α-酮戊二酸依赖型双加氧酶催化特性及反应耦联辅因子对其催化羟基化反应的影响
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摘要
【目的】以重组大肠杆菌表达的枯草芽孢杆菌(Bacillus subtilis)L-异亮氨酸双加氧酶(L-isoleucine dioxygenase,IDO)为研究对象,考察其催化L-异亮氨酸(L-Ile)羟基化反应的影响因素,构建IDO催化合成羟基氨基酸的反应体系。【方法】通过Ni-NTA亲和层析法从重组大肠杆菌(Escherichia coli)BL21/p ET28a-ido中纯化获得重组IDO,以L-Ile为底物,考察重组IDO催化羟基化反应的影响因素,并进一步针对耦联反应优化α-酮戊二酸(α-KG)在重组IDO酶促转化体系中的添加浓度。【结果】基于重组IDO催化L-Ile羟基化的活性测定,计算该酶Km为0.247 mmol/L,kcat为1.260 s-1,kcat/Km为5.101 L/(mmol·s),与其他同源酶动力学参数比较分析表明,重组IDO的底物亲和性及催化效率较高。重组IDO催化反应的最适温度为20°C、最适p H为7.0;在35°C以下较为稳定;反应体系中Fe2+最适浓度为1 mmol/L。重组IDO可催化不同L-氨基酸反应,对L-异亮氨酸、L-正亮氨酸、L-甲硫氨酸的活性较高。通过优化α-KG浓度,反应体系中添加30 mmol/Lα-KG时,可将底物浓度提高至70 mmol/L,产物4-羟基异亮氨酸(4-HIL)的摩尔产率达66.20%,表明α-KG作为反应耦联辅因子,其浓度对重组IDO催化L-Ile羟基化具有显著影响。【结论】重组IDO的底物亲和性、催化效率、最适催化条件、稳定性等基本性质有利于催化L-Ile羟基化反应。在其催化反应体系中,α-KG作为反应耦联辅因子,对酶促转化效果影响显著。研究结果为4-HIL及其他羟基氨基酸的酶促转化提供了研究基础。
[Objective] In order to construct a biocatalytic system to synthesize 4-hydroxyl isoleucine(4-HIL), L-isoleucine(L-Ile) dioxygenase(IDO) from Bacillus subtilis expressed in recombinant Escherichia coli was purified and characterized. [Methods] The recombinant IDO was purified by Ni-NTA affinity chromatography from recombinant Escherichia coli BL21/p ET28a-ido. The enzyme was the characterized by using L-Ile as the substrate. As the necessary cofactor of IDO, the concentration of α-ketoglutaric acid(α-KG) in the enzymatic system was further optimized to improve the catalytic efficiency. [Results] Kinetic parameters of the enzyme were obtained as Km0.247 mmol/L, kcat 1.260 s-1, and kcat/Km 5.101 L/(mmol·s). Compared with other homologous enzymes, the recombinant IDO had higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 °C and p H 7.0, and more stable at the temperatures below 35 °C. The optimal concentration of Fe~(2+) was 1 mmol/L in the catalytic system. The recombinant IDO was active to a variety of L-amino acids, of which L-isoleucine, L-norleucine, and L-methionine were more suitable for the IDO catalyzing hydroxylation. By optimization of α-KG concentration in enzymatic catalysis, 4-HIL with the yield of 66.20% was achieved from 70 mmol/L L-Ile by adding 30 mmol/L α-KG in the reaction system. Thus, the addition of α-KG as the reaction-coupled cofactor had a significant impact on the reaction efficiency of recombinant IDO-mediated L-Ile hydroxylation. [Conclusion] This study provides the basis for enzymatic conversion of 4-HIL and other hydroxylated amino acids.
