亮氨酸脱氢酶催化底物偶联法合成α-酮异己酸

doi:10.7503/cjcu20180698

摘要/Abstract

摘要：

构建了亮氨酸脱氢酶(LeuDH)催化的底物偶联反应体系, 打破氧化脱氨反应平衡, 同时制得高附加值的α-酮异己酸(α-KIC)和L-2-氨基丁酸, 并实现辅酶NAD⁺的高效循环再生. 基于LeuDH的底物专一性和催化动力学参数, 考察了不同酮酸底物对于底物偶联反应效率的影响, 选择转化率最高的2-丁酮酸作为偶联底物, 使α-KIC产率由单步氧化反应的2.75%提高至66.82%. 通过考察底物浓度、 pH值、 N $H 4 +$ 浓度和辅酶NAD⁺浓度等反应条件对偶联反应效率的影响, 使α-KIC产率进一步提高至83.25%, 同时辅酶NAD⁺的总转化数(TTN)达到5.88×10⁵. 通过改变底物L-亮氨酸和2-丁酮酸的摩尔比, 能够将α-KIC的产率进一步提高至92.74%.

关键词: 亮氨酸脱氢酶, 底物偶联, 辅酶再生, α-酮异己酸;, L-2-氨基丁酸

Abstract:

In order to shift the balance of oxidative deamination, the substrate coupling reaction system catalyzed byleucine dehydrogenase(LeuDH) was constructed. The α-KIC and L-2-aminobutyrate with high valuable were synthesized. Meanwhile, the coenzyme was efficiently regenerated. Based on the studies of the substrates specificity and kinetic parameters of LeuDH, the efficiency of substrate coupling reaction was compared using a series of ketonic acids. The result showed that 2-oxobutyrate could be used as the best coupling substrate. The yield of α-KIC was improved from 2.75% of the single-step oxidation reaction to 66.82% of the coupling reaction. The reaction conditions were optimized, including pH values, concentrations of substrate, N $H 4 +$ and coenzyme. The yield of α-KIC and the total turnover number(TTN) of ketonic acid reached 83.25% and 5.88×10⁵, respectively. By adapting the molar ratios of the L-leucine and 2-oxobutyrate, the yield of α-KIC was increased to 92.74%. The substrate coupling reaction system was suggested to be the effective way for the production of α-KIC.

Key words: Leucine dehydrogenase, Substrate coupling, Coenzyme regeneration, α-Ketoisocaprate;, L-2-Aminobutyrate

中图分类号:

O621.25⁺4

TrendMD:

丰险, 穆晓清, 聂尧, 徐岩. 亮氨酸脱氢酶催化底物偶联法合成α-酮异己酸. 高等学校化学学报, 2019, 40(4): 698.

FENG Xian,MU Xiaoqing,NIE Yao,XU Yan. Synthesis of α-Ketoisocaproate Through Substrate Coupling Reaction Catalyzed by Leucine Dehydrogenase^†. Chem. J. Chinese Universities, 2019, 40(4): 698.

图/表 11

Scheme 1 Schematic illustration of substrate coupling reaction catalyzed by LeuDH for production of α-KIC and L-2-aminobutyrate

Fig.1 Time curves of oxidation of L-leucine and reduction of α-KIC catalyzed by LeuDHAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5). c(α-KIC)=10 mmol/L, c(L-Leucine)=10 mmol/L.

Table 1 Enzymatic oxidation and reduction properties of LeuDH for L-leucine and α-KIC

Substrate	Structural formula	Specific activity/ (U·mg^-1)	V_m/ (mol·min^-1·mg^-1)	K_m/ (mmol·L^-1)	(k_cat/K_m)/ (L·mmol^-1·s^-1)
α-KIC		11.14	695.4	2.38	26.14
L-Leucine		1.66	2.75	24.56	0.012

Fig.2 Effects of coupling substrates to the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising amino acid(10 mmol/L), ketonic acid(10 mmol/L) and NAD+(0.2 mmol/L). a. Trimethylpyruvic acid; b. 3-methyl-2-oxopentanoic acid; c. 2-oxobutyrate; d. pyruvic acid.

