CN107299096A - Preparation method and application of imidazole and derivative thereof modified surfactant-enzyme nano composite catalyst - Google Patents
Preparation method and application of imidazole and derivative thereof modified surfactant-enzyme nano composite catalyst Download PDFInfo
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- CN107299096A CN107299096A CN201710474949.9A CN201710474949A CN107299096A CN 107299096 A CN107299096 A CN 107299096A CN 201710474949 A CN201710474949 A CN 201710474949A CN 107299096 A CN107299096 A CN 107299096A
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- imidazole
- enzyme
- derivants
- composite catalyst
- surfactant
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- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 239000003054 catalyst Substances 0.000 title claims abstract description 39
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 28
- 239000004094 surface-active agent Substances 0.000 claims abstract description 25
- 239000007864 aqueous solution Substances 0.000 claims abstract description 16
- 238000003756 stirring Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000008367 deionised water Substances 0.000 claims abstract description 6
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 6
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 6
- 239000012266 salt solution Substances 0.000 claims abstract description 3
- 238000012986 modification Methods 0.000 claims description 29
- 230000004048 modification Effects 0.000 claims description 27
- 230000003213 activating effect Effects 0.000 claims description 26
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000006555 catalytic reaction Methods 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 11
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 10
- 102000015439 Phospholipases Human genes 0.000 claims description 10
- 108010064785 Phospholipases Proteins 0.000 claims description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 102000018120 Recombinases Human genes 0.000 claims description 4
- 108010091086 Recombinases Proteins 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 3
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 3
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 2
- WVULKSPCQVQLCU-BUXLTGKBSA-N glycodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 WVULKSPCQVQLCU-BUXLTGKBSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 229940045946 sodium taurodeoxycholate Drugs 0.000 claims description 2
- YXHRQQJFKOHLAP-FVCKGWAHSA-M sodium;2-[[(4r)-4-[(3r,5r,8r,9s,10s,12s,13r,14s,17r)-3,12-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 YXHRQQJFKOHLAP-FVCKGWAHSA-M 0.000 claims description 2
- 241000272814 Anser sp. Species 0.000 claims 1
- 239000012071 phase Substances 0.000 abstract description 10
- 230000003197 catalytic effect Effects 0.000 abstract description 6
- 239000008346 aqueous phase Substances 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 239000011259 mixed solution Substances 0.000 abstract 2
- 238000005406 washing Methods 0.000 abstract 1
- 125000002252 acyl group Chemical group 0.000 description 12
- 230000000694 effects Effects 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000013508 migration Methods 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 3
- 229960001231 choline Drugs 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 1
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011943 nanocatalyst Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229960001866 silicon dioxide Drugs 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002411 thermogravimetry Methods 0.000 description 1
Classifications
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- 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
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic 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
- 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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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Inorganic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Catalysts (AREA)
Abstract
The invention discloses a preparation method and application of an imidazole and its derivative modified surfactant-enzyme nano composite catalyst. The preparation method comprises the following steps: preparing a surfactant aqueous solution, and dropwise adding a free enzyme solution at room temperature while stirring until the solution is clear to obtain a mixed solution; dropwise adding a metal ion salt solution into the mixed solution while stirring, and stirring at room temperature for 30-60min to obtain an aqueous solution of the metal ion/surfactant-enzyme nano composite catalyst; and (3) adding the aqueous solution of imidazole and derivatives thereof into the aqueous solution obtained in the step (2), stirring for 20-40min, centrifuging, washing for 1-3 times by using deionized water, and carrying out vacuum freeze drying until the weight is constant. The catalyst is applied to water-oil two-phase biocatalysis. The invention is prepared by adopting an aqueous phase solution mixing method, has simple operation, short period, mild condition, low cost, high enzyme stability, high enzyme recovery rate and high carrier enzyme molecule bonding strength, and has both biological and physical catalytic activity.
Description
Technical field
The invention belongs to the technique for fixing field of enzyme, and in particular to and a kind of imidazole and its derivants modification of surfaces activating agent-
The preparation method of enzyme nano-composite catalyst and application.
