Heterologous Expression of Paeoniflorin Converting Enzyme G6046 and the Identification of Its Enzymatic Activities

doi:10.13560/j.cnki.biotech.bull.1985.2023-0653

Abstract

Abstract:

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that shows various protective effects on the cardio-cerebral vascular system and always used in traditional herbal medicine. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites and mainly metabolized in intestinal bacteria. In this paper，the induction expression and purification of paeoniflorin converting enzyme G6046 in Escherichia coli BL21 DE3 were studied. The induction reaction conditions were optimized to improve the enzyme yield and the optimal conditions for induction reaction were found to be 24 h induction reaction at 16℃. Enzyme production of bacterium reached 5.34 mg/g, representing a 51.69% increase in enzyme production. Based on the conditions of induction expression, the conditions（pH and temperature）for the reaction of G6046 with paeoniflorin were optimized, pH 9.0 and 45℃ were proved to be the optimized conditions, resulting in a specific enzyme activity of 14.56 U/mg. The reaction system was enlarged under this optimized condition, and the reaction scale was from G6046-substrate（1∶1; mg/mg）to G6046-substrate（1∶20; mg/mg）, the yield of the converted product significantly increased, which lays a foundation for the isolation of peonidin conversion product.

Key words: paeoniflorin, paeoniflorin converting enzyme, HPLC

HAN Ying-yi, LI Zhang-han, CAO Xue-li, PEI Hai-run. Heterologous Expression of Paeoniflorin Converting Enzyme G6046 and the Identification of Its Enzymatic Activities[J]. Biotechnology Bulletin, 2023, 39(12): 200-208.

Figures/Tables 7

Fig. 1 Expressions of G6046-NΔ82-22b and condition optimization A: SDS-PAGE analysis of expression and purification of G6046-NΔ82-22b（P: the precipitation of G6046-NΔ82-22b; lane S: the supernatant of G6046-NΔ82-22b; lane Ft: the flow through of G6046-NΔ82-22b; W1 and W2: samples from the buffer wash; E: the eluted samples from the resin; M: molecular weight marker）. B: Enzyme production per gram of bacterium under different induction conditions（temperature, time）. Different lower letters indicate significant differences at P < 0.05 level

Fig. 2 pH-enzyme activity curve of paeoniflorin converting enzyme G6046 Temperature：25℃;time：3 h; significant test results showed significant differences between pH 9 and pH 7（P = 0.018 4）, pH 9 and pH 8（P = 0.040 4）, and no significant differences among others

Fig. 3 Enzyme reaction curves of paeoniflorin converting enzyme G6046 at pH 9.0 and different temperatures A：15℃; B：25℃; C：35℃; D：45℃; E：55℃; F：65℃

Fig. 4 G6046 reaction curves at different temperatures A：G6046 reaction curves in the first 3 h at different temperatures，pH 9.0. B: Temperature-enzymatic activity curve of paeoniflorin converting enzyme G6046，pH 9.0，significant test results showed significant differences between 45℃ and 55℃（P=0.017 0）, 45℃ and 65℃（P=0.001 6）, and no significant differences among others

Fig. 5 HPLC analysis of paeoniflorin and paeoniflorin after conversion reaction under different conditions A：Standard of paeoniflorin；B: liquid analysis of enzyme reaction for 96 h at pH 8.0，and 25℃，enzyme-paeoniflorin = 1∶10，mg/mg；C：liquid analysis of enzyme reaction at pH 9.0 and 25℃，enzyme-paeoniflorin = 1∶10，mg/mg for 48 h；D：liquid analysisi of enzyme reaction for 28 h at pH 9.0 and 45℃，under the condition of enzyme-paeoniflorin = 1∶10，mg/mg

Fig. 6 HPLC analysis of 36 h conversion of paeoniflorin by G6046 Reaction system：G6046-paeoniflorin（1∶20, mg/mg），reaction conditions: pH 9.0, 45℃