Table 2 Enzymatic oxidation and reduction properties of LeuDH for different kinds of amino acids and ketonic acids

Substrate			Yield(%)
2-Oxobutyrate	21.32	64.22	73.73
L-2-aminobutyrate	0.61	0.01	1.14
α-KIC	52.77	143.69	66.82
L-valine	0.71	0.03	2.94
α-Ketoisocaproate	11.14	26.14	70.36
L-leucine	1.66	0.01	2.09
Trimethylpyruvic acid	7.99	1.84	65.46
L-tert-leucine	——	——	——

Fig.3 Effects of initial NAD+ concentration on TTN and the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+.

Fig.4 Effects of NH4+ concentration on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+(0.01 μmol/L).

Fig.5 Effects of pH value on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate(10 mmol/L) and NAD+(0.01 μmol/L).

Fig.6 Effects of substrate concentration on the yield of α-KIC All reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine, 2-oxobutyrate and NAD+(0.01 μmol/L).

Fig.7 Time curves of the substrate coupling reaction systemAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(100 mmol/L), 2-oxobutyrate(100 mmol/L) and NAD+ (0.01 μmol/L).

Fig.8 Effects of the molar ratios of L-leucine and 2-oxobutyrate on the yield of α-KICAll reactions were carried out in 2 mL Tris-HCl buffer(0.1 mol/L, pH=8.5) comprising L-leucine(10 mmol/L), 2-oxobutyrate and NAD+(0.01 μmol/L).

