CN1737135A - Method for nanometer fixed enzyme directionally producing TF2A crude extract in vitro - Google Patents
Method for nanometer fixed enzyme directionally producing TF2A crude extract in vitro Download PDFInfo
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Abstract
The invention relates to a method for nanometer fixed enzyme directionally producing TF2A crude extract in vitro which comprises, first preparing raw extract of tea polyphenol oxidase whose enzyme vitality is 0.32-0.34, then carrying out the other two steps of nano immobilized enzyme preparation and tea polyphenol directional oxidization.
Description
Technical field
The present invention relates to the method for the in-vitro directed production theoflavin of a kind of nano immobilized enzyme-3-gallic acid ester.
Technical background
Theoflavin is the stronger material of biologic activity in the tea pigment, and its health-care effect, extraction and isolation technique etc. are subjected to the great attention of Chinese and overseas scholars.Experiment showed, that theoflavin has high anticancer, resisting cardiovascular disease, various active such as antibiotic, antiviral, its action effect obviously is better than tea-polyphenol, tea pigment.Theoflavin is extensively regarded as in the tealeaves actively utilizes one of material with the largest potentiality, and its content low, be difficult to realize that batch production production is the main difficult problem of current existence.Contain multiple monomer in the theoflavin, because the textural difference between monomer, every kind of monomeric biologic activity is also different.The theoflavin crude extract that from black tea, extracts, the content of its each kinds of theaflavin monomer is average, and at present directed targetedly production with certain kinds of theaflavin monomer is that the technology of the theoflavin mixture of main component is not seen relevant patent and report.Theoflavin-3-gallic acid ester, being called for short TF2A (theaflavin-3-gallate) is active stronger kinds of theaflavin monomer, has strong anti-oxidant activity and other biological and learns active; Preparation has a good application prospect based on the theoflavin mixture of TF2A.
The vitro enzyme enzymatic oxidation is produced theoflavin, is the on purpose directed theoflavin of producing of oxidation characteristic that utilizes polyphenoloxidase.Nanotechnology is the emerging technology that is born late 1980s, and it is used widely and has promoted traditional biological research and enter brand-new developmental stage.2002 " American Chemical Society's proceedings " report, the researchist of U.S.'s Pacific Northwest National Laboratory successfully utilizes nano material with enzyme immobilization, and can keep its activity, stability simultaneously, this top is become the application of nano material zymetology and has been opened possibility, has also brought technological breakthrough for enzyme immobilization.So far, nano material is not seen relevant patent and report in the application aspect the immobilization of tea-polyphenol oxydase.
Summary of the invention
At the blank in the above research, it is simple to the invention provides a kind of technology, and the method for the in-vitro directed production theoflavin of nano immobilized enzyme-3-gallic acid ester (TF2A) crude extract of high-load TF2A can be provided.
The present invention is for reaching above purpose, it is to realize: the method that a kind of nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro is provided by such technical scheme, comprise and at first prepare tea-polyphenol oxydase crude extract, enzyme activity is 0.32～0.34 in the crude extract, and this method is also carried out following steps successively:
1), nano immobilized enzyme preparation: the ratio according to 0.70～0.75g/70～75ml adds the nano-calcium carbonate calcium powder in above-mentioned tea-polyphenol oxydase crude extract, stirred 1.5～2 hours down at 2～8 ℃; Above-mentioned miscellany is all put into centrifuge container, and 3500～4500 change, 2～8 ℃ centrifugal 15～20 minutes, abandon supernatant; Citric acid phosphoric acid damping fluid with pH5.6 washs 2～3 times again, and the gained throw out is nano immobilized enzyme;
2), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, epi-nutgall acid catechin and gallate content is greater than 45% tea-polyphenol, citric acid phosphoric acid damping fluid with pH5.6 is mixed with the solution that concentration is 0.4～0.6mg/ml, and, form tea-polyphenol solution with pH to 4.9～5.1 that the HCl of 1mol/L regulates described solution; With above-mentioned steps 1) in the nano immobilized enzyme of gained be that the ratio of 0.008～0.012g/0.8～1.2ml is suspended in the citric acid phosphoric acid damping fluid of pH5.6 according to weightmeasurement ratio, fully stir it suspended evenly, form nano immobilized enzyme suspension; In described tea-polyphenol solution, add nano immobilized enzyme suspension and form mixed solution, volume ratio 0.25～0.3/3.8～4.2 of described nano immobilized enzyme suspension and tea-polyphenol solution; Described mixed solution under 35-38 ℃ temperature, was heated 10～12 minutes; Pour the mixed solution after the above-mentioned heating into centrifuge container, 7500～8500 change 2～8 ℃ centrifugal 15～20 minutes, collect supernatant liquor, extract, vacuum-drying.
