酶法高效转化苯乙酮酸合成L-苯甘氨酸
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摘要
L-苯甘氨酸是合成多种抗生素和抗癌药物的重要中间体,目前主要通过化学法合成.利用蜡样芽孢杆菌来源的亮氨酸脱氢酶(LeuDH)催化苯乙酮酸的还原氨基化合成L-苯甘氨酸,并偶联甲酸脱氢酶(FDH)进行辅酶再生,建立了一种新型的苯甘氨酸生物合成方法.结果表明,该辅酶再生体系可有效地用于L-苯甘氨酸的合成,且没有副产物残留,辅底物甲酸铵还可提供还原氨基化所需NH4+,随后对酶转化条件进行优化,最适转化条件为苯乙酮酸60 g/L,甲酸铵50.4 g/L,LeuDH 4 U/mL,FDH 2 U/mL,NAD+浓度0.14 g/L,p H 8.0以及30℃.在最优条件下,1 L的转化体系中,转化反应5 h,苯乙酮酸转化率达到99%,L-苯甘氨酸产量60.2 g/L,ee值>99%.本研究为L-苯甘氨酸的工业生产提供了一种更加简单、高效、经济的生物合成途径.(图8表4参27)
L-phenylglycine, an important intermediate in the synthesis of various antibiotics and anticancer drugs, is currently mainly synthesized through chemical methods. In this study, a new biological synthesis method for L-phenylglycine was developed. Leucine dehydrogenase(LeuDH) from Bacillus cereus was used to catalyze the reductive amination of benzoylformic acid for L-phenylglycine synthesis, and formate dehydrogenase(FDH) was used for cofactor regeneration. The results showed that the cofactor regeneration system was effective for the synthesis of L-phenylglycine without any by-products, and the reaction cosubstrate ammonium formate further provided the ammonium ions(NH_4~+) required for reductive amination. The enzymatic transformation conditions were then optimized to improve conversion efficiency. The obtained optimal conversion conditions were: benzoylformic acid 60 g/L, ammonium formate 50.4 g/L, LeuDH 4 U/mL, FDH 2 U/mL, NAD+ concentration0.14 g/L, pH 8.0, and a temperature of 30 ℃. Finally, under these optimal conditions, a 99% conversion rate was achieved in 5 h with the yield of 60.2 g/L, and an ee value > 99% at 1 L scale. This study, therefore, provides a simplified, more efficient and economical biosynthetic method for L-phenylglycine production.
L-phenylglycine, an important intermediate in the synthesis of various antibiotics and anticancer drugs, is currently mainly synthesized through chemical methods. In this study, a new biological synthesis method for L-phenylglycine was developed. Leucine dehydrogenase(LeuDH) from Bacillus cereus was used to catalyze the reductive amination of benzoylformic acid for L-phenylglycine synthesis, and formate dehydrogenase(FDH) was used for cofactor regeneration. The results showed that the cofactor regeneration system was effective for the synthesis of L-phenylglycine without any by-products, and the reaction cosubstrate ammonium formate further provided the ammonium ions(NH_4~+) required for reductive amination. The enzymatic transformation conditions were then optimized to improve conversion efficiency. The obtained optimal conversion conditions were: benzoylformic acid 60 g/L, ammonium formate 50.4 g/L, LeuDH 4 U/mL, FDH 2 U/mL, NAD+ concentration0.14 g/L, pH 8.0, and a temperature of 30 ℃. Finally, under these optimal conditions, a 99% conversion rate was achieved in 5 h with the yield of 60.2 g/L, and an ee value > 99% at 1 L scale. This study, therefore, provides a simplified, more efficient and economical biosynthetic method for L-phenylglycine production.
