CN102174642B - Method for detecting enzymatic activity of ester type catechin hydrolysis enzyme - Google Patents
Method for detecting enzymatic activity of ester type catechin hydrolysis enzyme Download PDFInfo
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for detecting enzymatic activity of ester type catechin hydrolysis enzyme. The method comprises the following operation steps of: 1, preparing enzyme liquid by using fresh leaves of tea tree; 2, preparing reaction liquid and reference liquid by using the enzyme liquid; 3, respectively preparing reaction carbinol liquid and reference carbinol liquid by using the reaction liquid and the reference liquid; and 4, respectively detecting non-ester type catechin in the reaction carbinol liquid and the reference carbinol liquid by using an HPLC (High Performance Liquid Chromatography) technology. The method disclosed by the invention is simple, does not need to purify enzyme extract, has accurate detected results and high sensitivity, can be used for accurately detecting the reaction product of ester type catechin hydrolysis enzyme, which means that the enzymatic activity of ester type catechin hydrolysis enzyme in fresh tea leaves is detected; in addition, the method is convenient to operate, needs short detection time and can be completed within 1-2 hours, can avoid complicated experimental operation.
Description
Technical field
The invention belongs to biomaterial detection technique field, be specifically related to the detection method that a kind of ester catechin lytic enzyme enzyme is lived.
Background technology
Catechin in the tea tree is not only the important secondary metabolism composition of tea tree, also is the quality component that constitutes the tealeaves flavour.Clinical study simultaneously shows that catechin is an important function composition in the tea drink, has reducing blood-fat, hypotensive, strengthening immunity, multiple pharmacologically active such as antibiotic, antitumor, anti-oxidant, therefore becomes the focus of medicine and food research
[1-5]
Catechin belongs to the flavane 3-alcohols material in the flavonoid, according to whether connecting the Nutgalls group on the 3-OH position, can be divided into two types of ester type and non-ester catechins.Ester catechin comprises L-Epicatechin gallate (ECG) and NVP-XAA 723 (EGCG), and non-ester catechin comprises catechin (C), l-Epicatechol (EC), l-Epigallocatechol (GC) and hin (EGC).See that from structure ester catechin is the ester that non-ester catechin and gallic acid form
[6]
Many researchs show; The biosynthetic pathway of the intravital non-ester catechin of plant (flavane 3-alcohols) is comparatively clear; The katalaze(enzyme) that relates generally to has phenylalanine ammonia lyase, styracin hydroxylase, 4-coumaric acyl CoA ligase enzyme, chalcone synthetase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3 '-hydroxylase, flavonoid 3 ', 5 '-hydroxylase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase enzyme, cyanidin(e) synthetic enzyme, anthocyanin reductase etc.
[7-10]
Applicant of the present invention utilizes thin-layer chromatography, performance liquid chromatography, LC-MS technology first; From tea tree, identified enzyme (the Galloylated Catechins Hydrolase of a kind of ability catalysis ester catechin hydrolysis; GCH); Under its catalysis, the ester catechin hydrolytic reactions generates gallic acid and non-ester catechin.And utilize 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 ester catechin lytic enzyme detection method.Solid experimental technique basis is established in the separation and purification that is established as follow-up ester catechin lytic enzyme, zymoprotein structure elucidation, enzyme gene clone and the functional verification thereof of ester catechin lytic enzyme enzyme biopsy survey method, and for local tea variety improvement from now on, improve tea leaf quality and have most important theories value.
Summary of the invention
Establish solid experimental technique basis for the separation and purification, zymoprotein structure elucidation, enzyme gene clone and the functional verification thereof that solve the ester catechin lytic enzyme, the present invention provides a kind of enzymic activity detection method of tea tree ester catechin lytic enzyme.