A kind of improvement as the method for nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro of the present invention: in extraction step step 2), the ethyl acetate volume of use is described supernatant liquor volume 1/4～1/6, collects ethyl acetate layer then; In the vacuum drying step, drying temperature is 35～45 ℃.
Further improvements in methods as nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro of the present invention: nano-calcium carbonate particles of powder size is 70nm, is shaped as the spindle shape.
Further improvements in methods as nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro of the present invention: at first prepare tea-polyphenol oxydase crude extract, enzyme activity is 0.34 in the crude extract, carries out following steps more successively:
1), nano immobilized enzyme preparation: the ratio according to 0.75g/75ml adds the nano-calcium carbonate calcium powder in above-mentioned tea-polyphenol oxydase crude extract, stirred 2 hours down at 4 ℃; Above-mentioned miscellany is all put into centrifuge container, and 4000 change, 4 ℃ centrifugal 15 minutes, abandon supernatant; Be 5.6 citric acid phosphoric acid damping fluid washing 3 times with pH again, the gained throw out is nano immobilized enzyme;
2), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, epi-nutgall acid catechin and gallate content is greater than 45% tea-polyphenol, citric acid phosphoric acid damping fluid with pH5.6 is mixed with the solution that concentration is 0.5mg/ml, and regulate pH to 5.0 with the HCl of 1mol/L, form tea-polyphenol solution; Described volume ratio be the nano immobilized enzyme suspension of 0.3/4.0 and the formed mixed solution of tea-polyphenol solution under 37 ℃ temperature, heated 10 minutes; Pour the mixed solution after the above-mentioned heating into centrifuge container, 8000 change 4 ℃ centrifugal 15 minutes, collect supernatant liquor, use volume as the ethyl acetate of supernatant liquor volume 1/5 extract, vacuum-drying.
The theoflavin mixture that method of the present invention is prepared from, four kinds of main kinds of theaflavin monomer: TF1 (theaflavin), TF2A, TF2B (theaflavin-39-gallate), TF3 (theaflavin-3, total content 39-digallate) is higher; Wherein TF2A content has comparative advantage.Concrete method and data are as follows: accurately prepare TF1 with redistilled water, and TF2A, TF2B, TF3 concentration is the theoflavin solution of present method preparation of 0.4mg/ml for the kinds of theaflavin monomer standard specimen solution of 0.1mg/ml and concentration respectively, carries out HPLC and analyzes.
The HPLC analysis condition: ODS C-18 post, 35 ℃ of column temperatures detect wavelength: 280nm, flow velocity: 1.0ml/min, sample size: 10 μ l, moving phase: A pump: water-second eyeball-acetate: 96.5-3-0.5 (v/v/v), B pump: water-second eyeball-acetate: 69.5-30-0.5 (v/v/v); Linear gradient elution, 0-55min are 0-100%B; 55-60min is 100%A.
Calculation formula is:
The concentration sum that records four kinds of main kinds of theaflavin monomer in the theoflavin mixed solution that method of the present invention produces is 64.5%, and TF2A content is 43.3%.
Use preparation method of the present invention, the preparation method of nano immobilized enzyme is simple, easily reclaims after the oxidation, and the material therefor cost is low; Oxidizing condition is simple, and oxidization time is short; The product of preparation has good directional property, and content is higher.
Description of drawings
Fig. 1 is theoflavin-3-gallic acid ester, the i.e. structure iron of TF2A.
