亮氨酸脱氢酶与甲酸脱氢酶共表达菌株发酵产酶条件优化及其在L-2-氨基丁酸合成中的应用
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摘要
亮氨酸脱氢酶催化2-酮丁酸生成L-2-氨基丁酸需要辅酶NADH参与,构建Escherichia coli (Leu DH/FDH),通过共表达亮氨酸脱氢酶和甲酸脱氢酶实现了辅酶NADH胞内循环再生。通过产酶条件优化,提高该菌株催化制备L-2-氨基丁酸的效率。结果表明,在5 L发酵罐上,在诱导温度为22℃、诱导剂乳糖质量浓度为8. 0 g/L和诱导时间为17 h的条件下,亮氨酸脱氢酶和甲酸脱氢酶的酶活分别达到79. 2 U/g和216. 1 U/g,催化效果最佳。利用该菌株全细胞为催化剂,耦合苏氨酸脱氨酶,在1 L反应体系中进行了催化反应,L-苏氨酸质量浓度为180 g/L时,无需额外添加辅酶,8 h反应后底物转化率达到99%,L-2-氨基丁酸e. e.值99. 5%以上,时空产率19. 3 g/(L·h)。该研究为建立高效、低成本的L-2-氨基丁酸工业化生产方法提供了基础。
A recombinant Escherichia coli( LeuDH/FDH) that co-expressed leucine dehydrogenase and formate dehydrogenase was constructed to allow intracellular coenzyme NADH regeneration,and the cultivation condition was optimized. The results demonstrated that the highest catalytic efficiency was achieved in a 5 L fermenter at 22 ℃ with8. 0 g/L lactose and inducted for 17 h. Under this condition,the activities of leucine dehydrogenase and formate dehydrogenase reached 79. 2 U/g and 216. 1 U/g,respectively. By using the whole cells and coupled with threonine deaminase in a 1 L bioreactor,the conversation rate of L-2-aminobutyric acid reached 99% with the yield of 19. 3 g/( L·h)in 8 h from 180 g/L L-threonine without NADH added. The e. e. value of L-2-aminobutyric acid was over 99. 5%.This provides an efficient and low-cost method for industrial production of L-2-aminobutyric acid.
A recombinant Escherichia coli( LeuDH/FDH) that co-expressed leucine dehydrogenase and formate dehydrogenase was constructed to allow intracellular coenzyme NADH regeneration,and the cultivation condition was optimized. The results demonstrated that the highest catalytic efficiency was achieved in a 5 L fermenter at 22 ℃ with8. 0 g/L lactose and inducted for 17 h. Under this condition,the activities of leucine dehydrogenase and formate dehydrogenase reached 79. 2 U/g and 216. 1 U/g,respectively. By using the whole cells and coupled with threonine deaminase in a 1 L bioreactor,the conversation rate of L-2-aminobutyric acid reached 99% with the yield of 19. 3 g/( L·h)in 8 h from 180 g/L L-threonine without NADH added. The e. e. value of L-2-aminobutyric acid was over 99. 5%.This provides an efficient and low-cost method for industrial production of L-2-aminobutyric acid.
引文
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[9] ZHU L,TAO R S,WANG Y,et al. Removal of L-alanine from the production of L-2-aminobutyric acid by introduction of alanine racemase and D-amino acid oxidase[J].Applied Microbiology and Biotechnology,2011,90(3):903-910.
[10] AGER D J,LI T,PANTALEONE D P,et al. Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates[J]. Journal of Molecular Catalysis B:Enzymatic,2001,11(4-6):199-205.
[11] PARK E S,DONG G Y,SHIN J S.ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor[J]. Organic and biomolecular chemistry,2013,11(40):6 929-6 933.
[12] SHIN J S,KIM B G. Transaminase-catalyzed asymmetric synthesis of L-2-aminobutyric acid from achiral reactants[J]. Biotechnology Letters,2009,31(10):1 595-15 99.
[13] MALIK M S,PARK E S,SHIN J S.ω-Transaminasecatalyzed kinetic resolution of chiral amines using L-threonine as an amino acceptor precursor[J]. Green Chemistry,2012,14(8):2 137-2 140.
