CN106480111A - A kind of method and its device preparing natural perfume material using immobilization hydroperoxide lyase serialization - Google Patents
A kind of method and its device preparing natural perfume material using immobilization hydroperoxide lyase serialization Download PDFInfo
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- CN106480111A CN106480111A CN201510520665.XA CN201510520665A CN106480111A CN 106480111 A CN106480111 A CN 106480111A CN 201510520665 A CN201510520665 A CN 201510520665A CN 106480111 A CN106480111 A CN 106480111A
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Abstract
The invention discloses a kind of prepare the method for natural perfume material and its device it is characterised in that this preparation method comprises the steps using immobilization hydroperoxide lyase serialization：(1) preparation of fatty acid hydroperoxide；(2) extraction of Amaranthus mangostanus L. hydroperoxide lyase；(3) preparation of immobilization hydroperoxide lyase；(4) the serialization preparation of hexanal.This continuous production device is made up of pillar material container (1), conduit (2), peristaltic pump (3), circulator bath clamping device (4), reactor (5) and product collecting container (6).Immobilization hydroperoxide lyase of the present invention has compared with high stability of operation, and the unit volume yield during which thereby enhancing continuous prodution and substrate conversion efficiency.Column reactor designed by the present invention can achieve the serialization preparation of hexanal, preparation process simple to operate it is easy to control and cost is relatively low.
Description
Technical field
The present invention relates to unsaturated fatty acid is converted into by one kind using immobilization hydroperoxide lyase
The method of natural perfume material, belongs to bioflavours preparing technical field.
Background technology
The present invention relates to preparing the process of polybasic unsaturated fatty acid catabolite.These catabolites are to wave
The natural flavour mountaineous material of the property sent out, these materials mainly include trans -2- hexenoic aldehyde, cis- 3- hexenoic aldehyde, hexanal
Deng being the significant contribution person of fresh fruit of vegetables unique green fresh scent, be therefore otherwise known as " green ".
So far, C6 aldehyde has been widely used in the industries such as daily chemical industry, food service industry and perfume product
In.
The huge market demand promotes research worker to expand the research producing in a large number for C6 aldehyde.Typically
For, obtaining C6 aldehyde stable in a large number by the means of chemosynthesis is to be also the most simply the most direct
Method.But the additive obtaining in field of food, natural means or essence and flavoring agent product are more susceptible to
The favor of consumer.For same product, the market sale price of natural prodcuts is far above chemistry and closes
Finished product.
The traditional preparation methods of natural faint scent type essence and flavoring agent are directly to extract to obtain from plant material, example
Distillation as Peppermint essential oil.But this method is also faced with problems at present and is difficult to meet industrialization
The demand producing, so that the metabolic pathway in simulating plant body, enzyme law catalysis dissociate unsaturated fatty acidss
The oxidation reaction of acid and its subsequent degradation reaction are had with the method preparing the C6 aldehydes such as hexanal, hexenoic aldehyde
There is very strong captivation.
In recent years, people begin attempt to by enzyme process come synthesizing aldehyde material, such as Chinese patent
AN1563310A discloses a kind of method that enzyme catalysiss produce olefine aldehyde analog flavor, and this method is many with C18-C20
Remote unsaturated fatty acid is raw material, with the lipoxidase of extraction and hydroperoxidation in organizing from natural plants
The enzyme system of thing lyases composition is catalyst, under the conditions of room temperature 15-25 DEG C, makes polynary unsaturated fatty acidss
Acid transfer ground enzyme catalysiss are cracked into olefine aldehyde analog flavor.Chinese patent CN101225406A is related to a kind of utilization
The method of enzymatic synthesiss natural perfume material present in Fructus psidii guajavae immaturus tissue, the method provides one kind with linoleic acid, Asia
The polybasic unsaturated fatty acid that fiber crops acid and arachidonic acid etc. have along cis- Isosorbide-5-Nitrae-pentadiene structure is substrate,
Using lipoxidase present in Fructus psidii guajavae immaturus tissue and hydroperoxide lyase degraded substrate, synthesis tool
The method having natural flavour mountaineous spice.So far, the unstability of hydroperoxide lyase and its reaction
Complexity always lead to Product yields too low (<20mg/L) and restrict this biosynthesis pathway realize
Industrialized key factor.
