CN102174639A - Activity detection method of ester type catechin synthetase - Google Patents
Activity detection method of ester type catechin synthetase Download PDFInfo
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Abstract
The invention relates to an activity detection method of ester type catechin synthetase. The method comprises five steps, namely preparation of enzyme solution, preparation of reaction solution, preparation of contrast solution, detection of enzyme activity, and calculation of the enzyme activity. The ester type catechin serving as an enzyme reaction product is detected by using high performance liquid chromatography so as to detect the enzyme activity, and interference of oxidase and ester type catechin hydrolase on enzyme activity detection can be avoided by adding antioxidant and hydrolase inhibitor in the reaction system can be avoided. The method is simple, has strong accuracy and high sensitivity, and can accurately detect the reaction product of the ester type catechin synthetase; and the operation is convenient, the detection time is short, fussy experiment operation is avoided, and the detection can be finished in 2 to 3 hours.
Description
Technical field
The invention belongs to biomaterial detection technique field, be specifically related to a kind of activity test method of ester catechin synthetic enzyme.
Background technology
Ester catechin mainly contains L-Epicatechin gallate (ECG) and NVP-XAA 723 (EGCG) in the tea tree, and non-ester catechin mainly contains l-Epicatechol (EC) and epigallocatechin (EGC); On the structure, ester catechin (L-Epicatechin gallate (ECG) and NVP-XAA 723 (EGCG)) is on the 3-OH position of non-ester catechin (l-Epicatechol (EC), epigallocatechin (EGC)) structure and the acid esters of gallic acid (GA) formation.
Ester catechin content is higher in the tea tree, is about 55% of tea-polyphenol total amount, is the 12%-19% of bright leaf dry matter
[1]Ester catechin has stronger pharmaceutical use than non-ester catechin
[2-3], that ester catechin has is anti-oxidant, antibiotic, anti-inflammatory, multiple pharmacologically active such as anticancer, Parkinson disease is also had the curative effect of a bit
[4]In addition, ester catechin still determines one of key factor of tea drink quality
[5]
Ester catechin synthetic difficulty is bigger, and tealeaves remains the main source of ester catechin
[5]The biosynthetic pathway of the intravital non-ester catechin of plant is comparatively clear, and the main katalaze enzyme that relates to has dihydroflavonol 4-reductase, leucoanthocyanidin reductase enzyme, anthocyanidin synthetic enzyme, anthocyanin reductase etc.
[6-8]Ester catechin is synthetic to be key one ring in the flavonoid biosynthetic pathway, and the unsolved mystery that the ester catechin biosynthetic pathway is always
[9]The applicant utilizes thin-layer chromatography (TLC) first, high performance liquid chromatography (HPLC) and mass spectrum (MS) technology, from tea tree, identified a kind of energy catalysis ester catechin synthetic enzyme (epicatechin:1-O-galloyl--D-glucose O-galloyltransferase, ECGT), under its catalysis, 1-O-Nutgalls acyl-β-D-glucose (β G) and non-ester catechin (l-Epicatechol (EC) or epigallocatechin (EGC)) reaction forms corresponding ester catechin (L-Epicatechin gallate (ECG) or NVP-XAA 723 (EGCG)).The applicant utilizes ammonium sulfate precipitation, anionresin and gel permeation chromatography that this enzyme has been carried out preliminary purification.Up to now, the bibliographical information that does not still have the enzyme biopsy survey method of ester catechin synthetic enzyme.
Summary of the invention
Finding and identifying to the invention provides a kind of activity test method of ester catechin synthetic enzyme on the basis that tea tree ester catechin synthetic enzyme exists first.