Embodiment
Embodiment 1: a kind of method of nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro, and its preparation process is as follows:
1), preparation tea-polyphenol oxydase crude extract: the preparation method sees clock trailing plants work " tea leaf quality physico-chemical analysis " (Shanghai science tech publishing house, published in 1989), come the enzyme activity of regulatory enzyme solution (measuring method is seen the yellow joyous work of meaning " Tea Science experimental technique " by adjusting concentration, Beijing agricultural press, nineteen ninety-five publishes) (following enzyme activity numerical value is represented with absorbancy), make that enzyme activity reaches 0.32 in the crude extract.The concrete operations step is that the bright leaf 100.00g of tea cleaned in title, places in the tissue mashing machine, adds the 800ml cold acetone, the 20g polyvinylpyrrolidone is smashed 5min to pieces, or wears into slurry fast with mortar, suction filtration then, filter residue be with the drip washing repeatedly of 80% cold acetone, be washed till filtrate colourless till.The filter residue of gained is an acetone powder, and it is standby to put refrigerator.Acetone powder is placed mortar, add pH5.6 citrate buffer solution and a small amount of quartz sand of 1: 3 (weightmeasurement ratio), grind homogenate 20min on ice, get crude enzyme liquid with overstocked method and suction filtration then, the centrifugal 15min of 4000rpm gets clear enzyme solution, transfer to certain volume, carry out determination of activity.Get enzyme liquid 1ml, add reaction mixture 3ml[reaction mixture by pH5.6 citric acid-phosphoric acid buffer: 0.1% proline(Pro): 1% pyrocatechol (10: 2: 3) preparation] in centrifuge tube, at 37 ℃ of warm down 10min that bathe, add 6mol/L urea soln 3ml immediately, termination reaction, the centrifugal 10min of 4000rpm gets supernatant, in 460nm place colorimetric.Pyrocatechol in the blank reaction solution replaces with damping fluid, and other conditions are identical.The enzymic activity calculation formula is improved, and is specially:
In the formula: E
460: during reaction terminating with blank for to impinging upon the absorbancy at 460nm place;
T: reaction times (min).
2), nano immobilized enzyme preparation: according to adding 0.70g nanometer CaCO in the tea-polyphenol oxydase crude extract of every 72ml
3The ratio of powder is with tea-polyphenol oxydase crude extract and nanometer CaCO
3After the powder mixes, stirred 1.5 hours down at 2 ℃; The mixture of gained is all put into centrifuge tube, and 3500 change, 4 ℃ centrifugal 15 minutes, abandon supernatant.Citric acid phosphoric acid damping fluid with pH5.6 washs 3 times again, and the precipitation that obtains is nano immobilized enzyme.The nano-calcium carbonate particles of powder size of selecting for use is 70nm, is shaped as the spindle shape.
3), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, EGCG content is greater than 45% tea-polyphenol, the ratio that adds the above-mentioned tea-polyphenol of 0.4mg according to the citric acid phosphoric acid damping fluid of the pH5.6 of every 1ml, above-mentioned tea-polyphenol and above-mentioned citric acid phosphoric acid damping fluid are mixed back formation solution, and regulate this pH value of solution to 4.9 with the HCl of 1mol/L, form tea-polyphenol solution.With step 2) in the nano immobilized enzyme of gained be that the ratio of 0.008g/1ml is suspended in the citric acid phosphoric acid damping fluid of pH5.6 (being the nano immobilized enzyme of putting into 0.008g in the citric acid phosphoric acid damping fluid of pH5.6 of every 1ml) according to weightmeasurement ratio, fully stir and make its suspension evenly, form nano immobilized enzyme suspension.Ratio according to 0.25/3.8 (volume ratio of nano immobilized enzyme suspension/tea-polyphenol solution) adds nano immobilized enzyme suspension in tea-polyphenol solution, form mixed solution; Pour this mixed solution into beaker, be put in 35 ℃ of water-baths and heated 12 minutes, take out then, pour centrifuge tube into, 7500 change, and 8 ℃ centrifugal 15 minutes; Collect supernatant liquor, supernatant liquor is poured in the separating funnel, adding volume is the ethyl acetate of supernatant liquor volume 1/5, and ethyl acetate layer is collected in extraction, and 45 ℃ of vacuum-dryings get the glassy yellow powder, and being main component is the theoflavin crude extract of TF2A.
