CN106282272B - A method of utilizing lipase-catalyzed synthesis C-6 '-lauroyl geniposide - Google Patents
A method of utilizing lipase-catalyzed synthesis C-6 '-lauroyl geniposide Download PDFInfo
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- CN106282272B CN106282272B CN201610673420.5A CN201610673420A CN106282272B CN 106282272 B CN106282272 B CN 106282272B CN 201610673420 A CN201610673420 A CN 201610673420A CN 106282272 B CN106282272 B CN 106282272B
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- lipase
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- lauroyl
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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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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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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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)
Abstract
Lipase-catalyzed synthesis C-6 '-lauroyl geniposide method is utilized the invention discloses a kind of, geniposide and laurate are dissolved in solvent by it, are reacted by catalyst of immobilized lipase.Compared with prior art; the present invention has the advantage that the present invention utilizes derivative C-6 '-lauroyl geniposide of lipase-catalyzed synthesis natural products geniposide for the first time; relative to chemical modification method; the present invention has reaction condition mild; catalytic site is selectively strong; the features such as processing step is simple, environmental-friendly.Meanwhile the immobilized lipase that the present invention uses, it is easily separated with substrate and product, service life long reusable carry out continuous production reduces production cost.Products C-the 6 '-lauroyl geniposide that the present invention is prepared is fat-soluble compared with geniposide to have obtained biggish improvement, and application prospect is more wide.
Description
Technical field
The invention belongs to biocatalysis fields, and in particular to a kind of to utilize lipase-catalyzed synthesis C-6 '-lauroyl capital
The method of the flat glycosides of Buddhist nun.
Background technique
Geniposide is a kind of iridoid glucoside, is the main pharmacodynamics ingredient of cape jasmine.Geniposide is to Digestive
System, cardiovascular system and central nervous system disease have significant curative effect, in addition, there are also certain anti-inflammatory, antioxygens for geniposide
Change, antitumor and treatment soft tissue injury effect.Geniposide is also widely answered other than medicinal in other fields
With, such as can be used as plant promoter, biological detection agent.
As a kind of iridoid glycosides compound, the polar functional group that geniposide possesses keeps its hydrophily stronger, more
Add and be easily dissolved in water, in methanol, is soluble among ethyl alcohol, acetone and n-butanol, but is insoluble in chloroform, ether, benzene
Equal lipophylic organic solvents, fat-soluble difference, largely Shangdi limits the application of geniposide for this.In the application often by capital Buddhist nun
Flat glycosides is modified with some form, obtains more stable, fat-soluble better geniposide derivative.
Currently, the modification of geniposide is mainly carried out by chemical method.Tang Wenjian et al. is in CN102875617A with capital Buddhist nun
Flat glycosides is raw material, and by a series of acetylations, deacetylation, a series of complex reaction such as condensation has been made a variety of geniposides and has spread out
Biology.However chemical modification method there are processing step complexity, severe reaction conditions, reaction site poor selectivity and environmental pollution compared with
The problems such as big.
Summary of the invention
Lipase-catalyzed synthesis C-6 '-lauroyl Geniposide is utilized the technical problem to be solved in the present invention is to provide a kind of
The method of glycosides, to solve processing step complexity, severe reaction conditions, reaction site poor selectivity and environment of the existing technology
Pollute the problems such as larger
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
A method of lipase-catalyzed synthesis C-6 '-lauroyl geniposide being utilized, it includes the following steps:
Geniposide and laurate are dissolved in solvent, reacted by catalyst of immobilized lipase, is reacted
Liquid is after centrifugation, dissolution and column purification to get product.
Wherein, geniposide and the molar ratio of laurate are 1:1~10.
Wherein, the immobilized lipase is by dredging the thermophilic hyphomycete lipase of cotton like, rhizomucor miehei bacterium lipase, south
Any one in pole lipase from candida sp B or antarctic candidia lipase A, which is fixed on carrier, to be obtained.
Wherein, the carrier is silica gel, textile fabrics or resin.
Wherein,
The silica gel is preferably 5~20 μm of partial size of silica gel particle；
The resin is preferably the particulate resins of 0.1~1mm of partial size；
The textile fabrics are preferably the nylon 86 of 100 mesh.
The immobilized lipase can be by the way that lipase to be fixed on carrier and be prepared by absorption or chemical bonding
It obtains, lipase adsorption is fixed on carrier for example, by using water phase immobilization method, concrete operations are as follows: be dissolved in lipase slow
In fliud flushing, carrier is added, concussion or stirring after a certain period of time, through suction filtration, washing and drying, obtain immobilized lipase.
Wherein, the enzyme activity of the lipase is 250~10000U/g, lipase activity is defined as: at 40 DEG C,
Hydrolysis of Olive Oil in the phosphate buffer solution of pH8.0, enzyme amount needed for releasing 1mmol fatty acid per minute are an enzyme activity list
Position.Wherein, lipase and the mass ratio of geniposide are 0.5~5:1.
Wherein, the Determination of oil-water partition coefficient LogP of the solvent is -0.5~+4.0, preferably n-hexane, tert-pentyl alcohol, tetrahydro
Furans or acetone.