Background technology
Glycerolphosphocholine is generated by phosphatidyl choline by hydrolysis.Because glycerolphosphocholine is
The important choline of one class, it serves not only as the precursor of biosynthesis phosphatidyl choline, is also synthesis in human body
The precursor of neurotransmitter acetylcholine, possesses potential application value in the diseases such as treatment alzheimer senile dementia.It is single
Shifted with phospholipase A1 catalysis hydrolysis of phosphatidlycholine generation glycerolphosphocholine, it is necessary to undergo a step acyl group, from Sn-2-
LPC transfer generations Sn-1-LPC.The reason for this acyl group transfer occurs is Sn-1-LPC thermodynamic stability than Sn-2-
LPC is low.Acyl group transfer can be carried out always in the reaction from Sn-2-LPC to Sn-1-LPC, until reaction reaches dynamic equilibrium.Instead
The concentration of cushioning liquid in medium is answered not have great change for acyl migration.But, acid condition and alkalescence condition
The progress of acyl group transfer can be accelerated, wherein alkaline environment is more beneficial for the process of acyl group transfer and hydrolysis.
Surfactant can not only reduce the interfacial tension of organic/water two-phase system, additionally it is possible to influence the structure of enzyme molecule
As the stability of enzyme molecule can be improved to a certain extent.However, resolvase is usual in the solvent containing surfactant
It is used only once, limits its purposes.Although enzyme can pass through co-precipitation shape with metal ion and surfactant
Into nano-complex.But with organic/water two-phase system catalytic reaction carry out for a long time, enzyme-surfactant is received
Rice compound is still not able to keep higher vigor and stability.Need to change enzyme-surfactant nano-complex
Property lifts its catalytic performance in organic/water two-phase system.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of 2-methylimidazole modification of surfaces activating agent-
The preparation method of enzyme nano-composite catalyst and application, this method are prepared using aqueous phase solution mixing method, the simple to operate, cycle
Short, mild condition, with low cost, enzyme stability are high, the enzyme rate of recovery high, carrier enzyme molecule bond strength is high, have both biological and thing
Manage catalytic activity.
In order to solve the above technical problems, the technical scheme that the present invention takes is：
A kind of preparation method of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst, comprises the following steps：
Step 1, surfactant-modified resolvase
With 0.01-1 mmol/mL aqueous surfactant solutions, at room temperature, free enzyme liquid is added dropwise while stirring to clarifying, must mix
Liquid；
Step 2, metal ion/surfactant-enzyme nano-composite catalyst is prepared
Into mixed liquor, side stirring, which becomes, is added dropwise 0.01-1 mmol/mL metal ion salt solutions, stirs obtained after 30-60min at room temperature
The aqueous solution of metal ion/surfactant-enzyme nano-composite catalyst；
Step 3, imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst is prepared
The 0.01-1 mmol/mL imidazole and its derivants aqueous solution is added into the aqueous solution of step 2,20-40min, centrifugation is stirred
And with deionized water rinsing 1-3 times, vacuum freeze drying to constant weight.
It is that the surfactant is NaTDC, sodium taurodeoxycholate, glycodesoxycholic acid as improved
Receive, one kind in chenodesoxycholic acid sodium, the solute for enzyme liquid of dissociating is phospholipase A1。
Be as improved, in step 1 concentration of surfactant be 10 mmol/L, mixing speed be 100~
150rpm。
It is that metal ion described in step 2 is Co as improved2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+、
Mg2+In it is any, concentration is 20mmol/L, and mixing speed is 100~150rpm.
It is that imidazole and its derivants described in step 3 are 2-methylimidazole as improved.
Further improved to be, the concentration of the imidazole and its derivants aqueous solution is 50mmol/L in step 3, and centrifugal speed is
7000rpm, centrifugation time is 15min, and the vacuum of vacuum freeze drying is 1.3~13Pa, and temperature is -85 DEG C~-10 DEG C.
Above-mentioned imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst is in water phase and an oil phase living things catalysis
Application.
It is as improved, above-mentioned application comprises the following steps：The raw material containing phosphatidyl choline is weighed in centrifuge tube, plus
Enter deionized water and organic solvent, imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst is added, in water
Stirring reaction obtains glycerol production phosphatidyl choline afterwards for a period of time in bath.
Reaction mechanism of the present invention is：
Compared with prior art, preparation method of the present invention is simple to operate, the cycle is short, with low cost, reaction condition is gentle, carrier enzyme
Molecule bond strength is high.A kind of utilization imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst of the present invention
Preparation method and application modification of surfaces activating agent-Form generation of the enzyme nano-composite catalyst to precipitate, are improved nano combined
The stability and activity of enzyme in catalyst, while having biological and physics catalytic activity concurrently, are especially suitable for aqueous catalysis, organic
Mutually catalysis, biphasic catalysis react field.