With the theoflavin mixture of this kind method preparation, the concentration sum of four kinds of kinds of theaflavin monomer is 67%, and TF2A content is 48%.
Embodiment 2: a kind of method of nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro, and its preparation process is as follows:
1), preparation tea-polyphenol oxydase crude extract: make tea-polyphenol oxydase crude extract as embodiment 1 described method, enzyme activity reaches 0.34 in the crude extract.
2), nano immobilized enzyme preparation: according to adding 0.73g nanometer CaCO in the tea-polyphenol oxydase crude extract of every 75ml
3The ratio of powder is with tea-polyphenol oxydase crude extract and nanometer CaCO
3After the powder mixes, stirred 2 hours down at 8 ℃; The mixture of gained is all put into centrifuge tube, and 4000 change, 2 ℃ centrifugal 20 minutes, abandon supernatant.Citric acid phosphoric acid damping fluid with pH5.6 washs 2 times again, and the precipitation that obtains is nano immobilized enzyme.The nano-calcium carbonate particles of powder size of selecting for use is 70nm, is shaped as the spindle shape.
3), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, EGCG content is greater than 45% tea-polyphenol, the ratio that adds the above-mentioned tea-polyphenol of 0.6mg according to the citric acid phosphoric acid damping fluid of the pH5.6 of every 1ml, above-mentioned tea-polyphenol and above-mentioned citric acid phosphoric acid damping fluid are mixed back formation solution, and regulate this pH value of solution to 5.1 with the HCl of 1mol/L, form tea-polyphenol solution.With step 2) in the nano immobilized enzyme of gained be that the ratio of 0.01g/1.2ml is suspended in the citric acid phosphoric acid damping fluid of pH5.6 (being the nano immobilized enzyme of putting into 0.01g in the citric acid phosphoric acid damping fluid of pH5.6 of every 1.2ml) according to weightmeasurement ratio, fully stir and make its suspension evenly, form nano immobilized enzyme suspension.Ratio according to 0.3/4.2 (volume ratio of nano immobilized enzyme suspension/tea-polyphenol solution) adds nano immobilized enzyme suspension in tea-polyphenol solution, form mixed solution; Pour this mixed solution into beaker, be put in 37 ℃ of water-baths and heated 11 minutes, take out then, pour centrifuge tube into, 8000 change, and 2 ℃ centrifugal 15 minutes; Collect supernatant liquor, supernatant liquor is poured in the separating funnel, adding volume is the ethyl acetate of supernatant liquor volume 1/6, and ethyl acetate layer is collected in extraction, and 40 ℃ of vacuum-dryings get the glassy yellow powder, and being main component is the theoflavin crude extract of TF2A.
With the theoflavin mixture of this kind method preparation, the concentration sum of four kinds of kinds of theaflavin monomer is 65.9%, and TF2A content is 47%.
Embodiment 3: a kind of method of nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro, and its preparation process is as follows:
1), preparation tea-polyphenol oxydase crude extract: make tea-polyphenol oxydase crude extract as embodiment 1 described method, enzyme activity reaches 0.33 in the crude extract.
2), nano immobilized enzyme preparation: according to adding 0.75g nanometer CaCO in the tea-polyphenol oxydase crude extract of every 74ml
3The ratio of powder is with tea-polyphenol oxydase crude extract and nanometer CaCO
3After the powder mixes, stirred 1.8 hours down at 4 ℃; The mixture of gained is all put into centrifuge tube, and 4500 change, 8 ℃ centrifugal 18 minutes, abandon supernatant.Citric acid phosphoric acid damping fluid with pH5.6 washs 3 times again, and the precipitation that obtains is nano immobilized enzyme.The nano-calcium carbonate particles of powder size of selecting for use is 70nm, is shaped as the spindle shape.
3), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, EGCG content is greater than 45% tea-polyphenol, the ratio that adds the above-mentioned tea-polyphenol of 0.5mg according to the citric acid phosphoric acid damping fluid of the pH5.6 of every 1ml, above-mentioned tea-polyphenol and above-mentioned citric acid phosphoric acid damping fluid are mixed back formation solution, and regulate this pH value of solution to 5.0 with the HCl of 1mol/L, form tea-polyphenol solution.With step 2) in the nano immobilized enzyme of gained be that the ratio of 0.012g/0.8ml is suspended in the citric acid phosphoric acid damping fluid of pH5.6 (being the nano immobilized enzyme of putting into 0.012g in the citric acid phosphoric acid damping fluid of pH5.6 of every 0.8ml) according to weightmeasurement ratio, fully stir and make its suspension evenly, form nano immobilized enzyme suspension.Ratio according to 0.28/4.0 (volume ratio of nano immobilized enzyme suspension/tea-polyphenol solution) adds nano immobilized enzyme suspension in tea-polyphenol solution, form mixed solution; Pour this mixed solution into beaker, be put in 38 ℃ of water-baths and heated 10 minutes, take out then, pour centrifuge tube into, 8500 change, and 4 ℃ centrifugal 20 minutes; Collect supernatant liquor, supernatant liquor is poured in the separating funnel, adding volume is the ethyl acetate of supernatant liquor volume 1/4, and ethyl acetate layer is collected in extraction, and 35 ℃ of vacuum-dryings get the glassy yellow powder, and being main component is the theoflavin crude extract of TF2A.
With the theoflavin mixture of this kind method preparation, the concentration sum of four kinds of kinds of theaflavin monomer is 66.8%, and TF2A content is 46.3%.
The above only is several case study on implementation of the present invention, should be pointed out that for those of ordinary skill in the art, can also make many modification and improvement, churning time for example, all modification and improve and all should be considered as protection scope of the present invention.
Claims (4)
1, a kind of method of nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro comprises at first preparing tea-polyphenol oxydase crude extract that enzyme activity is 0.32～0.34 in the crude extract, it is characterized in that described method also carries out following steps successively:
1), nano immobilized enzyme preparation: the ratio according to 0.70～0.75g/70～75ml adds the nano-calcium carbonate calcium powder in above-mentioned tea-polyphenol oxydase crude extract, stirred 1.5～2 hours down at 2～8 ℃; Above-mentioned miscellany is all put into centrifuge container, and 3500～4500 change, 2～8 ℃ centrifugal 15～20 minutes, abandon supernatant; Citric acid phosphoric acid damping fluid with pH5.6 washs 2～3 times again, and the gained throw out is nano immobilized enzyme;
2), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, epi-nutgall acid catechin and gallate content is greater than 45% tea-polyphenol, citric acid phosphoric acid damping fluid with pH5.6 is mixed with the solution that concentration is 0.4～0.6mg/ml, and, form tea-polyphenol solution with pH to 4.9～5.1 that the HCl of 1mol/L regulates described solution; With above-mentioned steps 1) in the nano immobilized enzyme of gained be that the ratio of 0.008～0.012g/0.8～1.2ml is suspended in the citric acid phosphoric acid damping fluid of pH5.6 according to weightmeasurement ratio, fully stir it suspended evenly, form nano immobilized enzyme suspension; In described tea-polyphenol solution, add nano immobilized enzyme suspension and form mixed solution, volume ratio 0.25～0.3/3.8～4.2 of described nano immobilized enzyme suspension and tea-polyphenol solution; Described mixed solution under 35-38 ℃ temperature, was heated 10～12 minutes; Pour the mixed solution after the above-mentioned heating into centrifuge container, 7500～8500 change 2～8 ℃ centrifugal 15～20 minutes, collect supernatant liquor, extract, vacuum-drying.
2, the method for nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro according to claim 1, it is characterized in that: in the extraction step described step 2), the ethyl acetate volume that uses is described supernatant liquor volume 1/4～1/6, collects ethyl acetate layer then; In the described vacuum drying step, drying temperature is 35～45 ℃.