Wherein, the reaction temperature of the reaction is 35~55 DEG C, and the reaction time is 20~60h.
Wherein, when reaction, reaction system is placed on shaking table, and shaking speed is 150~200r/min.
Wherein,
Centrifugal method is to be centrifuged 5~10min with the revolving speed of 2000~5000r/min；
Dissolving method is to take centrifugation gained supernatant liquid, and after drying up solvent, gained grease is dissolved in acetonitrile；
Chromatographic column used in column purification is SSepax Bio-C1810 μm, 150mm x 21.2mm.Chromatographic condition are as follows:
Mobile phase: acetonitrile-water (80:20) column temperature: 25 DEG C of sample volumes: 1.5mL flow velocity: 5mL/min Detection wavelength: 210nm.
Reaction equation of the invention is as follows:
The utility model has the advantages that
Compared with prior art, the present invention has the advantage that the present invention is naturally produced using lipase-catalyzed synthesis for the first time
The derivative C-6 ' of object geniposide-lauroyl geniposide, relative to chemical modification method, the present invention has reaction condition temperature
With, catalytic site is selectively strong, and processing step is simple, it is environmental-friendly the features such as.Meanwhile the immobilized lipase that the present invention uses
Enzyme is easily separated with substrate and product, and service life long reusable carry out continuous production reduces production cost.System of the present invention
Standby obtained products C -6 '-lauroyl geniposide is fat-soluble compared with geniposide to have obtained biggish improvement, and application prospect is more
It is wide.
Detailed description of the invention
Fig. 1 is the positive ion mass spectrum figure for the C-6 '-lauroyl geniposide being prepared in embodiment 3；
Fig. 2 is the nucleus magnetic hydrogen spectrum figure for the C-6 '-lauroyl geniposide being prepared in embodiment 3；
Fig. 3 is the nuclear-magnetism carbon spectrogram for the C-6 '-lauroyl geniposide being prepared in embodiment 3.
Specific embodiment
The technology of the present invention is further illustrated below by way of specific embodiment.It should be appreciated that embodiment described herein
Only for the purpose of illustrating and explaining the present invention and is not intended to limit the present invention.
In following embodiments, chromatographic column used in column purification is SSepax Bio-C1810 μm, 150mm x
21.2mm.Chromatographic condition are as follows: mobile phase: acetonitrile-water (80:20) column temperature: 25 DEG C of sample volumes: 1.5mL flow velocity: 5mL/min detection
Wavelength: 210nm.
Embodiment 1
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the tetrahydrofuran of 10mL, reaction is fixed on the thermophilic hyphomycete of thin cotton like on the silica gel particle that average grain diameter is 10 μm with 0.1g
Lipase is catalyst, enzyme activity 250U/g.Under the conditions of 40 DEG C of temperature, shaking table shakes (200r/min), reacts 60 hours.Instead
Reaction solution is centrifugated out immobilised enzymes after answering, supernatant liquid is taken to dry up solvent, obtained grease is dissolved with acetonitrile,
Inverted preparation column purification obtains product.Product yield is up to 61.3%.
C-6'- lauroyl geniposide obtained and geniposide are subjected to fat-soluble experiment, the two is in water/n-octyl alcohol
Distribution ratio in phase is shown in Table 1.
Table 1
Embodiment 2
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the tert-pentyl alcohol of 10mL, reaction is immobilized in the antarctic candida rouge on the macroreticular resin that average grain diameter is 500 μm with 0.1g
Fat enzyme B is catalyst, enzyme activity 1000U/g.Under the conditions of 40 DEG C of temperature, shaking table shakes (200r/min), reacts 60 hours.Instead
Reaction solution is centrifugated out immobilised enzymes after answering, supernatant liquid is taken to dry up solvent, obtained grease is dissolved with acetonitrile,
Inverted preparation column purification obtains product.Reaction conversion ratio is up to 53.2%.
Embodiment 3
Two kinds of substrate 0.04g geniposides and 0.15g vinyl laurate (molar ratio about 1:6) are added to reaction media
In the tetrahydrofuran of 10mL, reaction is fixed on the thermophilic hyphomycete of thin cotton like on the silica gel particle that average grain diameter is 5 μm with 0.1g
Lipase is catalyst, enzyme activity 250U/g.Under the conditions of temperature 45 C, shaking table shakes (200r/min), reacts 60 hours.Instead
Reaction solution is centrifugated out immobilised enzymes after answering, supernatant liquid is taken to dry up solvent, obtained grease is dissolved with acetonitrile,
Inverted preparation column purification obtains product.Reaction conversion ratio is up to 78.7%.
Embodiment 4
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the n-hexane of 10mL, reaction is fixed on the thermophilic hyphomycete rouge of thin cotton like on the silica gel particle that average grain diameter is 10 μm with 0.1g
Fat enzyme is catalyst, enzyme activity 250U/g.Under the conditions of 40 DEG C of temperature, shaking table shakes (200r/min), reacts 30 hours.Reaction
After reaction solution is centrifugated out immobilised enzymes, take supernatant liquid to dry up solvent, obtained grease is dissolved with acetonitrile, is passed through
Reverse phase preparative column purifying obtains product.Reaction conversion ratio is up to 33.5%.
