耐有机溶剂氨基酸脱氢酶基因挖掘与非天然氨基酸的非水相合成

doi:10.11949/0438-1157.20201841

摘要/Abstract

摘要：

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。基于基因挖掘技术，获得了17个耐盐氨基酸脱氢酶的基因，并分析了其进化同源性和蛋白质稳定性热力学参数。选取来源于Natranaerobius thermophilus的苯丙氨酸脱氢酶（PheDH），进行了基因合成和表达、分离和纯化，获得了耐盐氨基酸脱氢酶，并检测了其有机溶剂耐受性。结果表明，对于催化L-苯丙氨酸的氧化脱氨体系，反应的最适温度为60℃，最适pH为 12。在含有30%的二甲亚砜反应体系中，催化活性是水相体系的1.2倍。而对于催化还原胺化制备L-高苯丙氨酸的体系，最适温度为70℃，最适pH为8.5。在含30%的甲基叔丁基醚和二甲亚砜反应体系中，催化活性分别是原始活性的101.3%和99.2%。研究表明，该耐盐酶具有较好的耐热、耐有机溶剂等抗逆性能。

关键词: 酶, 生物催化, 蛋白质稳定性, 基因挖掘, 有机溶剂耐受性

Abstract:

The oxidoreductase, which can efficiently catalyze asymmetric reduction reactions to prepare chiral compounds in non-aqueous systems, has important scientific significance and industrial application prospects. Based on gene mining technology, 17 salt-tolerant amino acid dehydrogenase genes were obtained, and their evolutionary homology and protein stability thermodynamic parameters were further analyzed. Bioinformatic identification combined with structure classification and thermodynamic parameters calculation led to a promising phenylalanine dehydrogenase from Natranaerobius thermophiles. Catalytic characteristics of phenylalanine dehydrogenase in the non-aqueous system were studied. This PheDH was highly stable for oxidative deamination of phenylalanine with optimal temperature of 60℃ and optimal pH of 12, which is rarely reported in such alkaline environment. Enzyme activity was enhanced by 1.2 folds with 30% dimethylsulfoxide. For biosynthesis of L-homophenylalanine by reductive ammoniation,the optimal reaction condition is 70℃ and pH 8.5. With 30% methyl tert-butyl ether and dimethylsulfoxide,the relative activity is 101.3% and 99.2%, respectively. The results indicated that this halotolerant PheDH has better resistance to heat and organic solvents. This paper offers a novel strategy for mining of halotolerant amino acid dehydrogenase based on sequenced-driven approaches, and thus provides robust key enzymes to biochemists.

Key words: enzyme, biocatalysis, protein stability, genome mining, organic solvent tolerance

中图分类号:

TQ 028.8

段凌暄, 姚光晓, 江亮, 王世珍. 耐有机溶剂氨基酸脱氢酶基因挖掘与非天然氨基酸的非水相合成[J]. 化工学报, 2021, 72(7): 3757-3767.

DUAN Lingxuan, YAO Guangxiao, JIANG Liang, WANG Shizhen. Genome mining of organic solvent tolerant amino acid dehydrogenase for biosynthesis of unnatural amino acids in non-aqueous system[J]. CIESC Journal, 2021, 72(7): 3757-3767.

图/表 17

表1 筛选获得的来源于极端微生物的氨基酸脱氢酶

Table 1 Screening of amino acid dehydrogenases from extremophiles

分类	菌株名称	分类	菌株名称
嗜热	Geobacillus kaustophilus	嗜盐	Halobacterium salinarum
	Natranaerobius thermophilus		Haloferax volcanii
	Methanococcus jannaschii		Haloferax lucentense
	Thermotoga maritima		Haloarcula japonica
	Geobacillus stearothermophilus 10		Halogranum rubrum
	Sulfobacillus thermosulfidooxidans		Halogeometyicum borinquense
嗜热嗜碱	Anaerobranca gottschalkii	嗜盐嗜碱	Euhalothece natronophila
嗜热嗜碱	Halothermothrix orenii	嗜盐嗜碱	Ferroplasma acidiphilum

