CN107164358A - A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst - Google Patents

A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst Download PDF

Info

Publication number
CN107164358A
CN107164358A CN201710504209.5A CN201710504209A CN107164358A CN 107164358 A CN107164358 A CN 107164358A CN 201710504209 A CN201710504209 A CN 201710504209A CN 107164358 A CN107164358 A CN 107164358A
Authority
CN
China
Prior art keywords
enzyme
dopamine
surfactant
composite catalyst
nano
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710504209.5A
Other languages
Chinese (zh)
Inventor
陈可泉
倪艳
曹逊
李辉
欧阳平凯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Tech University
Original Assignee
Nanjing Tech University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Tech University filed Critical Nanjing Tech University
Priority to CN201710504209.5A priority Critical patent/CN107164358A/en
Publication of CN107164358A publication Critical patent/CN107164358A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres

Landscapes

  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention discloses the preparation method and applications of a kind of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst, belong to the application field of immobilised enzymes.The present invention is using the crosslinking of dopamine autohemagglutination, it is attached to the surface of surfactant enzyme nano-composite catalyst, so as to which the embedding of surfactant enzyme nano-composite catalyst be got up, make obtained surfactant enzyme nano-composite catalyst that there is certain mechanical strength, strengthen its structural stability, recycling rate of waterused and tolerance, the use cost of enzyme is substantially reduced, is particluarly suitable for the application in pure organic phase or water phase and an oil phase living things catalysis.

