CN110218758A - A kind of enzymatic preparation method of carotenoid ester - Google Patents
A kind of enzymatic preparation method of carotenoid ester Download PDFInfo
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- CN110218758A CN110218758A CN201910392525.7A CN201910392525A CN110218758A CN 110218758 A CN110218758 A CN 110218758A CN 201910392525 A CN201910392525 A CN 201910392525A CN 110218758 A CN110218758 A CN 110218758A
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- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
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Abstract
The present invention relates to a kind of methods that enzymatic prepares carotenoid ester.The substrate transformation rate that lutein ester has 90% or more, obtained lutein ester purity >=99% are prepared using this method.The load factor of enzyme reaches 30% or more in prepared fixed lipase catalyzed dose, and enzymatic activity is consistent with native enzyme substantially；For the microspheric catalyst partial size in 15 microns, particle diameter distribution is more uniform, the nanostructure with porous multilayer, convenient for the substrate transmitting in enzymic catalytic reaction, is suitble to use in continuous filling bed type reactor or fluidized-bed reactor.Product separating-purifying prepared by this method is simple, and post processing cost is low, and entire process flow is mild, controllable and environmental-friendly.
Description
Technical field
The present invention relates to a kind of methods that enzymatic prepares carotenoid ester, belong to biological chemical field, are used to prepare
Medicine intermediate.
Background technique
Lutein, alias carotenoid, carotenol, Lutein, core yellow body, marigold florigen and leaves of plants are yellow
Element etc., English entitled Lutein, molecular formula C40H56O2, relative molecular weight 568.85.Orange-yellow powder, pulpous state or liquid,
It is not soluble in water, it is dissolved in the organic solvents such as hexane.Natural carotenol is a kind of excellent antioxidant, can resist free radical in human body
Cell and organ damage are inside caused, cardiovascula arteriosclerosis, coronary heart disease and the neoplastic disease etc. for thus preventing body aging to cause.Leaf is yellow
Element and zeaxanthin are only 2 Carotenoids in eyes, caused by preventing senile eyeball retina macular disease
Visual impairment and blindness are the unique functions of lutein.Lutein ester is a kind of important carotenoid aliphatic ester.It is wide
It is general to be present in the plants such as marigold flower, pumpkin, wild cabbage, clover.Wherein, content is the abundantest in marigold flower, up to
30%~40%.In marigold flower and other plants, lutein is generally combined into fatty acid (such as lauric acid, palm fibre puts acid)
The form of ester exists.Lutein ester in nature can be divided into trans-lutein ester and cis- lutein ester, be with alltrans point
Based on sub- configuration.Alltrans lutein vinegar can be divided into lutein monoesters and lutein diester again.At normal temperature, the leaf of high-purity is yellow
Plain ester (content 60%~95) is in golden yellow to orange red powder.It is soluble in the chloros such as chloroform, methylene chloride, carbon dichloride
Hydrocarbon dissolves in n-hexane, acetone, ethyl acetate, ethyl alcohol equal solvent.Its stability is stronger, only high temperature, strong acid, iron ion and
The factors such as oxygen destroy lutein ester larger.
Currently, the method for synthesis lutein ester is mainly by traditional methodology of organic synthesis.Its process is first prepared by oleic acid
Oleic acid acyl chlorides, then oleic acid acyl chlorides is prepared into oil anhydride again, and last oil anhydride reacts to obtain lutein ester with lutein.From reaction
From the point of view of process, need to use acyl chlorides, in addition the reaction process temperature is higher, is unfavorable for the stabilization of lutein.Enzymic catalytic reaction exists
It is a kind of required tool in organic chemistry, enzyme specificity makes many reactions that can synthesize other means by single step reaction to need multistep
The compound of synthesis.In addition, it is it in lipid synthesis that the Some features low temperature of enzymic catalytic reaction, highly selective and by-product are few etc.
It plays a significant role in industrialized production.Currently, the research about the synthesis of lipase-catalyzed esters is mainly used in mountain both at home and abroad
The synthesis of pears alcohol oleic acid, wax ester, VA lactate and flavor ester.It is had not been reported in terms of synthesizing xanthophyll derivatives.