Content of the invention
One of the object of the invention is a kind of method providing serialization to prepare linoleic acid lipid metabolite, especially
It is by the catalytic action of immobilization hydroperoxide lyase, linoleic acid hydroperoxide to be degraded to produce
Natural perfume material material.
Invention includes following step：
1st, the preparation of fatty acid hydroperoxide：Fatty acid hydroperoxide is prepared by LOX reaction
(HPOs).0.1M free linoleic acid is scattered in (0.2M) in the borate buffer solution of pH 9.0, in
It is stirring evenly and then adding into LOX, logical oxygen at 4 DEG C in fermentation tank and be forced into 0.3MPa and reacted.1.5
Terminate after h to react and take a certain amount of product to measure its ultraviolet absorptivity in 234nm after ethanol dilution,
Then concentration (ε=the 25000cm of HPOs in reactant liquor is calculated according to molar absorption coefficient-1M-1).?
Under the conditions of afterwards reaction product solution being placed in 4 DEG C, cold preservation is stand-by.
2nd, the extraction of Amaranthus mangostanus L. hydroperoxide lyase：After removing root and stem, fresh Amaranthus mangostanus L. leaf is cut
Broken and with pH 8.5 Tris-HCl buffer (0.1M simultaneously contains 0.5% (m/v) PVP K-30) is pressed
According to 1:3 ratio mixing, homogenate 1min (10s × 6).After four layers of filtered through gauze, gained filtrate is at 4 DEG C
Lower 18000 × g centrifugation 30min.Centrifugation a certain amount of phosphate buffer of addition (PBS, 0.02
M, pH 6.8), DTT containing 10mM and 0.5% (v/v) Triton X-100 in PBS.At 4 DEG C
The centrifugation 30min of 18000 × g at same temperature of stirring 1h, gained supernatant is through 30% ammonium sulfate
After precipitation, gained precipitation is dissolved in the above-mentioned PBS containing DTT and Triton X-100 again,
Obtain final product free HPL crude enzyme liquid.
3rd, the preparation method of shitosan composite aquogel is as follows：
A. in acetic acid solution shitosan and γ-carrageenan being added to and stir to being completely dissolved, mixed
Close solution.
B. using syringe, step a gained mixed solution is dropwise instilled in NaOH solution and stirs formation
The hydrogel microsphere of a diameter of 1 2mm.
C. add excessive glutaraldehyde in step b gained hydrogel microsphere, obtain aldehyde radical activation hydrogel micro-
Ball.
D. add 1,6- hexamethylene diamine in step c gained aldehyde radical activation hydrogel microsphere.
E. it is filtrated to get the shitosan composite aquogel microsphere that surface is connected to 1,6- hexamethylene diamine.
Shitosan 2.5wt%～7.5wt% is contained in mixed solution described in step a；γ-carrageenan 2.5
Wt%～7.5wt%；Acetic acid solution 5%～25%v/v.
The concentration of NaOH solution described in step b is 0.5M～2.5M, mixed solution and NaOH solution
Volume ratio 1:5～10；Described whipping process：Speed of agitator is 10～50rpm, and mixing time is 1～5 day.
The addition of 1,6- hexamethylene diamine described in step d is 1,6- hexamethylene diamine：- NH in shitosan2Mol ratio
=0.5～1.0:1.
4th, the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel：
(1) shitosan composite aquogel microsphere is immersed in EDC aqueous solution, be subsequently adding containing HPL
PBS solution, at 4～10 DEG C shake 10～30min, filter, obtain shitosan Compound Water
Gel micro-ball immobilization hydroperoxide lyase.
