CN102643886A - Method for preparing polymerized rutin by laccase mediator system catalysis - Google Patents
Method for preparing polymerized rutin by laccase mediator system catalysis Download PDFInfo
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- CN102643886A CN102643886A CN201210108955XA CN201210108955A CN102643886A CN 102643886 A CN102643886 A CN 102643886A CN 201210108955X A CN201210108955X A CN 201210108955XA CN 201210108955 A CN201210108955 A CN 201210108955A CN 102643886 A CN102643886 A CN 102643886A
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Abstract
The invention relates to a method for preparing polymerized rutin by laccase mediator system catalysis. The method comprises the following steps that: (1) methanol and acetic acid buffer solution are mixed to be used as a buffer system of laccase catalysis; (2) rutin is dissolved into the methanol, then, the methanol solution of the rutin is dissolved into the 20-50ml buffer system to obtain mixed solutions; (3) laccase/mediators are added into the mixed solution under the stirring condition, reaction solution is obtained, the continuous stirring is carried out at 50-60 DEG C, and the enzyme reaction lasts 4-12 hours; (4) the reaction solution is heated to 90-100 DEG C, and the temperature is maintained for 5 to 10 minutes so that the enzyme is inactivated; and (5) the reaction solution is subjected to filtering, dialysis and freeze-drying, and the polymerized rutin is obtained. The method has the advantages that the reaction conditions are mild, the reaction selectivity and the catalytic activity of the enzyme are high, the enzyme raw material per se is non-toxic, the biological degradation can be realized, in addition, the recovery and the utilization are realized, and the like. The operation is simple, the time consumption is little, and the method is more suitable for industrial production.
Description
Technical field
The invention belongs to the preparation field of polymerization rutin, particularly a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin.
Background technology
Recently because the biology and the pharmacologically active of flavonoid, comprise anti-oxidant, anticancer, probiotics, antibiotic and anti-infective isoreactivity, people increase the research interest of flavonoid greatly.Flavonoid compound is made up of a large amount of low-molecular-weight polyphenols, and rutin is one of modal flavonol glucosides, it is reported to have clinical correlation function; Comprise anti-oxidant, hypertension, anti-infective; Reinforce capillary vessel and the permeability of regulating capillary vessel, stabilised blood platelet etc.These antioxygenations that act on it are relevant.
On basis to Flavonoid substances research; People have been concerned about sight line on the flavonoid polymkeric substance at present; Compare with monomer; It has good water-soluble, high free radical scavenging activity, and aspects such as activity not oxidized at the protection low-density lipoprotein and the inhibition XOD show except very big raising.And very few for the study on the synthesis that gathers Flavonoid substances,
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin, and this method is utilized gentle reaction conditions, the high reaction preference and the catalytic activity of enzyme; Proenzyme material itself is nontoxic in addition; Can biological degradation, and advantage such as recyclable utilization, simple to operate; Consuming time less, more be applicable to suitability for industrialized production.
A kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin of the present invention comprises:
(1) methyl alcohol was mixed as the catalytic buffer system of laccase according to volume ratio with acetate buffer solution in 1: 3～1: 5;
(2) rutin is dissolved in the methyl alcohol by mass volume ratio 0.1-0.3g: 1-3ml, the dissolve with methanol solution with rutin gets mixing solutions in above-mentioned 20-50ml buffer system then;
(3) under agitation condition, laccase/amboceptor is added above-mentioned mixing solutions and obtain reaction solution, continue to stir enzyme reaction 4-12h in 50-60 ℃;
(4) reaction solution is heated to 90-100 ℃, keeps 5-10min, make enzyme deactivation;
(5) with reacting liquid filtering, dialysis 6-12h, freeze-drying 24-48h obtains the polymerization rutin then.
Acetate buffer solution concentration in the said step (1) is 0.05-0.10mol/L, and the pH value is 5-7.
Laccase in the said step (3) is Denilite IIS dhdps enzyme (a no amboceptor), and amboceptor is I-hydroxybenzotriazole (HBT), and the mass ratio of Denilite IIS and I-hydroxybenzotriazole is 10: 1-15: 1.
The add-on of the laccase/amboceptor in the said step (3) (total mass of Denilite IIS and I-hydroxybenzotriazole) is 0.1-0.3g.
Stir speed (S.S.) in the said step (3) is 100-150r/min.
Employing
quantitative paper that is filtered in the said step (5) filters.
Dialysis in the said step (5) is the dialysis membrane dialysis of 700-1000 for adopting molecular weight cut-off.
The present invention utilizes Denilite IIS/HBT as the enzyme reaction system, react with the monomer rutin, through each correlation parameter in the adjustment reaction process, success prepare the rutin polymkeric substance.
Beneficial effect
(1) enzyme reaction of the present invention has gentle condition and high reaction preference, and proenzyme material itself is nontoxic, can biological degradation.