[14]苏金环,郭皓,曾聪明,等.生物催化一锅法制备2-氨基丁酸[J].精细化工,2012,29(9):921-924; 936.
[15] TAO R S,JIANG Y,ZHU F Y,et al. 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]. Biotechnology Letters,2014,36(4):835-841.
[16] GALKIN A,KULAKOVA L,YOSHIMURA T,et al.Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes[J]. Applied and Environmental Microbiology,1997,63(12):4 651-4 656.
[17] YASUKAWA K,HASEMI R,ASANO Y. Dynamic kineticresolution of alpha-aminonitriles to form chiral alphaamino acids[J]. Advanced Synthesis and Catalysis,2011,353(13):2 328-2 332.
[18]徐建妙,赵哲,陈策,等.甲酸脱氢酶外源表达发酵条件优化及在L-2-氨基丁酸合成中的应用[J].食品与发酵工业,2018,44(7):63-68.
[19]张玉彬.生物催化的手性合成[M].北京:化学工业出版社,2001:170-176.
[20]徐娴,贾红华,何冰芳,等.甲酸脱氢酶基因在大肠杆菌Rosetta中的高效表达[J].食品与发酵工业,2007,33(5):5-8.
[21]黄志华,刘铭,王宝光,等.甲酸脱氢酶用于辅酶NADH再生的研究进展[J].过程工程学报,2006,(6):1 011-1 016.
[22] WANG Y H,JING C F,YANG B,et a1. Production of a new sea anemone neurotoxin by recombinant Escherichia coli:optimization of culture conditions using response surface methodology[J]. Process Biochem,2005,40(8):2 721-2 728.
[23] XU G,JIANG Y,TAO R S. A recyclable biotransformation system for L-2-aminobutyric acid production based on immobilized enzyme technology[J]. Biotechnol Lett,2015,38(1):123-129.
[24]余龙,陈寅,周丽,等.双酶偶联催化马来酸生成L-天冬氨酸[J].食品与发酵工业,2018,44(8):20-26.
[25] PARK E,KIM M,SHINA J S. One-pot conversion of Lthreonine into L-homoalanine:biocatalytic production of an unnatural amino acid from a natural one[J]. Adv Synth Catal,2010,352(18):3 391-3 398.
[26] ANSORGE M B,KULA M R. Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli(p IET98)[J]. Biotechnol Bioeng,2000,68(5):557-562.
[27]刘维明,杨震炯,罗积杏,等.重组大肠杆菌共表达亮氨酸脱氢酶和甲酸脱氢酶发酵条件优化[J].生物加工过程,2015,13(4):23-28.
[28]刘珊,李元,祝俊.偶联基于酮还原酶的NADH再生系统一锅法酶催化合成L-2-氨基丁酸[J].中国生物工程杂志,2017,37(1):64-70.
[29]郭红颜,梁蕊,李航,等.用于合成L-2-氨基丁酸基因工程菌的构建[J].中国医药工业杂志,2015,46(10):1 076-1 079.
[2]曹炜,郑小玲.抗癫痫药物左乙拉西坦的合成研究进展[J].齐鲁药事,2012,31(10):602-604.
[3]黄红霞,林原斌. 3,4-亚甲二氧基苯乙胺的合成新方法[J].精细与专用化学品,2007,15(12):11-13.
[4] CHAN L,STEFANAC T,TURCOTTE N,et al. Design and evaluation of dihydroisoquinolines as potent and orally bioavailable human cytomegalorirus inhibitor[J]. J Bioorg Med Chem Lett,2000,10(13):1 477-1 480.
[5]白国义,陈立功,邢鹏,等.乙胺丁醇的合成[J].精细化工,2004,21(12):943-945; 949.
[6]奚强,林丫丫,王冲,等. 2-氨基丁酸的合成研究[J].武汉工程大学学报,2007,29(4):15-17.
[7] FOTHERINGHAM I G,GRINTER N,PANTALEONE D P,et al. Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12[J]. Bioorganic and Medicinal Chemistry,1999,7(10):2 209-2 213.
[8] FOTHERINGHAM I G. Transaminase biotransformation process:US,6197558B1[P]. 2001-03-06.
