化工学报 ›› 2021, Vol. 72 ›› Issue (7): 3757-3767.doi: 10.11949/0438-1157.20201841

• 生物化学工程与技术 • 上一篇    下一篇

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

段凌暄(),姚光晓,江亮,王世珍()   

  1. 厦门大学化学化工学院化学工程与生物工程系,福建 厦门 361005
  • 收稿日期:2020-12-16 修回日期:2021-01-27 出版日期:2021-07-05 发布日期:2021-07-05
  • 通讯作者: 王世珍 E-mail:duanlingxuan@126.com;szwang@xmu.edu.cn
  • 作者简介:段凌暄(1998—),女,硕士研究生,duanlingxuan@126.com
  • 基金资助:
    国家自然科学基金面上项目(21776233);福建省自然科学基金面上项目(2018J01013);中央高校基本科研业务费专项资金(20720200038)

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

DUAN Lingxuan(),YAO Guangxiao,JIANG Liang,WANG Shizhen()   

  1. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China
  • Received:2020-12-16 Revised:2021-01-27 Published:2021-07-05 Online:2021-07-05
  • Contact: WANG Shizhen E-mail:duanlingxuan@126.com;szwang@xmu.edu.cn

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

表1

筛选获得的来源于极端微生物的氨基酸脱氢酶"

分类菌株名称分类菌株名称
嗜热Geobacillus kaustophilus嗜盐Halobacterium salinarum
Natranaerobius thermophilusHaloferax volcanii
Methanococcus jannaschiiHaloferax lucentense
Thermotoga maritimaHaloarcula japonica
Geobacillus stearothermophilus 10Halogranum rubrum
Sulfobacillus thermosulfidooxidansHalogeometyicum borinquense
嗜热嗜碱Anaerobranca gottschalkii嗜盐嗜碱Euhalothece natronophila
Halothermothrix oreniiFerroplasma 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"

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"

NumberAverage valueΔHm/(kcal/mol)ΔCp/(kcal/(mol·K))Tm/℃ΔGr/(kcal/mol)
1KJE28589.1-144.7-4.0267.1-7
2WP-010871127.1-127.1-3.6565-6
3WP-091347685.1-138-3.671.2-6.8
4WP-004590695.1-154-4.3665.9-7.4
5ALA71326.1-118.5-3.4963.4-5.6
6PSR36482.1-128-3.4269.6-6.3
7WP-012635747.1-95.9-2.7166-4.6
8NT2349-113.3-3.3463.5-5.3

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)"

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"

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"

图5

AaDH 催化L-苯丙氨酸(L-Phe)的氧化脱氨反应"

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"

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"

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"

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"

图10

NT2349 催化EOPB的还原胺化反应制备高苯丙氨酸"

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"

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"

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"

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"

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"

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