Summary of the invention
The purpose of the present invention is needing to use acyl chlorides when overcoming existing methodology of organic synthesis synthesis lutein ester, in addition this has
The defects of machine synthetic method reaction process temperature is higher, is unfavorable for the stabilization of lutein, and directly carry out enzymatic and prepare leaf Huang
Plain ester haves the defects that enzyme inactivation, provides a kind of enzymatic process that can efficiently prepare lutein ester, solves lutein at this stage
The high problem of ester low output, price.Specifically, industrial lipase is dispersed in phosphate buffer, calcium chloride is then added dropwise
Solution obtains immobilized lipase-inorganic calcium phosphate crystal hybrid catalyst, with this hybrid catalyst catalysis lutein and fat
Acid carries out esterification and prepares lutein ester, obtains lutein ester sterling by purifying.
Below the step of present invention solves the above problems the technical solution of use, and enzymatic prepares lutein ester method:
(1) lipase immobilization: by the phosphorus that lipase Novozym435 is dissolved in 100~300mL, pH value is 7.0~8.0
In phthalate buffer, concentration is 1.0~5.0g/L, then with 60~120 drops/min under the stirring rate of 100~300r/min
Rate instill 0.1~2.0mol/L 100~300mL of calcium chloride water, immobilization is obtained by filtration after being stirred to react 2~6h
Lipase-inorganic calcium phosphate crystal hybrid catalyst；
(2) enzymatic prepares lutein ester: being in molar ratio in 0.5~1: 2~6 addition chloroforms by lutein and fatty acid
The step of being made into the solution of 10~20g/L, 1~2wt% of reactant is then added (1) prepared immobilized enzyme agent, in 20
Under~60 DEG C of nitrogen bath shaking tables, it is protected from light 10~20h of oscillating reactions, 4A ° of molecule of the activation of 1~2wt% of reactant is then added
Sieve, the reaction was continued 10~20h；Wherein, fatty acid is gallic acid, caffeic acid or palmitinic acid；
(3) purification of lutein ester: the lutein ester crude product that step (2) is prepared is mentioned with the method that column chromatographs
It is pure, used in eluant, eluent be petroleum ether, rotation obtains lutein ester sterling, purity >=99% except solvent.
The method that the enzymatic prepares lutein ester, this method immobilized lipase-inorganic calcium phosphate crystal hydridization
For catalyst particle size at 15~30 microns, particle diameter distribution is more uniform, is highly suitable for preparing fixed bed enzyme reactor, microspheric catalysis
Agent has the nanostructure of porous multilayer, and the substrate in enzymic catalytic reaction is facilitated to transmit.
The method that the enzymatic prepares lutein ester, the yield of this method immobilised enzymes reach 90% or more, fix
The protein load amount for changing enzyme is 30% or more.
The method that a kind of enzymatic prepares lutein ester, the lutein ester purity which is prepared >=
99%, the method that lutein ester is prepared in enzymatic has 90% or more the substrate transformation rate, and whole preparation process is mild, green
It is color, controllable.
Beneficial effects of the present invention: the substrate transformation rate that lutein ester has 90% or more is prepared using this method, is obtained
Lutein ester purity >=99%.The load factor of enzyme reaches 30% or more in prepared fixed lipase catalyzed dose, and
Enzymatic activity is consistent with native enzyme substantially；For the microspheric catalyst partial size in 15 microns, particle diameter distribution is more uniform, has
The nanostructure of porous multilayer is suitble to convenient for the substrate transmitting in enzymic catalytic reaction in continuous filling bed type reactor or stream
It is used in fluidized bed reactor.Product separating-purifying prepared by this method is simple, and post processing cost is low, and entire process flow is mild
It is controllable and environmental-friendly.
Specific embodiment
The embodiment of preparation method of the present invention introduced below, but following embodiment be for illustrating example of the invention, and
Any restriction to the claims in the present invention is not constituted.
Embodiment 1
Two gallate of lutein is prepared, reaction equation is shown in formula I.
(1) lipase Novozym435 lipase immobilization: is dissolved in the phosphate-buffered that the pH value of 100mL is 7.5
In liquid, then concentration 1.0g/L instills the chlorine of 0.1mol/L under the stirring rate of 100r/min with 60 drops/min rate
Change calcium aqueous solution 100mL, immobilized lipase-inorganic calcium phosphate crystal hybrid catalyst is obtained by filtration after being stirred to react 2h；
(2) enzymatic prepares lutein ester: matching in the lutein and gallic acid addition chloroform for being 1: 2 by the mass ratio of the material
At the solution of 10g/L, then the step of addition reactant 1wt% (1) prepared immobilized enzyme agent is shaken in 40 DEG C of nitrogen baths
Under bed, it is protected from light oscillating reactions 20h, 4A ° of molecular sieve of the activation of reactant 2wt%, the reaction was continued 10h is then added；
(3) purification of lutein ester: the lutein ester crude product that step (2) is prepared is mentioned with the method that column chromatographs
It is pure, used in eluant, eluent be petroleum ether, rotation obtains lutein ester sterling except solvent.