The technical solution that realizes above-mentioned purpose is as follows:
A kind of activity test method of ester catechin synthetic enzyme comprises following operation steps:
1.1, enzyme liquid preparation
Get 2 gram fresh leaves of tea plant, add liquid nitrogen, be ground into powder fast, add the phosphoric acid buffer liquor of 0.1 mol/L pH 7.4 of 2 gram polyvinylpyrrolidones (PVPP), 0.5 gram quartz sand, 18mL precooling and the ascorbic acid solution of 2mL 20mmol/L again, grind to form homogenate under 4 ℃ of conditions, under 12000 rev/mins of centrifuge speeds, 4 ℃ of conditions of temperature, centrifugation 15 minutes, get supernatant liquor, promptly get the enzyme liquid of concentration 1.2mg/mL;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Cumulative volume is that the reaction solution of 1.5mL comprises following raw materials according:
The enzyme liquid of enzyme liquid: 1.2mg/mL,
Antioxidant: the ascorbic acid solution of 20 mmol/L,
Hydrolysis inhibitor: the salicylic acid solution of 30.4 mmol/L,
Reaction substrate: l-Epicatechol (EC) solution of the epigallocatechin of 3.8 mmol/L (EGC) solution or 3.8 mmol/L,
9.6 1-O-Nutgalls acyl-β-D-glucose (β G) solution of mmol/L,
Damping fluid: the phosphoric acid buffer of 0.1 mol/L, pH=6.0,
0.46mL enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate l-Epicatechol (EC) solution or 0.15mL epigallocatechin (EGC), solution 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) solution and 0.59mL phosphoric acid buffer are mixed, 30 ℃ of water-baths of temperature 60 minutes get reaction solution;
1.2.2, the contrast liquid preparation
Reaction solution comprises following raw materials according:
Enzyme liquid: 100 ℃ of temperature boil 5 minutes enzyme liquid,
Antioxidant: the ascorbic acid solution of 20 mmol/L,
Hydrolysis inhibitor: the salicylic acid solution of 30.4 mmol/L,
Reaction substrate: l-Epicatechol (EC) solution of the epigallocatechin of 3.8 mmol/L (EGC) solution or 3.8 mmol/L,
9.6 1-O-Nutgalls acyl-β-D-glucose (β G) solution of mmol/L,
Damping fluid: the phosphoric acid buffer of 0.1 mol/L, pH=6.0;
100 ℃ of enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EGC) solution or 0.15mL l-Epicatechol (EC) solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) solution and 0.59mL phosphoric acid buffers that boiled 5 minutes of temperature of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add the 3mL ethyl acetate respectively, extractive reaction product at room temperature respectively, re-extract three times, collect respectively merge reaction solution ethyl acetate mutually and the ethyl acetate that contrasts liquid mutually; With the ethyl acetate of reaction solution mutually and the ethyl acetate of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure are 10 minutes respectively, respectively with dissolve with methanol and be settled to 500 microlitres, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the high performance liquid chromatography (HPLC) of enzyme reaction product analyzes
The content of NVP-XAA 723 (EGCG) or L-Epicatechin gallate (ECG) in employing high-efficient liquid phase chromatogram technology difference detection reaction methanol solution and the contrast methanol solution; In methanol of reaction liquid, detect NVP-XAA 723 (EGCG) or L-Epicatechin gallate (ECG), and in the contrast methanol solution, detect less than NVP-XAA 723 (EGCG) or L-Epicatechin gallate (ECG), can prove that promptly the ester catechin synthetic enzyme enzyme that detects in the bright leaf of tea is alive.
Utilize enzyme work calculation method, calculate ester catechin synthetic enzyme vigor, promptly with the enzyme product NVP-XAA 723 (EGCG) that forms in every milligram of enzyme catalysis of 30 ℃ of following per minutes or the amount of L-Epicatechin gallate (ECG), unit is mg/ minute/milligram albumen;
M/60 minute/0.55 milligram albumen of ester catechin synthetic enzyme enzyme work=△,
In the following formula: △ m represents the enzyme product NVP-XAA 723 (EGCG) of formation in 60 minutes or the amount of L-Epicatechin gallate (ECG); Expression enzyme reaction in 60 minutes 60 minutes; 0.55 milligram albumen represents to add in the 1.5mL reaction solution enzyme content.
