CN1629303A - Enzymatic synthesis method for L-ascorbic acid unsaturated fatty acid ester - Google Patents
Enzymatic synthesis method for L-ascorbic acid unsaturated fatty acid ester Download PDFInfo
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- CN1629303A CN1629303A CN 200410053976 CN200410053976A CN1629303A CN 1629303 A CN1629303 A CN 1629303A CN 200410053976 CN200410053976 CN 200410053976 CN 200410053976 A CN200410053976 A CN 200410053976A CN 1629303 A CN1629303 A CN 1629303A
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Abstract
The invention discloses an snzymatic synthesis method for L-ascorbic acid unsaturated fatty acid ester which comprises the steps of, using immobilization lipolytic enzyme as catalyst in reaction medium for the L-ascorbic acid unsaturated fatty acid ester in the reaction system, then collecting L-ascorbic acid unsaturated fatty acid ester from the reactant through the conventional methods. The process provided by the invention can achieve high conversion rate within a short period of reaction time.
Description
Technical field
The present invention relates to a kind of preparation method of L-xitix unsaturated fatty acid ester, be specifically related to a kind of L-xitix unsaturated fatty acid ester enzyme and urge synthetic method.
Technical background
L-xitix (Vc) is a kind of natural anti-reflecting oxide of widespread use, is one of human indispensable nutritive factor.It is (reactive oxygen species, inhibitor ROS), and the generation of ROS and various diseases is closely related of active oxidation material in the organism.
Unsaturated fatty acids such as arachidonic acid AA, linolenic acid ALA, can not synthesize in the human bodies such as timnodonic acid EPA and docosahexenoic acid DHA, need from food, absorb, so be human indispensable fatty acid, they have physiological function and biological effect more widely in body, and two keys the more, degree of unsaturation is higher, nutritive value is also higher, and unsaturated fatty acids has the 26S Proteasome Structure and Function of biometric safeguard film, the treatment cardiovascular disorder, anti-inflammatory, anticancer and promote brain development, fat-reducing, physiological functions such as farrowing rate of increase animal and surviving rate.
For L-xitix palmityl ester function, particularly Antioxidation Effects, Recent study is a lot, and people such as Jian-Wen Liu report this kind material because of its antioxygenation, thus the transfer of anticancer.
With respect to L-xitix palmityl ester, L-xitix unsaturated fatty acid ester nonaqueous phase enzyme urges study on the synthesis few at home and abroad.Unsaturated fatty acids widespread use in makeup, medicine, food; itself just has the character of oxidation protection; if it is connected with Vc; to give new molecule better character; than the bad hematic acid palmityl of anti-L-ester; the easier permeate through cell membranes of L-xitix unsaturated fatty acid ester; in lipid layer, distribute easilier; ROS there is stronger restraining effect; studies show that of early stage, L-xitix unsaturated fatty acid ester have extraordinary two-phase solvability, strong anti-oxidation protection effect etc.Again because unsaturated fatty acids and palmitinic acid are bigger in aspect difference such as physicochemical property such as solvabilities, therefore its synthesis mechanism and technology urge synthesizing with the enzyme of L-xitix palmityl ester very big difference, and the enzyme of L-xitix palmityl ester urges synthetic method can not be used for L-xitix unsaturated fatty acid ester.
The nonaqueous phase enzyme catalysis is a research field that develops rapidly, since Zaks andKlibanov in 1984 research be reported in can carry out the catalyzed reaction of enzyme in the nonaqueous phase since, research to the nonaqueous phase enzymic catalytic reaction has obtained a large amount of progress, lipase is a kind of enzyme that is most widely used in the nonaqueous phase enzyme catalysis research, the lipase kind is a lot, its function difference of lipase that the different sources different microorganisms produces, its catalytic transesterification and ester synthesis reaction respectively have characteristics.
At present, the synthetic method of L-xitix unsaturated fatty acid ester generally adopts as Journal ofMolecular Catalysis B:Enzymatic, 5 (1998) 19-23 document disclosed methods, therefore this method reaction conversion rate of fatty acid less than 20% can not satisfy the needs of the parties concerned.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of L-xitix unsaturated fatty acid ester enzyme to urge synthetic method, to overcome the above-mentioned defective that prior art exists, satisfy the needs of relevant field development.
The present invention synthesizes corresponding ester by enzymic catalytic reaction with unsaturated fatty acids and the reaction of L-xitix, and it is water-soluble that this ester can be stablized, thereby can add this anthropoid important nutritive substance of unsaturated fatty acids in water-soluble drink and food.
The general structure of said L-xitix unsaturated fatty acid ester is as follows:
R represents C
17H
33, C
17H
31, C
17H
29, C
19H
31, C
19H
29Or C
21H
31
Method of the present invention comprises the steps:
With L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, in reaction medium, with the immobilized lipase is catalyzer, in the reaction system, water-content is 0.01～0.06% by weight, 40～60 ℃ were reacted 1～16 hour, adopted conventional method to collect L-xitix unsaturated fatty acid ester then from reaction product.
