4-草酰巴豆酸互变异构酶催化合成肉桂醛的条件优化及其应用潜力分析

doi:10.19586/j.2095-2341.2024.0209

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 296-304.DOI: 10.19586/j.2095-2341.2024.0209

• 研究论文 • 上一篇

4-草酰巴豆酸互变异构酶催化合成肉桂醛的条件优化及其应用潜力分析

陈熙¹^,²(), 黄火清², 柏映国², 王苑², 夏呈强¹(), 罗会颖², 姚斌², 涂涛², 刘晓青²^,³()

^1.山西农业大学动物科学学院，山西晋中 030801
^2.中国农业科学院北京畜牧兽医研究所，畜禽营养与饲养全国重点实验室，北京 100193
^3.中国农业科学院生物技术研究所，北京 100081

收稿日期:2024-12-31 接受日期:2025-02-12 出版日期:2025-03-25 发布日期:2025-04-29
通讯作者: 夏呈强,刘晓青
作者简介:陈熙 E-mail: chenxi7592@163.com
基金资助:
国家重点研发计划项目(2022YFD1300703);中国农业科学院农业科技创新计划项目(CAAS-2DRW202305);财政部和农业农村部现代农业产业技术体系建设项目(CARS-41)

Reaction System Optimization and Application Potential Analysis of 4-Oxalocrotonate Tautomerase in Catalytic Synthesis of Cinnamaldehyde

Xi CHEN¹^,²(), Huoqing HUANG², Yingguo BAI², Yuan WANG², Chengqiang XIA¹(), Huiying LUO², Bin YAO², Tao TU², Xiaoqing LIU²^,³()

^1.College of Animal Science，Shanxi Agricultural University，Shanxi Jinzhong 030600，China
^2.State Key Laboratory of Animal Nutrition and Feeding，Institute of Animal Sciences，Chinese Academy of Agricultural Sciences，Beijing 100193，China
^3.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2024-12-31 Accepted:2025-02-12 Online:2025-03-25 Published:2025-04-29
Contact: Chengqiang XIA,Xiaoqing LIU

摘要/Abstract

摘要：

肉桂醛是一种重要的天然产物，广泛应用于医药、食品及饲料添加剂领域，然而传统化学合成方法存在高能耗和环境污染等问题，限制了其工业化应用。酶催化作为一种绿色合成途径，具有专一性高、环境友好的优点，对于探索酶催化合成肉桂醛具有重要意义。利用4-OT构建pET-20b-4-OT表达载体，在大肠杆菌中表达并纯化4-草酰巴豆酸互变异构酶(4-oxalocrotonate tautomerase，4-OT)，验证以苯甲醛和乙醛为底物合成肉桂醛的能力，并在不同pH、温度、酶浓度、盐浓度及底物浓度下研究其合成性能，通过高效液相色谱检测肉桂醛产量并分析优化条件对肉桂醛生成的影响。结果表明，4-OT在最适pH 7.3，最适温度37 ℃，酶浓度为15 mg·mL^-1时肉桂醛产量较初始条件提高181%，125 mmol·L^-1 K₂HPO₄-KH₂PO₄能显著提升酶活性；底物苯甲醛和乙醛的最优浓度分别为0.5和500 mmol·L^-1。扩大反应体系至400 mL后，肉桂醛产量最终提升719倍，转化率提高143倍。研究优化了4-OT催化合成肉桂醛的条件，显著提升了其催化效率和产量，为4-OT在肉桂醛绿色合成中的工业应用提供了有力支持。

关键词: 4-草酰巴豆酸互变异构酶, 肉桂醛, 酶催化, 条件优化, 绿色合成

Abstract:

Cinnamaldehyde is an important natural compound widely used in the pharmaceutical， food， and feed additive industries. However， traditional chemical synthesis methods involve high energy consumption and environmental pollution， limiting their industrial application. Enzymatic catalysis， as a green synthesis pathway， offers high specificity and environmental advantages， making the exploration of enzymatic cinnamaldehyde synthesis highly significant. Using 4-OT to construct the pET-20b-4-OT expression vector， the 4-oxalocrotonate tautomerase （4-OT） was expressed and purified in Escherichia coli to verify its ability to catalyze cinnamaldehyde synthesis from benzaldehyde and acetaldehyde as substrates， and its performance under different pH， temperature， enzyme concentration， salt concentration， and substrate concentration conditions was evaluated. The high-performance liquid chromatography was used to measure cinnamaldehyde yield， and the effects of optimized conditions on enzyme activity and product formation were analyzed. The optimal pH and temperature for 4-OT were determined to be pH 7.3 and 37 ℃， respectively. At an enzyme concentration of 15 mg·mL^-1， cinnamaldehyde yield increased by 181% compared to the initial conditions. The K₂HPO₄-KH₂PO₄ buffer system and a salt concentration of 125 mmol·L^-1 significantly enhanced enzyme activity. Optimal substrate concentrations were determined to be 0.5 mmol·L^-1 for benzaldehyde and 500 mmol·L^-1 for acetaldehyde. Expanding the reaction system to 400 mL increased the cinnamaldehyde yield by 719-fold and the conversion rate by 143-fold. This study optimized the conditions for 4-OT-catalyzed cinnamaldehyde synthesis， significantly improving catalytic efficiency and yield， thus providing strong support for the industrial application of 4-OT in green cinnamaldehyde synthesis.

Key words: 4-oxalocrotonate tautomerase, cinnamaldehyde, enzymatic catalysis, condition optimization, green synthesis

中图分类号:

Q814.9

陈熙, 黄火清, 柏映国, 王苑, 夏呈强, 罗会颖, 姚斌, 涂涛, 刘晓青. 4-草酰巴豆酸互变异构酶催化合成肉桂醛的条件优化及其应用潜力分析[J]. 生物技术进展, 2025, 15(2): 296-304.

Xi CHEN, Huoqing HUANG, Yingguo BAI, Yuan WANG, Chengqiang XIA, Huiying LUO, Bin YAO, Tao TU, Xiaoqing LIU. Reaction System Optimization and Application Potential Analysis of 4-Oxalocrotonate Tautomerase in Catalytic Synthesis of Cinnamaldehyde[J]. Current Biotechnology, 2025, 15(2): 296-304.

图/表 8

图1 肉桂醛的合成方法

Fig. 1 Synthesis method of cinnamaldehyde.

图2 表达质粒pET-20b-4-OT示意图注：RBS—核糖体结合位点。

Fig. 2 Diagram of expression plasmid pET-20b-4-OT

图3 重组4-OT的表达纯化及合成肉桂醛功能鉴定A：纯化的重组4-OT的SDS-PAGE分析，M—蛋白marker；1—粗酶液，2—纯化酶液；B：重组4-OT催化反应产物的HPLC检测

Fig. 3 Expression, purification, and functional identification of recombinant 4-OT

图4 pH和温度对4-OT催化合成肉桂醛的影响

Fig. 4 Effect of pH and temperature on the cinnamaldehyde synthesis catalyzed by 4-OT

图5 酶浓度对4-OT催化合成肉桂醛的影响A：不同酶浓度4-OT催化合成肉桂醛的产量；B：不同浓度4-OT催化反应产物的HPLC检测

Fig. 5 Effect of enzyme concentration on the cinnamaldehyde synthesis catalyzed by 4-OT

图6 盐离子类型和浓度对4-OT催化合成肉桂醛的影响A：不同的盐离子类型对肉桂醛产量的影响；B：不同K2HPO4-KH2PO4浓度对肉桂醛产量的影响

Fig. 6 Effect of salt ion type and concentration on the cinnamaldehyde synthesis catalyzed by 4-OT

图7 底物苯甲醛和乙醛浓度对4-OT催化合成肉桂醛的影响

Fig. 7 Effect of substrate concentrations of benzaldehyde and acetaldehyde on the cinnamaldehyde synthesis catalyzed by 4-OT