The inventive method has the advantage of following several respects:
1, this method is simple to operate, does not need the purifying enzyme extracting solution;
2, accuracy is strong, highly sensitive, can accurately detect ester catechin synthetic enzyme reaction product, proves that the ester catechin lytic enzyme enzyme that detects in the bright leaf of tea is alive;
3, easy to operate, detection time is short, and can finish at 2-3 hour detection time, avoided loaded down with trivial details experimental implementation;
4, solid experimental technique basis is established in the separation and purification that is established as follow-up ester catechin synthetic enzyme of ester catechin synthetic enzyme enzyme biopsy survey method, zymoprotein structure elucidation, enzyme gene clone and functional verification thereof; And to carrying out the biosynthetic metabolic gene engineering research of catechin, developing tea tree breeding new way and improve tea leaf quality and all have most important theories and be worth.
Description of drawings
Fig. 1 for reaction substrate be the ester catechin synthetic enzyme reaction product of epigallocatechin (EGC) high-efficient liquid phase chromatogram (on: reaction system, down: reaction pair according to).
Fig. 2 for reaction substrate be the ester catechin synthetic enzyme reaction product of l-Epicatechol (EC) high-efficient liquid phase chromatogram (on: reaction system, down: reaction pair according to).
Fig. 3 is the substrate EGC optimal concentration figure of ester catechin synthetic enzyme reaction.
Fig. 4 is the substrate β G optimal concentration figure of ester catechin synthetic enzyme reaction.
Fig. 5 is the optimal pH figure of ester catechin synthetic enzyme reaction.
Fig. 6 is the optimal reaction time diagram of ester catechin synthetic enzyme reaction.
Fig. 7 is the optimum temperuture figure of ester catechin synthetic enzyme reaction.
Embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1:
Major equipment: 1. high performance liquid chromatograph (HPLC); 2. thermostat water bath; 3. Rotary Evaporators; 4. whizzer; 5.pH meter;
Material and reagent:
1, detected material: fresh leaves of tea plant (80 ℃ of preservations are standby);
2, main agents
(1) polyvinylpyrrolidone (PVPP) is purchased in Shanghai Suo Laibao bio tech ltd, is used for polyphenols in the adsorptive enzyme liquid, keeps the greater activity of zymoprotein;
(2) quartz sand (commercially available): increase frictional force, improve the extraction yield of zymoprotein;
(3) 0.1 mol/L pH7.4 phosphoric acid buffers
A solution: take by weighing SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
42H
2O) 7.8 grams are dissolved in the distilled water of 500mL;
B solution: take by weighing Sodium phosphate dibasic (Na
2HPO
43H
2O) 17.9 grams are dissolved in the distilled water of 500mL, and are standby;
Before facing usefulness, get A solution or B solution certain volume, adjust pH to 7.4 is 0.1 mol/L pH7.4 phosphoric acid buffer mutually.4 ℃ of preservations are standby;
(4) 0.1 mol/L pH 6.0 phosphoric acid buffers
A solution: take by weighing SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
42H
2O) 7.8 grams are dissolved in the distilled water of 500mL;
B solution: take by weighing Sodium phosphate dibasic (Na
2HPO
43H
2O) 17.9 grams are dissolved in the distilled water of 500mL, and are standby;
Before facing usefulness, get A solution or B solution certain volume, adjust pH to 6.0 is 0.1 mol/L pH6.0 phosphoric acid buffer mutually.4 ℃ of preservations;
(5) 20 mmol/L xitix (purchasing the gold promise chemical industry company limited in the Wuhan City) solution: take by weighing 0.0605 milligram of xitix, add 25mL distilled water, vibration is preserved standby to dissolving fully below 4 ℃;
(6) 3.8 mmol/L epigallocatechins (EGC) (purchasing) solution: take by weighing 0.0023 gram EGC and be dissolved in the 2mL water, preserve standby below 4 ℃ in the medical company limited of last Hiroad standing grain;
(7) 9.7 mmol/L 1-O-Nutgalls acyl-β-D-glucose (β G purchases in Hong Kong modern technique Industrial Co., Ltd) solution: take by weighing 0.0012 gram 1-O-Nutgalls acyl-β-D-glucose and be dissolved in the 1mL water, preserve standby below 4 ℃;
(8) 30.4 mmol/L Whitfield's ointments (purchasing one bio tech ltd) solution: take by weighing 0.0042 gram Whitfield's ointment and be dissolved in the 1mL water, preserve standby below 4 ℃ in Shanghai China.
