CN106755145A - Method for catalytically synthesizing citrate by utilizing nano composite material immobilized lipase - Google Patents
Method for catalytically synthesizing citrate by utilizing nano composite material immobilized lipase Download PDFInfo
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- CN106755145A CN106755145A CN201611196411.8A CN201611196411A CN106755145A CN 106755145 A CN106755145 A CN 106755145A CN 201611196411 A CN201611196411 A CN 201611196411A CN 106755145 A CN106755145 A CN 106755145A
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- composite material
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- 239000000463 material Substances 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 26
- 108090001060 Lipase Proteins 0.000 title claims abstract description 23
- 239000004367 Lipase Substances 0.000 title claims abstract description 23
- 102000004882 Lipase Human genes 0.000 title claims abstract description 23
- 235000019421 lipase Nutrition 0.000 title claims abstract description 23
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 23
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 title claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 8
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 32
- 239000012621 metal-organic framework Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 7
- 239000002994 raw material Substances 0.000 claims abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229910021393 carbon nanotube Inorganic materials 0.000 claims abstract description 4
- 239000002041 carbon nanotube Substances 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 235000019441 ethanol Nutrition 0.000 claims description 15
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 11
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000006555 catalytic reaction Methods 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 102100021851 Calbindin Human genes 0.000 claims description 7
- 101000898082 Homo sapiens Calbindin Proteins 0.000 claims description 7
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 claims description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000000337 buffer salt Substances 0.000 claims description 6
- 150000007524 organic acids Chemical class 0.000 claims description 6
- 239000002798 polar solvent Substances 0.000 claims description 6
- 238000005215 recombination Methods 0.000 claims description 6
- 230000006798 recombination Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- -1 citric acid ester Chemical class 0.000 claims description 5
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims description 4
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 claims description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 3
- 229910001431 copper ion Inorganic materials 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 229910001428 transition metal ion Chemical class 0.000 claims description 3
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 229910001429 cobalt ion Inorganic materials 0.000 claims description 2
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 2
- 229910001437 manganese ion Inorganic materials 0.000 claims description 2
- 229910021645 metal ion Inorganic materials 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- 239000013105 nano metal-organic framework Substances 0.000 claims 1
- 239000013289 nano-metal-organic framework Substances 0.000 claims 1
- 229910052723 transition metal Inorganic materials 0.000 claims 1
- 150000003624 transition metals Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 2
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 238000003541 multi-stage reaction Methods 0.000 abstract 1
- 150000003138 primary alcohols Chemical class 0.000 abstract 1
- 235000015165 citric acid Nutrition 0.000 description 11
- 239000000047 product Substances 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- QMKYBPDZANOJGF-UHFFFAOYSA-N benzene-1,3,5-tricarboxylic acid Chemical compound OC(=O)C1=CC(C(O)=O)=CC(C(O)=O)=C1 QMKYBPDZANOJGF-UHFFFAOYSA-N 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- 108010091086 Recombinases Proteins 0.000 description 3
- 102000018120 Recombinases Human genes 0.000 description 3
- DOOTYTYQINUNNV-UHFFFAOYSA-N Triethyl citrate Chemical compound CCOC(=O)CC(O)(C(=O)OCC)CC(=O)OCC DOOTYTYQINUNNV-UHFFFAOYSA-N 0.000 description 3
- 239000001069 triethyl citrate Substances 0.000 description 3
- VMYFZRTXGLUXMZ-UHFFFAOYSA-N triethyl citrate Natural products CCOC(=O)C(O)(C(=O)OCC)C(=O)OCC VMYFZRTXGLUXMZ-UHFFFAOYSA-N 0.000 description 3
- 235000013769 triethyl citrate Nutrition 0.000 description 3
- 238000005292 vacuum distillation Methods 0.000 description 3
- QZCLKYGREBVARF-UHFFFAOYSA-N Acetyl tributyl citrate Chemical compound CCCCOC(=O)CC(C(=O)OCCCC)(OC(C)=O)CC(=O)OCCCC QZCLKYGREBVARF-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000005909 ethyl alcohol group Chemical group 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000004014 plasticizer Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- MQIUGAXCHLFZKX-UHFFFAOYSA-N Di-n-octyl phthalate Natural products CCCCCCCCOC(=O)C1=CC=CC=C1C(=O)OCCCCCCCC MQIUGAXCHLFZKX-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 108010084311 Novozyme 435 Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- WRLRISOTNFYPMU-UHFFFAOYSA-N [S].CC1=CC=CC=C1 Chemical compound [S].CC1=CC=CC=C1 WRLRISOTNFYPMU-UHFFFAOYSA-N 0.000 description 1
- 239000003377 acid catalyst Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 150000003639 trimesic acids Chemical class 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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- Molecular Biology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for catalytically synthesizing citrate by immobilized lipase of a nano composite material, which comprises the following steps: (1) preparing a nano composite material by carrying out a composite reaction on a carboxylated carbon nanotube and a metal organic framework raw material, and drying for later use; (2) immobilizing lipase by using the nano composite material prepared in the step (1); (3) catalyzing citric acid and primary alcohol by using the immobilized lipase prepared in the step (1) to generate citrate. The method can obviously improve the activity of the lipase, enhances the structural stability of the immobilized enzyme, and greatly increases the repeated use times.
