理性改造光脱羧酶并用于反式脂肪酸选择性脱羧反应

doi:10.3866/PKU.DXHX202210002

Abstract

Abstract:

Photoenzymatic synthesis and rational engineering of enzymes are among the most emerging research areas. Herein, a highly selective photodecarboxylation of trans fatty acid was achieved by rational repurposing of the photodecarboxylase. Specifically, via molecular docking, a mutant of the photodecarboxylase CvFAP, namely V453E-CvFAP, was rationaly designed, constructed and subjected to the photoactivated decarboxylation of fatty acids. It turned out that the mutant was highly selective towards elaidic acid, thereby enriching oleic acid. Experimental trainings like cells culturing, protein expression, reaction design, and qualitative and quantitative analysis of products were included. The experiment is easy to conduct and safe for undergraduate chemistry experiment.

Key words: Rational evolution of enzyme, Photodecarboxylase CvFAP, Trans fatty acid, Photobiocatalysis, Decarboxylation

Senhai Zeng, Yuyan Bao, Ao Cao, Xiaoqiang Huang. Rational Evolution of a Fatty Acid Photodecarboxylase for Highly Selective Photodecarboxylation of trans Fatty Acid[J].University Chemistry, 2023, 38(4): 243-252.

Figures/Tables 14

References 14

1	Schmermund L. ; Jurkaš V. ; Özgen F. F. ; Barone G. D. ; Büchsenschütz H. C. ; Winkler C. K. ; Schmidt S. ; Kourist R. ; Kroutil W. ACS Catal. 2019, 9 (5), 4115. doi: 10.1021/acscatal.9b00656
2	Amer M. ; Wojcik E. Z. ; Sun C. ; Hoeven R. ; Hughes J. M. X. ; Faulkner M. ; Yunus I. S. ; Tait S. ; Johannissen L. O. ; Hardman S. J. O. ; et al Energy Environ. Sci. 2020, 13 (6), 1818. doi: 10.1039/D0EE00095G
3	Li D. ; Han T. ; Xue J. ; Xu W. ; Xu J. ; Wu Q. Angew. Chem. Int. Ed. 2021, 60 (38), 20695. doi: 10.1002/anie.202107694
4	Wang Y. ; Xue P. ; Cao M. ; Yu T. ; Lane S. T. ; Zhao H. Chem. Rev. 2021, 121 (20), 12384. doi: 10.1021/acs.chemrev.1c00260
5	Harrison W. ; Huang X. ; Zhao H. Acc. Chem. Res. 2022, 55 (8), 1087. doi: 10.1021/acs.accounts.1c00719
6	Orsavova J. ; Misurcova L. ; Ambrozova J. V. ; Vicha R. ; Mlcek J. Int. J. Mol. Sci. 2015, 16 (6), 12871.
7	Micha R. ; Mozaffarian D. Nat. Rev. Endocrinol. 2009, 5 (6), 335. doi: 10.1038/nrendo.2009.79
8	Visvalingam J. ; Vahmani P. ; Rolland D. C. ; Dugan M. E. R. ; Yang X. Lipids 2017, 52 (7), 651. doi: 10.1007/s11745-017-4267-9
9	中国营养学会. 中国居民膳食指南, 北京: 人民卫生出版社, 2016, 134
10	Sorigué D. ; Légeret B. ; Cuiné S. ; Blangy S. ; Moulin S. ; Billon E. ; Richaud P. ; Brugière S. ; Couté Y. ; Nurizzo D. ; et al Science 2017, 357 (6354), 903. doi: 10.1126/science.aan6349
11	Sorigué D. ; Hadjidemetriou K. ; Blangy S. ; Gotthard G. ; Bonvalet A. ; Coquelle N. ; Samire P. ; Aleksandrov A. ; Antonucci L. ; Benachir A. ; et al Science 2021, 372 (6538), eabd5687. doi: 10.1126/science.abd5687
12	Zhang W. ; Ma M. ; Huijbers M. M. E. ; Filonenko G. A. ; Pidko E. A. ; van Schie M. ; de Boer S. ; Burek B. O. ; Bloh J. Z. ; van Berkel W. J. H. ; et al J. Am. Chem. Soc. 2019, 141 (7), 3116. doi: 10.1021/jacs.8b12282
13	Cha H.-J. ; Hwang S.-Y. ; Lee D.-S. ; Kumar A. R. ; Kwon Y.-U. ; Voß M. ; Schuiten E. ; Bornscheuer U. T. ; Hollmann F. ; Oh D.-K. ; et al Angew. Chem. Int. Ed. 2020, 59 (18), 7024. doi: 10.1002/anie.201915108
14	Xu J. ; Hu Y. ; Fan J. ; Arkin M. ; Li D. ; Peng Y. ; Xu W. ; Lin X. ; Wu Q. Angew. Chem. Int. Ed. 2019, 58 (25), 8474. doi: 10.1002/anie.201903165