[Objective] In order to construct a biocatalytic system to synthesize 4-hydroxyl isoleucine(4-HIL), L-isoleucine(L-Ile) dioxygenase(IDO) from Bacillus subtilis expressed in recombinant Escherichia coli was purified and characterized. [Methods] The recombinant IDO was purified by Ni-NTA affinity chromatography from recombinant Escherichia coli BL21/p ET28a-ido. The enzyme was the characterized by using L-Ile as the substrate. As the necessary cofactor of IDO, the concentration of α-ketoglutaric acid(α-KG) in the enzymatic system was further optimized to improve the catalytic efficiency. [Results] Kinetic parameters of the enzyme were obtained as Km0.247 mmol/L, kcat 1.260 s-1, and kcat/Km 5.101 L/(mmol·s). Compared with other homologous enzymes, the recombinant IDO had higher substrate affinity and catalytic efficiency. The recombinant IDO was more active at 20 °C and p H 7.0, and more stable at the temperatures below 35 °C. The optimal concentration of Fe~(2+) was 1 mmol/L in the catalytic system. The recombinant IDO was active to a variety of L-amino acids, of which L-isoleucine, L-norleucine, and L-methionine were more suitable for the IDO catalyzing hydroxylation. By optimization of α-KG concentration in enzymatic catalysis, 4-HIL with the yield of 66.20% was achieved from 70 mmol/L L-Ile by adding 30 mmol/L α-KG in the reaction system. Thus, the addition of α-KG as the reaction-coupled cofactor had a significant impact on the reaction efficiency of recombinant IDO-mediated L-Ile hydroxylation. [Conclusion] This study provides the basis for enzymatic conversion of 4-HIL and other hydroxylated amino acids.
引文
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[14]Qiao YY,Fan DD,Deng JJ,et al.The purification of recombinant human-like colleagen by Ni(Ⅱ)-based immobilized metal ion affinity chromatography[J].Highlights of Sciencepaper Online,2013,6(5):402-406(in Chinese)乔媛媛,范代娣,邓建军,等.镍离子亲和层析纯化重组类人胶原蛋白[J].中国科技论文在线,2013,6(5):402-406
[15]Hibi M,Kawashima T,Kodera T,et al.Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids[J].Applied and Environmental Microbiology,2011,77(19):6926-6930
[16]Zhang CL,Liu Y,Xue N,et al.Characterization of recombinant L-isoleucine-4-hydroxylase from Bacillus thuringiensis and its application in 4-hydroxyisoleucine biosynthesis[J].Acta Microbiologica Sinica,2014,54(8):889-896(in Chinese)张成林,刘远,薛宁,等.苏云金芽孢杆菌重组L-异亮氨酸羟化酶的酶学性质及其在4-羟基异亮氨酸合成中的应用[J].微生物学报,2014,54(8):889-896
[17]Wang A,Zeng YH,Han HJ,et al.Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR:ferredoxin NADP+reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions[J].Biochemistry,2007,46(43):12198-12211
[18]Hoffart LM,Barr EW,Guyer RB,et al.Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV)complex in a prolyl-4-hydroxylase[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(40):14738-14743
[19]Ogawa J,Yamanaka H,Mano J,et al.Synthesis of4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626[J].Bioscience,Biotechnology,and Biochemistry,2007,71(7):1607-1615
[2]Liu JH,Wu XF,Li FM,et al.Recent progress in oxygenase-catalyzed biotransformations[J].Chemical Production and Technology,2006,13(4):36-39(in Chinese)刘均洪,吴小飞,李凤梅,等.加氧酶催化生物转化研究最新进展[J].化工生产与技术,2006,13(4):36-39
[3]Wang Q,Ouazzani J,Sasaki NA,et al.A practical synthesis of(2S,3R,4S)-4-hydroxyisoleucine,a potent insulinotropicα-amino acid from fenugreek[J].European Journal of Organic Chemistry,2002,2002(5):834-839