A kind of activity test method of ester catechin synthetic enzyme comprises following operation steps:
1.1, enzyme liquid preparation
Claim 2 gram fresh leaves of tea plant, after adding liquid nitrogen, be ground into powder fast, at the phosphoric acid buffer of the 0.1 mol/L pH 7.4 that adds 2 gram polyvinylpyrrolidones (PVPP), 0.5 gram quartz sand, 18mL precooling and the ascorbic acid solution of 2mL 20mmol/L, grind to form homogenate under 4 ℃ of conditions, under 12000 rev/mins of centrifuge speeds, 4 ℃ of conditions of temperature, centrifugation 15 minutes, get supernatant liquor, promptly get 1.2mg/mL enzyme liquid;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Cumulative volume comprises following raw materials according for the 1.5mL reaction solution:
Enzyme liquid: 1.2mg/mL enzyme liquid,
Antioxidant: 20 mmol/L ascorbic acid solutions,
Hydrolysis inhibitor: 30.4 mmol/L salicylic acid solutions,
Reaction substrate: 3.8 mmol/L epigallocatechin (EGC) solution,
9.6 mmol/L 1-O-Nutgalls acyl-β-D-glucose (β G) solution,
Damping fluid: 0.1 mol/L pH=6.0 phosphoric acid buffer,
Enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EGC) solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose solution and the 0.59mL phosphoric acid buffer of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes get reaction solution;
Epigallocatechin (EGC) and 1-O-Nutgalls acyl-β-D-glucose structural formula is as follows respectively:
1.2.2, the contrast liquid preparation
Reaction solution comprises following raw materials according:
Enzyme liquid: 100 ℃ boil 5 minutes enzyme liquid,
Antioxidant: 20 mmol/L ascorbic acid solutions,
Hydrolysis inhibitor: 30.4 mmol/L salicylic acid solutions,
Reaction substrate: 3.8 mmol/L epigallocatechin (EGC) solution,
9.6 mmol/L 1-O-Nutgalls acyl-β-D-glucose (β G) solution,
Damping fluid: 0.1 mol/L pH=6.0 phosphoric acid buffer,
100 ℃ of enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EGC) solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) solution and 0.59mL phosphoric acid buffers that boiled 5 minutes of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add the 3mL ethyl acetate respectively, extractive reaction product at room temperature respectively, re-extract three times, collect respectively merge reaction solution ethyl acetate mutually and the ethyl acetate that contrasts liquid mutually; With the ethyl acetate of reaction solution mutually and the ethyl acetate of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure are 10 minutes respectively, respectively with dissolve with methanol and be settled to 500 microlitres, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
Utilize known high-efficient liquid phase chromatogram technology (HPLC) detection reaction methanol solution and the content that contrasts NVP-XAA 723 (EGCG) in the methanol solution respectively.In methanol of reaction liquid, detect NVP-XAA 723 (EGCG), and in the contrast methanol solution, detect, can prove that promptly the ester catechin synthetic enzyme enzyme that detects in the bright leaf of tea is alive less than NVP-XAA 723 (EGCG).
NVP-XAA 723 (EGCG) structural formula is as follows:
Utilize typical curve to calculate the content of ester catechin (NVP-XAA 723 (EGCG)) in the methanol of reaction liquid, see Fig. 1.
HPLC chromatographic condition: chromatogram pump: water these 600 (Waters 600); Controller: water these 600 (Waters 600); Chromatographic column: the dark 250*4.6mm of the holy and pure blue 4u spoke of F door (Phenomenex Synergi 4u Fusion 250*4.6mm); Detector: this (Waters) 2487 of UV water; Sensitivity: 0.01; Detect wavelength: 280 nm; Flow velocity: 1.2 mL/minute; Sample size: 5 microlitres; Before each sample sample introduction, with moving phase balance 15 minutes.Adopt condition of gradient elution A phase: 1% acetate, the B phase: trifluoroacetic acid aqueous solution, gradient are that A is by 90% to 87% in preceding 20 minutes, and B is by 10% to 13%; 20 minutes to 40 minutes A are by 87% to 70%, and B is by 13% to 30%.Utilizing the catechin standard substance to carry out qualitative and quantitative calculates.