(2) the present invention utilizes laccase catalyzed polymerization Polyphenols or polyamines class to produce to be difficult to traditional method synthetic functional polymer, and this preparation method is simple to operate, and is efficient, energy-conservation province consumption, and environmental friendliness has a good application prospect.
Description of drawings
Fig. 1 is the UV spectrum (A: monomer, B: polymkeric substance) of the rutin polymkeric substance of monomer rutin and preparation;
Fig. 2 is the infared spectrum (A: monomer, B: polymkeric substance) of the rutin polymkeric substance of monomer rutin and preparation;
Fig. 3 is the hydrogen spectrum of rutoside monomer;
Fig. 4 is the hydrogen spectrum of the rutin polymkeric substance of preparation;
Fig. 5 is the molecular weight (A: dimer, B: tripolymer) of mass spectroscopy rutin polymkeric substance.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Methyl alcohol is mixed as the catalytic buffer system of laccase according to 1: 4 volume ratio with 0.10mol/L acetate buffer solution (pH=5); The 0.2g rutin is dissolved in the 2ml methyl alcohol, then with the rutin dissolve with methanol solution in above-mentioned 20ml buffer system; Under the 150r/min agitation condition, 0.2g Denilite IIS/HBT (Denilite IIS and HBT mass ratio are 10: 1) is slowly added in the above-mentioned mixing solutions, in 60 ℃ of lasting stirring reaction 4h; Reaction system is heated to 90 ℃, keeps 10min, make enzyme deactivation; Reaction solution is filtered with
; Using molecular weight cut-off again is 1000 dialysis membrane dialysis 12h; Freeze-drying 48h obtains product polymerization rutin then.
Embodiment 2
Methyl alcohol is mixed as the catalytic buffer system of laccase according to 1: 5 volume ratio with 0.05mol/L acetate buffer solution (pH=6); The 0.3g rutin is dissolved in the 3ml methyl alcohol, then with the rutin dissolve with methanol solution in above-mentioned 40ml buffer system; Under the 100r/min agitation condition, 0.3g Denilite IIS/HBT (Denilite IIS and HBT mass ratio are 15: 1) is slowly added in the above-mentioned mixing solutions, in 50 ℃ of lasting stirring reaction 12h; Reaction system is heated to 100 ℃, keeps 10min, make enzyme deactivation; Reaction solution is filtered with
; Using molecular weight cut-off again is 700 dialysis membrane dialysis 6h; Freeze-drying 24h obtains product polymerization rutin then.
Embodiment 3
Methyl alcohol is mixed as the catalytic buffer system of laccase according to 1: 3 volume ratio with 0.08mol/L acetate buffer solution (pH=7); The 0.1g rutin is dissolved in the 1ml methyl alcohol, then with the rutin dissolve with methanol solution in above-mentioned 50ml buffer system; Under the 110r/min agitation condition, 0.1gDenilite IIS/HBT (Denilite IIS and HBT mass ratio are 12: 1) is slowly added in the above-mentioned mixing solutions, in 55 ℃ of lasting stirring reaction 8h; Reaction system is heated to 95 ℃, keeps 7min, make enzyme deactivation; Reaction solution is filtered with
; Using molecular weight cut-off again is 800 dialysis membrane dialysis 9h; Freeze-drying 36h obtains product polymerization rutin then.
Embodiment 4
(1) through measuring (see figure 1), can see, because the π-π * transition of aromatic ring fragment with UV-2102PC type ultraviolet-visible pectrophotometer; Monomer occurs two peaks 255 with the 350nm place; As for gathering (rutin), previous peak can see that also then one then becomes broad peak; This possibly be because of polymkeric substance generation conjugated cause, and it is the oxidation coupling formation through rutin that the aromatic ring C-C between monomer links.
(2) through with the NEXUS-670 type infrared-Raman appearance IR spectroscopy (see figure 2); For the monomer rutin; All peak shapes of rutin polymkeric substance have all broadened, but rutin is similar with the peak shape of gathering rutin, in gathering the spectrum of rutin; Because the vibration that phenolic group is connected with O-H between hydroxyl, a broad peak is centered close to 3340cm
-1The place, 1650cm
-1The peak at place belongs to the vibration of carbonyl, 1595cm
-1The peak at place belongs to the C=C vibration of aromatic ring, and 1521,1121 and 971cm
-1The peak at place diminishes, and characteristic peak does not occur again.
(3) be respectively Fig. 3 and shown in Figure 4 through the molecular structure with Avance 400 nuclear magnetic resonance spectrometer test unit rutins and polymerization rutin, rutin is at solvent DMSO-d with the hydrogen spectrum of gathering rutin
6Middle measurement, in the wave spectrum of polymkeric substance, at δ 2.5-4.0,4.0-5.6,6.2-8.5,10.5-11 and 12-13 place observe broad peak, and monomer is unimodal in corresponding zone.