3, the method for nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro according to claim 2 is characterized in that: described nano-calcium carbonate particles of powder size is 70nm, is shaped as the spindle shape.
4, the method for nanometer fixed enzyme directionally producing TF 2 A crude extract in vitro according to claim 3 is characterized in that: at first prepare tea-polyphenol oxydase crude extract, enzyme activity is 0.34 in the crude extract, carries out following steps more successively:
1), nano immobilized enzyme preparation: the ratio according to 0.75g/75ml adds the nano-calcium carbonate calcium powder in above-mentioned tea-polyphenol oxydase crude extract, stirred 2 hours down at 4 ℃; Above-mentioned miscellany is all put into centrifuge container, and 4000 change, 4 ℃ centrifugal 15 minutes, abandon supernatant; Be 5.6 citric acid phosphoric acid damping fluid washing 3 times with pH again, the gained throw out is nano immobilized enzyme;
2), the selective paraffin oxidation of tea-polyphenol: get catechin content greater than 98%, epi-nutgall acid catechin and gallate content is greater than 45% tea-polyphenol, citric acid phosphoric acid damping fluid with pH5.6 is mixed with the solution that concentration is 0.5mg/ml, and regulate pH to 5.0 with the HCl of 1mol/L, form tea-polyphenol solution; Described volume ratio be the nano immobilized enzyme suspension of 0.3/4.0 and the formed mixed solution of tea-polyphenol solution under 37 ℃ temperature, heated 10 minutes; Pour the mixed solution after the above-mentioned heating into centrifuge container, 8000 change 4 ℃ centrifugal 15 minutes, collect supernatant liquor, using volume is that the ethyl acetate of described supernatant liquor volume 1/5 extracts, vacuum-drying.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101443370B (en) * | 2006-03-31 | 2012-01-04 | 科学工业研究委员会 | A solid state matrix, process of preparation thereof, and process of preparation of theaflavins |
| WO2016143223A1 (en) * | 2015-03-09 | 2016-09-15 | 株式会社島津製作所 | Immobilized protease with improved resistance to change in external environment |
| CN106047851A (en) * | 2016-06-14 | 2016-10-26 | 南京农业大学 | Enzymatic method for preparing theaflavin digallate |
| CN109097414A (en) * | 2018-08-23 | 2018-12-28 | 江苏天晟药业股份有限公司 | A kind of preparation method of theaflavin-3-gallate |
| CN109536544A (en) * | 2018-12-19 | 2019-03-29 | 江苏德和生物科技有限公司 | A method of theaflavin is produced using immobile polyphenol oxidase |
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| CN1164745C (en) * | 2001-05-15 | 2004-09-01 | 厦门大学 | Method for immobilizing penicillin amidase and cell containing penicillin amidase |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101443370B (en) * | 2006-03-31 | 2012-01-04 | 科学工业研究委员会 | A solid state matrix, process of preparation thereof, and process of preparation of theaflavins |
| WO2016143223A1 (en) * | 2015-03-09 | 2016-09-15 | 株式会社島津製作所 | Immobilized protease with improved resistance to change in external environment |
| CN107406841A (en) * | 2015-03-09 | 2017-11-28 | 株式会社岛津制作所 | The immobilization proteinase improved to the patience of outside environmental change |
| JPWO2016143223A1 (en) * | 2015-03-09 | 2017-12-28 | 株式会社島津製作所 | Immobilized protease with improved resistance to external environmental changes |
| CN106047851A (en) * | 2016-06-14 | 2016-10-26 | 南京农业大学 | Enzymatic method for preparing theaflavin digallate |
| CN109097414A (en) * | 2018-08-23 | 2018-12-28 | 江苏天晟药业股份有限公司 | A kind of preparation method of theaflavin-3-gallate |
| CN109536544A (en) * | 2018-12-19 | 2019-03-29 | 江苏德和生物科技有限公司 | A method of theaflavin is produced using immobile polyphenol oxidase |
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