Utilize known enzyme work calculation method to calculate ester catechin synthetic enzyme vigor, promptly measure with the enzyme product ester catechin (NVP-XAA 723 (EGCG)) that forms in every milligram of enzyme catalysis of 30 ℃ of following per minutes, unit is mg/ minute/milligram albumen.
M/60 minute/0.55 milligram albumen of ester catechin synthetic enzyme enzyme work=△
In the formula: △ m represents enzyme product ester catechin (NVP-XAA 723 (the EGCG)) amount of formation in 60 minutes; Expression enzyme reaction in 60 minutes 60 minutes; 0.55 milligram albumen represents to add in the 1.5mL reaction solution enzyme content.
Embodiment 2
Major equipment is with embodiment 1.
Material and reagent:
1. detected material: with embodiment 1;
2. main agents:
Reaction substrate: 3.8 mmol/L epigallocatechin (EC) solution: take by weighing 0.0011 gram EC and be dissolved in the 1mL water, preserve standby below 4 ℃.Other reagent is with embodiment 1;
A kind of activity test method of ester catechin synthetic enzyme comprises following operation steps:
1.1, enzyme liquid preparation
With embodiment 1;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EC) solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) solution and the 0.59mL phosphoric acid buffer of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes get reaction solution;
Epigallocatechin (EC) and 1-O-Nutgalls acyl-β-D-glucose (β G) structural formula are as follows respectively:
1.2.2, the contrast liquid preparation
100 ℃ of enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin (EC) solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) solution and 0.59mL phosphoric acid buffers that boiled 5 minutes of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add the 3mL ethyl acetate respectively, extractive reaction product at room temperature respectively, re-extract three times, collect respectively merge reaction solution ethyl acetate mutually and the ethyl acetate that contrasts liquid mutually; With the ethyl acetate of reaction solution mutually and the ethyl acetate of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure are 10 minutes respectively, respectively with dissolve with methanol and be settled to 500 microlitres, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
Utilize known high-efficient liquid phase chromatogram technology (HPLC) detection reaction methanol solution and the content that contrasts L-Epicatechin gallate (ECG) in the methanol solution respectively.In methanol of reaction liquid, detect L-Epicatechin gallate (ECG), and in the contrast methanol solution, detect, can prove that promptly the ester catechin synthetic enzyme enzyme that detects in the bright leaf of tea is alive less than L-Epicatechin gallate (ECG).
L-Epicatechin gallate (ECG) structural formula is as follows:
Utilize typical curve to calculate the content of ester catechin (L-Epicatechin gallate (ECG)) in the methanol of reaction liquid, see Fig. 2.
HPLC chromatographic condition: with embodiment 1.
Utilize known enzyme work calculation method to calculate ester catechin synthetic enzyme vigor, promptly measure with the enzyme product ester catechin (L-Epicatechin gallate (ECG)) that forms in every milligram of enzyme catalysis of 30 ℃ of following per minutes, unit is mg/ minute/milligram albumen.
M/60 minute/0.55 milligram albumen of ester catechin synthetic enzyme enzyme work=△
In the formula: △ m represents enzyme product ester catechin (L-Epicatechin gallate (the ECG)) amount of formation in 60 minutes; Expression enzyme reaction in 60 minutes 60 minutes; 0.55 milligram albumen represents to add in the 1.5mL reaction solution enzyme content.
The technical characterstic explanation
1. reaction substrate epigallocatechin (EGC) concentration
Fig. 3 shows that ester catechin synthetic enzyme optimal reaction substrate EGC concentration is 0.38 mmol/L.Along with reaction substrate EGC concentration increases, product EGCG amount presents fast rise trend.After EGC concentration reached 0.38mmol/L, the reaction product amount was to maximum (mensuration of working method 1.2 enzymic activitys among the embodiment 1).
2. reaction substrate 1-O-Nutgalls acyl-β-D-glucose (β G) concentration
Fig. 4 shows that ester catechin synthetic enzyme optimal reaction substrate β G concentration is 0.8mmol/L.Along with reaction substrate β G concentration increases, product EGCG amount presents fast rise trend.After β G concentration reached 0.8mmol/L, the reaction product amount reached maximum (working method 1.2 enzyme assaies among the embodiment 1).