Brief description of the drawings
Fig. 1 is a kind of sweeping for the imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst of gained of embodiment 1
Electron microscope is retouched, wherein, a is phospholipase A1Surfactant nano-complex, b is the phospholipase A that imidazole and its derivants are modified1
Surfactant nano-complex
Fig. 2 is the projection electron microscope of the imidazole and its derivants modification of surfaces activating agent of embodiment 1-enzyme nano-composite catalyst, a
For phospholipase A1Surfactant nano-complex, b is the phospholipase A that imidazole and its derivants are modified1Surfactant nanometer is multiple
Compound；
Fig. 3 is the phosphatidase that the surfactant nano-complex that imidazole and its derivants are modified is modified with imidazole and its derivants
A1Fourier's infared spectrum of surfactant nano-complex；
Fig. 4 is the surfactant nano-complex and imidazole and its derivants modification of surfaces activity that imidazole and its derivants are modified
The thermogravimetric analysis of agent-enzyme nano-composite catalyst；
Fig. 5 is that surfactant nano-complex, imidazole and its derivants modification of surfaces that imidazole and its derivants are modified are active
Agent-enzyme nano-composite catalyst, free phospholipase A1Catalytic efficiency comparison diagram；
The phospholipase A of Fig. 6 imidazole and its derivants modification1The recycling rate of waterused figure of surfactant nano-complex.
Embodiment
The preferred technical solution of the present invention is described in detail with reference to invention and accompanying drawing.
Embodiment 1
A kind of preparation method of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst, comprises the following steps：
Step 1, surfactant-modified resolvase
With 10 mmol/mL aqueous surfactant solutions, at room temperature, free enzyme liquid is added dropwise while stirring to clarifying, mixed liquor is obtained,
Wherein, mixing speed is 100rpm；
Step 2, metal ion/surfactant-enzyme nano-composite catalyst is prepared（Hmim@MSNC）
Into mixed liquor, side stirring becomes the cobalt chloride solution that 20mmol/L is added dropwise, and stirs metal ion/table is obtained after 30min at room temperature
The aqueous solution of face activating agent-enzyme nano-composite catalyst, wherein, mixing speed is 120rpm.
Step 3, imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst is prepared（Hmim@PLA1-
MSNC）
The 50mmol/L imidazole and its derivants aqueous solution is added into the aqueous solution of step 2,30min is stirred, centrifuges and spend
Ionized water is rinsed 3 times, and vacuum freeze drying to constant weight, wherein centrifugal speed are 7000rpm, and centrifugation time is 15min, very
The vacuum of vacuum freecing-dry is 1.3Pa, and temperature is -85 DEG C.
Imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst manufactured in the present embodiment（Hmim@
PLA1-MSNC）Scanning figure under Electronic Speculum is as shown in figure 1, it can be seen that phospholipase A1Surfactant is nano combined
Thing（PLA1-MSNC）Particle diameter in 100 below nm, and the phospholipase A of imidazole and its derivants modification1Surfactant nanometer is multiple
Compound（Hmim@PLA1-MSNC）Particle diameter in 200 below nm, this is due to that imidazole and its derivants can be prepared with material
The cobalt ions reaction of solution in journey, in MSNC or PLA1- MSNC Surface Creation crystal, and these crystal can will be multiple
MSNC nano particles or PLA1- MSNC nano particles are crosslinked together.Compared with MSNC and Hmim@MSNC, PLA1- MSNC and
Hmim@PLA1- MSNC pattern is changed, and this is due to the PLA during prepared by immobilised enzymes1To carrier structure institute shadow
Loud.
In order to further verify PLA1Whether it is successfully embedded in inside Hmim@MSNC, tests Hmim@MSNC, Hmim@
PLA1- MSNC Fourier's infared spectrum.As shown in Fig. 2 Hmim@PLA1- MSNC is in 1645cm-1Place occurs in that acyl group C=O's
Vibration peak, it was demonstrated that PLA1Successfully it has been fixed on inside Hmim@MSNC.
As shown in figure 3, Hmim@MSNC are in pyrolysis second stage, its weight have dropped 61.93%, and Hmin@PLA1-MSNC
65.07% is have dropped, PLA has been also demonstrated that1Successfully it has been fixed on inside Hmim@MSNC.
Imidazole and its derivants are 2-methylimidazole in the present embodiment.
Embodiment 2
With imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst of the gained of embodiment 1 in water-oil phase
Using the research for catalyzing and synthesizing glycerolphosphocholine.
