亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能

曹文斌; 李昊; 陈飞飞; 潘江; 郑高伟; 许建和

doi:10.14135/j.cnki.1006-3080.20210505001

亚胺还原酶的改造及其催化合成1-苯基-1,2,3,4-四氢异喹啉的性能

doi: 10.14135/j.cnki.1006-3080.20210505001

华东理工大学生物工程学院、省部共建上海生物制造产业协同创新中心，上海 200237

基金项目: 国家自然科学基金项目(21878085，21871085)

详细信息

作者简介:
曹文斌（1993-），男，硕士生，主要研究方向为生物催化。E-mail：494186758@qq.com

通讯作者:
许建和，E-mail：jianhexu@ecust.edu.cn

中图分类号: Q81
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- 被引次数: 0
出版历程
- 收稿日期: 2021-05-05
- 网络出版日期: 2021-07-13

Molecular Engineering and Characterization of Imine Reductases for the Synthesis of 1-Phenyl-1,2,3,4-tetrahydro-isoquinoline

Shanghai Collaborative Innovation Center for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China

摘要

摘要: 对亚胺还原酶酶库的筛选获得了性能优良的候选酶AdIR1，基于蛋白结构分析对其进行分子改造获得催化效率更优的突变体酶F172Y，并对其进行动力学与热稳定性表征；最后利用所开发的突变酶实现了(S)-1-Ph-THIQ克级制备，证明了该酶法合成路线的实用性。
- 四氢异喹啉 /
- 手性胺 /
- 亚胺还原酶 /
- 不对称合成 /
- 生物催化
Abstract: 1,2,3,4-Tetrahydro-isoquinolines are a significant class of building blocks used in the pharmaceutical and agrochemical industries, and existed widely in a variety of chiral amine drugs. Among them, (S)-1-Phenyl-1,2,3,4-tetrahydro-isoquinoline ((S)-1-Ph-THIQ) is the key precursor for the synthesis of Solifenacin, a drug for the treatment of overactive bladder. Imine reductase (IRED)-catalyzed asymmetric reduction of 1-phenyl-3,4-dihydroisoquinoline (1-Ph-DHIQ) is a green and promising route towards chiral 1-Ph-THIQ. However, currently there is only a limited number of reported IREDs that could catalyze the synthesis of chiral 1-Ph-THIQ from 1-Ph-DHIQ, and they may suffer from issues including low activity, poor stereoselectivity, and substrate inhibition. In this study, we first discovered an IRED AdIR1 with considerable properties by screening a panel of IREDs and identified key residues which may affect the activity via homo-modelling and structure comparison. Protein engineering was performed to generate mutant F172Y with elevated catalytic efficiency, which was then characterized in terms of kinetic parameters and thermostability. Finally, preparative synthesis of (S)-1-Ph-THIQ on gram-scale was achieved employing mutant F172Y, demonstrating the considerable applicability of this biocatalytic route in the synthesis of (S)-1-Ph-THIQ.
- tetrahydro-isoquinolines /
- chiral amines /
- imine reductases /
- asymmetric synthesis /
- biocatalysis

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图 1 亚胺还原酶催化的不对称还原反应

Figure 1. Asymmetric reduction by IRED

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图 2 SnIR与AdIR1的结构比对结果

Figure 2. Results for structural alignment of AdIR1 with SnIR

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图 3 pH对母本(A)与F172Y(B)酶活力的影响

Figure 3. Effect of pH on the activity of WT(A) and F172Y(B). Activity-pH data was measured using the following buffer

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图 4 母本(a)与F172Y(b)突变体的热稳定性

Figure 4. Thermostability of WT (a) and F172Y (b)

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图 5 母本与突变体F172Y的动力学特征曲线

Figure 5. Kinetic characteristic curves of the wild type and F172Y of AdIR1

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图 6 母本(黄色)与突变体F172Y(绿色)的反应进程曲线

Figure 6. Reaction process curves of Wild Type (yellow) and F172Y (blue) of AdIR1

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图 7 不同助溶剂浓度下的反应进程曲线

Figure 7. Reaction process curves with different cosolvent concentrations

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图 8 克级制备反应的反应进程曲线

Figure 8. Reaction process curve of gram-scale preparative reaction.

