具有重要的意义。以包含D-扁桃酸脱氢酶Lh DMDH编码基因的重组大肠杆菌全细胞为催化剂,考察了它在无辅酶和辅底物添加的条件下对D-扁桃酸生物转化的效果,并对催化产物进行了纯化和鉴定。结果表明,本研究成功实现了在无辅酶和辅底物添加条件下苯乙酮酸的生物合成,产物的得率和纯度分别为45%和99%左右。成果也为外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成奠定了基础。
Phenylglyoxylic acid(PGA) is key building block in the chemical synthesis, which could be used to synthesize a variety of important pharmaceutical intermediate, thus exploiting the green synthesis process of phenylacetone acid has significant economic value. The recombinant Escherichia coli whole cells containing the encoding gene of Lh DMDH was used as catalyst in this study. The biotransformation of D-mandelic acid was researched under the condition of no coenzyme and cosubstrate addition. Subsequently, the transformed products were purified and identified. These results indicated that biosynthesis of phenylglyoxylic acid was realized under the condition of no coenzyme and cosubstrate addition. The yield and purity of PGA were 45% and 99%, respectively. In addition, this study established a solid foundation for chiral resolution of racemic mandelic acid and the biosynthesis of PGA.
Phenylglyoxylic acid(PGA) is key building block in the chemical synthesis, which could be used to synthesize a variety of important pharmaceutical intermediate, thus exploiting the green synthesis process of phenylacetone acid has significant economic value. The recombinant Escherichia coli whole cells containing the encoding gene of Lh DMDH was used as catalyst in this study. The biotransformation of D-mandelic acid was researched under the condition of no coenzyme and cosubstrate addition. Subsequently, the transformed products were purified and identified. These results indicated that biosynthesis of phenylglyoxylic acid was realized under the condition of no coenzyme and cosubstrate addition. The yield and purity of PGA were 45% and 99%, respectively. In addition, this study established a solid foundation for chiral resolution of racemic mandelic acid and the biosynthesis of PGA.
引文
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[4]BAER E,KATES M.A new method of preparing phenylglyoxylic acid and its methyl ester[J].J.Am.Chem.Soc.,1945,67(9):1482-1483.
[5]CRICH D,ZOU Y.Catalytic oxidation adjacent to carbonyl groups and at benzylic positions with a fluorous seleninic acid in the presence of iodoxybenzene[J].J.Org.Chem.,2005,70(8):3309-3311.
[6]BORNSCHEUER U T,HUISMAN G W,KAZLAUSKAS R J,et al.Engineering the third wave of biocatalysis[J].Nature,2012,485(7397):185-194.
[7]DE CARVALHO C C C R.Enzymatic and whole cell catalysis:finding new strategies for old processes[J].Biotechnol.Adv.,2011,29(1):75-83.
[8]CARDILLO A B,PERASSOLO M,SARTUQUI M,et al.Production of tropane alkaloids by biotransformation using recombinant Escherichia coli whole cells[J].Biochem.Eng.J.,2017,125(Suppl.C):180-189.
[9]WACHTMEISTER J,ROTHER D.Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale[J].Curr.Opin.Biotech.,2016,42(Suppl.C):169-177.
[10]HUMMEL W,SCHUTTE H,KULA M R.D-Mandelic acid dehydrogenase from Lactobacillus curvatus[J].Appl.Microbiol.Biotechnol.,1988,28(4):433-439.
[11]马晨露,唐存多,史红玲,等.头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成[J].中国生物工程杂志,2015,(12):65-71.MA C L,TANG C D,SHI H L,et al.Semi rational modification of cephalosporin C acylase and biosynthesis of 7-ACA[J].China Biotechnology,2015,(12):65-71.
[12]GAO S J,WANG J Q,WU M C,et al.Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N-terminus substitution[J].Biotechnol.Bioeng.,2013,110(4):1028-1038.
[13]FAN C W,XU G C,MA B D,et al.A novel D-mandelate dehydrogenase used in three-enzyme cascade reaction for highly efficient synthesis of non-natural chiral amino acids[J].J.Biotechnol.,2015,195:67-71.
[14]ZHANG J,CUI Z,CHANG H,et al.Conversion of glycerol to1,3-dihydroxyacetone by glycerol dehydrogenase co-expressed with an NADH oxidase for cofactor regeneration[J].Biotechnol.Lett.,2016,38(9):1559-1564.
[15]RESCH V,FABIAN W M F,KROUTIL W.Deracemisation of mandelic acid to optically pure non-natural L-phenylglycine via a redox-neutral biocatalytic cascade[J].Adv.Synth.Catal.,2010,352(6):993-997.