The technical solution that realizes above-mentioned purpose is following:
A kind of enzymic activity detection method of ester catechin lytic enzyme comprises following operation steps:
1.1, enzyme liquid preparation
Claim 2 gram fresh leaves of tea plant, behind the adding liquid nitrogen, be ground into powder fast; At the phosphoric acid buffer liquor of the 0.1 mol/L pH 7.4 that adds 2 gram Vinylpyrrolidone polymers (PVPP), 0.5 gram silica sand, 18mL precooling and the ascorbic acid solution of 2mL concentration 20mmol/L; Under 4 ℃ of conditions of temperature, grinding to form homogenate, is centrifugal 15 min under 12000 rev/mins, the 4 ℃ conditions in centrifuge speed; Get supernatant, get 1.2mg/mL enzyme liquid;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Reaction solution comprises following raw materials according:
Zyme extract: 1.2mg/mL enzyme liquid
Damping fluid: 0.1 mol/L pH6.5 phosphoric acid buffer
Inhibitor: 40mmol/L xitix
Reaction substrate: 4mmol/L NVP-XAA 723 (EGCG) solution or nutgall catechin gallic acid ester (GCG) solution or L-Epicatechin gallate (ECG) solution;
Enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 (EGCG) solution or nutgall catechin gallic acid ester (GCG) solution or L-Epicatechin gallate (ECG) solution and 1.795mL phosphoric acid buffer with 0.33mL; Mixing; Place 30 ℃ of water-baths to react 30min, get reaction solution;
1.2.2, the contrast liquid preparation
Contrast liquid comprises following raw materials according:
Zyme extract: 100 ℃ of enzyme liquid that boil 5 minutes
Damping fluid: 0.1 mol/L pH6.5 phosphoric acid buffer
Inhibitor: 40mmol/L ascorbic acid solution
Reaction substrate: 4mmol/L NVP-XAA 723 (EGCG) solution or nutgall catechin gallic acid ester (GCG) solution or L-Epicatechin gallate (ECG) solution;
100 ℃ of 0.33mL are boiled enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 (EGCG) solution or nutgall catechin gallic acid ester (GCG) solution or L-Epicatechin gallate (ECG) solution and the 1.795mL phosphoric acid buffer of 5min; Mixing is placed in 30 ℃ of water-baths and reacts 30min, must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add 5mL ETHYLE ACETATE respectively, difference is the extractive reaction product at room temperature, re-extract three times, the ETHYLE ACETATE of the ETHYLE ACETATE phase of collection merging reaction solution and contrast liquid is mutually respectively; With the ETHYLE ACETATE of reaction solution mutually with the ETHYLE ACETATE of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure of temperature 10 minutes, use dissolve with methanol more respectively, and be settled to 500 microlitres respectively respectively, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the performance liquid chromatography (HPLC) of enzyme reaction product analyzes
Utilize the content of l-Epicatechol (EC) or hin (EGC) or l-Epigallocatechol (GC) in the known high-efficient liquid phase chromatogram technology detection reaction of catechin methanol solution and the contrast methanol solution; In methanol of reaction liquid, detect enzyme reaction product l-Epicatechol (EC) or hin (EGC) or l-Epigallocatechol (GC); And in the contrast methanol solution, detect less than enzyme reaction product l-Epicatechol (EC) or hin (EGC) or l-Epigallocatechol (GC), promptly can prove to detect ester catechin lytic enzyme enzyme work in the fresh leaves of tea plant.
Utilize enzyme work calculation method; Calculate ester catechin lytic enzyme vigor; Promptly with the enzyme product l-Epicatechol (EC) that forms in every milligram of enzyme catalysis of 30 ℃ of following PMs or the amount of hin (EGC) or l-Epigallocatechol (GC), unit is mg/ minute/milligram albumen;
M/30 minute/0.40 milligram albumen of ester catechin lytic enzyme enzyme work=△
In the formula: △ m representes the enzyme product l-Epicatechol (EC) of formation in 30 minutes or the amount of hin (EGC) or l-Epigallocatechol (GC); Expressed enzyme reaction in 30 minutes 30 minutes; 0.40 milligram albumen representes to add in the 2.5mL reaction solution enzyme content.