The application comprises the following steps：Weigh 1.5 g egg yolk lecithin（Contain 70% phosphatidyl choline）Load 10
In mL plastic tube, 2.5 mL deionized water and 2.5 mL organic solvent are added, 100 μ L free PLA is eventually adding1
Or the Hmim@PLA containing equal protein content1/MSNC（15 mg）.The magnetic agitation in 40 °C of water-bath（600
rpm）30 min, the yield of glycerolphosphocholine is determined using high performance liquid chromatography.
200 μ L are extracted reaction solution to be dissolved in 1 mL methanol-n-hexyl alcohol mixed liquor（v:v=99:1）, and use 0.22 μm
Nylon membrane filtered dilutions, to remove insoluble matter.Filtrate is dried and analyzed in HPLC-ELSD in nitrogen atmosphere
Determine.Chromatographic column is silicagel column（250 mm×4.6 mm,5 μm）, mobile phase is methanol and the aqueous solution（v:v=90:10）, flow velocity
For 1.0 mL/min, post case temperature is controlled at 25 °C.
Embodiment 3
In embodiment 2 using organic solvent dissolve egg yolk lecithin, wherein organic solvent be respectively isooctane, normal heptane, just oneself
Alkane, n-octyl alcohol, butanol, methanol, N,N-dimethylformamide, dimethyl sulfoxide (DMSO).Compare different organic solvents to PLA1With Hmim@
PLA1- MSNC is catalyzed the influence of yield, as a result as shown in table 1.
When reacting 30 min, dissociate PLA1Phosphatidyl choline generation glycerophosphatide acyl courage can not be catalyzed in all organic solvents
Alkali.And Hmim@PLA1- MSNC can be catalyzed phosphatidyl choline and be generated respectively in isooctane, normal heptane, n-hexane, n-octyl alcohol
341.02 μm of ol/L, 559.84 μm of ol/L, 919.96 μm of ol/L, 239.73 μm of ol/L glycerolphosphocholine, and
Generation glycerolphosphocholine can not be catalyzed in butanol, methanol, N,N-dimethylformamide, dimethyl sulfoxide (DMSO).With organic
The trend for first increasing and reducing afterwards is presented in the rise of solvent polarity, the yield of glycerolphosphocholine.Because working as organic solvent
Polarity it is relatively low when, although the damage to enzyme activity is smaller, but the solubility to substrate is relatively low, is unfavorable for Hmim@PLA1-MSNC
Catalytic reaction.With the rise of organic solvent polarity, the solubility of substrate also increases therewith, but the higher solvent of polarity holds
Easily cause the inactivation of enzyme.In these organic solvents, substrate phosphatidyl choline can be dissolved in n-hexane well, and just oneself
Injury of the alkane to enzyme activity is smaller, therefore Hmim@PLA1- MSNC is catalyzed phosphatidyl choline generation glycerophosphatide acyl courage in n-hexane
The yield highest of alkali.
Embodiment 4
The free PLA of research1With Hmim@PLA1The otherness of-MSNC catalytic efficiencies, investigation compared for PLA1、PLA1-MSNC、
PLA1- MSNC and imidazole and its derivants and Hmim@PLA1- MSNC catalysis phosphatidyl choline generation glycerolphosphocholines
Yield.
As shown in figure 5, after 30 min of reaction, dissociate PLA1Phosphatidyl choline generation glycerolphosphocholine can not be catalyzed
Yield, reason is probably that one side reaction system is organic water two-phase, and larger interfacial tension is to the PLA that dissociates1Conformation exist
Destruction, the overlong time that another aspect acyl migration needs, causes the generation without glycerolphosphocholine.And work as
PLA1When being fixed in MSNC, the PLA generated1- MSNC has the function of surfactant, can reduce organic water two-phase
Interfacial tension, and the stability of enzyme can be kept.But it is due to that now acyl migration stills need a period of time, so
PLA1The glycerolphosphocholine yield of-MSNC catalysis generations only has 203.86 μm of ol/L.When the 2-methylimidazole for adding 2 mg
When, PLA1The glycerolphosphocholine yield of-MSNC catalysis generations reaches 521.27 μm of ol/L, because 2-methylimidazole makes
Obtain reaction solution pH and be in alkalescence, and subalkaline environmental benefits are in acyl migration.During nanocatalyst is prepared,
PLA is modified using 2-methylimidazole1- MSNC may make Hmim@PLA1- MSNC turns into subalkaline catalyst, and causes therein
PLA1Conformation more stablize.After 30 min are reacted, Hmim@PLA1The glycerolphosphocholine yield of-MSNC catalysis generations
919.96 μm of ol/L can be reached.