Description
Technical field
The present invention relates to enzyme technology field, and in particular to one kind is fixed lipase catalyzed using nano composite material
The method of synthesizing citric acid ester.
Background technology
Citrate is the nontoxic plasticizer of the internationally recognized environmental protection of a class, is widely used in food, medicine, health care
The fields such as product, toy, cosmetics, are the potential substitutes of phthalic ester plasticizer.
At present, the production method of citrate is mainly citric acid and corresponding alcohol, with the strong acid such as concentrated sulfuric acid, to toluene sulphur
Acid etc. are catalyst, with the polar solvent high such as alcohol as narrow aqua, crude product are obtained by controlling reaction temperature and reaction time, then lead to
Cross means of purification and obtain the preferable product of purity.Said process severe reaction conditions, wherein the strong acid catalyst for largely using is easy
Serious corrosion is produced to equipment, and the wastewater treatment difficulty for producing easily causes environmental pollution.
Enzymatic clarification citrate has that reaction condition is gentle, equipment durability is high, it is environment-friendly the characteristics of.Existing report
The method for catalyzing and synthesizing triethyl citrate using commercially available immobilized lipase Novozym435, the technique is eliminated in product
With acid number and purification decolorization process, process is simple, suitable industrialized production, but the method still suffer from the immobilized enzyme catalysis for using
Vigor is not high, reuses the relatively low problem of number of times.Above mentioned problem also turns into restriction enzymatic clarification citrate large-scale industry
The key that metaplasia is produced.
The content of the invention
The invention discloses a kind of method of the fixed lipase catalyzed synthesizing citric acid ester of utilization nano composite material.Should
Process for fixation is remarkably improved the vigor of lipase, and structural stability enhancing is reused number of times and is substantially increased.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of method of the fixed lipase catalyzed synthesizing citric acid ester of utilization nano composite material, comprises the following steps：
(1) prepared with metal organic framework (MOF) raw material recombination reaction by carboxylic carbon nano-tube (MWNT-COOHs) and received
Nano composite material, drying for standby；
(2) using nano composite material immobilized lipase obtained in step (1)；
(3) fixed lipase catalyzed citric acid and primary alconol the generation citrate for being prepared using step (1).
In step (1), described metal organic framework raw material includes the inorganic salts of organic acid part and transition metal ions；
Wherein, described organic acid part is equal phthalic acid, trimesic acid, any one (preferably equal benzene of terephthalic acid (TPA) weight
Dioctyl phthalate or trimesic acid)；The inorganic salts of described metal ion are appointing in copper ion, cobalt ions, zinc ion, manganese ion
The inorganic salts of meaning a kind of (preferably copper ion)；Described inorganic salts include nitrate, sulfate or villaumite；Wherein, carboxylated carbon
The reaction mass of the inorganic salts of nanotube, organic acid part and transition metal ions than 3~4: 1~2: 1~3, preferred mass ratio
It is any one ratio in 4: 1: 2,3: 1: 3,4: 2: 2,3: 2: 3,4: 1: 1, most preferably 4: 1: 2.
In step (1), the reaction dissolvent of recombination reaction is polar solvent, the mixed solution of second alcohol and water, wherein, it is described
Any one (preferably DMF) in polar solvent DMF, DMSO or methyl alcohol；Polar solvent, the volume ratio of second alcohol and water are 1~2: 1
~2: 1~2, preferred volume ratio is any one ratio in 1: 1: 1,1: 2: 1,2: 1: 1,1: 1: 2,1: 2: 2,2: 1: 2,2: 2: 1
Example, most preferably 1: 1: 1.