Useful technique effect of the present invention is embodied in following several respects:
1, detection method of the present invention is simple, does not need the purifying enzyme extracting solution;
2, the detected result accuracy of the inventive method is strong, highly sensitive, can accurately detect ester catechin lytic 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, can accomplish detection at 1-2 hour, has 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 lytic enzyme of ester catechin lytic enzyme enzyme biopsy survey method, zymoprotein structure elucidation, enzyme gene clone and functional verification thereof, and for local tea variety improvement from now on, improve tea leaf quality and have most important theories value.
Description of drawings
Fig. 1 for reaction substrate be the ester catechin lytic enzyme reaction product of L-Epicatechin gallate (ECG) high-efficient liquid phase chromatogram (on: reaction system, down: reaction pair according to).
Fig. 2 for reaction substrate be the ester catechin lytic enzyme reaction product of NVP-XAA 723 (EGCG) high-efficient liquid phase chromatogram (on: reaction system, down: reaction pair according to).
Fig. 3 for reaction substrate be the ester catechin lytic enzyme reaction product of nutgall catechin gallic acid ester (GCG) high-efficient liquid phase chromatogram (on: reaction system, down: reaction pair according to).
Fig. 4 is the optimal reactive temperature figure of ester catechin lytic enzyme reaction.
Fig. 5 is the ph optimum figure of ester catechin lytic enzyme reaction.
Fig. 6 is the optimal reaction time diagram of ester catechin lytic enzyme reaction.
Fig. 7 is the righttest substrate EGCG concentration map of ester catechin lytic enzyme reaction.
Embodiment
Below in conjunction with accompanying drawing, the present invention is done to describe further through embodiment.
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. material to be detected: fresh leaves of tea plant (80 ℃ of preservations are subsequent use);
2. main agents
(1) Vinylpyrrolidone polymer (PVPP) is purchased the Suo Laibao bio tech ltd in Shanghai.Be used for polyphenols in the adsorption precipitation crude enzyme liquid, keep the greater activity of zymoprotein;
(2) silica 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 zero(ppm) water of 500mL;
B solution: take by weighing Sodium phosphate, dibasic (Na
2HPO
43H
2O) 17.9 grams are dissolved in the zero(ppm) water of 500mL, and are subsequent use;
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 subsequent use;
(4) 0.1 mol/L pH 6.5 phosphoric acid buffers
A solution: take by weighing SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
42H
2O) 7.8 grams are dissolved in the zero(ppm) water of 500mL;
B solution: take by weighing Sodium phosphate, dibasic (Na
2HPO
43H
2O) 17.9 grams are dissolved in the zero(ppm) water of 500mL, and are subsequent use;
Before facing usefulness, get A solution or B solution certain volume, adjust pH to 6.5 is 0.1 mol/L pH6.5 phosphoric acid buffer mutually.(4 ℃ of preservations)
(5) 40mmol/L xitix (purchasing the gold promise chemical industry ltd in the Wuhan City) solution: take by weighing 0.007 gram xitix, add 1mL zero(ppm) water, vibration is to dissolving fully, 4 ℃ of preservations;
(6) 4mmol/L NVP-XAA 723 (EGCG) (purchasing in the medical ltd of last Hiroad standing grain) solution: take by weighing 0.0018 gram EGCG, add 1mL zero(ppm) water, vibration is placed for 4 ℃ and is preserved to dissolving fully.