Fig. 7 Schematic of G6046-paeoniflorin reaction system by step-by-step amplification A：The reaction volume of paeoniflorin conversion under the original reaction conditions（pH 8.0, room temperature），reaction scale：1 mg paeoniflorin. B：The reaction volume of paeoniflorin conversion under the optimized reaction conditions（pH 9.0，45℃）, reaction scale：10 mg paeoniflorin added to 1 mg G6046. C：The reaction volume of paeoniflorin conversion under the optimized reaction conditions（pH 9.0，45℃）, reaction scale：100 mg paeoniflorin was added to 5 mg of G6046

References 36

[1]	黄山君, 王瑞, 等. 硫磺熏制白芍的安全性评价初步研究[J]. 药学学报, 2012, 47(4): 486-491.
	Huang SJ, Wang R, et al. Primary safety evaluation of sulfated Paeoniae Radix Alba[J]. Acta Pharm Sin, 2012, 47(4): 486-491.
[2]	Li YB, Sun YX, Ma XL, et al. Effects of Sini San used alone and in combination with fluoxetine on central and peripheral 5-HT levels in a rat model of depression[J]. J Tradit Chin Med, 2013, 33(5): 674-681. pmid: 24660595
[3]	Zhu XX, Jing LL, Chen C, et al. Danzhi Xiaoyao San ameliorates depressive-like behavior by shifting toward serotonin via the downregulation of hippocampal indoleamine 2, 3-dioxygenase[J]. J Ethnopharmacol, 2015, 160: 86-93. doi: 10.1016/j.jep.2014.11.031 URL
[4]	Ma X, Zhao YL, Zhu Y, et al. Paeonia lactiflora Pall. protects against ANIT-induced cholestasis by activating Nrf2 via PI3K/Akt signaling pathway[J]. Drug Des Devel Ther, 2015, 9: 5061-5074.
[5]	Wang JS, Huang Y, Zhang SP, et al. A protective role of paeoniflorin in fluctuant hyperglycemia-induced vascular endothelial injuries through antioxidative and anti-inflammatory effects and reduction of PKC β 1[J]. Oxid Med Cell Longev, 2019, 2019: 5647219.
[6]	张燕丽, 田园, 付起凤, 等. 白芍的化学成分和药理作用研究进展[J]. 中医药学报, 2021, 49(2): 104-109.
	Zhang YL, Tian Y, Fu QF, et al. Research progress of chemical constituents and pharmacological action of Paeonia tactilora pall[J]. Acta Chin Med Pharmacol, 2021, 49(2): 104-109.
[7]	Cheng J, Chen M, Wan HQ, et al. Paeoniflorin exerts antidepressant-like effects through enhancing neuronal FGF-2 by microglial inactivation[J]. J Ethnopharmacol, 2021, 274: 114046. doi: 10.1016/j.jep.2021.114046 URL
[8]	Wang K, Zhu L, Zhu X, et al. Protective effect of paeoniflorin on Aβ25-35-induced SH-SY5Y cell injury by preventing mitochondrial dysfunction[J]. Cell Mol Neurobiol, 2014, 34(2): 227-234. doi: 10.1007/s10571-013-0006-9 pmid: 24263411
[9]	Gu XS, Wang F, Zhang CY, et al. Neuroprotective effects of paeoniflorin on 6-OHDA-lesioned rat model of Parkinson's disease[J]. Neurochem Res, 2016, 41(11): 2923-2936. doi: 10.1007/s11064-016-2011-0 URL
[10]	Zheng MZ, Liu CM, Fan YJ, et al. Neuroprotection by Paeoniflorin in the MPTP mouse model of Parkinson's disease[J]. Neuropharmacology, 2017, 116: 412-420. doi: S0028-3908(17)30009-6 pmid: 28093210