(2) the microsphere immobilized hydroperoxides of shitosan composite aquogel are cleaned with PBS and deionized water
Lyases.The purpose of cleaning is to wash away other impurity, and therefore wash number can adjust as needed,
Generally clean twice.
(3) the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel after cleaning, again
It is suspended in PBS, stored for future use under the conditions of 4 DEG C.
In the described complex liquid of step (1), shitosan composite aquogel microsphere and the mol ratio of EDC are
1:1～10；HPL is 1 with the mol ratio of shitosan composite aquogel microsphere:1.
5th, the serialization preparation of hexanal：Packed bed column reactor such as Fig. 1 for hexanal serialization preparation
Shown.The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel of 4～20U is filled in length
200～500mm, in the glass column of diameter 10.5～20mm.The temperature of reactor passes through outside circulation
Water-bath chuck is controlled to 30～40 DEG C.The 13-HPOD solution (0.02～0.2 that concentration is 10～50mM
M PBS, pH 4.0～6.5 contain 1～3mM DTT) it is continuously added in reaction column by peristaltic pump.
Start the yield collected effluent and measure wherein C6 aldehyde when system reaches stable.
Product quantitative analyses：Measure the aldehyde material generating using headspace gas chromatography (HS-GC).
Specific detecting step is as follows：With the insulation through 2min for the spectrophotomelric assay method identical reaction solution it
Afterwards rapidly by enzyme-deactivating.Product is measured using gas chromatograph, and that is, testing sample shifts rapidly
To gas phase head-space sampler, insulation balance 30min auto injection at 70 DEG C.Blank is containing only free
Crude enzyme liquid or the PBS of immobilized enzyme.The hexanal concentrations that reaction generates are calculated by below equation：
Cs=As/A0×C0
Wherein, AsFor response sample peak area；A0For standard sample peak area；CsFor response sample concentration；
C0For standard sample concentration.
The two of the object of the invention there is provided a kind of packed bed column reactor, by shitosan composite aquogel
Microsphere immobilized hydroperoxide lyase is used for the serialization preparation of armaticity flavor components C6 aldehyde, should
Device by pillar material container (1), conduit (2), peristaltic pump (3), circulator bath clamping device (4),
Reactor (5) and product collecting container (6) composition.
Material container (1) top is connected with peristaltic pump (3) afterbody by conduit (2), peristaltic pump (3)
Top is connected with the bottom of reactor (5) by conduit (2), and the outside of reactor (5) accompanies circulation
Water-bath clamping device (4), conduit (2) and product collecting container (6) are passed through in the top of reactor (5)
Top be connected.
Reactor (5) is low pressure glass column, its a length of 200～500mm, a diameter of 10.5～20mm.
The present invention compared with prior art has advantages below：
1) the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel of the present invention shows
Write the unit volume yield that improve product.
2) compared with resolvase stirring-type batch reactive mode, hexanal serialization preparation of the present invention
Method can make hexanal unit volume yield reach 3560 ± 102.1 in raising reacton volumetric production highest
And reduce and need enzyme amount in course of reaction.
3) column reactor designed by the present invention can achieve the serialization preparation of hexanal, entirely prepared
Journey simple to operate it is easy to control and cost is relatively low.
Brief description
Fig. 1 uses the sketch of immobilization hydroperoxide lyase continuous prodution hexanal.
The impact to hexanal unit volume yield for the enzyme dosage in Fig. 2 column reactor.
The impact to substrate conversion efficiency for the enzyme dosage in Fig. 3 column reactor.
In Fig. 4 column reactor, concentration of substrate is to the impact needing enzyme amount.
The impact to conversion ratio for the concentration of substrate in Fig. 5 column reactor.