A kind of activity test method of ester catechin lytic 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 liquor of the 0.1 mol/L pH 7.4 that adds 2 gram Vinylpyrrolidone polymers (PVPP), 0.5 gram silica sand, 18mL precooling and the ascorbic acid solution of 2mL 20mmol/L; Grind to form homogenate under 4 ℃ of conditions; In centrifuge speed is centrifugal 15 min under 15000g, the 4 ℃ of conditions, gets supernatant, 1.2mg/mL enzyme liquid;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
TV is that 2.5mL ester catechin lytic enzyme reaction system comprises following raw materials according
Enzyme liquid: 1.2mg/mL enzyme liquid
Inhibitor: 40mmol/L ascorbic acid solution
Reaction substrate: 4mmol/L NVP-XAA 723 (EGCG) solution
Damping fluid: 0.1 mol/L pH6.5 phosphoric acid buffer
With enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 (EGCG) solution and the 1.795mL phosphoric acid buffer of 0.33mL, mixing is placed in 30 ℃ of water-baths and reacts 30min, gets reaction solution;
NVP-XAA 723 (EGCG) structural formula is following:
1.2.2, the contrast liquid preparation
Reaction solution comprises following raw materials according:
Enzyme liquid: 100 ℃ of enzyme liquid that boil 5 minutes
Inhibitor: 40mmol/L ascorbic acid solution
Reaction substrate: 4mmol/L NVP-XAA 723 (EGCG) solution
Damping fluid: 0.1 mol/L pH6.5 phosphoric acid buffer
100 ℃ of 0.33mL are boiled enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 (EGCG) solution and the 1.795mL phosphoric acid buffer of 5min, and mixing is placed in 30 ℃ of water-baths and reacts 30min, must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid; Add 5mL ETHYLE ACETATE respectively, difference is the extractive reaction product at room temperature, re-extract three times; Collect ETHYLE ACETATE phase that merges reaction solution and the ETHYLE ACETATE that contrasts liquid mutually; Respectively behind 60 ℃ of concentrating under reduced pressure 10min, respectively with dissolve with methanol and be settled to 500 microlitres, respectively methanol of reaction liquid and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
Utilize the known high-efficient liquid phase chromatogram technology of catechin (HPLC) detection reaction methanol solution and the content that contrasts hin (EGC) in the methanol solution respectively; In methanol of reaction liquid, detect hin (EGC), and in the contrast methanol solution, detect, can prove that promptly the ester catechin lytic enzyme enzyme that detects in the bright leaf of tea is alive less than hin (EGC);
Under inhibitor (xitix) protection, ester catechin lytic enzyme catalysis NVP-XAA 723 (EGCG) hydrolytic reactions generates hin (EGC) and gallic acid (GA).
Hin (EGC) and gallic acid (GA) structural formula are distinguished as follows:
Utilize typical curve to calculate the content of hin (EGC) 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 catechin and gallic acid standard substance to carry out qualitative and quantitative calculates.
Utilize known enzyme work calculation method to calculate ester catechin lytic enzyme vigor, promptly measure with the enzyme product hin (EGC) that forms in every milligram of enzyme catalysis of 30 ℃ of following PMs, unit is mg/ minute/milligram albumen.
M/30 minute/0.40 milligram albumen of ester catechin lytic enzyme enzyme work=△
In the formula: △ m representes enzyme product hin (EGC) amount of formation in 30 minutes; Expressed enzyme reaction in 30 minutes 30 minutes; 0.40 milligram albumen representes to add in the 2.5mL reaction solution enzyme content.
Embodiment 2
Major equipment: with embodiment 1.
Material and reagent:
1, material to be detected: with embodiment 1
2, main agents:
Reaction substrate: 4mmol/L L-Epicatechin gallate (ECG) (purchasing) in the medical ltd of last Hiroad standing grain: take by weighing 0.0035 gram ECG,, adding 2mL zero(ppm) water, vibration is placed for 4 ℃ and is preserved to dissolving fully.