图1 筛选获得极端微生物的氨基酸脱氢酶的亲缘关系分析图

Fig.1 Phylogenetic analysis of genetic relationship among screened AaDHs

表2 不同极端菌株来源的氨基酸脱氢酶的热力学参数比较

Table 2 Comparison of thermodynamic parameters of amino acid dehydrogenases from extremophiles

Number	Average value	ΔH_m/(kcal/mol)	ΔC_p/(kcal/(mol·K))	T_m/℃	ΔG_r/(kcal/mol)
1	KJE28589.1	-144.7	-4.02	67.1	-7
2	WP-010871127.1	-127.1	-3.65	65	-6
3	WP-091347685.1	-138	-3.6	71.2	-6.8
4	WP-004590695.1	-154	-4.36	65.9	-7.4
5	ALA71326.1	-118.5	-3.49	63.4	-5.6
6	PSR36482.1	-128	-3.42	69.6	-6.3
7	WP-012635747.1	-95.9	-2.71	66	-4.6
8	NT2349	-113.3	-3.34	63.5	-5.3

图2 NT2349的结构和表面电荷分布和带电性能图(a) NT2349的三维结构; (b) 酸性和碱性氨基酸残基分布(红色代表酸性氨基酸,蓝色代表碱性氨基酸); (c) NT2349的表面电荷分布(红色代表负电荷,蓝色代表正电荷)

Fig.2 Structure of NT2349 and surface charged residues distribution(a) NT2349 three-dimensional structure; (b) acidic and basic amino acid residue distribution (red represents acidic amino acids, blue represents basic amino acids); (c) the surface charge distribution of NT2349 (red represents negative charge, blue represents positive charge)

图3 NT2349与底物的二级结构相互作用图(a) NT2349的氨基酸残基与苯丙氨酸相互作用图;(b) NT2349活性位点与苯丙氨酸相互作用的Heatmap图

Fig.3 Interactions of secondary structure of NT2349 with L-phenylalanine(a) key residues interact with L-phenylalanine; (b) interaction Heatmap of active site with L-phenylalanine

图4 NT2349与高苯丙氨酸对接结果(a) NT2349与高苯丙氨酸的对接;(b) 3D相互作用力;(c) 2D相互作用力示意图

Fig.4 Docking of NT2349 with L- homophenylalanine

图5 AaDH 催化L-苯丙氨酸(L-Phe)的氧化脱氨反应

Fig.5 AaDH catalyzes the oxidative deamination of L- phenyalanine

图6 温度对NT2349的氧化脱氨活性影响Reaction conditions: L-Phe, 2 mmol/L; glycine-NaOH buffer, 160 mmol/L (pH 10); NAD+, 0.2 mmol/L

Fig.6 Effect of temperature on oxidative deamination activity of NT2349

图7 pH对NT2349的氧化脱氨活性影响Reaction conditions: L-Phe, 2 mmol/L; 0.2 mol/L potassium phosphate buffer, pH 5—6; 0.2 mol/L Tris-HCl buffer, pH 7—8; 0.2mol/L glycine-NaOH buffer, pH 9—13.6 ;NAD+, 0.2 mmol/L

Fig.7 The effect of pH on oxidative deamination activity of NT2349

图8 有机溶剂对NT2349的氧化脱氨反应酶活影响Reaction conditions: L-Phe, 2 mmol/L; glycine-NaOH buffer, 160 mmol/L (pH 10); NAD+, 0.2 mmol/L

Fig.8 Effect of organic solvent on oxidative deamination activity of NT2349

图9 有机溶剂体系中酶催化氧化脱氨的酶活稳定性Reaction conditions: L-Phe, 2 mmol/L; glycine-NaOH buffer, 160 mmol/L (pH 10); NAD+,0.2 mmol/L

Fig.9 Enzyme activity stability of oxidative deamination of NT2349 in organic solvents

图10 NT2349 催化EOPB的还原胺化反应制备高苯丙氨酸

Fig.10 NT2349 catalyzes the synthesis of L-homophenylalanine by reductive amination of EOPB