Preferable methods of the present invention comprises the steps:
With L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, in reaction medium, with the immobilized lipase is catalyzer, in the reaction system, water-content is 0.04%-0.06% by weight, 40～60 ℃ of reactions 1～3 hour, and then be reaction 8-16 hour under 0.01～0.04% the condition at water-content, adopt conventional method from reaction product, to collect L-xitix unsaturated fatty acid ester.
The control of water-content can realize that in the gross weight of L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, the add-on of molecular sieve is 5～20wt% by add molecular sieve in reaction system;
Or the moisture content in the dry backflow hierarchy of control of employing reduction vaporization.
Term " reduction vaporization is dry to reflux " refers to reactor pressure and reduces, the evaporation of reaction solvent entrapped moisture, and through condensation, through the siccative drying, moisture is absorbed when flowing back to.Vacuum-pumping at vacuum tightness 0.08～0.1Mpa, makes the moisture evaporation in the system with the pressure-controlling of system, the condensation after drying, and the solvent partial reflux is to reach the purpose of water-content in the hierarchy of control.
The mol ratio of L-xitix and unsaturated fatty acids or unsaturated fatty acid ester is 1: 1～5;
Said reaction response medium comprises a kind of in tertiary amyl alcohol, acetonitrile or the tetrahydrofuran (THF);
Said unsaturated fatty acids comprises oleic acid, linolic acid, a kind of in linolic acid in distress, arachidonic acid, alpha-linolenic acid, timnodonic acid or the docosahexenoic acid (DHA) etc. altogether;
Said unsaturated fatty acid ester comprises a kind of in Witconol 2301, ethyl oleate, methyl linoleate, ethyl linoleate, EPA-EE, the docosahexenoic acid ethyl ester etc.;
Said immobilized lipase is selected from the lipase that derives from candiyeast, can adopt Journal ofMolecular Catalysis B:Enzymatic, 18 (2002) 261-266. document disclosed methods are prepared, or employing commercially available prod, the trade mark of producing as Novozymes Company is the immobilized lipase of Novozym 435, and this enzyme is that the Type B lipase of Candida antarctica is fixed on the propylene type macroporous resin.
In the nonaqueous phase enzyme catalysis, moisture is an important factors, and the enzyme molecule is kept its active configuration needs certain moisture, in addition, but polarity or wetting ability because of reaction solvent and carrier under identical water activity are different, and water activity is also different in the microenvironment of enzyme molecule.In the enzyme process of L-xitix linoleate is synthetic, we find, no matter be in transesterification or in ester synthesis reaction, water activity is all very big to the influence of productive rate, when containing a certain amount of moisture in the reaction system, water activity not only influences the activity of lipase in the transesterification reaction, and to the hydrolysis of the hydrolysis of substrate methyl linoleate, product and synthetic all influential, moisture can produce complicated influence to entire reaction.
The contriver is through a large amount of tests, find, the content of moisture is very remarkable to the influence of transformation efficiency and reaction efficiency in the reaction system, to moisture contents different in the reaction system, as be respectively 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% etc. and test, the initial velocity of assaying reaction and reach balance after transformation efficiency.The result shows: moisture content is the initial velocity maximum when 0.04%-0.06%, and less than 0.03% o'clock transformation efficiency maximum.
Therefore, during reaction, at 0.04%-0.06%, and be controlled at below 0.03% after two hours, can under the situation that reduces reaction time, obtain maximum conversion rate like this preceding two hours moisture controlled
Adopt method of the present invention, in unsaturated fatty acids, transformation efficiency is more than 35%, and the reaction times can shorten to below 16 hours.
This shows that method of the present invention can obtain high transformation efficiency in the short reaction times, be a kind of method for preparing L-xitix unsaturated fatty acid ester with industrializing implementation prospect.
Description of drawings
Fig. 1 be in the reaction system moisture content to the influence of initial velocity of reaction and productive rate.
Fig. 2 is that different in moisture content influences the product synthetic.
Embodiment
Embodiment 1
Get the tool plug triangular flask of 50ml, in bottle, take by weighing the vitamins C of about 0.3g, take by weighing the 0.6g methyl linoleate.Add the 10ml tertiary amyl alcohol, add molecular sieve 1.0g.Sealing.Triangular flask is put into high temperature shaking table preheating 30 minutes (50 ℃ of high temperature shaking table temperature, 200rps), the trade mark that adds Novozymes Company's production is Novozym 435 immobilized lipase 0.3g, whole system moisture content is controlled at 0.06%, react after 2 hours, add molecular sieve 3g, whole system moisture content is controlled at 0.03%, reacted 10 hours, reaction finishes, and column chromatography for separation obtains the linolic acid Vc ester of 0.15g purity 92%.
Embodiment 2
Get the tool plug triangular flask of 50ml, in bottle, take by weighing the vitamins C of about 0.3g, take by weighing 0.6g oleic acid.Add the 10ml acetonitrile, add molecular sieve 2g.Sealing.Triangular flask is put into high temperature shaking table preheating 30 minutes (60 ℃ of high temperature shaking table temperature, 200rps), the trade mark that adds Novozymes Company's production is Novozym 435 immobilized lipase 0.3g, whole system moisture content is controlled at 0.05%, react after 3 hours, add molecular sieve 1.5g, whole system moisture content is controlled at 0.02%, reacted 8 hours, reaction finishes, and column chromatography for separation obtains 0.12g purity 92% above oleic acid Vc ester.