This hair imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst method is simple, and catalytic production is sweet
Oily phosphatidyl choline yield can reach 919.96 μm of ol/L, and the immobilised enzymes is catalyzed phosphatidyl choline generation in n-hexane
The yield highest of glycerolphosphocholine.
In actual production process, immobilised enzymes is not only cost-effective, shorten the reaction time, and also to meet can be more
The demand of secondary recycling.Fig. 6 has investigated Hmim@PLA1The situation that-MSNC is reused in above-mentioned reaction condition.Such as Fig. 6
It is shown：After the recycling of 10 times, Hmim@PLA1- MSNC enzyme activity is reduced only by 60%, shows in this reaction system
Middle Hmim@PLA1- MSNC possesses good recycling performance.
Claims (9)
1. a kind of preparation method of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst, it is characterised in that
Comprise the following steps：
Step 1, surfactant-modified resolvase
With 0.01-1 mmol/mL aqueous surfactant solutions, at room temperature, free enzyme liquid is added dropwise while stirring to clarifying, must mix
Liquid；
Step 2, metal ion/surfactant-enzyme nano-composite catalyst is prepared
Into mixed liquor, side stirring, which becomes, is added dropwise 0.01-1 mmol/mL metal ion salt solutions, stirs obtained after 30-60min at room temperature
The aqueous solution of metal ion/surfactant-enzyme nano-composite catalyst；
Step 3, imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst is prepared
The 0.01-1 mmol/mL imidazole and its derivants aqueous solution is added into the aqueous solution of step 2,20-40min is stirred, from
The heart and with deionized water rinsing 1-3 times, vacuum freeze drying to constant weight.
2. the preparation of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst according to claim 1
Method, it is characterised in that the surfactant is NaTDC, sodium taurodeoxycholate, glycodesoxycholic acid is received, goose
Any one in NaTDC, the solute for enzyme liquid of dissociating is phospholipase A1。
3. the preparation of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst according to claim 1
Method, it is characterised in that the concentration of surfactant is 10mmol/L in step 1, mixing speed is 100~150rpm.
4. the preparation of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst according to claim 1
Method, it is characterised in that metal ion described in step 2 is Co2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+、Mg2+In
Any, concentration is 20mmol/L, and mixing speed is 100~150rpm.
5. the preparation of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst according to claim 1
Method, it is characterised in that imidazole and its derivants described in step 3 are 2-methylimidazole.
6. the preparation of imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst according to claim 5
Method, it is characterised in that the concentration of the 2-methylimidazole aqueous solution is 50mmol/L in step 3, and centrifugal speed is 7000rpm, from
The heart time is 15min, and the vacuum of vacuum freeze drying is 1.3~13Pa, and temperature is -85 DEG C~-10 DEG C.
7. based on imidazole and its derivants modification of surfaces activating agent-enzyme nano-composite catalyst obtained by claim 1 in water oil
Application in two-phase living things catalysis.
8. application according to claim 7, it is characterised in that comprise the following steps：Weigh the raw material containing phosphatidyl choline
In centrifuge tube, deionized water and organic solvent are added, imidazole and its derivants modification of surfaces activating agent-enzyme nanometer is added multiple
Catalyst is closed, stirring reaction obtains glycerol production phosphatidyl choline afterwards for a period of time in water-bath.
9. application according to claim 8, it is characterised in that the organic solvent be isooctane, normal heptane, n-hexane,
N-octyl alcohol, butanol, methanol, N,N-dimethylformamide or dimethyl sulfoxide (DMSO).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107829323A (en) * | 2017-11-30 | 2018-03-23 | 英泰时尚服饰（苏州）有限公司 | Compound soaping agent of imidazoles modification laccase and preparation method thereof |
| CN110257362A (en) * | 2019-06-04 | 2019-09-20 | 开平牵牛生化制药有限公司 | Preparation method and application of cholic acid and derivative thereof surfactant modified metal organic framework nano composite catalyst |
| CN110484527A (en) * | 2019-08-21 | 2019-11-22 | 清华大学 | A kind of deficiency metal organic framework-multienzyme complex and preparation method thereof and its application |
| CN110643592A (en) * | 2019-10-29 | 2020-01-03 | 南京工业大学 | Method for modifying whole cells by cholate-metal ion composite and application thereof |
| CN113387889A (en) * | 2021-06-17 | 2021-09-14 | 湖南文理学院 | Novel porous imidazole metal compound nano powder and preparation method thereof |
| CN113387889B (en) * | 2021-06-17 | 2023-05-09 | 湖南文理学院 | Preparation method of porous imidazole metal compound nano-powder |
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