Specific embodiment
Further illustrate the present invention below by specific embodiment.It should be understood that the enforcement of the present invention
Example is only used for the present invention, rather than limitation of the present invention, to this under the concept thereof of the present invention
Being improved or simplified of invention preparation method broadly falls into the scope of protection of present invention.
The preparation method of fatty acid hydroperoxide of the present invention is：Fatty acid hydroperoxide is prepared by LOX reaction
Peroxide (HPOs).0.1M free linoleic acid is scattered in (0.2 in the borate buffer solution of pH 9.0
M), it is stirring evenly and then adding into LOX, logical oxygen at 4 DEG C in the fermentation tank and be forced into 0.3MPa and carry out
Reaction.Terminate after 1.5h to react and take a certain amount of product to measure its purple in 234nm after ethanol dilution
Outer absorbance, then calculates concentration (ε=25000 of HPOs in reactant liquor according to molar absorption coefficient
cm-1M-1).Under the conditions of finally reaction product solution being placed in 4 DEG C, cold preservation is stand-by.
The extracting method of Amaranthus mangostanus L. hydroperoxide lyase of the present invention is：After removing root and stem, will be fresh
The chopping of Amaranthus mangostanus L. leaf and with the Tris-HCl buffer of pH 8.5 (0.1M containing 0.5% (m/v) PVP
K-30) according to 1:3 ratio mixing, homogenate 1min (10s × 6).Gained after four layers of filtered through gauze
Filtrate 18000 × g centrifugation 30min at 4 DEG C.Centrifugation adds a certain amount of phosphate buffer
(PBS, 0.02M, pH 6.8), DTT containing 10mM and 0.5% (v/v) Triton X-100 in PBS.
The centrifugation 30min of 18000 × g at same temperature of 1h is stirred, gained supernatant is through 30% at 4 DEG C
After ammonium sulfate precipitation, gained precipitation be dissolved in again above-mentioned containing DTT and Triton X-100's
In PBS, obtain final product free HPL crude enzyme liquid.
The method of C6 aldehyde product quantitative analyses of the present invention is：Using headspace gas chromatography (HS-GC)
Measure the aldehyde material generating.Specific detecting step is as follows：React with spectrophotomelric assay method identical
Solution through after the insulation of 2min rapid by enzyme-deactivating.Product is measured using gas chromatograph,
I.e. testing sample is transferred quickly to gas phase head-space sampler, and insulation at 70 DEG C balances 30min and automatically enters
Sample.Blank is the PBS containing only free crude enzyme liquid or immobilized enzyme.Reaction generate hexanal concentrations pass through with
Lower formula is calculated：
Cs=As/A0×C0
Wherein, AsFor response sample peak area；A0For standard sample peak area；CsFor response sample concentration；
C0For standard sample concentration.
Embodiment 1
Prepare shitosan composite aquogel：2.5g shitosan and 2.5g γ-carrageenan are added to 100g
In acetic acid solution containing 5%v/v and stir to being completely dissolved, obtain mixed solution.Subsequently using injection
Stir in the device NaOH solution that dropwise Deca 50ml mixed solution is 0.5M to 500ml concentration and slowly
Mix (50rpm) 1 day, to form the hydrogel microsphere of a diameter of 1 2mm.Using excessive glutaraldehyde
The amino on activation hydrogel microsphere surface, to introduce aldehyde radical, is subsequently added 1, the 6- hexamethylene diamine of 7.75mmol,
After reaction, to be connected shitosan composite aquogel by active aldehyde radical micro- for 1,6- hexamethylene diamine (HMDA)
The surface of ball.
The immobilization of hydroperoxide lyase：15.5mmol surface is connected to 1,6- hexamethylene diamine
(HMDA) shitosan composite aquogel microsphere immersion 300ml contains the water-soluble of 155mmol EDC
In liquid, be subsequently adding the PBS solution that 100ml contains 15.5mmol HPL, after 4 DEG C shake
30min, filters, obtains the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel；Use PBS
And the deionized water cleaning microsphere immobilized hydroperoxide lyase of shitosan composite aquogel is twice；Cleaning
Later the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel, is resuspended in PBS
In, stored for future use under the conditions of 4 DEG C.The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel
Stability be greatly improved.