引文
1 Van Langen LM,Van Rantwijk F,?vedas VK,Sheldon RA.Penicillin acylase-catalyzed peptide synthesis:a chemo-enzymatic route to stereoisomers of 3,6-diphenylpiperazine-2,5-dione[J].Tetrahedron Asymmetry,2000,11(5):1077-1083
2 Ningsih F,Kitani S,Fukushima E,Nihira T.VisG is essential for biosynthesis of virginiamycin S,a streptogramin type B antibiotic,as a provider of the nonproteinogenic amino acid phenylglycine[J].Microbiology,2011,157(11):3213-3220
3 Mast YJ,Wohlleben W,Schinko E.Identif ication and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis[J].JBiotechnol,2011,155(1):63-67
4 Al Toma RS,Brieke C,Cryle MJ,Süssmuth RD.Structural aspects of phenylglycines,their biosynthesis and occurrence in peptide natural products[J].Nat Prod Rep,2015,32(8):1207-1235
5 Croteau R,Ketchum REB,Long RM,Kaspera R,Wildung MR.Taxol biosynthesis and molecular genetics[J].Phytochem Rev,2006,5(1):75-97
6 Landini D,Montanari F,Rolla F.Synthesis ofα-aminoarylacetic acids from chloroform,arylaldehydes,and aqueous ammonia under two-phase conditions[J].Synthesis,1979,10(16):26-27
7徐亚荣.苯甘氨酸、对氯苯甘氨酸合成工艺的研究[D].南京:南京工业大学,2005[Xu YR.Study on synthetic technology of phenylglycine andρ-chlorophenylglyci ne[D].Nanji ng:Nanji ng Un iversit y of Technology,2005]
8 Liu SP,Liu RX,El-Rotail AAMM,Ding ZY,Gu ZH,Zhang L,Shi GY.Heterologous pathway for the production of L-phenylglycine from glucose by E.coli[J].J Biotechnol,2014,186:91-97
9 Resch V,Fabian WMF,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
10 Fan CW,Xu GC,Ma BD,Bai YP,Zhang J,Xu JH.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
11 Cheng J,Xu G C,Ha n R Z,Dong JJ,Ni Y.Ef f icient a ccess to L-phenylglycine using a newly identified amino acid dehydrogenase from Bacillus clausii[J].RSC Adv,2016,6(84):80557-80563
12黄春辉,林陈水.亮氨酸脱氢酶研究进展及其工业应用[J].氨基酸和生物资源,2012,34(2):16-20[Hang CH,Lin CS.Research progress of leucine dehydrogenase and its industrial application[J].Amino Acids Biotic Res,2012,34(2):16-20]
13 Li J,Pan J,Zhang J,Xu JH.Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum[J].J Mol Catal B Enzym,2014,105:11-17
14 Tao RS,Jiang Y,Zhu FY,Yang S.A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH-regeneration system based on L-leucine dehydrogenase and formate dehydrogenase[J].Biotechnol Lett,2014,36(4):835-841
15戚云龙,杨套伟,周俊平,郑俊贤,徐美娟,张显,饶志明.多酶催化拆分DL-正缬氨酸生产L-正缬氨酸[J].应用与环境生物学报,2017,23(6):1015-1021[Qi YL,Yang TW,Zhou JP,Zheng JX,Xu MJ,Zhang X,Rao ZM.Multi-enzymatic resolution of DL-norvaline for L-norvaline production[J].Chin J Appl Environ Biol,2017,23(6):1015-1021]
16 Zhou JP,Wang YL,Chen JJ,Xu MJ,Yang TW,Zheng JX,Zhang X,Rao ZM.Rational engineering of Bacillus cereus leucine dehydrogenase towardsα-keto acid reduction for improving unnatural amino acid production[J].Biotechnol J,2019,14(3):1800253
17 Liu WM,Ma HM,Luo JX,Shen WH,Xu X,Li S,Hu Y,Huang H.Efficient synthesis of L-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E.coli[J].Biochem Eng J,2019,14(3):204-209
18 Kragl U,Vasic-Racki D,Wandrey C.Continuous production of L-tertleucine in series of two enzyme membrane reactors[J].Bioprocess Eng,1996,14(6):291-297