3. reaction assay time
Fig. 5 shows that the ester catechin synthetic enzyme optimal reaction time is 60 minutes.Along with the prolongation in reaction times, reaction product EGCG amount increases gradually.After the reaction times surpasses 60 minutes, reaction system color burn, reaction product (working method 1.2 enzyme assaies among the embodiment 1) on a declining curve.
4. pH value in reaction
Fig. 6 shows that ester catechin synthetic enzyme optimal reaction pH value is 6.0.The pH value is obvious to the reaction influence, and pH value in reaction was greater than or less than 6.0 o'clock, and product EGCG obviously reduces.
5. temperature of reaction
Fig. 7 shows the rising with temperature of reaction, and the amount of ester catechin synthetic enzyme reaction product EGCG is slow ascendant trend, and when temperature of reaction reached 30 ℃, it is maximum that the reaction product amount reaches.But when temperature of reaction surpasses 30 ℃, the reaction system color is obviously deepened, side reaction aggravation, the amount of ester catechin synthetic enzyme reaction product EGCG obviously descend (working method 1.2 enzyme assaies among the embodiment 1).
6. utilize high performance liquid chromatography (HPLC) analytical procedure simultaneously qualitative and quantitative detection ester catechin building-up reactions substrate and product catechin (Fig. 1, Fig. 2).
Antioxidant can effectively protective reaction system in reaction substrate and reaction product, avoid oxidized oxydasis.
8. reaction product ester catechin (L-Epicatechin gallate (ECG) or NVP-XAA 723 (EGCG)) in the effective protective reaction system of hydrolysis inhibitor is avoided by the hydrolysis of ester catechin lytic enzyme.
Reference
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(Wolfram?S,?Wang?Y,?Thielecke?F.?Anti-obesity?effects?of?green?tea:?from?bedside?to?bench.?Mol?Nutr?Food?Res.?2006,?50(2):?176-187.)
[3] examine YH, long HH, Lee MJ, etc. tea, obesity and diabetes. molecular nutrition is learned. and 2006,50 (2): 188-210.
(Kao?YH,?Chang?HH,?Lee?MJ?et?al.?Tea,?obesity,?and?diabetes.?Mol?Nutr?Food?Res.?2006,?50(2):?188-210.)
[4] Melky Cabrera C, the tall R of ell tower, Ji Menneisi R. is about the summary of green tea beneficial effect. AAN's proceedings. 2006,25:79-99.
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Claims (2)
1. the activity test method of an ester catechin synthetic enzyme is characterized in that comprising following operation steps:
1.1, enzyme liquid preparation
Get 2 gram fresh leaves of tea plant, add liquid nitrogen, be ground into powder fast, add the phosphoric acid buffer liquor of 0.1 mol/L pH 7.4 of 2 gram polyvinylpyrrolidones (PVPP), 0.5 gram quartz sand, 18mL precooling and the ascorbic acid solution of 2mL 20mmol/L again, grind to form homogenate under 4 ℃ of conditions, under 12000 rev/mins of centrifuge speeds, 4 ℃ of conditions of temperature, centrifugation 15 minutes, get supernatant liquor, promptly get the enzyme liquid of concentration 1.2mg/mL;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Cumulative volume is that the reaction solution of 1.5mL comprises following raw materials according:
The enzyme liquid of enzyme liquid: 1.2mg/mL
Antioxidant: the ascorbic acid solution of 20 mmol/L
Hydrolysis inhibitor: the salicylic acid solution of 30.4 mmol/L
Reaction substrate: the epigallocatechin solution of 3.8 mmol/L or the l-Epicatechol solution of 3.8 mmol/L,
9.6 1-O-Nutgalls acyl-β-D-glucose solution of mmol/L
Damping fluid: the phosphoric acid buffer of 0.1 mol/L, pH=6.0
0.46mL enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate l-Epicatechol solution or 0.15mL epigallocatechin, solution 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose solution and 0.59mL phosphoric acid buffer are mixed, 30 ℃ of water-baths of temperature 60 minutes get reaction solution;