参考文献 39

[1]	Zhou Y. S., Jetton T. L., Goshorn S., Lynch C. J., She P. X., J. Biol.Chem.,2010, 285(44), 33718—33726
[2]	Yang J. C., Chi Y. J., Burkhardt B. R., Guan Y. F., Wolf B. A., Nutr.Rev.,2010, 68(5), 270—279
[3]	Taschetto L., Scaini G., Zapelini H. G., Ramos Â. C., Strapazzon G., Andrade V. M., Réus G. Z., Michels M., Dal-Pizzol F., Quevedo J.,Metab. Brain Dis.,2017, 32(5), 1—12
[4]	Heissig H., Urban K. A., Hastedt K., Zünkler B. J., Panten U., Mol.Pharmacol.,2005, 68(4), 1097—1105
[5]	Wisniewski M. S. W., Carvalho-Silva M., Gomes L. M., Zapelini H. G., Schuck P. F., Ferreira G. C., Scaini G., Streck E. L., Metab. Brain Dis.,2016, 31(2), 377—383
[6]	Girón M. D., Vílchez J. D., Rafael S., Manuel M., Natalia S., Nefertiti C., Argilés J. M., Ricardo R., López-Pedrosa J. M., J. Cachexia Sarcopeni.,2016, 7(1), 68—78
[7]	van Someren K. A., Edwards A. J., Howatson G., Int. J. Sport Nutr. Exerc. Metab.,2005, 15(4), 413—424
[8]	Escobar J., Frank J. W., Suryawan A., Nguyen H. V., van Horn C. G., Hutson S. M., Davis T. A., J.Nutr.,2010, 140(8), 1418—1424
[9]	Ogo S., Uehara K., Abura T., Fukuzumi S., J. Am. Chem.Soc.,2004, 126(10), 3020—3021
[10]	Hidalgo F. J., Delgado R. M., Zamora R.,Food Chem.,2013, 141(2), 1140—1146
[11]	Aparicio M., Bellizzi V., Chauveau P., Cupisti A., Ecder T., Fouque D., Garneata L., Lin S., Mitch W. E., Teplan V., J. Ren.Nutr.,2012, 22(2), S1—S21
[12]	Mou S., Li J., Yu Z., Wang Q., Ni Z., J. Int. Med. Res. ,2013, 41(1), 129—137
[13]	Holecek M., Nutr., 2010, 26(5), 482—490
[14]	Norton L. E., Wilson G. J., Layman D. K., Moulton C. J., Garlick P. J., Nutr.Metab.,2012, 9(1), 67—76
[15]	Cann A. F., Liao J. C., Appl. Microbiol.Biotechnol.,2010, 85(4), 893—899
[16]	Freiding S., Ehrmann M. A., Vogel R. F., Food Microbiol.,2012, 29(2), 205—214
[17]	Geueke B., Hummel W., Enzyme Microb.Tech.,2002, 31(1/2), 77—87
[18]	Pollegioni L., Molla G., Sacchi S., Rosini E., Verga R., Pilone M. S., Appl. Microbiol.Biotechnol.,2008, 78(1), 1—16
[19]	Du X. Y., Clemetson K. J., Toxicon,2002, 40(6), 659—665
[20]	Baud D., Jeffries J. W. E., Moody T. S., Ward J. M., Hailes H. C., Green Chem.,2017, 19(4), 1134—1143
[21]	Jin J. Z., Chang D. L., Zhang J., Appl. Biochem.Biotechnol.,2011, 164(3), 376—385
[22]	Farnberger J. E., Lorenz E., Richter N., Wendisch V. F., Kroutil W., Microb. Cell Fact.,2017, 16(1), 132—148
[23]	Chen X., Gao X. Z., Zhu D. M., Acta Microbiol.Sinica,2017, 57(8), 1249—1261
	(陈曦, 高秀珍, 朱敦明. 微生物学报, 2017, 57(8), 1249—1261)
[24]	Xue Y. P., Cao C. H., Zheng Y. G., Chem. Soc.Rev.,2018, 47(4), 1516—1561
[25]	Katoh R., Ngata S., Ozawa A., Ohshima T., Kamekura M., Misono H., J. Mol. Catal. B: Enzym.,2003, 23(2—6), 231—238
[26]	Zhu D. M., Hua L., J.Biotechnol.(,2009, 410), 1420—1431
[27]	Miao W. J., Wang P., Zhang S. P., Chinese J. Proc.Eng.,2008, 8(1), 102—108
	(苗维娟, 王平, 张松平. 过程工程学报, 2008, 8(1), 102—108)
[28]	Seelbach K., Riebel B., Hummel W., Kula M. R., Tishkov V. I., Egorov A. M., Wandrey C., Kragl U., Tetrahedron Lett.,1996, 37(9), 1377—1380
[29]	Kara S., Spickermann D., Schrittwieser J. H., Leggewie C., Berkel W. J. H. V., Arends I. W. C. E., Hollmann F., Green Chem.,2013, 15(2), 330—335
[30]	Lu J., Zhang Y., Sun D., Jiang W., Wang S., Fang B., Appl. Biochem.Biotechnol.,2016, 180(6), 1180—1195
[31]	Kajiwara S., Maeda H., Agric. Biol.Chem.,2006, 51(11), 2873—2879
[32]	Katoh R., Nagata S., Misono H., J. Mol. Catal. B: Enzym.,2003, 23(2—6), 239—247
[33]	Akhteruzzaman S., Kato Y., Kouzuki H., Suzuki H., Hisaka A., Stieger B., Meier P. J., Sugiyama Y., Phramacol. Exp.Ther.,1999, 290(3), 1107—1115
[34]	Li J., Genome Data Mining of Leucine Dehydrogenase and Its Catalytic Performance in Reductive Amination of Trimethylpyruvic Acid to L-tert-Leucine, East China University of Science and Technology, Guangzhou, 2014
	(李静. 亮氨酸脱氢酶的基因发掘、催化性能及其应用研究,广州: 华南理工大学, 2014)
[35]	Wa V. D. D., Zhao H., Curr. Opin.Biotechnol.,2003, 14(4), 421—426
[36]	Chenault H. K., Whitesides G. M., Appl. Biochem.Biotechnol.,1987, 14(2), 147—197
[37]	Sun T. Q., Li B., Nie Y., Wang D., Xu Y., Chem. J. Chinese Universities,2017, 38(10), 1772—1777
	(孙太强, 李斌, 聂尧, 王栋, 徐岩. 高等学校化学学报, 2017, 38(10), 1772—1777)
[38]	Xu C., Yin X., Zhang C., Chen H., Huang H., Hu Y., Chem. Res. Chinese Universities,2018, 34(2), 279—284
[39]	Zhang J. L., Tao S. S., Zhang B. J., Wu X. R., Chen Y. J., ACS Catal.,2014, 4(5), 1584—1587