19 Ansorge MB,Kula MR.Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E.coli[piet98][J].Biotechnol Bioeng,2015,68(5):557-562
20 Zheng JX,Yang TW,Zhou JP,Xu MJ,Zhang X,Rao ZM.Elimination of a free cysteine by creation of a disulfide bond increases the activity and stability of Candida boidinii formate dehydrogenase[J].Appl Environ Microbiol,2017,83(2):1-12
21 Fujita Y,Ramaley R,Freese E.Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis[J].JBacteriol,1977,132(1):282
22 Sanwal BD,Zink MW.L-leucine dehydrogenase of Bacillus cereus[J].Arch Biochem Biophys,1961,94(3):430-435
23 Schütte H,Flossdorf J,Sahm H,Kula MR.Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii[J].FEBS J,1976,62(1):151
24 Menzel A,Werner H,Altenbuchner J,Gr?ger H.From enzymes to“Designer bugs”in reductive amination:a new process for the synthesis of L-tertleucine using a whole cell-catalyst[J].Eng Life Sci,2004,4(6):573-576
25 Zhang CQ,Chen XX,Zou RY,Zhou K,Stephanopoulos G,Too HP.Combining genotype improvement and statistical media optimization for isoprenoid production in E.coli[J].PloS ONE,2013,8(10):1-11
26 Qi YL,Yang TW,Zhou JP,Zheng JX,Xu MJ,Zhang X,Rao ZM,Yang ST.Development of a multi-enzymatic desymmetrization and its application for the biosynthesis of L-norvaline from DL-norvaline[J].Process Biochem,2017,55:104-109
27 Ou ZM,Shi HB,Sun XY,Shen WH.Synthesis of S-licarbazepine by asymmetric reduction of oxcarbazepine with Saccharomyces cerevisiae CGMCC No.2266[J].J Mol Catal B Enzyme,2011,72(3):294-297
2 Ningsih F,Kitani S,Fukushima E,Nihira T.VisG is essential for biosynthesis of virginiamycin S,a streptogramin type B antibiotic,as a provider of the nonproteinogenic amino acid phenylglycine[J].Microbiology,2011,157(11):3213-3220
3 Mast YJ,Wohlleben W,Schinko E.Identif ication and functional characterization of phenylglycine biosynthetic genes involved in pristinamycin biosynthesis in Streptomyces pristinaespiralis[J].JBiotechnol,2011,155(1):63-67
4 Al Toma RS,Brieke C,Cryle MJ,Süssmuth RD.Structural aspects of phenylglycines,their biosynthesis and occurrence in peptide natural products[J].Nat Prod Rep,2015,32(8):1207-1235
5 Croteau R,Ketchum REB,Long RM,Kaspera R,Wildung MR.Taxol biosynthesis and molecular genetics[J].Phytochem Rev,2006,5(1):75-97
6 Landini D,Montanari F,Rolla F.Synthesis ofα-aminoarylacetic acids from chloroform,arylaldehydes,and aqueous ammonia under two-phase conditions[J].Synthesis,1979,10(16):26-27
7徐亚荣.苯甘氨酸、对氯苯甘氨酸合成工艺的研究[D].南京:南京工业大学,2005[Xu YR.Study on synthetic technology of phenylglycine andρ-chlorophenylglyci ne[D].Nanji ng:Nanji ng Un iversit y of Technology,2005]
8 Liu SP,Liu RX,El-Rotail AAMM,Ding ZY,Gu ZH,Zhang L,Shi GY.Heterologous pathway for the production of L-phenylglycine from glucose by E.coli[J].J Biotechnol,2014,186:91-97
9 Resch V,Fabian WMF,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
10 Fan CW,Xu GC,Ma BD,Bai YP,Zhang J,Xu JH.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