The purity of its two gallate of Lutein is 99%, and two gallate of lutein is prepared in enzymatic
Method has 92% or more the substrate transformation rate.
Embodiment 2
Two caffeic acid ester of lutein is prepared, reaction equation is as shown in Formula II.
(1) lipase Novozym435 lipase immobilization: is dissolved in the phosphate-buffered that the pH value of 100mL is 7.5
In liquid, then concentration 1.0g/L instills the chlorine of 0.1mol/L under the stirring rate of 200r/min with 100 drops/min rate
Change calcium aqueous solution 100mL, immobilized lipase-inorganic calcium phosphate crystal hybrid catalyst is obtained by filtration after being stirred to react 6h；
(2) enzymatic prepares lutein ester: being made into the lutein and caffeic acid addition chloroform for being 1: 2 by the mass ratio of the material
The solution of 10g/L, then the step of addition reactant 1wt% (1) prepared immobilized enzyme agent, bathes shaking tables in 40 DEG C of nitrogen
Under, it is protected from light oscillating reactions 12h, 4A ° of molecular sieve of the activation of reactant 2wt%, the reaction was continued 12h is then added；
(3) purification of lutein ester: the lutein ester crude product that step (2) is prepared is mentioned with the method that column chromatographs
It is pure, used in eluant, eluent be petroleum ether, rotation obtains lutein ester sterling except solvent.
The purity of its two caffeic acid ester of Lutein is 99%, the method that two caffeic acid ester of lutein is prepared in enzymatic
With 93% or more the substrate transformation rate.
Embodiment 3
Lutein dipalmitate is prepared, reaction equation is as shown in formula III.
(1) lipase Novozym435 lipase immobilization: is dissolved in the phosphate-buffered that the pH value of 100mL is 7.5
In liquid, then concentration 1.0g/L instills the chlorine of 0.1mol/L under the stirring rate of 200r/min with 60 drops/min rate
Change calcium aqueous solution 100mL, immobilized lipase-inorganic calcium phosphate crystal hybrid catalyst is obtained by filtration after being stirred to react 3h；
(2) enzymatic prepares lutein ester: being made into the lutein and palmitinic acid addition chloroform for being 1: 2 by the mass ratio of the material
The solution of 10g/L, then the step of addition reactant 1wt% (1) prepared immobilized enzyme agent, bathes shaking tables in 40 DEG C of nitrogen
Under, it is protected from light oscillating reactions 12h, 4A ° of molecular sieve of the activation of reactant 2wt%, the reaction was continued 12h is then added；
(3) purification of lutein ester: the lutein ester crude product that step (2) is prepared is mentioned with the method that column chromatographs
It is pure, used in eluant, eluent be petroleum ether, rotation obtains lutein ester sterling except solvent.
The purity of its two caffeic acid ester of Lutein is 99%, the method that two caffeic acid ester of lutein is prepared in enzymatic
With 92% or more the substrate transformation rate.
Embodiment 4
Lutein dipalmitate is prepared, reaction equation is as shown in formula III.
(1) lipase immobilization: with embodiment 3；
(2) enzymatic prepares lutein ester: being made into the lutein and palmitinic acid addition chloroform for being 1: 2 by the mass ratio of the material
The solution of 10g/L, then the step of addition reactant 1wt% (1) prepared immobilized enzyme agent, bathes shaking tables in 60 DEG C of nitrogen
Under, it is protected from light oscillating reactions 12h, 4A ° of molecular sieve of the activation of reactant 2wt%, the reaction was continued 12h is then added；
(3) purification of lutein ester: with embodiment 3.
The purity of its two caffeic acid ester of Lutein is 99%, the method that two caffeic acid ester of lutein is prepared in enzymatic
With 90% or more the substrate transformation rate.
Embodiment 5
Lutein dipalmitate is prepared, reaction equation is as shown in formula III.
(1) lipase immobilization: with embodiment 3；
(2) enzymatic prepares lutein ester: being made into the lutein and palmitinic acid addition chloroform for being 1: 2 by the mass ratio of the material
The solution of 10g/L, then the step of addition reactant 1wt% (1) prepared immobilized enzyme agent, bathes shaking tables in 60 DEG C of nitrogen
Under, it is protected from light oscillating reactions 20h, 4A ° of molecular sieve of the activation of reactant 2wt%, the reaction was continued 20h is then added；
(3) purification of lutein ester: with embodiment 3.