课时	实验内容
第一次课 8 h	1) 接种WT–CvFAP、V453E–CvFAP菌种、扩大培养、诱导表达； 2) 使用AutoDock分子对接，理解突变位点的选择； 3) 菌体收集
第二次课 8 h	1) 获得粗酶溶液； 2) 用FAD辅基定量分析酶浓度； 3) 光酶反应，并使用GC-MS进行表征
第三次课 8 h	1) 绘制底物标准曲线，计算转化率； 2) 自主探索实验条件对结果的影响； 3) 对实验结果和分子对接结果分析讨论

组别	m(油酸)/mg	m(反油酸)/mg	V(MeOH)/mL	酶液
第一组	10.11	0	1.00	WT-CvFAP
第二组	0	9.97	1.00	WT-CvFAP
第三组	10.11	0	1.00	V453E-CvFAP
第四组	0	9.97	1.00	V453E-CvFAP

底物	酶	d ^*/Å
油酸	野生型	5.3
反油酸	野生型	5.2
油酸	突变体	5.1
反油酸	突变体	4.5

酶液	A ^b	A₀ ^c	A − A₀	c(FAD)/(mmol∙L⁻¹)	c(粗酶)/(mmol∙L⁻¹)
WT-CvFAP	0.742	0.647	0.095	0.0201	0.100
V453E-CvFAP	0.764	0.647	0.117	0.0247	0.124

酶液	c(酶) ^a/(μmol∙L⁻¹)	底物	c(底物) ^a/(mmol∙L⁻¹)	α ^b/%	TON ^c
V453E-CvFAP	30.0	反油酸	3.53	100	118
V453E-CvFAP	30.0	油酸	3.58	78.1	93
V453E-CvFAP	22.5	反油酸	3.53	100	157
V453E-CvFAP	22.5	油酸	3.58	50.2	80
V453E-CvFAP	10.0	反油酸	3.53	100	353
V453E-CvFAP	10.0	油酸	3.58	12.1	43
V453E-CvFAP	5.00	反油酸	3.53	97.3	697
V453E-CvFAP	5.00	油酸	3.58	3.40	26
V453E-CvFAP	2.00	反油酸	3.53	28.7	505
V453E-CvFAP	2.00	油酸	3.58	0.3	6.3
WT-CvFAP	5.00	反油酸	3.53	100	706
WT-CvFAP	5.00	油酸	3.58	100	716

Rational Evolution of a Fatty Acid Photodecarboxylase for Highly Selective Photodecarboxylation of trans Fatty Acid

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酶液	c(酶)/(μmol∙L⁻¹)	底物	c(底物)/(mmol∙L⁻¹)	α/%	TON
WT-CvFAP	5.00	反油酸	3.53	100	706
WT-CvFAP	5.00	油酸	3.58	100	716
V453E-CvFAP	5.00	反油酸	3.53	97.3	697
V453E-CvFAP	5.00	油酸	3.58	3.40	26

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