Description

A kind of dopamine and its derivative Quick cross-linking surfactant-enzyme is nano combined is urged The preparation method and applications of agent
Technical field
The invention belongs to fixation techniques for enzyme field, and in particular to a kind of dopamine and its derivative Quick cross-linking surface The preparation method and applications of activating agent-enzyme nano-composite catalyst.
Background technology
Enzyme immobilization can not only significantly improve the stability of enzyme, and can realize the recycling of enzyme, drop significantly The use cost of low enzyme.During enzyme immobilization, often use glutaraldehyde as crosslinking agent, however, there are some researches show:It is fixed Change enzyme activity to be inversely proportional with glutaraldehyde consumption, excessive glutaraldehyde is crosslinked between causing unnecessary enzyme molecule, and excessive friendship Joint conference distorts the structure of enzyme, causes enzyme activity to reduce, in addition, the bio-toxicity of glutaraldehyde, limits its application.Therefore, It is badly in need of finding the efficient green immobilization that a kind of simple, nontoxic, gentle method carries out enzyme.
Dopamine is a kind of biological neural mediator, auto polymerization reaction can occur in alkaline aerobic solution, so as to form one Poly-dopamine layer of the layer strongly adherent in solid material surface.Because dopamine has good dispersion, biocompatibility and temperature And reaction condition, it is used for field of enzyme immobilization as a kind of preferable bonding material.
The Chinese patent of application number 201110460474.0 discloses a kind of using glutaraldehyde cross-linking immobilization modification fat Enzyme Novozyme435 method, the preparation method is with glutaraldehyde as cross linker, in 20-50 by lipase Novozyme 435 DEG C, lipase Novozy after cross-linking reaction 15-120min, the secondary modification obtained after chemical crosslinking is carried out under 40-240rpm 435 stability is improved, and gained yield is more uncrosslinked after the Novozyme 435 after secondary modification is reused 5 times The lipase Novozyme 435 of modification improves 49.3%.
On to address the main crosslinking agent of existing cross-linked immobilized enzyme catalyst be glutaraldehyde solution, preparation-obtained two Immobilised enzymes after secondary modification, although recycling rate of waterused is improved, but immobilised enzymes is in the presence of glutaraldehyde, the knot of enzyme Structure is distorted, and causes enzyme activity to reduce, in addition, the bio-toxicity of glutaraldehyde, limits its application, therefore, is badly in need of seeking Look for a kind of efficient green secondary modification of simple, nontoxic, gentle method being fixed enzyme.
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 dopamine and its derivative Quick cross-linking table The preparation method and applications of face activating agent-enzyme nano-composite catalyst, this method is crosslinking agent with dopamine or derivatives thereof, Dissolved oxygen is provided in the presence of magnetic agitation to be aoxidized from poly- crosslinking, be it is a kind of it is simple, nontoxic, gently, it is efficient The secondary modification immobilised enzymes of green.
To solve prior art problem, the technical scheme that the present invention takes is:
A kind of preparation method of dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst, exists first Room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst, is then with dopamine or derivatives thereof Crosslinking agent, stirring is lower to be carried out from poly- crosslinking, when surfactant-enzyme nano-composite catalyst outer wrapping black poly-dopamine, Centrifugation freeze-drying, produces the surfactant-enzyme nano-composite catalyst of " nucleocapsid " shape after dopamine crosslinking embedding.
It is that described dopamine or derivatives thereof is Dopamine hydrochloride as improved.
A kind of preparation side of above-mentioned dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst Method, comprises the following steps:
Step 1, room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst
1)0.01-1 mmol/mL aqueous surfactant solutions are prepared, at room temperature, are dripped while stirring with 100 ~ 150 rpm speed Plus free enzyme aqueous solution obtains mixed liquor to clarifying;
2)Mixed liquor is added dropwise to 0.01-1 mmol metal ion salt solutions while stirring, at room temperature with 100 ~ 150 rpm speed Spend after stirring reaction 30-60 min and obtain the nano-composite catalyst aqueous solution of surfactant-enzyme;
3)The nano-composite catalyst aqueous solution of surfactant-enzyme is centrifuged, and with deionized water rinsing 1-3 times, washed away not The resolvase of absorption, vacuum freeze drying to constant weight is that can obtain surfactant-enzyme nano-composite catalyst;
Step 2, dopamine is from poly- crosslinking
20-200mg surfactants-enzyme nano-composite catalyst is taken to be dispersed in 10-100 mL moles under ultrasonication Concentration is in 10-100 mmol Tris-HCl buffer solution, addition 10-40 mg dopamine or derivatives thereof, at room temperature magnetic force Stir and centrifuged after 24h, and be washed with deionized 1-3 times, vacuum freeze drying to constant weight is produced after dopamine crosslinking embedding The surfactant of " nucleocapsid " shape-enzyme nano-composite catalyst.