11 Cheng J,Xu G C,Ha n R Z,Dong JJ,Ni Y.Ef f icient a ccess to L-phenylglycine using a newly identified amino acid dehydrogenase from Bacillus clausii[J].RSC Adv,2016,6(84):80557-80563
12黄春辉,林陈水.亮氨酸脱氢酶研究进展及其工业应用[J].氨基酸和生物资源,2012,34(2):16-20[Hang CH,Lin CS.Research progress of leucine dehydrogenase and its industrial application[J].Amino Acids Biotic Res,2012,34(2):16-20]
13 Li J,Pan J,Zhang J,Xu JH.Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum[J].J Mol Catal B Enzym,2014,105:11-17
14 Tao RS,Jiang Y,Zhu FY,Yang S.A one-pot system for production of L-2-aminobutyric acid from L-threonine by L-threonine deaminase and a NADH-regeneration system based on L-leucine dehydrogenase and formate dehydrogenase[J].Biotechnol Lett,2014,36(4):835-841
15戚云龙,杨套伟,周俊平,郑俊贤,徐美娟,张显,饶志明.多酶催化拆分DL-正缬氨酸生产L-正缬氨酸[J].应用与环境生物学报,2017,23(6):1015-1021[Qi YL,Yang TW,Zhou JP,Zheng JX,Xu MJ,Zhang X,Rao ZM.Multi-enzymatic resolution of DL-norvaline for L-norvaline production[J].Chin J Appl Environ Biol,2017,23(6):1015-1021]
16 Zhou JP,Wang YL,Chen JJ,Xu MJ,Yang TW,Zheng JX,Zhang X,Rao ZM.Rational engineering of Bacillus cereus leucine dehydrogenase towardsα-keto acid reduction for improving unnatural amino acid production[J].Biotechnol J,2019,14(3):1800253
17 Liu WM,Ma HM,Luo JX,Shen WH,Xu X,Li S,Hu Y,Huang H.Efficient synthesis of L-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E.coli[J].Biochem Eng J,2019,14(3):204-209
18 Kragl U,Vasic-Racki D,Wandrey C.Continuous production of L-tertleucine in series of two enzyme membrane reactors[J].Bioprocess Eng,1996,14(6):291-297
19 Ansorge MB,Kula MR.Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E.coli[piet98][J].Biotechnol Bioeng,2015,68(5):557-562
20 Zheng JX,Yang TW,Zhou JP,Xu MJ,Zhang X,Rao ZM.Elimination of a free cysteine by creation of a disulfide bond increases the activity and stability of Candida boidinii formate dehydrogenase[J].Appl Environ Microbiol,2017,83(2):1-12
21 Fujita Y,Ramaley R,Freese E.Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis[J].JBacteriol,1977,132(1):282
22 Sanwal BD,Zink MW.L-leucine dehydrogenase of Bacillus cereus[J].Arch Biochem Biophys,1961,94(3):430-435
23 Schütte H,Flossdorf J,Sahm H,Kula MR.Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii[J].FEBS J,1976,62(1):151
24 Menzel A,Werner H,Altenbuchner J,Gr?ger H.From enzymes to“Designer bugs”in reductive amination:a new process for the synthesis of L-tertleucine using a whole cell-catalyst[J].Eng Life Sci,2004,4(6):573-576
25 Zhang CQ,Chen XX,Zou RY,Zhou K,Stephanopoulos G,Too HP.Combining genotype improvement and statistical media optimization for isoprenoid production in E.coli[J].PloS ONE,2013,8(10):1-11
26 Qi YL,Yang TW,Zhou JP,Zheng JX,Xu MJ,Zhang X,Rao ZM,Yang ST.Development of a multi-enzymatic desymmetrization and its application for the biosynthesis of L-norvaline from DL-norvaline[J].Process Biochem,2017,55:104-109
27 Ou ZM,Shi HB,Sun XY,Shen WH.Synthesis of S-licarbazepine by asymmetric reduction of oxcarbazepine with Saccharomyces cerevisiae CGMCC No.2266[J].J Mol Catal B Enzyme,2011,72(3):294-297