[11]	Nizamutdinova IT, Jin YC, Kim JS, et al. Paeonol and paeoniflorin, the main active principles of Paeonia albiflora, protect the heart from myocardial ischemia/reperfusion injury in rats[J]. Planta Med, 2008, 74(1): 14-18. doi: 10.1055/s-2007-993775 pmid: 18203054
[12]	Chen C, Du P, Wang JJ. Paeoniflorin ameliorates acute myocardial infarction of rats by inhibiting inflammation and inducible nitric oxide synthase signaling pathways[J]. Mol Med Rep, 2015, 12(3): 3937-3943. doi: 10.3892/mmr.2015.3870 pmid: 26035555
[13]	Li P, Li ZH. Neuroprotective effect of paeoniflorin on H₂O₂-induced apoptosis in PC12 cells by modulation of reactive oxygen species and the inflammatory response[J]. Exp Ther Med, 2015, 9(5): 1768-1772. doi: 10.3892/etm.2015.2360 URL
[14]	Yu JB, Zhao ZX, Peng R, et al. Gut microbiota-based pharmacokinetics and the antidepressant mechanism of paeoniflorin[J]. Front Pharmacol, 2019, 10: 268. doi: 10.3389/fphar.2019.00268 URL
[15]	Lin YT, Huang WS, Tsai HY, et al. In vivo microdialysis and in vitro HPLC analysis of the impact of paeoniflorin on the monoamine levels and their metabolites in the rodent brain[J]. BioMedicine, 2019, 9(2): 11. doi: 10.1051/bmdcn/2019090211 URL
[16]	Zhao ZX, Fu J, Ma SR, et al. Gut-brain axis metabolic pathway regulates antidepressant efficacy of albiflorin[J]. Theranostics, 2018, 8(21): 5945-5959. doi: 10.7150/thno.28068 URL
[17]	刘玉峰, 孙珊珊, 等. 赤芍中芍药苷和芍药内酯苷的代谢及药动学研究进展[J]. 辽宁大学学报: 自然科学版, 2018, 45(4): 296-303.
	Liu YF, Sun SS, et al. Research progress on the metabolism and pharmacokinetics of paeoniflorin and albiflorin in Radix paeoniae Rubra[J]. J Liaoning Univ Nat Sci Ed, 2018, 45(4): 296-303.
[18]	Heikal OA, Akao T, Takeda S, et al. Pharmacokinetic study of paeonimetabolin I, a major metabolite of paeoniflorin from paeony roots[J]. Biol Pharm Bull, 1997, 20(5): 517-521. pmid: 9178932
[19]	Akao T, Shu YZ, Matsuda Y, et al. Metabolism of paeoniflorin and related compounds by human intestinal bacteria. IV. Formation and structures of adducts of a metabolic intermediate with sulfhydryl compounds by Lactobacillus brevis[J]. Chem Pharm Bull, 1988, 36(8): 3043-3048. doi: 10.1248/cpb.36.3043 URL
[20]	He JX, Goto E, Akao T, et al. Interaction between Shaoyao-Gancao-Tang and a laxative with respect to alteration of paeoniflorin metabolism by intestinal bacteria in rats[J]. Phytomedicine, 2007, 14(7-8): 452-459. doi: 10.1016/j.phymed.2006.09.014 URL
[21]	Takeda S, Isono T, et al. Absorption and excretion of paeoniflorin in rats[J]. J Pharm Pharmacol, 1995, 47(12A): 1036-1040. pmid: 8932691
[22]	Gao H, Zhang LS, Song JN, et al. Absorption and biotransformation of four compounds in the Guizhi Decoction in the gastrointestinal tracts of rats[J]. J Tradit Chin Med, 2019, 39(3): 332-338. pmid: 32186005
[23]	Zhan JX, Guo HZ, Dai JG, et al. Microbial transformations of artemisinin by Cunninghamella echinulata and Aspergillus niger[J]. Tetrahedron Lett, 2002, 43(25): 4519-4521.