图11 温度对NT2349的还原胺化活性影响Reaction conditions: EOPB, 2 mmol/L; NH4Cl-NH3·H2O buffer, 160 mmol/L (pH 8.5); NADH 0.2 mmol/L

Fig.11 The effect of temperature on the reductive amination activity of NT2349

图12 pH对NT2349的还原胺化活性影响Reaction conditions: EOPB, 2 mmol/L; NH4Cl-NH3·H2O buffer, 160 mmol/L (pH 7.5—11); NADH 0.2 mmol/L

Fig.12 The effect of pH on the reductive amination activity of NT2349

图13 有机溶剂对NT2349的还原胺化反应酶活影响Reaction conditions: EOPB, 2 mmol/L; NH4Cl-NH3·H2O buffer, 160 mmol/L (pH 8.5); NADH 0.2 mmol/L

Fig.13 Effect of organic solvent on the activity of reductive ammoniation reactivity

图14 有机溶剂中NT2349催化还原胺化的酶活稳定性Reaction conditions: EOPB, 2 mmol/L; NH4Cl-NH3·H2O buffer, 160 mmol/L (pH 8.5); NADH, 0.2 mmol/L

Fig.14 Enzyme activity stability of NT2349 reductive amination in organic solvents

图15 NT2349在30%有机溶剂下的圆二色谱图(a)乙醇; (b)正丁醇; (c)环己烷; (d)甲基叔丁基醚

Fig.15 Circular dichroism spectra of NT2349 in 30% organic solvents(a) alcohol; (b) n-butyl alcohol; (c) cyclohexane; (d) MTBE