1.2.2, the contrast liquid preparation
Reaction solution comprises following raw materials according:
Enzyme liquid: 100 ℃ of enzyme liquid that boil 5 minutes of temperature
Antioxidant: the ascorbic acid solution of 20 mmol/L
Hydrolysis inhibitor: the salicylic acid solution of 30.4 mmol/L
Reaction substrate: the epigallocatechin solution of 3.8 mmol/L or the l-Epicatechol solution of 3.8 mmol/L,
9.6 1-O-Nutgalls acyl-β-D-glucose solution of mmol/L
Damping fluid: the phosphoric acid buffer of 0.1 mol/L, pH=6.0
100 ℃ of enzyme liquid, 0.3mL antioxidant, 0.074mL hydrolysis inhibitor, 0.15mL reaction substrate epigallocatechin solution or 0.15mL l-Epicatechol solution, 0.075mL reaction substrate 1-O-Nutgalls acyl-β-D-glucose solution and 0.59mL phosphoric acid buffers that boiled 5 minutes of temperature of 0.46mL are mixed, 30 ℃ of water-baths of temperature 60 minutes must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add the 3mL ethyl acetate respectively, extractive reaction product at room temperature respectively, re-extract three times, collect respectively merge reaction solution ethyl acetate mutually and the ethyl acetate that contrasts liquid mutually; With the ethyl acetate of reaction solution mutually and the ethyl acetate of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure are 10 minutes respectively, respectively with dissolve with methanol and be settled to 500 microlitres, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
The content of NVP-XAA 723 or L-Epicatechin gallate in employing high-efficient liquid phase chromatogram technology difference detection reaction methanol solution and the contrast methanol solution; In methanol of reaction liquid, detect NVP-XAA 723 or L-Epicatechin gallate, and in the contrast methanol solution, detect less than NVP-XAA 723 or L-Epicatechin gallate, can prove that promptly the ester catechin synthetic enzyme enzyme that detects in the bright leaf of tea is alive.
2. the activity test method of a kind of ester catechin synthetic enzyme according to claim 1 is characterized in that:
Utilize enzyme work calculation method, calculate ester catechin synthetic enzyme vigor, promptly with the enzyme product NVP-XAA 723 that forms in every milligram of enzyme catalysis of 30 ℃ of following per minutes or the amount of L-Epicatechin gallate, unit is mg/ minute/milligram albumen;
M/60 minute/0.55 milligram albumen of ester catechin synthetic enzyme enzyme work=△,
In the following formula: △ m represents the enzyme product NVP-XAA 723 of formation in 60 minutes or the amount of L-Epicatechin gallate; Expression enzyme reaction in 60 minutes 60 minutes; 0.55 milligram albumen represents to add in the 1.5mL reaction solution enzyme content.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103740740A (en) * | 2014-01-28 | 2014-04-23 | 湖南农业大学 | Tea tree epicatechin galloyl transferase gene CsECGT as well as coded protein and application thereof |
| CN108181416A (en) * | 2017-12-15 | 2018-06-19 | 中国农业科学院茶叶研究所 | A kind of method for measuring 1,2,6 three nutgall acyl-β of tealeaves-D-Glucose and application thereof |
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| TAKASHI AKAGI等: "Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon(Diospyros kaki thunb.) fruit", 《PLANTA》 * |
| 夏涛 等: "类黄酮及茶儿茶素生物合成途径及其调控研究进展", 《中国农业科学》 * |
| 高可君: "茶叶中二氢黄酮醇还原酶/无色花青素还原酶性质研究", 《中国优秀硕士学位论文全文数据库 农业科技辑（月刊）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740740A (en) * | 2014-01-28 | 2014-04-23 | 湖南农业大学 | Tea tree epicatechin galloyl transferase gene CsECGT as well as coded protein and application thereof |
| CN108181416A (en) * | 2017-12-15 | 2018-06-19 | 中国农业科学院茶叶研究所 | A kind of method for measuring 1,2,6 three nutgall acyl-β of tealeaves-D-Glucose and application thereof |
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