Preferably, the 1 of step 1)Described in surfactant be NaTDC, sodium taurodeoxycholate, sweet ammonia take off Oxycholic acid receive or chenodesoxycholic acid sodium in it is any;Resolvase is antarctic candidia lipase A, antarctic candidia lipase B, fold lipase from candida sp, porcine pancreatic lipase, dredge cotton like thermophilic silk embrace bacterium lipase, papain, pepsin or One or more combinations in trypsase, the free enzyme aqueous solution middle reaches are 0.1 ~ 1 from the mass ratio of enzyme and water:50.
Preferably, the 1 of step 1)In using aqueous surfactant solution as object of reference, the addition of the free enzyme aqueous solution Measure as 0.005 ~ 1 mL/mL.
Preferably, the 2 of step 1)Described in metal ion be Co2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+ Or Mg2+In it is any.
Preferably, the 3 of step 1)5 ~ 15 min of middle centrifugation, rotating speed is 4000 ~ 8000 rpm during centrifugation, and vacuum refrigeration is done Dry vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Preferably, the pH of Tris-HCl buffer solutions is 8 in step 2, and magnetic stirring speed is 100 ~ 150 rpm, stirring Time is 1-36h, and rotating speed is that 4000 ~ 8000 rpm centrifuge 5-15min during centrifugation, and the vacuum of vacuum freeze drying is 1.3 ~ 13 Pa, temperature is -85 DEG C ~ -10 DEG C.
Above-mentioned dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst is in pure organic phase or water Application in oily two-phase living things catalysis.
Compared with prior art, the present invention is attached to surfactant-enzyme nano combined using dopamine from poly- crosslinking The surface of catalyst, so that the embedding of surfactant-enzyme nano-composite catalyst be got up, so as to get surfactant-enzyme Nano-composite catalyst has certain mechanical strength, strengthens its structural stability, recycling rate of waterused and tolerance, substantially reduces The use cost of enzyme, is particluarly suitable for the application in pure organic phase biological catalysis.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of embodiment 1, wherein,(a)For CRL-MSNC,(b)For PDA@CRL-MSNC;
Fig. 2 is the transmission electron microscope picture of embodiment 1, wherein,(a)For CRL-MSNC,(b)For PDA@CRL-MSNC;
The organic solvent-resistant stable that Fig. 3 is CRL, CRL-MSNC and PDA@CRL-MSNC in embodiment 1;
Fig. 4 is the scanning electron microscope (SEM) photograph of embodiment 2, wherein,(a)For Papain-MSNC,(b)For PDA@Papain-MSNC;
Fig. 5 is CRL-MSNC and the recycling rates of waterused of PDA@CRL-MSNC in aqueous in embodiment 1;
Fig. 6 catalyzes and synthesizes the conversion ratio of VE-succinate for CRL, CRL-MSNC and PDA@CRL-MSNC in embodiment 1;
Fig. 7 is recycling rates of waterused of the CRL-MSNC and PDA@CRL-MSNC in DMSO in embodiment 1.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1
A kind of Dopamine hydrochloride Quick cross-linking is modified deoxycholic acid-lipase from candida sp-Co2+The system of nano-composite catalyst Preparation Method, comprises the following steps:
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1)Surfactant-modified resolvase
The mL of 0.1 mmol/mL deoxycholic acids sodium water solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1 mL/s Drop rate be added dropwise the 20 mg/mL lipase from candida sp aqueous solution(CRL)2 mL, stirring obtains mixed liquor to solution clarification;
2)Mixed liquor is added into the 0.2 mmol cobalt chloride hexahydrate aqueous solution with 1 mL/s drop rate while stirring, at room temperature To obtain deoxycholic acid-lipase from candida sp-Co after the 120 rpm min of speed stirring reaction 302+Nano-composite catalyst water Solution;
3)By deoxycholic acid-lipase from candida sp-Co2+8000 rpm of nano-composite catalyst aqueous solution centrifugation, And with deionized water rinsing 2 times, unadsorbed resolvase is washed away, vacuum freeze drying to constant weight is that can obtain deoxycholic acid-vacation Silk Yeast-lipase-Co2+Nano-composite catalyst(CRL-MSNC), wherein, vacuum be 1.3 ~ 13 Pa, temperature be -85 DEG C ~ - 10℃。
Step 2, dopamine is from poly- cross-linking modified
100 mg CRL-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM(pH 8.0) In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature;
By centrifugation of the CRL-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted DOPA Amine, vacuum freeze drying to constant weight produces cross-linking modified rear " nucleocapsid " shape deoxycholic acid-lipase from candida sp-Co of dopamine2+ Nano-composite catalyst(PDA@CRL-MSNC), wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Scanning figures of the CRL-MSNC and PDA@CRL-MSNC manufactured in the present embodiment under Electronic Speculum as shown in Figure 1-2, from figure In as can be seen that CRL-MSNC be a kind of spherical particle, its average grain diameter is in 80 nm or so.And after poly-dopamine modified lithium Similar shape is also presented in the PDA@CRL-MSNC of " nucleocapsid " shape of generation, and its particle size range shows to gather in 100 nm or so Dopamine successfully sticks to its surface, and thickness is about 20 nm.
