CN106282272A - Method for catalytically synthesizing C-6' -lauroyl geniposide by using lipase - Google Patents

Method for catalytically synthesizing C-6' -lauroyl geniposide by using lipase Download PDF

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CN106282272A
CN106282272A CN201610673420.5A CN201610673420A CN106282272A CN 106282272 A CN106282272 A CN 106282272A CN 201610673420 A CN201610673420 A CN 201610673420A CN 106282272 A CN106282272 A CN 106282272A
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lipase
geniposide
lauroyl
reaction
synthesis method
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CN106282272B (en
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姚忠
陆怡欣
倪芳
朱本伟
孙芸
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Nanjing Tech University
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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Abstract

The invention discloses a method for synthesizing C-6' -lauroyl geniposide by lipase catalysis, which dissolves geniposide and vinyl laurate in a solvent and takes immobilized lipase as a catalyst for reaction. Compared with the prior art, the invention has the following advantages: the invention firstly utilizes lipase to catalyze and synthesize the derivative C-6' -lauroyl geniposide of the natural product geniposide, and compared with a chemical modification method, the invention has the characteristics of mild reaction conditions, strong catalytic site selectivity, simple process steps, environmental friendliness and the like. Meanwhile, the immobilized lipase used in the invention is easy to separate from a substrate and a product, has long service life, can be repeatedly used for continuous production, and reduces the production cost. Compared with geniposide, the fat solubility of the product C-6' -lauroyl geniposide prepared by the invention is greatly improved, and the application prospect is wider.