Embodiment 3
Get the reactor of 50ml, take by weighing the vitamins C of about 0.3g, take by weighing 0.6gDHA (purity is more than 85%).Add the 15ml tetrahydrofuran (THF), 40 ℃, 200 rev/mins stirrings, 30 minutes, the trade mark that adds Novozymes Company's production was Novozym 435 immobilized lipase 0.3g, utilized the dry control moisture that refluxes of reduction vaporization, the pressure of system is vacuum tightness 0.08～0.1Mpa, the system moisture content of making is controlled at 0.02%, reacts 16 hours, and reaction finishes, column chromatography for separation obtains 0.1g purity 92% above DHAVc ester.
Claims (9)
1.L-xitix unsaturated fatty acid ester enzyme is urged synthetic method, it is characterized in that, comprise the steps: L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, in reaction medium, with the immobilized lipase is catalyzer, and in the reaction system, water-content is 0.01～0.06% by weight, 40～60 ℃ were reacted 1～16 hour, adopted conventional method to collect L-xitix unsaturated fatty acid ester then from reaction product.
2. method according to claim 1, it is characterized in that, comprise the steps: L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, in reaction medium, with the immobilized lipase is catalyzer, and in the reaction system, water-content is 0.04%-0.06% by weight, 40～60 ℃ of reactions 1～3 hour, and then be reaction 2-16 hour under 0.01～0.04% the condition at water-content.
3. method according to claim 1 is characterized in that, adds molecular sieve in reaction system, and in the gross weight of L-xitix and unsaturated fatty acids or unsaturated fatty acid ester, the add-on of molecular sieve is 5～20wt%.
4. method according to claim 1 is characterized in that, adopts the moisture content in the dry backflow hierarchy of control of reduction vaporization.
5. method according to claim 1 is characterized in that, the mol ratio of L-xitix and unsaturated fatty acids or unsaturated fatty acid ester is 1: 1～5.
6. according to each described method of claim 1～5, it is characterized in that said reaction response medium comprises a kind of in tertiary amyl alcohol, acetonitrile or the tetrahydrofuran (THF).
7. according to each described method of claim 1～5, it is characterized in that said unsaturated fatty acids comprises oleic acid, linolic acid, a kind of in linolic acid in distress, arachidonic acid, alpha-linolenic acid, timnodonic acid or the docosahexenoic acid etc. altogether.
8. according to each described method of claim 1～5, it is characterized in that said unsaturated fatty acid ester comprises a kind of in Witconol 2301, ethyl oleate, methyl linoleate, ethyl linoleate, EPA-EE, the docosahexenoic acid ethyl ester.
9. according to each described method of claim 1～5, it is characterized in that said immobilized lipase is selected from the lipase that derives from candiyeast.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010130139A1 (en) * | 2009-05-11 | 2010-11-18 | 江南大学 | Ascorbic acid aryl acetate/propionate ester, preparation method and pharmaceutical composition thereof |
| CN104177318A (en) * | 2014-09-02 | 2014-12-03 | 石家庄康诺生物技术有限公司 | L-ascorbic acid-6-(10-hydroxyl-2-caproleic acid) esters or derivatives thereof and application of esters or derivatives |
| CN104262302A (en) * | 2014-09-02 | 2015-01-07 | 石家庄康诺生物技术有限公司 | L-ascorbic acid-6-(E-2-caproleic acid)ester or derivatives thereof and application thereof |
| CN104610201A (en) * | 2014-12-19 | 2015-05-13 | 暨南大学 | L-ascorbyl ricinoleate, and preparing method and applications thereof |
-
2004
- 2004-08-24 CN CN 200410053976 patent/CN1629303A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010130139A1 (en) * | 2009-05-11 | 2010-11-18 | 江南大学 | Ascorbic acid aryl acetate/propionate ester, preparation method and pharmaceutical composition thereof |
| CN104177318A (en) * | 2014-09-02 | 2014-12-03 | 石家庄康诺生物技术有限公司 | L-ascorbic acid-6-(10-hydroxyl-2-caproleic acid) esters or derivatives thereof and application of esters or derivatives |
| CN104262302A (en) * | 2014-09-02 | 2015-01-07 | 石家庄康诺生物技术有限公司 | L-ascorbic acid-6-(E-2-caproleic acid)ester or derivatives thereof and application thereof |
| CN104177318B (en) * | 2014-09-02 | 2016-05-04 | 石家庄康诺生物技术有限公司 | L-AA-6-(10-hydroxyl-2-decylenic acid) ester or derivatives thereof and their application |
| CN104262302B (en) * | 2014-09-02 | 2016-05-11 | 石家庄康诺生物技术有限公司 | L-AA-6-(E-2-decylenic acid) ester or derivatives thereof and their application |
| CN104610201A (en) * | 2014-12-19 | 2015-05-13 | 暨南大学 | L-ascorbyl ricinoleate, and preparing method and applications thereof |
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Open date: 20050622 |