Other reagent is with embodiment 1
A kind of activity test method of ester catechin lytic 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
With enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate L-Epicatechin gallate (ECG) solution and the 1.795mL phosphoric acid buffer of 0.33mL, mixing is placed in 30 ℃ of water-baths and reacts 30min, gets reaction solution;
L-Epicatechin gallate (ECG) structural formula is following:
1.2.2, the contrast liquid preparation
100 ℃ of 0.33mL are boiled enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate L-Epicatechin gallate (ECG) solution and the 1.795mL damping fluid of 5min, and mixing is placed in 30 ℃ of water-baths and reacts 30min, must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid; Add 5mL ETHYLE ACETATE respectively, difference is the extractive reaction product at room temperature, re-extract three times; Collect ETHYLE ACETATE phase that merges reaction solution and the ETHYLE ACETATE that contrasts liquid mutually; Respectively behind 60 ℃ of concentrating under reduced pressure 10min, respectively with dissolve with methanol and be settled to 500 microlitres, respectively methanol of reaction liquid and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
Utilize the known high-efficient liquid phase chromatogram technology of catechin (HPLC) detection reaction methanol solution and the content that contrasts l-Epicatechol (EC) in the methanol solution respectively; In methanol of reaction liquid, detect l-Epicatechol (EC), and in the contrast methanol solution, detect, promptly can prove to detect ester catechin lytic enzyme enzyme work in the fresh leaves of tea plant less than l-Epicatechol (EC).
Under inhibitor (xitix) protection, ester catechin lytic enzyme catalysis L-Epicatechin gallate (ECG) hydrolytic reactions generates l-Epicatechol (EC) and gallic acid (GA).
L-Epicatechol (EC) and gallic acid (GA) structural formula are following:
Utilize typical curve to calculate the content of l-Epicatechol (EC) 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 lytic enzyme vigor, promptly measure with the enzyme product l-Epicatechol (EC) that forms in every milligram of enzyme catalysis of 30 ℃ of following PMs, unit is mg/ minute/milligram albumen.
M/30 minute/0.40 milligram albumen of ester catechin lytic enzyme enzyme work=△
In the formula: △ m representes enzyme product l-Epicatechol (EC) amount of formation in 30 minutes; Expressed enzyme reaction in 30 minutes 30 minutes; 0.40 milligram albumen representes to add in the 2.5mL reaction solution enzyme content.
Embodiment 3:
Major equipment is with embodiment 1.
Material and reagent:
1, material to be detected: with embodiment 1
2, main agents:
Reaction substrate: 4mmol/L nutgall catechin gallic acid ester (GCG) (purchasing) in the medical ltd of last Hiroad standing grain.Take by weighing 0.0018 gram nutgall catechin gallic acid ester (GCG), add 1mL zero(ppm) water, vibration is placed for 4 ℃ and is preserved to dissolving fully.
Other reagent is with embodiment 1.
A kind of activity test method of ester catechin lytic enzyme comprises following operation steps:
1.1, enzyme liquid prepares with embodiment 1
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
TV is that 2.5mL ester catechin lytic enzyme reaction system comprises following raw materials according
Enzyme liquid: the 1.2mg/mL enzyme liquid that step (1) obtains
Inhibitor: 40mmol/L ascorbic acid solution
Reaction substrate: 4mmol/L nutgall catechin gallic acid ester (GCG) solution
Damping fluid: 0.1 mol/L pH6.5 phosphoric acid buffer
With enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate nutgall catechin gallic acid ester (GCG) solution and the 1.795mL damping fluid of 0.33mL, mixing is placed in 30 ℃ of water-baths and reacts 30min, gets reaction solution;
Nutgall catechin gallic acid ester (GCG) structural formula is distinguished as follows:
1.2.2, the contrast liquid preparation
100 ℃ of 0.33mL are boiled enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate nutgall catechin gallic acid ester (GCG) solution and the 1.795mL phosphoric acid buffer of 5min, and mixing is placed in 30 ℃ of water-baths and reacts 30min, must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid; Add 5mL ETHYLE ACETATE respectively, difference is the extractive reaction product at room temperature, re-extract three times; Collect ETHYLE ACETATE phase that merges reaction solution and the ETHYLE ACETATE that contrasts liquid mutually; Respectively behind 60 ℃ of concentrating under reduced pressure 10min, respectively with dissolve with methanol and be settled to 500 microlitres, respectively methanol of reaction liquid 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-Epigallocatechol (GC) in the methanol solution respectively.In methanol of reaction liquid, detect l-Epigallocatechol (GC), and in the contrast methanol solution, detect, promptly can prove to detect ester catechin lytic enzyme enzyme work in the fresh leaves of tea plant less than l-Epigallocatechol (GC).