Description

Method for catalytically synthesizing C-6' -lauroyl geniposide by using lipase
Technical Field
The invention belongs to the field of biocatalysis, and particularly relates to a method for catalytically synthesizing C-6' -lauroyl geniposide by using lipase.
Background
Geniposide is an iridoid glucoside, and is the main effective component of fructus Gardeniae. Geniposide has obvious curative effect on diseases of digestive system, cardiovascular system and central nervous system, and has certain anti-inflammatory, antioxidant, antitumor and soft tissue injury treating effects. Geniposide is widely applied to other fields besides medicine, such as plant yield increasing agents, biological detection agents and the like.
As an iridoid glycoside compound, geniposide has a polar functional group so that the geniposide has stronger hydrophilicity, is more easily dissolved in water and methanol, can be dissolved in ethanol, acetone and n-butyl alcohol, but is insoluble in lipophilic organic solvents such as chloroform, diethyl ether, benzene and the like, and has poor fat solubility, thereby greatly limiting the application of the geniposide. In application, geniposide is modified in a certain form to obtain a more stable geniposide derivative with better fat solubility.
Currently, geniposide modification is mainly performed by chemical methods. CN102875617A Tangwenjian et al take geniposide as a raw material, and prepare various geniposide derivatives through a series of complex reactions such as acetylation, deacetylation, condensation and the like. However, the chemical modification method has the problems of complex process steps, harsh reaction conditions, poor reaction site selectivity, great environmental pollution and the like.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for catalytically synthesizing C-6' -lauroyl geniposide by using lipase, so as to solve the problems of complex process steps, harsh reaction conditions, poor reaction site selectivity, large environmental pollution and the like in the prior art
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for synthesizing C-6' -lauroyl geniposide by lipase catalysis comprises the following steps:
dissolving geniposide and vinyl laurate in a solvent, reacting by taking immobilized lipase as a catalyst, and centrifuging, dissolving and purifying reaction liquid by a column to obtain a product.
Wherein the molar ratio of geniposide to vinyl laurate is 1: 1 to 10.
Wherein the immobilized lipase is obtained by immobilizing any one of thermomyces lanuginosus lipase, trichoderma oryzae lipase, candida antarctica lipase B or candida antarctica lipase A on a carrier.
Wherein, the carrier is silica gel, textile products or resin.
Wherein,
the silica gel is preferably silica gel particles with the particle size of 5-20 mu m;
the resin is preferably granular resin with the particle size of 0.1-1 mm;
the textile product is preferably 100 mesh nylon 86.
The immobilized lipase can be prepared by immobilizing lipase on a carrier through adsorption or chemical bonding, for example, the lipase is adsorbed and immobilized on the carrier by an aqueous phase immobilization method, which comprises the following specific operations: dissolving lipase in buffer solution, adding carrier, oscillating or stirring for a certain time, suction filtering, washing and drying to obtain immobilized lipase.
Wherein the enzyme activity of the lipase is 250-10000U/g, and the definition of the enzyme activity of the lipase is as follows: the enzyme amount required for hydrolysis of olive oil at 40 ℃ in a phosphate buffer solution of pH8.0 to release 1mmol of fatty acid per minute is one enzyme activity unit. Wherein the mass ratio of the lipase to the geniposide is 0.5-5: 1.
wherein the oil-water distribution coefficient LogP of the solvent is-0.5 to +4.0, and n-hexane, tert-amyl alcohol, tetrahydrofuran or acetone is preferred.
Wherein the reaction temperature is 35-55 ℃, and the reaction time is 20-60 h.
Wherein, during the reaction, the reaction system is arranged on a shaking table, and the rotating speed of the shaking table is 150-200 r/min.
Wherein,
the centrifugation method is to centrifuge for 5-10 min at the rotating speed of 2000-5000 r/min;
the dissolving method comprises taking the upper layer liquid obtained by centrifugation, drying the solvent by blowing, and dissolving the obtained oily substance in acetonitrile;
the chromatographic column used for the column purification is SSepax Bio-C1810 μm, 150mm x21.2 mm. The chromatographic conditions are as follows: mobile phase: acetonitrile-water (80:20) column temperature: sample introduction at 25 ℃: 1.5mL flow rate: 5mL/min detection wavelength: 210 nm.
The reaction formula of the invention is as follows:
has the advantages that:
compared with the prior art, the invention has the following advantages: the invention firstly utilizes lipase to catalyze and synthesize the derivative C-6' -lauroyl geniposide of the natural product geniposide, and compared with a chemical modification method, the invention has the characteristics of mild reaction conditions, strong catalytic site selectivity, simple process steps, environmental friendliness and the like. Meanwhile, the immobilized lipase used in the invention is easy to separate from a substrate and a product, has long service life, can be repeatedly used for continuous production, and reduces the production cost. Compared with geniposide, the fat solubility of the product C-6' -lauroyl geniposide prepared by the invention is greatly improved, and the application prospect is wider.
Drawings
FIG. 1 is a positive ion mass spectrum of C-6' -lauroyl geniposide prepared in example 3;
FIG. 2 is a nuclear magnetic resonance spectrum of C-6' -lauroyl geniposide prepared in example 3;
FIG. 3 is a nuclear magnetic carbon spectrum of C-6' -lauroyl geniposide prepared in example 3.
Detailed Description
The present technology is further illustrated by the following specific examples. It is to be understood that the embodiments described herein are merely illustrative and explanatory of the invention and are not restrictive thereof.
In the following examples, the column used for column purification was SSepax Bio-C1810 μm, 150 mm. times.21.2mm. The chromatographic conditions are as follows: mobile phase: acetonitrile-water (80:20) column temperature: sample introduction at 25 ℃: 1.5mL flow rate: 5mL/min detection wavelength: 210 nm.
Example 1
Two substrates, 0.04g geniposide and 0.1g vinyl laurate (molar ratio about 1:4), were added to 10mL tetrahydrofuran in the reaction medium, and the enzyme activity was 250U/g in the presence of 0.1g Thermomyces lanuginosus lipase immobilized on silica gel particles having an average particle size of 10 μm as a catalyst. Shaking the shaking table (200r/min) at the temperature of 40 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The product conversion rate reaches 61.3 percent.
The prepared C-6' -lauroyl geniposide and geniposide are subjected to fat-soluble experiments, and the distribution ratio of the two in a water/n-octanol phase is shown in table 1.
TABLE 1
Example 2
Two substrates, 0.04g geniposide and 0.1g vinyl laurate (molar ratio about 1:4), are added into 10mL of tertiary amyl alcohol of a reaction medium, 0.1g candida antarctica lipase B immobilized on macroporous resin with the average particle size of 500 mu m is used as a catalyst for reaction, and the enzyme activity is 1000U/g. Shaking the shaking table (200r/min) at the temperature of 40 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 53.2 percent.
Example 3
Two substrates, 0.04g of geniposide and 0.15g of vinyl laurate (molar ratio about 1:6), were added to 10mL of tetrahydrofuran in the reaction medium, and 0.1g of Thermomyces lanuginosus lipase immobilized on silica gel particles having an average particle size of 5 μm was used as a catalyst for the reaction, and the enzyme activity was 250U/g. Shaking the shaking table (200r/min) at the temperature of 45 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 78.7 percent.
Example 4
Two substrates, 0.04g of geniposide and 0.1g of vinyl laurate (the molar ratio is about 1:4), are added into 10mL of n-hexane of a reaction medium, and 0.1g of thermomyces lanuginosus lipase immobilized on silica gel particles with the average particle size of 10 mu m is used as a catalyst for the reaction, and the enzyme activity is 250U/g. Shaking the shaking table (200r/min) at the temperature of 40 ℃ to react for 30 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 33.5 percent.
Example 5
Two substrates, 0.04g of geniposide and 0.1g of vinyl laurate (the molar ratio is about 1:4), are added into 10mL of tetrahydrofuran of a reaction medium, 0.2g of candida antarctica lipase A immobilized on macroporous resin with the average particle size of 1mm is used as a catalyst for reaction, and the enzyme activity is 500U/g. Shaking the shaking table (200r/min) at the temperature of 40 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 12.3 percent.
Example 6
Two substrates, 0.04g geniposide and 0.1g vinyl laurate (molar ratio about 1:4), were added to 10mL acetone in the reaction medium, and the reaction was carried out using 0.1g thermomyces lanuginosus lipase immobilized on silica gel particles having an average particle size of 5 μm as a catalyst and the enzyme activity was 250U/g. Shaking the shaking table (200r/min) at the temperature of 35 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 58.7 percent.
Example 7
Two substrates, 0.04g of geniposide and 0.1g of vinyl laurate (the molar ratio is about 1:4), are added into 10mL of tetrahydrofuran of a reaction medium, 0.1g of Rhizomucor miehei lipase fixed on 100-mesh nylon 86 is used as a catalyst for reaction, and the enzyme activity is 3000U/g. Shaking the shaking table (150r/min) at the temperature of 40 ℃ to react for 60 hours. And after the reaction is finished, centrifugally separating the immobilized enzyme from the reaction liquid, drying the solvent by drying the upper layer liquid, dissolving the obtained oily substance by using acetonitrile, and purifying by using a reverse-phase preparation column to obtain the product. The reaction conversion rate reaches 17.2 percent.
Example 8
Immobilization of lipase: the D311 ion exchange resin as the carrier is soaked and swelled with deionized water to remove impurities, then soaked with 4% NaOH and 4% HCl under stirring alternately in a magnetic stirrer for 2 hours, and washed with deionized water to neutrality. Subsequently, the pretreated carrier resin was vacuum-filtered, and 1.0g of the resin was placed in a Erlenmeyer flask, 5mL of a buffer (pH 10) containing 1.5mL of Candida antarctica lipase B enzyme solution was added, and the mixture was adsorbed and fixed in a constant temperature shaker at 35 ℃ and 110rpm for 10 hours. And after the fixation is finished, washing with deionized water, carrying out vacuum filtration, and drying to obtain the immobilized enzyme.