As shown in figure 5, the present embodiment CRL-MSNC and PDA@CRL-MSNC are in aqueous by 10 recyclings Afterwards, CRL-MSNC relative enzyme activity is less than 80%, and PDA@CRL-MSNC relative enzyme activity remains to maintain more than 93%.Natural CRL Optimal pH with PDA CRL-MSNC is 7.0, but is due to protective effect of the nano-carrier to CRL so that PDA CRL- MSNC is more extensive to pH accommodation, its in aqueous optimal catalytic pH range be 6.0-8.0.
As shown in figure 3, after ethanol, methanol and dmso treatment, CRL-MSNC and PDA@CRL-MSNC phase 68%, more than 88% can be maintained at respectively to vigor, and under identical treatment conditions, free CRL can only keep less vigor (<10%).
Embodiment 2
A kind of Dopamine hydrochloride Quick cross-linking is modified deoxycholic acid-papain-Mn2+The preparation side of nano-composite catalyst Method, comprises the following steps:
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1)Surfactant-modified resolvase
The mL of 0.1 mmol/mL deoxycholic acids sodium water solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1 mL/s Drop rate be added dropwise 20 mg/mL Papain enzyme aqueous solutions(Papain)2 mL, stirring obtains mixed liquor to solution clarification;
2)Mixed liquor is added into the chloride hydrate manganese aqueous solution of 0.2 mmol tetra- with 1 mL/s drop rate while stirring, at room temperature To obtain deoxycholic acid-papain-Mn after the 120 rpm min of speed stirring reaction 302+The nano-composite catalyst aqueous solution;
3)By deoxycholic acid-papain-Mn2+The nano-composite catalyst aqueous solution 8000 rpm centrifugation, is used in combination Deionized water rinsing 2 times, washes away unadsorbed resolvase, and vacuum freeze drying to constant weight is that can obtain deoxycholic acid-pawpaw egg White enzyme-Mn2+Nano-composite catalyst(Papain-MSNC), wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Step 2, dopamine is from poly- cross-linking modified
100 mg Papain-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM(pH 8.0)In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature;
By centrifugation of the Papain-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted Dopamine, vacuum freeze drying to constant weight produces cross-linking modified rear " nucleocapsid " shape deoxycholic acid-papain-Mn of dopamine2+ Nano-composite catalyst(PDA@Papain-MSNC), wherein, vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
Scanning electron microscope (SEM) photograph such as Fig. 2 institutes of the Papain-MSNC and PDA@Papain-MSNC manufactured in the present embodiment under Electronic Speculum Show, it can be seen that Papain-MSNC is the spherical particle that average grain diameter is 50 nm, PDA@Papain-MSNC are portions Scattered besom shape is decomposed, in the aqueous solution after 10 times reuse, Papain-MSNC relative enzyme activity is less than 83%, and PDA@ Papain-MSNC relative enzyme activity remains to maintain more than 94%, and natural Papain and PDA@Papain-MSNC optimal pH is equal For 5.0, PDA@Papain-MSNC, optimal catalytic pH range is 3.5-8.0 in aqueous.
Embodiment 3
A kind of Dopamine hydrochloride Quick cross-linking is modified glycodesoxycholic acid-papain-Mn2+The system of nano-composite catalyst Preparation Method, comprises the following steps:
Step 1, room temperature aqueous phase Rapid coprecipitation prepares surfactant-enzyme nano-composite catalyst
1)Surfactant-modified resolvase
The mL of the 0.1 mmol/mL Glycodeoxrycholic acids aqueous solution 10 is prepared, at room temperature, with 120 rpm speed while stirring with 1 The mL of 20 mg/mL Papains enzyme aqueous solution 2 is added dropwise in mL/s drop rate, and stirring obtains mixed liquor to solution clarification;
2)Mixed liquor is added into the chloride hydrate manganese aqueous solution of 0.2 mmol tetra- with 1 mL/s drop rate while stirring, at room temperature To obtain glycodesoxycholic acid-papain-Mn after the 120 rpm min of speed stirring reaction 302+Nano-composite catalyst water Solution;
3)By glycodesoxycholic acid-papain-Mn2+8000 rpm of nano-composite catalyst aqueous solution centrifugation, And with deionized water rinsing 2 times, unadsorbed resolvase is washed away, vacuum freeze drying to constant weight is that can obtain sweet ammonia deoxidation courage Acid-papain-Mn2+Nano-composite catalyst(Papain- glycodesoxycholic acids-Mn2+).
Step 2, dopamine is from poly- cross-linking modified
100 mg Papain-MSNC, the min of ultrasonication 5 is taken to make the Tris-HCl that it is dispersed in 10 mL 10mM(pH 8.0)In buffer solution, 20 mg Dopamine hydrochloride is added, 24 h are crosslinked with 120 rpm rotating speed magnetic agitation at room temperature;
By centrifugation of the Papain-MSNC aqueous solution with 8000 rpm, and it is washed with deionized 2 times, washes away unreacted Dopamine, vacuum freeze drying to constant weight can obtain cross-linking modified rear " nucleocapsid " shape glycodesoxycholic acid-Papain of dopamine Enzyme-Mn2+Nano-composite catalyst(PDA@Papain- glycodesoxycholic acids-Mn2+).