[24]	Gonçalves MD, Tomiotto-Pellissier F, de Matos RLN, et al. Recent advances in biotransformation by Cunninghamella species[J]. Curr Drug Metab, 2021, 22(13): 1035-1064. doi: 10.2174/1389200222666211126100023 pmid: 34825868
[25]	刘鑫鑫, 马骁驰, 霍长虹, 等. 芍药苷和芍药内酯苷的微生物转化[J]. 中国中药杂志, 2010, 35(7): 872-875.
	Liu XX, Ma XC, Huo CH, et al. Microbiological transformation of paeoniflorin and albiflorin[J]. China J Chin Mater Med, 2010, 35(7): 872-875.
[26]	施敏, 马晓彤, 等. 基于短刺小克银汉霉的芍药苷转化芍药内酯苷研究[J]. 中国医药生物技术, 2018, 13(2): 178-184.
	Shi M, Ma XT, et al. Study on the transformation of paeoniflorin from paeoniflorin to paeoniflorin based on Hyphantria brevispi-nosa[J]. Chin Med Biotechnol, 2018, 13(2): 178-184.
[27]	Ye YH, Pei HR, Cao XL, et al. The study of a novel paeoniflorin-converting enzyme from Cunninghamella blakesleeana[J]. Molecules, 2023, 28(3): 1289. doi: 10.3390/molecules28031289 URL
[28]	Buchan DWA, Jones DT. The PSIPRED protein analysis workbench: 20 years on[J]. Nucleic Acids Res, 2019, 47(W1): W402-W407. doi: 10.1093/nar/gkz297
[29]	Kelley LA, Mezulis S, Yates CM, et al. The Phyre2 web portal for protein modeling, prediction and analysis[J]. Nat Protoc, 2015, 10(6): 845-858. doi: 10.1038/nprot.2015.053 pmid: 25950237
[30]	Mao ZJ, Yu L, et al. Expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyti-cus[J]. Acta Biochim Biophys Sin, 2007, 39(10): 763-769. doi: 10.1111/j.1745-7270.2007.00339.x URL
[31]	Wu WP, Shen QY, Zhang RL, et al. The structure of the MICU1-MICU2 complex unveils the regulation of the mitochondrial calcium uniporter[J]. EMBO J, 2020, 39(19): e104285. doi: 10.15252/embj.2019104285 URL
[32]	Lu ZF, Wang BY, et al. YdfD, a Lysis protein of the Qin prophage, is a specific inhibitor of the IspG-catalyzed step in the MEP pathway of Escherichia coli[J]. Int J Mol Sci, 2022, 23(3): 1560. doi: 10.3390/ijms23031560 URL
[33]	Pena-Francesch A, Demirel MC. Squid-inspired tandem repeat proteins: functional fibers and films[J]. Front Chem, 2019, 7: 69. doi: 10.3389/fchem.2019.00069 pmid: 30847338
[34]	Wang WB, Liu JX, Guo SS, et al. Identification of Vibrio para-haemolyticus and Vibrio spp. specific outer membrane proteins by reverse vaccinology and surface proteome[J]. Front Microbiol, 2021, 11: 625315. doi: 10.3389/fmicb.2020.625315 URL
[35]	曲均革, 姚晓敏, 等. 产纤维素酶海洋细菌的筛选鉴定和产酶条件优化[J]. 上海海洋大学学报, 2012, 21(6): 1053-1057.
	Qu JG, Yao XM, et al. Screening and identification of a cellulase-producing marine bacterium and optimization of its fermentation conditions[J]. J Shanghai Ocean Univ, 2012, 21(6): 1053-1057.
[36]	张大智, 詹儒林, 柳凤, 等. 芒果细菌性角斑病菌细胞壁降解酶反应条件优化[J]. 广东农业科学, 2015, 42(8): 72-76.
	Zhang DZ, Zhan RL, Liu F, et al. Reaction conditions optimization of cell wall degrading enzymes from mango bacterial leaf spot pathogen[J]. Guangdong Agric Sci, 2015, 42(8): 72-76.