[9] ZHU L,TAO R S,WANG Y,et al. Removal of L-alanine from the production of L-2-aminobutyric acid by introduction of alanine racemase and D-amino acid oxidase[J].Applied Microbiology and Biotechnology,2011,90(3):903-910.
[10] AGER D J,LI T,PANTALEONE D P,et al. Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates[J]. Journal of Molecular Catalysis B:Enzymatic,2001,11(4-6):199-205.
[11] PARK E S,DONG G Y,SHIN J S.ω-Transaminase-catalyzed asymmetric synthesis of unnatural amino acids using isopropylamine as an amino donor[J]. Organic and biomolecular chemistry,2013,11(40):6 929-6 933.
[12] SHIN J S,KIM B G. Transaminase-catalyzed asymmetric synthesis of L-2-aminobutyric acid from achiral reactants[J]. Biotechnology Letters,2009,31(10):1 595-15 99.
[13] MALIK M S,PARK E S,SHIN J S.ω-Transaminasecatalyzed kinetic resolution of chiral amines using L-threonine as an amino acceptor precursor[J]. Green Chemistry,2012,14(8):2 137-2 140.
[14]苏金环,郭皓,曾聪明,等.生物催化一锅法制备2-氨基丁酸[J].精细化工,2012,29(9):921-924; 936.
[15] TAO R S,JIANG Y,ZHU F Y,et al. 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]. Biotechnology Letters,2014,36(4):835-841.
[16] GALKIN A,KULAKOVA L,YOSHIMURA T,et al.Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes[J]. Applied and Environmental Microbiology,1997,63(12):4 651-4 656.
[17] YASUKAWA K,HASEMI R,ASANO Y. Dynamic kineticresolution of alpha-aminonitriles to form chiral alphaamino acids[J]. Advanced Synthesis and Catalysis,2011,353(13):2 328-2 332.
[18]徐建妙,赵哲,陈策,等.甲酸脱氢酶外源表达发酵条件优化及在L-2-氨基丁酸合成中的应用[J].食品与发酵工业,2018,44(7):63-68.
[19]张玉彬.生物催化的手性合成[M].北京:化学工业出版社,2001:170-176.
[20]徐娴,贾红华,何冰芳,等.甲酸脱氢酶基因在大肠杆菌Rosetta中的高效表达[J].食品与发酵工业,2007,33(5):5-8.
[21]黄志华,刘铭,王宝光,等.甲酸脱氢酶用于辅酶NADH再生的研究进展[J].过程工程学报,2006,(6):1 011-1 016.
[22] WANG Y H,JING C F,YANG B,et a1. Production of a new sea anemone neurotoxin by recombinant Escherichia coli:optimization of culture conditions using response surface methodology[J]. Process Biochem,2005,40(8):2 721-2 728.
[23] XU G,JIANG Y,TAO R S. A recyclable biotransformation system for L-2-aminobutyric acid production based on immobilized enzyme technology[J]. Biotechnol Lett,2015,38(1):123-129.
[24]余龙,陈寅,周丽,等.双酶偶联催化马来酸生成L-天冬氨酸[J].食品与发酵工业,2018,44(8):20-26.
[25] PARK E,KIM M,SHINA J S. One-pot conversion of Lthreonine into L-homoalanine:biocatalytic production of an unnatural amino acid from a natural one[J]. Adv Synth Catal,2010,352(18):3 391-3 398.
[26] ANSORGE M B,KULA M R. Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli(p IET98)[J]. Biotechnol Bioeng,2000,68(5):557-562.
[27]刘维明,杨震炯,罗积杏,等.重组大肠杆菌共表达亮氨酸脱氢酶和甲酸脱氢酶发酵条件优化[J].生物加工过程,2015,13(4):23-28.
[28]刘珊,李元,祝俊.偶联基于酮还原酶的NADH再生系统一锅法酶催化合成L-2-氨基丁酸[J].中国生物工程杂志,2017,37(1):64-70.
[29]郭红颜,梁蕊,李航,等.用于合成L-2-氨基丁酸基因工程菌的构建[J].中国医药工业杂志,2015,46(10):1 076-1 079.