Claims (9)

1. A method for synthesizing C-6' -lauroyl geniposide by lipase catalysis is characterized by comprising the following steps:
dissolving geniposide and vinyl laurate in a solvent, reacting by taking immobilized lipase as a catalyst, and centrifuging, dissolving and purifying reaction liquid by a column to obtain a product.
2. The synthesis method according to claim 1, wherein the molar ratio of geniposide to vinyl laurate is 1: 1 to 10.
3. The synthetic method according to claim 1, wherein the immobilized lipase is obtained by immobilizing any one of Thermomyces lanuginosus lipase, Rhizomucor miehei lipase, Candida antarctica lipase B or Candida antarctica lipase A on a carrier.
4. A synthesis method according to claim 3, characterized in that the carrier is silica gel, textile or resin.
5. The synthetic method according to claim 3, wherein the lipase has an enzyme activity of 250 to 10000U/g; wherein, the lipase activity is defined as: the enzyme amount required for hydrolysis of olive oil at 40 ℃ in a phosphate buffer solution of pH8.0 to release 1mmol of fatty acid per minute is one enzyme activity unit.
6. The synthesis method according to claim 1, wherein the mass ratio of the lipase to the geniposide is 0.5-5: 1.
7. the synthesis method according to claim 1, wherein the solvent has a water-oil distribution coefficient Log P of-0.5 to + 4.0.
8. The synthesis method according to claim 1, wherein the solvent is n-hexane, t-amyl alcohol, tetrahydrofuran or acetone.
9. The synthesis method according to claim 1, wherein the reaction temperature is 35-55 ℃ and the reaction time is 20-60 h.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107955822A (en) * 2017-12-21 2018-04-24 浙江工业大学 A kind of lipase-catalyzed online synthesis S-（4- methyl-benzyls）The method of laurate thioesters

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102875617A (en) * 2012-09-12 2013-01-16 安徽医科大学 Geniposide derivative, preparation method thereof and application of geniposide derivative to inflammation resistance

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
CN102875617A (en) * 2012-09-12 2013-01-16 安徽医科大学 Geniposide derivative, preparation method thereof and application of geniposide derivative to inflammation resistance

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万丽花，等: "两相体系中β-葡萄糖苷酶催化栀子苷水解制备京尼平", 《化工学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107955822A (en) * 2017-12-21 2018-04-24 浙江工业大学 A kind of lipase-catalyzed online synthesis S-（4- methyl-benzyls）The method of laurate thioesters

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