Papain- glycodesoxycholic acids-the Mn that the present embodiment is prepared2+With PDA@Papain- glycodesoxycholic acids- Mn2+Scanning figure under Electronic Speculum shows, Papain- glycodesoxycholic acids-Mn2+It is the spherical particle that average grain diameter is 80 nm, PDA@Papain- glycodesoxycholic acids-Mn2+It is the besom shape that part is dismissed, in the aqueous solution after 10 times reuse, Papain- glycodesoxycholic acids-Mn2+Relative enzyme activity be less than 76%, and PDA@Papain- glycodesoxycholic acids-Mn2+It is relative Enzyme activity remains to maintain more than 90%, and natural Papain and PDA@Papain- glycodesoxycholic acids-Mn2+Optimal pH be 5.0, PDA@Papain- glycodesoxycholic acids-Mn2+Optimal catalytic pH range is 4.0-8.0 in aqueous.
Embodiment 4
Vitamin E amber is catalyzed and synthesized in pure organic phase with gained CRL-MSNC and PDA the@CRL-MSNC of embodiment 1 and natural CRL The application of amber acid esters, comprises the following steps:
The first step, takes 10 mL plastic tube, sequentially adds 0.2 mmol vitamin E, 1 mmol succinic anhydride, 400 μ L Natural CRL or CRL-MSNC containing equal protein content and PDA@CRL-MSNC, 5 mL organic solvent;
Second step, by the mixture obtained in step 1, closed processes are stirred after 55 °C of heating water baths with 120 rpm speed Reaction;
After 3rd step, 72 h of reaction, the content of substrate and product is determined using U.S.'s Agilent highly effective liquid phase chromatographic system.
VE succinic acid is catalyzed and synthesized to calculate by the measure substrate of highly effective liquid phase chromatographic system and the content of product Ester conversion rate, when reaction temperature be 55 °C, stir speed (S.S.) be 800 rpm when, under identical reaction conditions, CRL-MSNC with PDA@CRL-MSNC, which catalyze and synthesize VE-succinate conversion ratio, can reach 55%(Fig. 6), natural CRL catalyzes and synthesizes More than 4 times, demonstrate the PDA@CRL-MSNC of the invention prepared and possess same with CRL-MSNC superior urge in pure organic phase Change efficiency.
Wherein highly effective liquid phase chromatographic system, chromatographic column is C-18 reversed-phase columns(250 mm×4.6 mm,5 μm), mobile phase is Methanol and acetic acid solution(Volume ratio is 50:0.3), flow velocity is 1.0 mL/min, and post case temperature is controlled at 36 °C, and detector is two Pole pipe array and detector, Detection wavelength are 285 nm.
Embodiment 5
With CRL-MSNC the and PDA@CRL-MSNC of the gained of embodiment 1, determine the CRL-MSNC containing equal protein content with PDA@CRL-MSNC catalyze and synthesize the recycling rate of waterused of VE-succinate in DMSO, comprise the following steps:
The first step, takes 10 mL plastic tube, sequentially adds 0.2 mmol vitamin E, 1 mmol succinic anhydride, contains phase The organic solvent of CRL-MSNC and PDA@CRL-MSNC, 5 mL Deng protein content;
Second step, by the mixture obtained in step 1, closed processes are stirred after 55 °C of heating water baths with 120rpm speed Reaction;
After 3rd step, the h of stirring reaction 4, the mixture reacted in step 2 is centrifuged into 10 min under 10000 rpm, height is used The content of substrate and product in effect liquid phase chromatogram system measurement supernatant;
4th step, will centrifuge obtained precipitation, is washed with deionized 2 times, for next group enzyme reaction, enters repeatedly in step 3 Row 10 times catalysis calculates its recycling rate of waterused.
As a result closed as shown in fig. 7, calculating catalysis by the measure substrate of highly effective liquid phase chromatographic system and the content of product Into VE-succinate conversion ratio, when reaction temperature is 55 °C, and stir speed (S.S.) is 800 rpm, in identical reaction condition Under, after 10 times reuse, PDA@CRL-MSNC enzyme activity is reduced only by 55%, shows the PDA@in this reaction system CRL-MSNC possesses good recycling performance.And by comparison, CRL-MSNC react 30 min after just by DMSO is completely dissolved, and causing CRL-MSNC, only its enzyme activity after primary first-order equation is just greatly reduced.Further demonstrate PDA@CRL-MSNC prepared by the present invention possess more superior catalytic efficiency in pure organic phase.
The present invention can be seen that using dopamine from poly- crosslinking by embodiment 1-3, be attached to surfactant-enzyme and receive The surface of rice composite catalyst, so that the embedding of surfactant-enzyme nano-composite catalyst be got up, so as to get surface-active Agent-enzyme nano-composite catalyst has certain mechanical strength, strengthens its structural stability, recycling rate of waterused and tolerance, greatly The use cost of big reduction enzyme, is particluarly suitable for the application in pure organic phase biological catalysis.Pass through 10 repetitions in aqueous After utilization, the relative enzyme activity that PDA@CRL-MSNC relative enzyme activity can maintain more than 93%, PDA@Papain-MSNC can be maintained More than 94%, PDA@Papain- glycodesoxycholic acids-Mn2+Relative enzyme activity can maintain more than 90%;Pass through embodiment 4-5 As can be seen that " nucleocapsid " shape surfactant-enzyme is nano combined that the present invention is prepared using dopamine after cross-linking modified is urged Agent has more superior catalytic efficiency in water-oil phase living things catalysis, before especially being had a wide range of applications in pure organic phase Scape.