In step (1), the reaction temperature of recombination reaction is 80-150 DEG C (preferably 85 DEG C), and the reaction time is that 12-36h is (excellent
Select 24h)；Drying temperature is 80-120 DEG C (preferably 90 DEG C).
The nano composite material that step (1) is prepared is MWNT-COOHs surface modifications MOF.
In step (2), described lipase is CALB or P450, certainly the invention is not limited in both lipase,
The enzyme of other fatty enzyme families can also be used.
Lipase activity used in the present invention is generally 2000-10000U/g, and enzyme activity is defined as what is synthesized for esters
Vigor is represented with the propyl laurate ester unit (PLU/g) of generation in every 1 gram of enzyme unit time.
In step (2), specially be dissolved in lipase in buffer salt solution by the way of absorption by immobilization, and addition is received
Stirring and adsorbing 0.5-3h (preferably 1h) after nano composite material；Lipase is 1: 10~20 (excellent with the mass ratio of nano composite material
Select 1: 10).Wherein, described buffer salt solution is PBS or PBK.
In step (3), catalytic reaction condition is pH5.5-8, and catalytic temperature is 30-70 DEG C, and catalysis time is 6-18h, is urged
Change buffer salt solution and use PBS or PBK；Immobilised enzymes is 1: 25~50 (preferably 1: 30), primary alconol excess with citric acid mass ratio.
In step (3), primary alconol position ethanol, butanol, any one in octanol.
In step (3), the primary alconol of the narrow aqua as selection for using.
Beneficial effect：The inventive method can significantly improve the vigor of lipase, the enhancing of immobilised enzymes structural stability, weight
Multiple access times are substantially increased.
Brief description of the drawings
Fig. 1 is the TEM photos of raw material MWNT-COOHs；
The TEM photos of the nano composite material MWNT-COOHs-MOF that Fig. 2 embodiments 1 are prepared.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should also without limitation on sheet described in detail in claims
Invention.
The detection method of following examples is as follows：
The measure of citrate uses general gas chromatography.DB-1301(15m*0.53mm*1μm)；Column temperature is adopted
Use temperature programming：First 80 DEG C of holding 0.5min, then increase to 220 DEG C and keep 10min with the speed of 20 DEG C/min；Using hydrogen
Flame ionization detector (FID), detector temperature：275℃；Injector temperature：225℃；Carrier gas：Nitrogen, flow velocity：2.3mL/
min；Split ratio：20∶1；Sample size：1.0μL.
Immobilised enzymes measures fixed：The total amount of resolvase before and after immobilization is determined on the premise of total enzyme amount is certain respectively, is subtracted each other
Being fixed enzyme amount.Enzyme assay method uses Bradford's colorimetric method.
Embodiment 1：
5g trimesic acids and 10gCu (NO3)2It is dissolved in 500ml solvents (DMF: ethanol: water=1: ultrasonically treated in 1: 1)
30min.20g MWNT-COOHs are dissolved in ultrasonically treated 30min in 150mlDMF.85 DEG C of reaction 24h after the mixing of above two solution
90 DEG C of drying afterwards are made nano composite material.1g CALB (enzyme activity 5000U/g) is dissolved in 50mL PBS solutions, adds above-mentioned material
10g, is centrifuged after stirring 1h and reclaims nano composite material, and uses PBS, being fixed CALB, by 100g citric acids,
8h, filtering removal immobilised enzymes, 50 DEG C of decompressions are reacted at 100mL absolute ethyl alcohols and 50 DEG C of 3g immobilizations CALB, pH7 heating stirring
Excessive ethanol is distilled out, triethyl citrate finished product 141.0g, molar yield 98.1% is obtained.Immobilised enzymes recycling 20
Secondary, catalysis activity still keeps more than 85%.
Fig. 1 is the TEM photos of raw material MWNT-COOHs, and surface is smooth, zymoprotein limited amount that can be immobilized and insecure.
Fig. 2 is the TEM photos of nano composite material MWNT-COOHs-MOF, and protrusion of surface is the position of MOF modifications, structure specific surface area
Increase, immobilization enzyme amount is more, and Stability Analysis of Structures.