The serialization preparation of hexanal：Packed bed column reactor such as Fig. 1 institute for hexanal serialization preparation
Show.The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel of 18U is filled in long 200
Mm, in the glass column of diameter 10.5mm.The temperature of reactor passes through outside circulator bath chuck control
It is made as 30 DEG C.By the 13-HPOD solution for 50mM for the concentration, (0.02M PBS, pH 6.5 contains 1
MM DTT) it is continuously added in reaction column by peristaltic pump.Start when system reaches stable
Collect effluent and measure the yield of wherein C6 aldehyde.
As shown in Table 1, compared with resolvase stirring-type batch reactive mode, pillar of the present invention
Continuous reaction improves the unit volume yield of product in course of reaction and unit enzyme activity yield and reduces
Enzyme amount is needed in course of reaction.
Table 1 generates the Amaranthus mangostanus L. raw material required for 1g hexanal
Embodiment 2
Prepare shitosan composite aquogel：7.5g shitosan and 7.5g γ-carrageenan are added to 100g
In acetic acid solution containing 25%v/v and stir to being completely dissolved, obtain mixed solution.Subsequently using note
In the emitter NaOH solution that dropwise Deca 50ml mixed solution is 2.5M to 250ml concentration and slow
Stirring (10rpm) 5 days, to form the hydrogel microsphere of a diameter of 1 2mm.Using excessive penta 2
Aldehyde activates the amino on hydrogel microsphere surface to introduce aldehyde radical, and 1, the 6- being subsequently added 46.56mmol is own
Diamidogen, after reaction, 1,6- hexamethylene diamine (HMDA) is connected shitosan Compound Water by active aldehyde radical
The surface of gel micro-ball.
The immobilization of hydroperoxide lyase：46.56mmol surface is connected to 1,6- hexamethylene diamine
(HMDA) shitosan composite aquogel microsphere immerses the water that 300ml contains 46.56mmol EDC
In solution, be subsequently adding the PBS solution that 100ml contains 46.56mmol HPL, after 10 DEG C shake
Swing 10min, filter, obtain the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel；With
PBS and the deionized water cleaning microsphere immobilized hydroperoxide lyase of shitosan composite aquogel are twice；
The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel after cleaning, is resuspended in
In PBS, stored for future use under the conditions of 4 DEG C.The microsphere immobilized hydroperoxidation of gained shitosan composite aquogel
The stability of thing lyases is greatly improved.
The serialization preparation of hexanal：Packed bed column reactor such as Fig. 1 institute for hexanal serialization preparation
Show.The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel of 20U is filled in long 500
In the low pressure glass column of mm, diameter 20mm.The temperature of reactor passes through outside circulator bath chuck
It is controlled to 40 DEG C.By the 13-HPOD solution for 10mM for the concentration, (0.2M PBS, pH 4.0 contains
3mM DTT) it is continuously added in reaction column by peristaltic pump.Open when system reaches stable
Beginning collects effluent and measures the yield of wherein C6 aldehyde.
Embodiment 3
Prepare shitosan composite aquogel：3.0g shitosan and 4.0g γ-carrageenan are added to 100g
In acetic acid solution containing 10%v/v and stir to being completely dissolved, obtain mixed solution.Subsequently using note
In the emitter NaOH solution that dropwise Deca 50ml mixed solution is 1.0M to 400ml concentration and slow
Stirring (45rpm) 1.5 days, to form the hydrogel microsphere of a diameter of 1 2mm.Using excessive penta
Dialdehyde activates the amino on hydrogel microsphere surface to introduce aldehyde radical, is subsequently added the 1,6- of 18.63mmol
Hexamethylene diamine, after reaction, 1,6- hexamethylene diamine (HMDA) is connected shitosan by active aldehyde radical and is combined
The surface of hydrogel microsphere.
