D-氨基酸氧化酶的分子改造及应用研究进展

doi:10.16085/j.issn.1000-6613.2020-1933

摘要/Abstract

摘要：

D-氨基酸氧化酶是一类含有黄素腺嘌呤二核苷酸的氧化还原酶，能够催化D-氨基酸氧化脱氢，生成相应的α-酮酸、过氧化氢和氨。该类酶在自然界中分布广泛，主要来源于真核生物和少数原核生物。作为一种经典的生物催化剂，D-氨基酸氧化酶具有反应条件温和、底物谱广泛、对映体选择性好等特点，在合成医药、农药和精细化学品等方面具有重要的应用价值。本文综述了D-氨基酸氧化酶的基本蛋白结构特征及其催化机制，重点介绍了D-氨基酸氧化酶底物特异性和热稳定性分子改造的策略和代表性成果以及该类酶在生物催化中的应用，例如制备7-氨基头孢烷酸、手性氨基酸、胺类化合物和α-酮酸。最后探讨了D-氨基酸氧化酶目前在生物催化应用过程中存在的问题。后续的研究可围绕新酶的挖掘与改造展开工作。基于对映体选择性和底物识别的分子机制，理性设计酶的催化性能，并以挖掘或改造获得的D-氨基酸氧化酶作为新酶元件，用于构建功能化学品生物合成新途径。

关键词: D-氨基酸氧化酶, 分子改造, 生物催化, 手性氨基酸, 手性胺

Abstract:

D-amino acid oxidase is a class of flavine adenine dinucleotide containing oxidordeuctase that catalyzes the oxidative dehydrogenation of D-amino acids, which results in corresponding α-keto acids, hydrogen peroxide, and ammonia. D-amino acid oxidases are widely distributed in nature, mainly found in multiple eukaryotic and few prokaryotic organisms. As a classic biocatalyst, D-amino acid oxidases have played important roles in the synthesis of various medicine, pesticides as well as fine chemicals, owing to the mild reaction conditions, broad substrate spectrum and excellent enantioselectivity. Herein, we review the structural characteristics, catalytic mechanism, and protein engineering of D-amino acid oxidase, as well as its application in biocatalysis. Different molecular modification strategies for the engineering of D-amino acid oxidase and the representative achievements are further described. The preparation of 7-aminocephalosporanic acid,chiral amino acids,amines and α-keto acids by using D-amino acid oxidase is also summarized. Finally, the current problems in the biocatalytic application of this enzyme are discussed. The subsequent research could focus on the mining and engineering of novel D-amino acid oxidase. Based on the molecular mechanism of enantioselectivity and substrate recognition, the rational design could be applied to modify the catalytic performance. Furthermore, the newly obtained enzymes could be utilized to construct novel biosynthetic pathways for functional chemicals.

Key words: D-amino acid oxidase, molecular modification, biocatalysis, chiral amino acids, chiral amines

中图分类号:

Q554⁺.4

居述云, 吴坚平, 杨立荣. D-氨基酸氧化酶的分子改造及应用研究进展[J]. 化工进展, 2021, 40(3): 1215-1225.

JU Shuyun, WU Jianping, YANG Lirong. Advances in the molecular modification and application of D-amino acid oxidase[J]. Chemical Industry and Engineering Progress, 2021, 40(3): 1215-1225.

图/表 10

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参数	RgDAAO	pkDAAO	hDAAO
单亚基氨基酸数量	368	347	347
与RgDAAO的序列一致性	100%	28.73%	27.73%
亚基分子量	40000	39600	39600
亚基聚合状态	同源二聚体+2FAD	同源二聚体+2FAD	同源二聚体+2FAD
配体	D-三氟丙氨酸	苯甲酸	2-氨基苯甲酸
与配体相互作用的关键氨基酸残基	Y223,Y238,R285,S335, M213,F58	Y224,Y228,R283, G313,I230	Y224,Y228,R283,G313, I230,L215
PDB登录号	1C0L	1VE9	2E4A

