Genome mining of organic solvent tolerant amino acid dehydrogenase for biosynthesis of unnatural amino acids in non-aqueous system

doi:10.11949/0438-1157.20201841

Abstract

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

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

CLC Number:

TQ 028.8

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.

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.

Figures/Tables 17

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

Fig.1 Phylogenetic analysis of genetic relationship among screened AaDHs

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

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)

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

Fig.4 Docking of NT2349 with L- homophenylalanine

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

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

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

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

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

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

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

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

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

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

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

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