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表 1 目的基因克隆所用的引物

Table 1. Primers used in gene cloning

Entry	Enzyme	Primers (5’ to 3’)	Restriction site
1	AdIR1	GGAATTCCATATGATCACACTGATCGGGCTCGGT CCGGAATTCCTACTTGCGGTCCGCCTTGATG	Nde I EcoR I
2	SeIR2	GGGAATTCCATATGAGCACGCCGCTGACGCTGAT CCGGAATTCCTACTTCGCGGGCTTGATC	Nde I EcoR I

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表 2 目的基因定点突变所用PCR引物

Table 2. Primers used for site-directed mutagenesis.

Entry	Name	Primers (5’ to 3’)	Length
1	118_FP	GGCGTGATGNDTCCTGCCGAGTTGGTGGGCAAGGAG	36
2	118_RP	CTCGGCAGGAHNCATCACGCCGCCCGCGACGAACCG	36
3	172_FP	CTGGACATCNDTCTGACCTCGCTTTCGGTGTTCATG	36
4	172_RP	CGAGGTCAGAHNGATGTCCAGCTGCGCCTGGTAGAA	36
5	216_FP	GCCGAGCAGNDTGAAAAGGGCGAGCACCCCGGCGAC	36
6	216_RP	GCCCTTTTCAHNCTGCTCGGCCGCGGCGTCCAGGTA	36

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表 3 亚胺还原酶不对称还原1-苯基-3,4-二氢异喹啉的转化率和ee值

Table 3. Asymmetric reduction of 1-phenyl-3,4-dihydroisoquinoline by AdIR1and SeIR2.

Entry	Enzyme	Time/h	C/%	ee/%
1	AdIR1	24	98	98 (S)
2	SeIR2	24	54	94 (S)

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表 4 亚胺还原酶的关键位点比对结果

Table 4. Alignment of key residues of the imine reductases.

Entry	Enzyme	1^st site	2^nd site	3^rd site
1	SnIR	T123	F178	G228
2	AdIR1	V118	F172	I216

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表 5 AdIR1突变体的测活结果

Table 5. Activity assay results of AdIR1 mutants.

Entry	Enzyme	Mutation site	Specific activity/(mU·mg⁻¹)	Fold
1	WT	−	7.90	1.0
2	M1	118	8.81	1.1
3	M2	118	5.92	0.7
4	M3	118	8.88	1.2
5	M4	172	15.3	1.9

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表 6 突变体F172Y与母本的半衰期对比

Table 6. Half-lives of WT and F172Y at different temperatures.

Enzyme	t_1/2/h
Enzyme	30 ℃	40 ℃	50 ℃
WT	70.0	50.4	1.0
F172Y	324.0	314.0	3.9

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表 7 母本与F172Y的催化动力学参数

Table 7. Kinetic parameters of WT and F172Y.

Enzyme	Temperature/ ℃	K_m/ (mmol·L⁻¹)	k_cat/ (s⁻¹)	k_cat/K_m/ (s⁻¹·mmol/L⁻¹)
WT	30	0.05 ± 0.01	0.005 ± 0.000	0.10
F172Y	30	0.05 ± 0.01	0.017 ± 0.000	0.34
F172Y	40	0.07 ± 0.01	0.027 ± 0.000	0.38

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表 8 亚胺还原酶突变体F172Y在30 ℃与40 ℃下的反应转化结果

Table 8. Reaction conversion of F172Y under 30 ℃ and 40 ℃.

T/℃	Time/h	Conversion/ %	ee/%
30	6	69	99
30	12	84	99
40	6	84	99
40	12	91	99

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