Under inhibitor (xitix) protection, ester catechin lytic enzyme catalysis nutgall catechin gallic acid ester (GCG) hydrolytic reactions generates l-Epigallocatechol (GC) and gallic acid (GA).
L-Epigallocatechol (GC) and gallic acid (GA) structural formula are distinguished as follows:
Utilize typical curve to calculate the content of l-Epigallocatechol (GC) in the methanol of reaction liquid, see Fig. 3.
HPLC chromatographic condition: with embodiment 1
Utilize known enzyme work calculation method to calculate ester catechin lytic enzyme vigor, promptly measure with the enzyme product l-Epigallocatechol (GC) that forms in every milligram of enzyme catalysis of 30 ℃ of following PMs, unit is mg/ minute/milligram albumen.
M/30 minute/0.40 milligram albumen of ester catechin lytic enzyme enzyme work=△
In the formula: △ m representes enzyme product l-Epigallocatechol (GC) amount of formation in 30 minutes; Expressed enzyme reaction in 30 minutes 30 minutes; 0.40 milligram albumen representes to add in the 2.5mL reaction solution enzyme content.
The technical characterstic explanation
1. temperature of reaction
Fig. 4 shows the rising with temperature of reaction, and the amount of ester catechin lytic enzyme reaction product EGC is slow ascendant trend, and when temperature of reaction reached 35 ℃, it is maximum that the reaction product amount reaches.But when temperature of reaction surpasses 30 ℃, the reaction system color is obviously deepened, and the side reaction aggravation is so select 30 ℃ as ester catechin lytic enzyme temperature of reaction comparatively suit (working method 1.2 enzyme activity determinations among the embodiment 1).
2. react pH
Fig. 5 shows that ester catechin lytic enzyme optimal reaction pH is 6.5.The pH value is obvious to the reaction influence, and when pH value in reaction was 6.5, product EGC measured maximum.PH value in reaction was greater than 6.5 o'clock, and product EGC obviously reduces (working method 1.2 enzyme activity determinations among the embodiment 1).
3. reaction assay time
Fig. 6 shows that the ester catechin lytic enzyme optimal reaction time is 30 minutes.Along with the prolongation in reaction times, reaction product EGC amount increases gradually.After the reaction times surpasses 30 minutes, reaction product (working method 1.2 enzyme activity determinations among the embodiment 1) on a declining curve.
4. reaction substrate concentration
Fig. 7 shows that ester catechin lytic enzyme optimal reaction substrate EGCG concentration is 0.2 mmol/L.Along with reaction substrate EGCG concentration increases, product EGC totally presents fast rise trend.After EGCG concentration reached 0.2mmol/L, the reaction product ascendant trend eased up.Experiment confirm repeatedly, reaction system EGCG concentration is set to 0.2mmol/L, comparatively suitable (working method 1.2 enzyme activity determinations among the embodiment 1).
5. utilize performance liquid chromatography (HPLC) analytical procedure qualitative and quantitative detection ester catechin building-up reactions substrate and product catechin and gallic acid (GA) (Fig. 1, Fig. 2 and Fig. 3) simultaneously.
Inhibitor can effectively protective reaction system in reaction substrate and reaction product, avoid oxidized oxydasis.
Reference:
[1] 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.
?(Cabrera?C,?Artacho?R,?Gimenez?R.?Beneficial?effects?of?green?tea--a?review.?J?Am?Coll?Nutr.?2006,25:?79-99.)
[2] what HY, journey ML, father-in-law SF, etc. NVP-XAA 723 is to the influence of enteroviral antiviral pharmacology. american agriculture and food chemistry magazine. and 2009,57:6140-6147.
(Ho?HY,?Cheng?ML,?Weng?SF,et?al.?Antiviral?effect?of?epigallocatechin?gallate?on?enterovirus?71.?J?Agric?Food?Chem?.?2009,57:6140-6147.)