参数	RgDAAO	pkDAAO	hDAAO
单亚基氨基酸数量	368	347	347
与RgDAAO的序列一致性	100%	28.73%	27.73%
亚基分子量	40000	39600	39600
亚基聚合状态	同源二聚体+2FAD	同源二聚体+2FAD	同源二聚体+2FAD
配体	D-三氟丙氨酸	苯甲酸	2-氨基苯甲酸
与配体相互作用的关键氨基酸残基	Y223,Y238,R285,S335, M213,F58	Y224,Y228,R283, G313,I230	Y224,Y228,R283,G313, I230,L215
PDB登录号	1C0L	1VE9	2E4A

原始酶	改造策略	突变体	k_cat /min^-1	K_m /mmol·L^-1	(k_cat/K_m) /mL·mol^-1·min^-1	参考文献
RgDAAO	改变底物结合口袋处氨基酸残基的极性和带电性质，稳定底物结合构象	M213R	235(29)	2(18)	118(1.6)	[20]
			975(60)	33(77.3)	29.5(0.8)
			630(5000)	17.8(0.8)	35.4(6100)
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	M213G	870(125)	0.03(0.04)	29000(3100)	[22]
			31(6)	0.03(0.01)	1035(600)
			92(53)	0.05(0.33)	1840(160)
			950(180)	1.6(2.1)	600(90)	[23]
	定向进化	L118H	3620(3900)	0.4(0.9)	8415(4330)	[28]
		L118H	31(40)	12.8(33.1)	2.4(1.2)
		T60A/Q144R /K152E	53(40)	7.9(33.1)	6.7(1.2)
TvDAAO	基于同源建模，改变底物结合口袋处氨基酸残基的极性，稳定底物结合构象	F54Y	2200(370)	4.8(1.6)	470(230)	[27]
pkDAAO	突变与底物（配体）羧基相互作用的精氨酸残基，重塑底物结合口袋	Y228L/R283G	444.0(-)	7.0(-)	63.4(-)	[21,25]
	突变与底物（配体）羧基相互作用的精氨酸残基，重塑底物结合口袋	Y228L/R283G	46.8(-)	1.5(-)	31.2(-)	[21,25]
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	Y228L/R283G /F242I	180.0(-)	3.6(-)	50.0(-)	[25]
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	I230A/R283G	360.6(-）	2.94(-)	122.7(-)	[26]

原始酶	改造策略	突变体	k_cat /min^-1	K_m /mmol·L^-1	(k_cat/K_m) /mL·mol^-1·min^-1	参考文献
RgDAAO	改变底物结合口袋处氨基酸残基的极性和带电性质，稳定底物结合构象	M213R	235(29)	2(18)	118(1.6)	[20]
			975(60)	33(77.3)	29.5(0.8)
			630(5000)	17.8(0.8)	35.4(6100)
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	M213G	870(125)	0.03(0.04)	29000(3100)	[22]
			31(6)	0.03(0.01)	1035(600)
			92(53)	0.05(0.33)	1840(160)
			950(180)	1.6(2.1)	600(90)	[23]
	定向进化	L118H	3620(3900)	0.4(0.9)	8415(4330)	[28]
		L118H	31(40)	12.8(33.1)	2.4(1.2)
		T60A/Q144R /K152E	53(40)	7.9(33.1)	6.7(1.2)
TvDAAO	基于同源建模，改变底物结合口袋处氨基酸残基的极性，稳定底物结合构象	F54Y	2200(370)	4.8(1.6)	470(230)	[27]
pkDAAO	突变与底物（配体）羧基相互作用的精氨酸残基，重塑底物结合口袋	Y228L/R283G	444.0(-)	7.0(-)	63.4(-)	[21,25]
	突变与底物（配体）羧基相互作用的精氨酸残基，重塑底物结合口袋	Y228L/R283G	46.8(-)	1.5(-)	31.2(-)	[21,25]
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	Y228L/R283G /F242I	180.0(-)	3.6(-)	50.0(-)	[25]
	减小底物结合口袋处氨基酸残基的空间位阻，扩大底物结合口袋	I230A/R283G	360.6(-）	2.94(-)	122.7(-)	[26]

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