[4]Smirnov SV,Sokolov PM,Kodera T,et al.A novel family of bacterial dioxygenases that catalyse the hydroxylation of free L-amino acids[J].FEMS Microbiology Letters,2012,331(2):97-104
[5]Aouadi K,Jeanneau E,Msaddek M,et al.1,3-Dipolar cycloaddition of a chiral nitrone to(E)-1,4-dichloro-2-butene:a new efficient synthesis of(2S,3S,4R)-4-hydroxyisoleucine[J].Tetrahedron Letters,2012,53(23):2817-2821
[6]Kodera T,Smirnov SV,Samsonova NN,et al.A novel L-isoleucine hydroxylating enzyme,L-isoleucine dioxygenase from Bacillus thuringiensis,produces(2S,3R,4S)-4-hydroxyisoleucine[J].Biochemical and Biophysical Research Communications,2009,390(3):506-510
[7]Smirnov SV,Kodera T,Samsonova NN,et al.Metabolic engineering of Escherichia coli to produce(2S,3R,4S)-4-hydroxyisoleucine[J].Applied Microbiology and Biotechnology,2010,88(3):719-726
[8]HaefeléC,Bonfils C,Sauvaire Y.Characterization of a dioxygenase from Trigonella foenum-graecum involved in4-hydroxyisoleucine biosynthesis[J].Phytochemistry,1997,44(4):563-566
[9]Fowden L,Pratt HM,Smith A.4-Hydroxyisoleucine from seed of Trigonella foenum-graecum[J].Phytochemistry,1973,12(7):1707-1711
[10]Hausinger RP.Fe(II)/α-ketoglutarate-dependent hydroxylases and related enzymes[J].Critical Reviews in Biochemistry and Molecular Biology,2004,39(1):21-68
[11]Ogawa J,Kodera T,Smirnov SV,et al.A novel L-isoleucine metabolism in Bacillus thuringiensis generating(2S,3R,4S)-4-hydroxyisoleucine,a potential insulinotropic and anti-obesity amino acid[J].Applied Microbiology and Biotechnology,2011,89(6):1929-1938
[12]Smirnov SV,Samsonova NN,Novikova AE,et al.A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine:identification of an enzyme possessing HMKP(4-hydroxy-3-methyl-2-keto-pentanoate)aldolase activity[J].FEMS Microbiology Letters,2007,273(1):70-77
[13]Fu MJ,Nie Y,Mu XQ,et al.A novel isoleucine dioxygenase and its expression in recombinant Escherichia coli for synthesis of4-hydroxyisoleucine[J].Chemical Industry and Engineering Progress,2014,33(11):3037-3044(in Chinese)付敏杰,聂尧,穆晓清,等.新型异亮氨酸双加氧酶及其重组大肠杆菌合成羟基异亮氨酸[J].化工进展,2014,33(11):3037-3044
[14]Qiao YY,Fan DD,Deng JJ,et al.The purification of recombinant human-like colleagen by Ni(Ⅱ)-based immobilized metal ion affinity chromatography[J].Highlights of Sciencepaper Online,2013,6(5):402-406(in Chinese)乔媛媛,范代娣,邓建军,等.镍离子亲和层析纯化重组类人胶原蛋白[J].中国科技论文在线,2013,6(5):402-406
[15]Hibi M,Kawashima T,Kodera T,et al.Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids[J].Applied and Environmental Microbiology,2011,77(19):6926-6930
[16]Zhang CL,Liu Y,Xue N,et al.Characterization of recombinant L-isoleucine-4-hydroxylase from Bacillus thuringiensis and its application in 4-hydroxyisoleucine biosynthesis[J].Acta Microbiologica Sinica,2014,54(8):889-896(in Chinese)张成林,刘远,薛宁,等.苏云金芽孢杆菌重组L-异亮氨酸羟化酶的酶学性质及其在4-羟基异亮氨酸合成中的应用[J].微生物学报,2014,54(8):889-896
[17]Wang A,Zeng YH,Han HJ,et al.Biochemical and structural characterization of Pseudomonas aeruginosa Bfd and FPR:ferredoxin NADP+reductase and not ferredoxin is the redox partner of heme oxygenase under iron-starvation conditions[J].Biochemistry,2007,46(43):12198-12211
[18]Hoffart LM,Barr EW,Guyer RB,et al.Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV)complex in a prolyl-4-hydroxylase[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(40):14738-14743
[19]Ogawa J,Yamanaka H,Mano J,et al.Synthesis of4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626[J].Bioscience,Biotechnology,and Biochemistry,2007,71(7):1607-1615