(4) utilize Agilent 1260with Agilent 6224Accurate-Mass TOF LC/MS LC-MS to measure composition and the molecular weight (see figure 5) of polymerization rutin; Discovery has two kinds of dimers and two kinds of tripolymers; The dimer molecule amount is respectively 1216 and 911; The tripolymer molecular weight is respectively 1824 and 1519, and small molecular weight material possibly be that the macromolecule material is sloughed due to the glucosides.
Claims (7)
1. method of utilizing laccase mediator systems catalytic preparation polymerization rutin comprises:
(1) methyl alcohol was mixed as the catalytic buffer system of laccase according to volume ratio with acetate buffer solution in 1: 3～1: 5;
(2) rutin is dissolved in the methyl alcohol by mass volume ratio 0.1-0.3g: 1-3ml, the dissolve with methanol solution with rutin gets mixing solutions in above-mentioned 20-50ml buffer system then;
(3) under agitation condition, laccase/amboceptor is added above-mentioned mixing solutions and obtain reaction solution, continue to stir enzyme reaction 4-12h in 50-60 ℃;
(4) reaction solution is heated to 90-100 ℃, keeps 5-10min, make enzyme deactivation;
(5) with reacting liquid filtering, dialysis 6-12h, freeze-drying 24-48h obtains the polymerization rutin then.
2. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1, it is characterized in that: the acetate buffer solution concentration in the said step (1) is 0.05-0.10mol/L, the pH value is 5-7.
3. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1; It is characterized in that: the laccase in the said step (3) is a Denilite IIS dhdps enzyme; Amboceptor is an I-hydroxybenzotriazole, and the mass ratio of Denilite IIS and I-hydroxybenzotriazole is 10: 1-15: 1.
4. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1, it is characterized in that: the add-on of the laccase/amboceptor in the said step (3) is 0.1-0.3g.
5. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1, it is characterized in that: the stir speed (S.S.) in the said step (3) is 100-150r/min.
6. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1 is characterized in that: employing
quantitative paper that is filtered in the said step (5) filters.
7. a kind of method of utilizing laccase mediator systems catalytic preparation polymerization rutin according to claim 1 is characterized in that: the dialysis in the said step (5) is the dialysis membrane dialysis of 700-1000 for adopting molecular weight cut-off.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726504A (en) * | 2015-03-27 | 2015-06-24 | 南京理工大学 | Method for laccase catalytic synthesis of 5,5'-dehydrodiacetovanillone |
| CN110669799A (en) * | 2019-11-05 | 2020-01-10 | 常熟浸大科技有限公司 | Method for synthesizing polypyrrole by laccase |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0058697A4 (en) * | 1980-08-25 | 1983-01-14 | American Cyanamid Co | Novel rutin nona- and deca(h-)sulfate salts, methods of preparing novel salts, and use of novel salts as complement inhibitors. |
| CN101423538A (en) * | 2008-12-06 | 2009-05-06 | 广西禅方药业有限公司 | Production process for extracting high-purity birutan from flos sophora |
| CN102226213A (en) * | 2011-04-21 | 2011-10-26 | 浙江大学 | Method for synthesizing betulinic acid by catalyzing betulin with laccase |
-
2012
- 2012-04-13 CN CN201210108955XA patent/CN102643886A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0058697A4 (en) * | 1980-08-25 | 1983-01-14 | American Cyanamid Co | Novel rutin nona- and deca(h-)sulfate salts, methods of preparing novel salts, and use of novel salts as complement inhibitors. |
| CN101423538A (en) * | 2008-12-06 | 2009-05-06 | 广西禅方药业有限公司 | Production process for extracting high-purity birutan from flos sophora |
| CN102226213A (en) * | 2011-04-21 | 2011-10-26 | 浙江大学 | Method for synthesizing betulinic acid by catalyzing betulin with laccase |
Non-Patent Citations (1)
| Title |
|---|
| MOTOICHI KURISAWA等: "Enzymatic Synthesis and Antioxidant Properties of Poly(rutin)", 《BIOMACROMOLECULES》, vol. 4, no. 5, 26 October 2011 (2011-10-26), pages 1394 - 1399, XP002547747, DOI: doi:10.1021/BM034136B * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726504A (en) * | 2015-03-27 | 2015-06-24 | 南京理工大学 | Method for laccase catalytic synthesis of 5,5'-dehydrodiacetovanillone |
| CN104726504B (en) * | 2015-03-27 | 2018-08-10 | 南京理工大学 | Laccase catalytic synthesis method of 5, 5' -dehydro-divanillyl acetone |
| CN110669799A (en) * | 2019-11-05 | 2020-01-10 | 常熟浸大科技有限公司 | Method for synthesizing polypyrrole by laccase |
| CN110669799B (en) * | 2019-11-05 | 2023-08-25 | 常熟浸大科技有限公司 | Method for synthesizing polypyrrole by laccase |
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Application publication date: 20120822 |