The immobilization of hydroperoxide lyase：18.63mmol surface is connected to 1,6- hexamethylene diamine
(HMDA) shitosan composite aquogel microsphere immerses the water that 300ml contains 93.15mmol EDC
In solution, be subsequently adding the PBS solution that 100ml contains 18.63mmol HPL, after 5 DEG C shake
Swing 25min, filter, obtain the microsphere immobilized hydroperoxide lyase of shitosan composite aquogel；With
PBS and the deionized water cleaning microsphere immobilized hydroperoxide lyase of shitosan composite aquogel are twice；
The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel after cleaning, is resuspended in
In PBS, stored for future use under the conditions of 4 DEG C.The microsphere immobilized hydroperoxidation of gained shitosan composite aquogel
The stability of thing lyases is greatly improved.
The serialization preparation of hexanal：Packed bed column reactor such as Fig. 1 institute for hexanal serialization preparation
Show.The microsphere immobilized hydroperoxide lyase of shitosan composite aquogel of 4U is filled in long 250
Mm, in the glass column of diameter 15mm.The temperature of reactor is passed through outside circulator bath chuck and is controlled
For 35 DEG C.By the 13-HPOD solution for 30mM for the concentration, (0.03M PBS, pH 6.0 contains 1.5
MM DTT) it is continuously added in reaction column by peristaltic pump.Start when system reaches stable
Collect effluent and measure the yield of wherein C6 aldehyde.
From Fig. 2 and Fig. 3, in the starting stage of hexanal synthetic reaction, Product yields and substrate convert
Rate increases rapidly with the increase of shitosan composite aquogel microsphere immobilized hydroperoxide lyase consumption
Greatly, be 16U in enzyme dosage, concentration of substrate be during 10mM substrate conversion efficiency up to 100%.
From Fig. 4 and Fig. 5, this microsphere immobilized hydroperoxide lyase of shitosan composite aquogel
During herein described continuous prodution, when concentration of substrate is relatively low, continuous operation stability is preferable.
Claims (9)
1. a kind of prepare the method for natural perfume material it is characterised in that comprising the steps using immobilization hydroperoxide lyase serialization：
(1) preparation of fatty acid hydroperoxide；
(2) extraction of Amaranthus mangostanus L. hydroperoxide lyase；
(3) preparation of immobilization hydroperoxide lyase；
(4) the serialization preparation of hexanal.
2. a kind of according to claim 1 prepare the method for natural perfume material it is characterised in that the preparation method of the described immobilization hydroperoxide lyase of step (3) is using immobilization hydroperoxide lyase serialization：
(1) hydrogel microsphere is immersed in EDC aqueous solution, hydrogel microsphere is 1 with the mol ratio of EDC:1～10, it is subsequently adding the PBS solution containing HPL, HPL is 1 with the mol ratio of hydrogel microsphere:1, at 4～10 DEG C shake 10～30min, filter, obtain hydrogel microsphere immobilization hydroperoxide lyase；
(2) clean hydrogel microsphere immobilization hydroperoxide lyase with PBS and deionized water；
(3) the hydrogel microsphere immobilization hydroperoxide lyase after cleaning, is resuspended in PBS, stored for future use under the conditions of 4 DEG C.