Embodiment 5
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the tetrahydrofuran of 10mL, reaction is immobilized in average grain diameter with 0.2g as the antarctic candida rouge on the macroreticular resin of 1mm
Fat enzyme A is catalyst, enzyme activity 500U/g.Under the conditions of 40 DEG C of temperature, shaking table shakes (200r/min), reacts 60 hours.Instead
Reaction solution is centrifugated out immobilised enzymes after answering, supernatant liquid is taken to dry up solvent, obtained grease is dissolved with acetonitrile,
Inverted preparation column purification obtains product.Reaction conversion ratio is up to 12.3%.
Embodiment 6
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the acetone of 10mL, the thin cotton like that reaction is fixed on the silica gel particle that average grain diameter is 5 μm with 0.1g is thermophilic, and hyphomycete is fatty
Enzyme is catalyst, enzyme activity 250U/g.Under the conditions of 35 DEG C of temperature, shaking table shakes (200r/min), reacts 60 hours.Reaction knot
Reaction solution is centrifugated out immobilised enzymes after beam, takes supernatant liquid to dry up solvent, obtained grease is dissolved with acetonitrile, through anti-
Mutually preparation column purification obtains product.Reaction conversion ratio is up to 58.7%.
Embodiment 7
Two kinds of substrate 0.04g geniposides and 0.1g vinyl laurate (molar ratio about 1:4) are added to reaction media
In the tetrahydrofuran of 10mL, reaction is fixed on the rhizomucor miehei bacterium lipase on 100 mesh nylon 8s 6 as catalyst using 0.1g, enzyme
Living is 3000U/g.Under the conditions of 40 DEG C of temperature, shaking table shakes (150r/min), reacts 60 hours.After reaction by reaction solution
It is centrifugated out immobilised enzymes, supernatant liquid is taken to dry up solvent, obtained grease is dissolved with acetonitrile, inverted preparation column purification
Obtain product.Reaction conversion ratio is up to 17.2%.
Embodiment 8
Fatty enzyme immobilizatio: D311 ion exchange resin as carrier first impregnates swollen profit with deionized water, impurity elimination, so
With 4%NaOH and 4%HCl, alternately stirring is impregnated 2 hours under magnetic stirring apparatus afterwards, is washed with deionized water to neutrality.Then will
It takes 1.0g to be put into conical flask after pretreated vector resin vacuum filtration, is added and contains 1.5mL antarctic candida rouge
Buffer (pH 10) 5mL of fat enzyme B enzyme solution, 35 DEG C of constant temperature, revolving speed 110rpm, absorption fixes 10 hours in constant temperature oscillator.
It is washed with deionized, is filtered by vacuum after the completion of fixed, it is dry, obtain immobilised enzymes.
Claims (9)
1. a kind of utilize lipase-catalyzed synthesis C-6 '-lauroyl geniposide method, which is characterized in that it includes such as
Lower step:
Geniposide and laurate are dissolved in solvent, reacted by catalyst of immobilized lipase, reaction solution warp
To get product after centrifugation, dissolution and column purification；
The lipase is to dredge the thermophilic hyphomycete lipase of cotton like, rhizomucor miehei bacterium lipase, antarctic candida fat
Any one in enzyme B or antarctic candidia lipase A.
2. the synthetic method according to claim 1, which is characterized in that geniposide and the molar ratio of laurate are
1:1 ~ 10.
3. the synthetic method according to claim 1, which is characterized in that the immobilized lipase is thermophilic by dredging cotton like
Hyphomycete lipase, rhizomucor miehei bacterium lipase, candida antarctica lipase B or antarctic candidia lipase A
In any one be fixed on carrier and obtain.
4. the synthetic method according to claim 3, which is characterized in that the carrier is silica gel, textile fabrics or tree
Rouge.
5. the synthetic method according to claim 3, which is characterized in that the enzyme activity of the lipase be 250 ~
10000U/g；Wherein, the lipase activity is defined as: at 40 DEG C, in the phosphate buffer solution of pH8.0 water
Olive oil is solved, enzyme amount needed for releasing 1 mmol fatty acid per minute is an enzyme-activity unit.
6. the synthetic method according to claim 1, which is characterized in that lipase and the mass ratio of geniposide be 0.5 ~
5:1.
7. the synthetic method according to claim 1, which is characterized in that the Determination of oil-water partition coefficient Log P of the solvent
It is -0.5 ~+4.0.
8. the synthetic method according to claim 1, which is characterized in that the solvent is n-hexane, tert-pentyl alcohol, tetrahydro
Furans or acetone.
9. the synthetic method according to claim 1, which is characterized in that the reaction temperature of the reaction is 35 ~ 55 DEG C, instead
It is 20 ~ 60h between seasonable.
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| 两相体系中β-葡萄糖苷酶催化栀子苷水解制备京尼平;万丽花，等;《化工学报》;20140930;第65卷(第9期);第3583-3591页 |
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