[3] Hu J, all D, old Y. are rich in the preparation and the oxidation-resistance of the green tea extract of L-Epicatechin gallate and NVP-XAA 723. american agriculture and food chemistry magazine. and 2009,57:1349-1353.
(Hu?J,?Zhou?D,?Chen?Y.?Preparation?and?antioxidant?activity?of?green?tea?extract?enriched?in?epigallocatechin?(EGC)?and?epigallocatechin?gallate?(EGCG).?J?Agric?Food?Chem.?2009,?57:?1349-1353.)
[4] Ke CH, Liu KM, beam PC. tea catechin, hin, l-Epigallocatechol and nutgall catechin gallic acid ester influence bone metabolism. american agriculture and food chemistry magazine. 2009,57:7293-7297.
(Ko?CH,?Lau?KM,?Choy?WY,?Leung?PC.?Effects?of?tea?catechins,?epigallocatechin,?gallocatechin,?and?gallocatechin?gallate,?on?bone?metabolism.?J?Agric?Food?Chem.?2009,57:?7293-7297.)
[5] Sai Like F, the latent effect-summary of bachmann M. green tea catechins in the prevention of metabolic syndromes. vegetable chemistry. 2009,70:11-24.
(Thielecke?F,?Boschmann?M.?The?potential?role?of?green?tea?catechins?in?the?prevention?of?the?metabolic?syndrome-a?review.?Phytochemistry?.?2009,70:11-24.)
[6] Singh K draws girl A, Borrow A, etc. the relation of clone's anthocyanin reductase gene expression difference and l-Epicatechol concentration in the tea tree. tree physiology. 2009,29:837-846.
(Singh K, Rani A, Paul A, etc. Differential display mediated cloning of anthocyanidin reductase gene from tea (
Camellia sinensis) and its relationship with the concentration of epicatechins. Tree Physiol. 2009,29:837-846.)
[7] Wen Keer-sherry B. Vitamin P complex. a various pattern for genetics, biological chemistry, cytobiology and biotechnology. plant physiology. 2001,126:485-493.
(Winkel-Shirley?B.?Flavonoid?biosynthesis.?A?colorful?model?for?genetics,?biochemistry,?cell?biology,?and?biotechnology.?Plant?Physiol.?2001,126:485-493.)
[8] thank to DY, Sa agate SB, the NL of Pavia, etc. by the effect of anthocyanin reductase in the biosynthesizing of plant flavone class material of BANYULS coding. science. 2003,299:396-399
(Xie?DY,?Sharma?SB,?Paiva?NL,?et?al.?Role?of?anthocyanidin?reductase,?encoded?by?BANYULS?in?plant?flavonoid?biosynthesis.?Science.?2003,299:?396-399.)
[9] thanking to DY, beautiful Sha A, Jackson etc. two cDNA to the dihydro Flavonol 4-reductase enzyme of in clover section plant, encoding clone the molecule of chains and the analysis on the biological chemistry level. plant physiology. 2004,134:979-994
(Xie?D?Y,?Lisa?A,?Jackson,?et?al.?Molecular?and?Biochemical?Analysis?of?Two?cDNA?Clones?Encoding?Dihydroflavonol-4-Reductase?from?Medicago?truncatula.?Plant?physiol.?2004,?134:?979-994.)