3. a kind of according to claim 2 prepare the method for natural perfume material it is characterised in that the preparation method of the described hydrogel microsphere of step () is using immobilization hydroperoxide lyase serialization：
A. γ-the carrageenan of the shitosan of 2.5wt.%～7.5wt.% and 2.5wt.%～7.5wt.% is added in the solution containing 5%～25%v/v acetic acid and stirs to being completely dissolved, obtain mixed solution；
B. using syringe, step a gained mixed solution is dropwise instilled in the NaOH solution that concentration is 0.5M～2.5M, the volume ratio 1 of mixed liquor and NaOH solution:5～10, then stirring forms the hydrogel microsphere of a diameter of 1 2mm；
C. add excessive glutaraldehyde in step b gained hydrogel microsphere, obtain aldehyde radical activation hydrogel microsphere；
D. add 1,6- hexamethylene diamine in step c gained aldehyde radical activation hydrogel microsphere, the addition of 1,6- hexamethylene diamine is 1,6- hexamethylene diamine：- NH in shitosan2Mol ratio=0.5～1.0:1；
E. it is filtrated to get the shitosan composite aquogel microsphere that surface is connected to 1,6- hexamethylene diamine.
4. a kind of according to claim 1 prepare the method for natural perfume material it is characterised in that the continuous preparation method of the described hexanal of step (4) is using immobilization hydroperoxide lyase serialization：
(A) immobilization hydroperoxide lyase is filled in the glass column of packed bed column reactor；
(B) fatty acid hydroperoxide is continuously added in reaction column by peristaltic pump；
(C) start to collect effluent when system reaches stable；
(D) measure the yield of C6 aldehyde.
5. according to claim 4 a kind of using immobilization hydroperoxide lyase serialization prepare natural perfume material method it is characterised in that described hexanal serialization preparation reaction temperature be 30～40 DEG C.
6. according to claim 4 a kind of using immobilization hydroperoxide lyase serialization prepare natural perfume material method it is characterised in that the described immobilization hydroperoxide lyase of step (A) loading be 4～20U.
7. a kind of method preparing natural perfume material using immobilization hydroperoxide lyase serialization according to claim 4, it is characterized in that, the 13-HPOD solution containing 1～3mM DTT, 0.02～0.2M PBS, pH4.0～6.5 that the described fatty acid hydroperoxide of step (B) is 10～50mM for concentration.
8. a kind of packed bed column reactor as used in described method arbitrary in claim 1 to 7, it is characterized in that, this device is made up of pillar material container (1), conduit (2), peristaltic pump (3), circulator bath clamping device (4), reactor (5) and product collecting container (6)；
Material container (1) top is connected with peristaltic pump (3) afterbody by conduit (2), peristaltic pump (3) top is connected with the bottom of reactor (5) by conduit (2), the outside of reactor (5) accompanies circulator bath clamping device (4), and the top of reactor (5) is connected with the top of product collecting container (6) by conduit (2).
9. according to claim 8 a kind of packed bed column reactor it is characterised in that described reactor (5) be low pressure glass column, its a length of 200～500mm, a diameter of 10.5～20mm.
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| CN103525803A (en) * | 2013-10-28 | 2014-01-22 | 江南大学 | Preparation method and application of natural material composite system immobilized bifunctional enzyme used for wine |
-
2015
- 2015-08-24 CN CN201510520665.XA patent/CN106480111A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995026413A1 (en) * | 1994-03-25 | 1995-10-05 | Reynolds Technologies, Inc. | Method for providing green note compounds |
| CN101717764A (en) * | 2009-12-11 | 2010-06-02 | 江南大学 | Method for preparing electrophoresis pure amaranth 13-hydroperoxide lyase |
| CN103525803A (en) * | 2013-10-28 | 2014-01-22 | 江南大学 | Preparation method and application of natural material composite system immobilized bifunctional enzyme used for wine |
Non-Patent Citations (2)
| Title |
|---|
| QINGQING LIU 等: "Covalent immobilization of hydroperoxide lyase on chitosan hybrid hydrogels and production of C6 aldehydes by immobilized enzyme", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| 刘庆庆: "苋菜氢过氧化物裂解酶的固定化及其在C6醛制备中的应用", 《中国博士学位论文全文数据库 基础科学辑》 * |
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Application publication date: 20170308 |