Embodiment 2：
10g terephthalic acid (TPA)s and 20gZn (NO3)2It is dissolved in 1L solvents (DMSO: ethanol: water=1: ultrasonically treated in 2: 1)
30min.40g MWNT-COOHs are dissolved in ultrasonically treated 30min in 300mlDMSO.100 DEG C of reactions after the mixing of above two solution
105 DEG C of drying are made nano composite material after 18h.1g P450 (4530U/g) are dissolved in 50mL PBK solution, add above-mentioned material
15g, is centrifuged after stirring 2h and reclaims nano composite material, and cleaned with PBK.200g citric acids, the anhydrous butanol of 200mL and 8g are consolidated
Surely change P450,12h is reacted at 60 DEG C of pH6.8 heating stirrings, filtering removal immobilised enzymes, 80 DEG C of vacuum distillations go out excessive fourth
Alcohol, obtains ATBC finished product 358.2g, molar yield 95.6%.Immobilised enzymes is reused 18 times, and catalysis activity is still
Keep more than 80%.
Comparative example 1：
By free CALB (the immobilization CALB prepared with 3g embodiments 1 of 100g citric acids, 100mL absolute ethyl alcohols and 0.3g
Total amount is identical), 12h is reacted at 50 DEG C of pH7 heating stirrings, centrifugation removal resolvase, 50 DEG C of vacuum distillations go out excessive ethanol, obtain
Triethyl citrate finished product 110.5g, molar yield 76.9%.
Comparative example 2：
The free P450 of 200g citric acids, the anhydrous butanol of 200mL and 1g is (total with immobilization P450 prepared by 8g embodiments 2
Amount is identical), 16h is reacted at 60 DEG C of pH6.8 heating stirrings, centrifugation removal resolvase, 80 DEG C of vacuum distillations go out excessive butanol, obtain
ATBC finished product 304.2g, molar yield 81.1%.
Claims (10)
1. the method for the fixed lipase catalyzed synthesizing citric acid ester of a kind of utilization nano composite material, it is characterised in that including
Following steps：
(1) nano composite material, drying for standby are prepared by carboxylic carbon nano-tube and metal organic framework raw material recombination reaction；
(2) using nano composite material immobilized lipase obtained in step (1)；
(3) fixed lipase catalyzed citric acid and primary alconol the generation citrate for being prepared using step (1).
2. method according to claim 1, it is characterised in that in step (1), described metal organic framework raw material includes
The inorganic salts of organic acid part and transition metal ions；Wherein, described organic acid part is equal phthalic acid, equal benzene front three
Any one of acid, terephthalic acid (TPA) weight；The inorganic salts of described metal ion are copper ion, cobalt ions, zinc ion, manganese ion
In the inorganic salts of any one.
3. method according to claim 2, it is characterised in that carboxylic carbon nano-tube, organic acid part and transition metal
The reaction mass of the inorganic salts of ion is than 3~4: 1~2: 1~3.
4. method according to claim 1, it is characterised in that in step (1), the reaction dissolvent of recombination reaction is molten for polarity
Agent, the mixed solution of second alcohol and water, wherein, any one in described polar solvent DMF, DMSO or methyl alcohol；Polar solvent,
The volume ratio of second alcohol and water is 1~2: 1~2: 1~2.
5. method according to claim 1, it is characterised in that in step (1), the reaction temperature of recombination reaction is 80-150
DEG C, the reaction time is 12-36h；Drying temperature is 80-120 DEG C.
6. method according to claim 1, it is characterised in that in step (2), described lipase is CALB or P450.
7. method according to claim 1, it is characterised in that in step (2), immobilization by the way of absorption, specifically
It is that lipase is dissolved in buffer salt solution, stirring and adsorbing 0.5-3h after addition nano composite material；Lipase with it is nano combined
The mass ratio of material is 1: 10~20.
8. method according to claim 7, it is characterised in that described buffer salt solution is PBS or PBK.
9. method according to claim 1, it is characterised in that in step (3), catalytic reaction condition is pH5.5-8, catalysis
Temperature is 30-70 DEG C, and catalysis time is 6-18h, and catalysis buffer salt solution uses PBS or PBK；Immobilised enzymes and citric acid quality
Than being 1: 25~50, primary alconol excess.
10. method according to claim 1, it is characterised in that in step (3), in primary alconol position ethanol, butanol, octanol
Any one.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107828772A (en) * | 2017-10-30 | 2018-03-23 | 陕西师范大学 | A kind of immobilized enzyme reactor for ratio fluorescent detection and preparation method thereof |
| CN107828772B (en) * | 2017-10-30 | 2021-04-27 | 陕西师范大学 | Immobilized enzyme reactor for ratio fluorescence detection and preparation method thereof |
| CN109569728A (en) * | 2018-12-18 | 2019-04-05 | 吉林大学 | A kind of biology base metal organic frame assembles esterase catalyzed dose, preparation method and its application in biodiesel synthesis |
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