图8 不同反应体积中肉桂醛产量随反应时间的变化

Fig. 8 Cinnamaldehyde yield over time in different reaction volumes

参考文献 28

1	SONG F, LI H, SUN J, et al.. Protective effects of cinnamic acid and cinnamic aldehyde on isoproterenol-induced acute myocardial ischemia in rats[J]. J. Ethnopharmacol., 2013, 150(1): 125-130.
2	WORRETH S, BIEGER V, ROHR N, et al.. Cinnamaldehyde as antimicrobial in cellulose-based dental appliances[J]. J. Appl. Microbiol., 2022, 132(2): 1018-1024.
3	REIS J H, GEBERT R R, BARRETA M, et al.. Effects of phytogenic feed additive based on thymol, carvacrol and cinnamic aldehyde on body weight, blood parameters and environmental bacteria in broilers chickens[J]. Microb. Pathog., 2018, 125: 168-176.
4	LV C, YUAN X, ZENG H W, et al.. Protective effect of cinnamaldehyde against glutamate-induced oxidative stress and apoptosis in PC12 cells[J]. Eur. J. Pharmacol., 2017, 815: 487-494.
5	TORRE J E D, GASSARA F, KOUASSI A P, et al.. Spice use in food: properties and benefits[J]. Crit. Rev. Food Sci. Nutr., 2017, 57(6): 1078-1088.
6	QU S, YANG K, CHEN L, et al.. Cinnamaldehyde, a promising natural preservative against Aspergillus flavus [J/OL]. Front. Microbiol., 2019, 10: 2895[2025-02-25]. .
7	LIU Y, ESPINOSA C D, ABELILLA J J, et al.. Non-antibiotic feed additives in diets for pigs: a review[J]. Anim. Nutr., 2018, 4(2): 113-125.
8	ROSSI B, TOSCHI A, PIVA A, et al.. Single components of botanicals and nature-identical compounds as a non-antibiotic strategy to ameliorate health status and improve performance in poultry and pigs[J]. Nutr. Res. Rev., 2020, 33(2): 218-234.
9	YANG C, DIARRA M S, CHOI J, et al.. Effects of encapsulated cinnamaldehyde on growth performance, intestinal digestive and absorptive functions, meat quality and gut microbiota in broiler chickens[J/OL]. Transl. Anim. Sci., 2021, 5(3): txab099[2025-02-25]. .
10	张立华,王一樊,张旭阳,等.响应面分析法优化酶法提取红景天苷的工艺研究[J].生物技术进展,2023,13(3):441-448.
	ZHANG L H, WANG Y F, ZHANG X Y, et al.. Optimization of enzymatic extraction process of salidroside by response surface analysis[J]. Curr. Biotechnol., 2023, 13(3): 441-448.
11	夏琳,徐向丽,王雪云,等.植物绿原酸生物合成研究进展[J].生物技术进展,2024,14(6):973-979.
	XIA L, XU X L, WANG X Y, et al.. Research progress on the biosynthesis of chlorogenic acid in plant[J]. Curr. Biotechnol., 2024, 14(6): 973-979.
12	YU M, WANG S, ZHU H, et al.. In situ reactive heat breaking cell wall by SO(3) hydration: innovative cell-wall breaking technique to enhance extraction of cinnamaldehyde from cinnamon[J]. Prep. Biochem. Biotechnol., 2021, 51(9): 833-841.
13	FAN Q, HUANG F, ZHOU L, et al.. Development of a strategy for a quick process for separation of volatile compounds in counter-current chromatography: purification of cinnamaldehyde from Cinnamomum cassia by high performance counter-current chromatography[J]. Prep. Biochem. Biotechnol., 2021, 51(10): 1056-1059.
14	FOUDAH A I, SHAKEEL F, ALQARNI M H, et al.. Simultaneous estimation of cinnamaldehyde and eugenol in essential oils and traditional and ultrasound-assisted extracts of different species of cinnamon using a sustainable/green HPTLC technique[J/OL]. Molecules, 2021, 26(7): 2054[2025-02-25]. .