The purity of its two caffeic acid ester of Lutein is 99%, the method that two caffeic acid ester of lutein is prepared in enzymatic
With 95% or more the substrate transformation rate.
Claims (1)
1. a kind of method that enzymatic prepares carotenoid ester, it is characterised in that: enzymatic prepares carotenoid ester method
The step of it is as follows:
(1) lipase immobilization: by the phosphate that lipase Novozym435 is dissolved in 100~300mL, pH value is 7.0~8.0
In buffer, concentration is 1.0~5.0g/L, then with 60~120 drops/min speed under the stirring rate of 100~300r/min
Rate instills 100~300mL of calcium chloride water of 0.1~2.0mol/L, and immobilized lipase is obtained by filtration after being stirred to react 2~6h
Enzyme-inorganic calcium phosphate crystal hybrid catalyst；
(2) enzymatic prepares lutein ester: being in molar ratio to be made into 0.5~1: 2~6 addition chloroforms by lutein and fatty acid
The solution of 10~20g/L, then the step of addition 1~2wt% of reactant (1) prepared immobilized enzyme agent, in 20~60
Under DEG C nitrogen bath shaking table, it is protected from light 10~20h of oscillating reactions, 4A ° of molecular sieve of the activation of 1~2wt% of reactant is then added, after
10~20h of continuous reaction；Wherein, fatty acid is gallic acid, caffeic acid or palmitinic acid；
(3) purification of lutein ester: the Methods For Purification for the lutein ester crude product column chromatography that step (2) is prepared,
Used in eluant, eluent be petroleum ether, rotation obtains lutein ester sterling, purity >=99% except solvent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269162A (en) * | 2020-03-31 | 2020-06-12 | 江苏佰岁通源健康管理有限公司 | Compound modified by lutein and more stable and having double functions as well as preparation method and application thereof |
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| US20050255541A1 (en) * | 2002-08-20 | 2005-11-17 | Sungene Gmbh & Co. Kgaa | Method for hydrolysing carotenoids esters |
| CN107338279A (en) * | 2017-09-01 | 2017-11-10 | 广州立达尔生物科技股份有限公司 | Lipase-catalyzed esterification produces lutein carboxylate |
| CN108342433A (en) * | 2018-02-02 | 2018-07-31 | 杭州师范大学 | A kind of compound enzyme crystal of lipase-calcium phosphate, preparation method and its method for catalyzing and synthesizing clindamycin palmitate |
| CN109402209A (en) * | 2018-11-09 | 2019-03-01 | 北京联合大学 | A method of carotenoid polybasic ester is prepared from free state hydroxyl carotenoid |
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2019
- 2019-05-13 CN CN201910392525.7A patent/CN110218758A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050255541A1 (en) * | 2002-08-20 | 2005-11-17 | Sungene Gmbh & Co. Kgaa | Method for hydrolysing carotenoids esters |
| CN107338279A (en) * | 2017-09-01 | 2017-11-10 | 广州立达尔生物科技股份有限公司 | Lipase-catalyzed esterification produces lutein carboxylate |
| CN108342433A (en) * | 2018-02-02 | 2018-07-31 | 杭州师范大学 | A kind of compound enzyme crystal of lipase-calcium phosphate, preparation method and its method for catalyzing and synthesizing clindamycin palmitate |
| CN109402209A (en) * | 2018-11-09 | 2019-03-01 | 北京联合大学 | A method of carotenoid polybasic ester is prepared from free state hydroxyl carotenoid |
Non-Patent Citations (1)
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| 杨云裳: "酶法合成没食子酸叶黄素酯的研究", 《食品科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111269162A (en) * | 2020-03-31 | 2020-06-12 | 江苏佰岁通源健康管理有限公司 | Compound modified by lutein and more stable and having double functions as well as preparation method and application thereof |
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Effective date of registration: 20210309 Address after: 100086 a002, east of the second floor of commercial and garage, No.17 Zhichun Road, Haidian District, Beijing Applicant after: Beijing Yueda Biotechnology Co.,Ltd. Address before: 710075 Room 501, building F, gazelle Valley, area C, 69 Jinye Road, high tech Zone, Xi'an City, Shaanxi Province Applicant before: XI'AN DAY NATURAL Inc. |
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Application publication date: 20190910 |