[10] Rod Dixon DA, shed is educated DY, husky agate SB. pycnogenols-be the final edge of flavonoid research. new plant is learned. and 2005,165:9-28
(Dixon?RA,?Xie?DY,?Sharma?SB.?Proanthocyanidins-a?final?frontier?in?flavonoid?research. New?Phytol?.?2005,165:?9-28)?。
Claims (2)
1. the enzymic activity detection method of an ester catechin lytic enzyme is characterized in that comprising following operation steps:
1.1, enzyme liquid preparation
Claim 2 gram fresh leaves of tea plant, behind the adding liquid nitrogen, be ground into powder fast; The phosphoric acid buffer liquor and the ascorbic acid solution of 2mL concentration 20mmol/L that add the 0.1mol/L pH 7.4 of 2 gram Vinylpyrrolidone polymers, 0.5 gram silica sand, 18mL precooling again; Under 4 ℃ of conditions of temperature, grinding to form homogenate, is centrifugal 15min under 12000 rev/mins, the 4 ℃ conditions in centrifuge speed; Get supernatant, get 1.2mg/mL enzyme liquid;
1.2, the mensuration of enzymic activity
1.2.1, the preparation of reaction solution
Reaction solution comprises following raw materials according:
Zyme extract: 1.2mg/mL enzyme liquid
Damping fluid: 0.1mol/L pH6.5 phosphoric acid buffer
Inhibitor: 40mmol/L xitix
Reaction substrate: 4mmol/L NVP-XAA 723 solution or nutgall catechin gallic acid ester solution or L-Epicatechin gallate solution;
Enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 solution or nutgall catechin gallic acid ester solution or L-Epicatechin gallate solution and 1.795mL phosphoric acid buffer with 0.33mL; Mixing; Place 30 ℃ of water-baths to react 30min, get reaction solution;
1.2.2, the contrast liquid preparation
Contrast liquid comprises following raw materials according:
Zyme extract: 100 ℃ of enzyme liquid that boil 5 minutes
Damping fluid: 0.1mol/L pH6.5 phosphoric acid buffer
Inhibitor: 40mmol/L ascorbic acid solution
Reaction substrate: 4mmol/L NVP-XAA 723 solution or nutgall catechin gallic acid ester solution or L-Epicatechin gallate solution;
100 ℃ of 0.33mL are boiled enzyme liquid, 0.25mL inhibitor, 0.125mL reaction substrate NVP-XAA 723 solution or nutgall catechin gallic acid ester solution or L-Epicatechin gallate (ECG) solution and the 1.795mL phosphoric acid buffer of 5min; Mixing; Place 30 ℃ of water-baths to react 30min, must contrast liquid;
1.2.3, methanol of reaction liquid and the contrast methanol solution preparation
In reaction solution and contrast liquid, add 5mL ETHYLE ACETATE respectively, difference is the extractive reaction product at room temperature, re-extract three times, the ETHYLE ACETATE of the ETHYLE ACETATE phase of collection merging reaction solution and contrast liquid is mutually respectively; With the ETHYLE ACETATE of reaction solution mutually with the ETHYLE ACETATE of contrast liquid mutually, 60 ℃ of concentrating under reduced pressure of temperature 10 minutes, use dissolve with methanol more respectively, and be settled to 500 microlitres respectively respectively, obtain methanol of reaction liquid respectively and contrast methanol solution;
1.2.4, the efficient liquid phase chromatographic analysis of enzyme reaction product
Utilize the content of l-Epicatechol or hin or l-Epigallocatechol in the known high-efficient liquid phase chromatogram technology detection reaction of catechin methanol solution and the contrast methanol solution; In methanol of reaction liquid, detect enzyme reaction product l-Epicatechol or hin or l-Epigallocatechol; And in the contrast methanol solution, detect less than enzyme reaction product l-Epicatechol or hin or l-Epigallocatechol, promptly can prove to detect ester catechin lytic enzyme enzyme work in the fresh leaves of tea plant.
2. the enzymic activity detection method of a kind of ester catechin lytic enzyme according to claim 1 is characterized in that:
Utilize enzyme work calculation method, calculate ester catechin lytic enzyme vigor, promptly with the enzyme product hin that forms in every milligram of enzyme catalysis of 30 ℃ of following PMs or the amount of l-Epicatechol or l-Epigallocatechol, unit is mg/ minute/milligram albumen;
M/30 minute/0.40 milligram albumen of ester catechin lytic enzyme enzyme work=△
In the formula: △ m representes the enzyme product hin of formation in 30 minutes or the amount of l-Epicatechol or l-Epigallocatechol; Expressed enzyme reaction in 30 minutes 30 minutes; 0.40 milligram albumen representes to add in the 2.5mL reaction solution enzyme content.
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