参考文献 31

1	Liszka M J, Clark M E, Schneider E, et al. Nature versus nurture: developing enzymes that function under extreme conditions[J]. Annual Review of Chemical and Biomolecular Engineering, 2012, 3: 77-102.
2	Chen F F, Zheng G W, Liu L, et al. Reshaping the active pocket of amine dehydrogenases for asymmetric synthesis of bulky aliphatic amines[J]. ACS Catalysis, 2018, 8(3): 2622-2628.
3	Dhake K P, Thakare D D, Bhanage B M. Lipase: a potential biocatalyst for the synthesis of valuable flavour and fragrance ester compounds[J]. Flavour and Fragrance Journal, 2013, 28(2): 71-83.
4	DasSarma S, DasSarma P. Halophiles and their enzymes: negativity put to good use[J]. Current Opinion in Microbiology, 2015, 25: 120-126.
5	Chen S, Engel P. An engineered mutant, L307V of phenylalanine dehydrogenase from Bacillus sphaericus: high activity and stability in organic-aqueous solvent mixtures and utility for synthesis of non-natural L-amino acids [J]. Journal of Biotechnology,2007, 131(2): 116.
6	Munawar N, Engel P C. Overexpression in a non-native halophilic host and biotechnological potential of NAD⁺-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014[J]. Extremophiles, 2012, 16(3): 463-476.
7	Alsafadi D, Paradisi F. Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii[J]. Extremophiles, 2013, 17(1): 115-122.
8	Munawar N, Engel P C. Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase[J]. Extremophiles, 2013, 17(1): 43-51.
9	Timpson L M, Alsafadi D, Mac Donnchadha C, et al. Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea[J]. Extremophiles, 2012, 16(1): 57-66.
10	Hartmann E M, Durighello E, Pible O, et al. Proteomics meets blue biotechnology: a wealth of novelties and opportunities[J]. Marine Genomics, 2014, 17: 35-42.
11	Nguyen V, Wilson C, Hoemberger M, et al. Evolutionary drivers of thermoadaptation in enzyme catalysis[J]. Science, 2017, 355(6322): 289-294.
12	Broom A, Trainor K, MacKenzie D W, et al. Using natural sequences and modularity to design common and novel protein topologies[J]. Current Opinion in Structural Biology, 2016, 38: 26-36.
13	Wang H Y, Qu G, Li J K, et al. Data mining of amine dehydrogenases for the synthesis of enantiopure amino alcohols[J]. Catalysis Science & Technology, 2020, 10(17): 5945-5952.
14	Dalmaso G Z, Ferreira D, Vermelho A B. Marine extremophiles: a source of hydrolases for biotechnological applications[J]. Marine Drugs, 2015, 13(4): 1925-1965.
15	Trincone A. Biocatalytic processes using marine biocatalysts: ten cases in point[J]. Current Organic Chemistry, 2013, 17(10): 1058-1066.
16	Mutti F G, Knaus T, Scrutton N S, et al. Conversion of alcohols to enantiopure amines through dual-enzyme hydrogen-borrowing cascades[J]. Science, 2015, 349(6255): 1525-1529.
17	Ducrot L, Bennett M, Grogan G, et al. NAD(P)H-dependent enzymes for reductive amination: active site description and carbonyl-containing compound spectrum[J]. Advanced Synthesis & Catalysis, 2021, 363(2): 328-351.
18	Liu Z N, Lei D W, Qiao B, et al. Integrative biosynthetic gene cluster mining to optimize a metabolic pathway to efficiently produce L-homophenylalanine in Escherichia coli[J]. ACS Synthetic Biology, 2020, 9(11): 2943-2954.
19	陈曦, 高秀珍, 朱敦明. 氨基酸脱氢酶的催化机理、分子改造及合成应用[J]. 微生物学报, 2017, 57(8): 1249-1261.
	Chen X, Gao X Z, Zhu D M. Catalysis, engineering and application of amino acid dehydrogenases[J]. Acta Microbiologica Sinica, 2017, 57(8): 1249-1261.
20	Vallenet D, Calteau A, Cruveiller S, et al. MicroScope in 2017: an expanding and evolving integrated resource for community expertise of microbial genomes[J]. Nucleic Acids Research, 2017, 45(D1): D517-D528.
21	Schaeffer D, Grishin N V. Identification of protein homologs and domain boundaries by iterative sequence alignment [J]. Computational Methods in Protein Evolution, 2019, 1851: 277-286.
22	Yang J Y, Zhang Y. I-TASSER server: new development for protein structure and function predictions[J]. Nucleic Acids Research, 2015, 43(W1): W174-W181.
23	Studer G, Rempfer C, Waterhouse A M, et al. QMEANDisCo—distance constraints applied on model quality estimation[J]. Bioinformatics, 2020, 36(6): 1765-1771.
24	Kikani B A, Singh S P. The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium, Anoxybacillus beppuensis TSSC-1[J]. Process Biochemistry, 2012, 47(12): 1791-1798.
25	Lin P, Zhang Y H, Yao G X, et al. Immobilization of formate dehydrogenase on polyethylenimine-grafted graphene oxide with kinetics and stability study[J]. Engineering in Life Sciences, 2020, 20(3/4): 104-111.
26	石云云, 李信志, 张桂敏. 微生物嗜盐酶的研究进展[J]. 微生物学报, 2017, 57(8): 1180-1188.
	Shi Y Y, Li X Z, Zhang G M. Advances in microbial halophilic enzymes[J]. Acta Microbiologica Sinica, 2017, 57(8): 1180-1188.
27	Kayikci M, Venkatakrishnan A J, Scott-Brown J, et al. Visualization and analysis of non-covalent contacts using the Protein Contacts Atlas[J]. Nature Structural & Molecular Biology, 2018, 25(2): 185-194.
28	Xu L S, Wang Z Y, Mao P T, et al. Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase[J]. Bioresource Technology, 2013, 133: 635-637.
29	Dumorné K, Córdova D C, Astorga-Eló M, et al. Extremozymes: a potential source for industrial applications[J]. Journal of Microbiology and Biotechnology, 2017, 27(4): 649-659.
30	Raddadi N, Cherif A, Daffonchio D, et al. Biotechnological applications of extremophiles, extremozymes and extremolytes[J]. Applied Microbiology and Biotechnology, 2015, 99(19): 7907-7913.
31	Jin M, Gai Y, Guo X, et al. Properties and applications of extremozymes from deep-sea extremophilic microorganisms: a mini review[J]. Marine Drugs, 2019, 17(12): 656.

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