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Biochemistry

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Published: October 2nd, 2018

DOI:

10.3791/58166

1Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, 2CONACYT, Facultad de Ingeniería Química, Campus de Ciencias Exactas e Ingeniería, Universidad Autónoma de Yucatán

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

Caffeine is a secondary metabolite that is biosynthesized by plants of the genus Coffea1. This alkaloid belongs to the methylxanthine family and is regarded as a chemical plant defense because it can act against the adverse effects of pathogens and herbivores2,3. In addition, this metabolite is responsible for the stimulating properties of the coffee drink, which is commonly consumed worldwide4,5. Due to its properties, several research groups are interested in studying the biosynthetic pathway and catabolism of caffein....

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1. Caffeine Extraction in Cell Suspensions of C. arabica L.

  1. Use C. arabica cell suspensions9. Maintain the suspensions by biweekly subcultures in Murashige and Skoog medium at pH 4.3 with constant 100 rpm shaking at 25 °C under continuous light (8.3 W/m2).
  2. Harvest the cells under vacuum filtration using 11 µm pore filter paper and a Buchner funnel.
  3. Register the fresh weight of the collected cells using a scale, wrap them in alu.......

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Caffeine extracts obtained via the process presented here were analyzed by TLC-densitometry by subjecting the samples to plate chromatography according to the scheme shown in Figure 1. To quantify the levels of caffeine in the cell extracts, a curve with various concentrations of commercial standard for this compound was used (Figure 2A). The pattern of absorbance for caffeine was analyzed in the visible light spectrum (UV-VIS) u.......

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We present here the optimal conditions for evaluating the caffeine content, CS activity and transcript levels in an in vitro plant tissue culture, such as cell suspensions of C. arabica. Previous reports have confirmed that maintaining cells under light irradiation and in the presence of theobromine in the culture medium are suitable parameters for increasing the level of caffeine, making it possible to evaluate the caffeine separation methods using reversed-phase high-performance liquid chromatography .......

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The work of our laboratory was funded by a grant from the Consejo Nacional de Ciencia y Tecnología (CONACyT 219893) to SMTHS. This research was also supported by a fellowship granted to RJPK (No. 37938) by CONACyT and the Sistema Nacional de Investigadores (4422). The authors thank CIATEJ for the use of its installations during the writing of this manuscript. Special thanks are extended to Dr. Víctor Manuel González Mendoza for all recommendations in the molecular biology section and Valentín Mendoza Rodríguez, IFC, UNAM for the facilities during the filming of this article.

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Name Company Catalog Number Comments
Murashige & Skoog Basal salt mixture PhytoTechnology Laboratories M524 Packge Size: 50 L
Reagent (mg/L)
Ammonium Nitrate (1650)
Boric acid (6.2)
Calcium chloride, anhydrous (322.2)
Cobalt Chloride•H2O (0.025)
Cupric Sulfate•5H2O (0.025)
Na2EDTA•2H2O (37.26)
Ferrous Sulfate•7H2O (27.8)
Magnesium Sulfate, Anhydrous (180.7)
Manganese Sulfate•H2O (16.9)
Molybdic Acid (Sodium Salt)• 2H2O (0.25)
Potassium Iodide (0.83)
Potassium Nitrate (1900)
Potassium Phosphate, Monobasic (170)
Zinc Sulfate•7H2O (8.6)
Supplemented with
myo-inositol (100)
thiamine (10)
cysteine (25)
sucrose (30000)
2,4-dichlorophenoxyacetic acid (3)
6-benzylamine purine (1)
Caffeine SIGMA C0750-5G STANDARD-5g
Theobromine SIGMA T4500 20 g
CAMAG TLC Scanner-4 CAMAG 27.62
WinCATS Planar Chromatography Manager software CAMAG 1.4.10 Software
Isoamyl alcohol (24:1) SIGMA C-0549 500 mL
Cyclohexane JALMEX C4375-13 1 L
Acetone J.T. BAKER 900643 4 L
Methanol J.T. BAKER 9093-03 4 L
Chloroform JALMEX C-4425-15 3.5 L
TLC silica gel 60 F254 Merck 1.05554.0001 TLC plate
β-mercaptoethanol M6250 SIGMA 100 mL
(+)-sodium L- ascorbate A4034 SIGMA 100 g
Trizma base SIGMA T6066 1 Kg
Hydrochloric acid 36.5-38% J.T. Baker 9535-05 2.5 L
Pierce BCA Protein Assay Kit Thermo scientific 232227 Kit
Methyl [3H]-S-adenosyl methionine Perkin Elmer NET155 Specific activity of 15 Ci/mmol
Liquid scintillation vials SIGMA Z253081
Thermostatic bath/circulator Cole Parmer 60714
Micro centrifugue tube Eppendorf Tube of 1.5 mL
Cryogenic vials Heathrow Scientific HS23202A 2 mL
Centrifuge 5804 Eppendorf 5804 000925
Vortex Thermolyne LR 5947
Porcelain mortar Fisherbrand FB961B
Filter paper Whatman Z274844 Porosity medium
Picofuge Stratagene 400550 2000 x g
Analytical balance AND HR-120 Model HR-120
Scintillation counter Beckman Coulter 6500
Gel photodocumentation system Bio-Rad Chemic XRS Model Chemic XRS
Compact UV lamp UVP 95002112 UVGL-25
Scienceware HDPE Buchner funnel SIGMA 2419907 Type 37600 mixer
TRIzol reagent Thermo scientific 15596-018 200 mL
ReverdAid Reverse transcriptase Thermo scientific #EP0441 10000 U
Oligo (dT)18 primer Thermo scientific #S0131 100 µM
DNase I, RNase-free Thermo scientific #EN0525 1000 U
Magnesium chloride Thermo scientific EN0525 1.25 mL
Ethylenediaminetetraacetic acid Thermo scientific EN0525 1 mL
dNTP mix Thermo scientific R0191 R0191
SYBR Green qPCR Master Mix (2X) Thermo scientific K0251 For 200 reactions of 25 µL
PikoReal Thermo scientific 2.2 Software
Phenol, pH 8.0, equilibrated, Molecular Biology Grade, Ultrapure USB J75829 100 mL
Isopropyl alcohol Karal 2040 1 L
Ethyl alcohol SIGMA 64175 1 L
Diethyl pyrocarbonate SIGMA D5758 100 mL
Lab Rotator LW Scientific Mod. LW210

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