15	余春平,刘淑龙.肉桂醛的合成研究进展[J].浙江化工,2023,54(1):18-22.
	YU C P, LIU S L. Research progress in synthesis of cinnamaldehyde[J]. Zhejiang Chem. Ind., 2023, 54(1): 18-22.
16	BANG H B, SON J, KIM S C, et al.. Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde[J]. Metab. Eng., 2023, 76: 63-74.
17	XU G, KUNZENDORF A, CROTTI M, et al.. Gene fusion and directed evolution to break structural symmetry and boost catalysis by an oligomeric C-C bond-forming enzyme[J/OL]. Angew. Chem. Int. Ed., 2022, 61(8): e202113970[2025-02-25]. .
18	VAN DER MEER J Y, PODDAR H, JBAAS B, et al.. Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases[J/OL]. Nat. Commun., 2016, 7: 10911[2025-02-25]. .
19	王全富,蔺一飞,苗苗,等.海冰微生物Pseudoalteromonas sp.ANT319耐盐性初步研究[J].生物技术进展,2015,5(3):240-245.
	WANG Q F, LIN Y F, MIAO M, et al.. Preliminary study on salt tolerance of sea-ice microorganism Pseudoalteromonas sp. ANT319[J]. Curr. Biotechnol., 2015, 5(3): 240-245.
20	TRIVEDI N, GUPTA V, KUMAR M, et al.. An alkali-halotolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca [J]. Carbohydr. Polym., 2011, 83(2): 891-897.
21	赵子琪,刘烨瑀,于爽,等.海洋来源低温果胶酶产生菌筛选、鉴定与酶学性质研究[J].食品工业科技,45(10):133-140.
	ZHAO Z Q, LIU Y Y, YU S, et al.. Screening, identification and enzymatic properties study of cold-adapted marine pectinase producing bacteria[J]. Sci. Technol. Food Ind., 2024, 45(10): 133-140.
22	黄丽娜,刘欢,张奋强,等.红景天苷生物合成机制及其基因工程研究进展[J].生物技术进展,2017,7(2):106-110.
	HUANG L N, LIU H, ZHANG F Q, et al.. Progress of biosynthesis mechanism and genetic engineering of salidroside[J]. Curr. Biotechnol., 2017, 7(2): 106-110.
23	RAHIMI M, VAN DER MEER J Y, GEERTSEMA E M, et al.. Engineering a promiscuous tautomerase into a more efficient aldolase for self-condensations of linear aliphatic aldehydes[J]. Chembiochem, 2017, 18(14): 1435-1441.
24	SAIFUDDIN M, GUO C, BIEWENGA L, et al.. Enantioselective aldol addition of acetaldehyde to aromatic aldehydes catalyzed by proline-based carboligases[J]. ACS Catal., 2020, 10(4): 2522-2527.
25	GUO C, SAIFUDDIN M, SARAVANAN T, et al.. Biocatalytic asymmetric Michael additions of nitromethane to α, β-unsaturated aldehydes via enzyme-bound iminium ion intermediates[J]. ACS Catal., 2019, 9(5): 4369-4373.
26	KUNZENDORF A, XU G, SAIFUDDIN M, et al.. Biocatalytic asymmetric cyclopropanations via enzyme-bound iminium ion intermediates[J]. Angew. Chem. Int. Ed., 2021, 60(45): 24059-24063.
27	RAHIMI M, VAN DER MEER J Y, GEERTSEMA E M, et al.. Mutations closer to the active site improve the promiscuous aldolase activity of 4-oxalocrotonate tautomerase more effectively than distant mutations[J]. Chembiochem, 2016, 17(13): 1225-1228.
28	赵晨辉.羟醛缩合合成肉桂醛工艺研究[D].北京:北京化工大学,2017.

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4-草酰巴豆酸互变异构酶催化合成肉桂醛的条件优化及其应用潜力分析

Reaction System Optimization and Application Potential Analysis of 4-Oxalocrotonate Tautomerase in Catalytic Synthesis of Cinnamaldehyde

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