Claims (9)

1. the preparation method of a kind of dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst, it is special Levy and be, surfactant-enzyme nano-composite catalyst is prepared into room temperature aqueous phase Rapid coprecipitation first, then with dopamine Or derivatives thereof be crosslinking agent, stirring is lower to be carried out from poly- crosslinking, when surfactant-enzyme nano-composite catalyst outer wrapping is black During color poly-dopamine, centrifugation freeze-drying, the surfactant-enzyme nanometer for producing " nucleocapsid " shape after dopamine crosslinking embedding is multiple Close catalyst.
2. a kind of dopamine according to claim 1 and its derivative Quick cross-linking surfactant-enzyme is nano combined urges The preparation method of agent, it is characterised in that described dopamine or derivatives thereof is Dopamine hydrochloride.
3. a kind of dopamine according to claim 1 and its derivative Quick cross-linking surfactant-enzyme is nano combined urges The preparation method of agent, it is characterised in that comprise the following steps:
Step 1, room temperature aqueous phase Rapid coprecipitation is prepared into surfactant-enzyme nano-composite catalyst
0.01-1 mmol/mL aqueous surfactant solutions are prepared, at room temperature, are added dropwise while stirring with 100 ~ 150 rpm speed Free enzyme aqueous solution obtains mixed liquor to clarifying;
Mixed liquor is added dropwise to 0.01-1 mmol metal ion salt solutions while stirring, at room temperature with 100 ~ 150 rpm speed The nano-composite catalyst aqueous solution of surfactant-enzyme is obtained after stirring reaction 30-60 min;
The nano-composite catalyst aqueous solution of surfactant-enzyme is centrifuged, and with deionized water rinsing 1-3 times, washes away and does not inhale Attached resolvase, vacuum freeze drying to constant weight is that can obtain surfactant-enzyme nano-composite catalyst;
Step 2, dopamine is from poly- crosslinking
20-200mg surfactants-enzyme nano-composite catalyst is taken to be dispersed in 10-100 mL moles under ultrasonication Concentration is in 10-100 mmol Tris-HCl buffer solution, addition 10-40 mg dopamine or derivatives thereof, at room temperature magnetic force Stir and centrifuged after 24h, and be washed with deionized 1-3 times, vacuum freeze drying to constant weight is produced after dopamine crosslinking embedding The surfactant of " nucleocapsid " shape-enzyme nano-composite catalyst.
4. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3 Method, it is characterised in that the 1 of step 1)Described in surfactant be NaTDC, sodium taurodeoxycholate, sweet ammonia deoxidation Cholic acid receive or chenodesoxycholic acid sodium in it is any;Resolvase be antarctic candidia lipase A, candida antarctica lipase B, Fold lipase from candida sp, porcine pancreatic lipase, the thermophilic silk of thin cotton like embrace bacterium lipase, papain, pepsin or pancreas One or more combinations in protease, the free enzyme aqueous solution middle reaches are 0.1 ~ 1 from the mass ratio of enzyme and water:50.
5. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3 Method, it is characterised in that the 1 of step 1)In using aqueous surfactant solution as object of reference, the addition of the free enzyme aqueous solution Measure as 0.005 ~ 1 mL/mL.
6. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3 Method, it is characterised in that the 2 of step 1)Described in metal ion be Co2+、Ca2+、Zn2+、Mn2+、Ba2+、Cu2+、Ni2+、Sn2+Or Mg2+In it is any.
7. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3 Method, it is characterised in that the 3 of step 1)5 ~ 15 min of middle centrifugation, rotating speed is 4000 ~ 8000 rpm during centrifugation, and vacuum refrigeration is done Dry vacuum is 1.3 ~ 13 Pa, and temperature is -85 DEG C ~ -10 DEG C.
8. a kind of preparation of dopamine Quick cross-linking surfactant-enzyme nano-composite catalyst according to claim 3 Method, it is characterised in that the pH of Tris-HCl buffer solutions is 8 in step 2, and magnetic stirring speed is 100 ~ 150 rpm, during stirring Between be 1-36h, rotating speed is that 4000 ~ 8000 rpm centrifuge 5-15min during centrifugation, and the vacuum of vacuum freeze drying is 1.3 ~ 13 Pa, temperature is -85 DEG C ~ -10 DEG C.
9. based on dopamine and its derivative Quick cross-linking surfactant-enzyme nano-composite catalyst obtained by claim 1 Application in pure organic phase or water phase and an oil phase living things catalysis.
CN201710504209.5A 2017-06-28 2017-06-28 A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst Pending CN107164358A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710504209.5A CN107164358A (en) 2017-06-28 2017-06-28 A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710504209.5A CN107164358A (en) 2017-06-28 2017-06-28 A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst

Publications (1)

Publication Number Publication Date
CN107164358A true CN107164358A (en) 2017-09-15

Family

ID=59827554

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710504209.5A Pending CN107164358A (en) 2017-06-28 2017-06-28 A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst

Country Status (1)

Country Link
CN (1) CN107164358A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107973919A (en) * 2017-11-02 2018-05-01 天津大学 A kind of preparation method of pickering emulsion amine stabilized based on DOPA and its immobilised enzymes application
CN109536479A (en) * 2018-12-05 2019-03-29 清华大学 A kind of crosslinking immobilized bi-enzyme-surfactant complex and preparation method thereof
CN109576257A (en) * 2018-12-04 2019-04-05 清华大学 A kind of enzyme catalyst and preparation method thereof of part photo-thermal effect
CN110862981A (en) * 2019-11-26 2020-03-06 南京工业大学 Method for immobilizing lipase by using nano material
CN115232784A (en) * 2022-07-25 2022-10-25 复旦大学附属中山医院 Medicine for inducing myocardial cell volume overload and preparation method of volume overload model of myocardial cell in vitro

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105349519A (en) * 2015-11-23 2016-02-24 浙江理工大学 Lipase immobilization carrier, enzyme immobilization method and method for improving resolution performance
CN105734039A (en) * 2016-04-09 2016-07-06 中国海洋大学 Preparation method of polydopamine/graphene oxide composite film with muramidase immobilized
CN105754985A (en) * 2016-04-15 2016-07-13 清华大学 Enzyme-metal ion nano compound and preparation method thereof
CN106750462A (en) * 2016-12-19 2017-05-31 大连理工大学 A kind of surface modifying method of dopamine and its derivative polymerization and crosslinking curing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105349519A (en) * 2015-11-23 2016-02-24 浙江理工大学 Lipase immobilization carrier, enzyme immobilization method and method for improving resolution performance
CN105734039A (en) * 2016-04-09 2016-07-06 中国海洋大学 Preparation method of polydopamine/graphene oxide composite film with muramidase immobilized
CN105754985A (en) * 2016-04-15 2016-07-13 清华大学 Enzyme-metal ion nano compound and preparation method thereof
CN106750462A (en) * 2016-12-19 2017-05-31 大连理工大学 A kind of surface modifying method of dopamine and its derivative polymerization and crosslinking curing

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAO X等: "Encapsulation of enzymes in metal ion-surfactant nanocomposites for catalysis in highly polar solvents", 《CHEM. COMMUN.》 *
SHILIN CAO等: "Recent advances in immobilized enzymes on nanocarriers", 《CHINESE JOURNAL OF CATALYSIS》 *
XUN CAO等: "Increasing the hydrolytic activity of lipase in oil/water two-phase system using surfactant–enzyme nanocomposite", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 *
YANNING QU等: "Interfacial Polymerization of Dopamine in a Pickering Emulsion:Synthesis of Cross-Linkable Colloidosomes and Enzyme Immobilization at Oil/Water Interfaces", 《ACS APPLIED MATERIALS & INTERFACES》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107973919A (en) * 2017-11-02 2018-05-01 天津大学 A kind of preparation method of pickering emulsion amine stabilized based on DOPA and its immobilised enzymes application
CN109576257A (en) * 2018-12-04 2019-04-05 清华大学 A kind of enzyme catalyst and preparation method thereof of part photo-thermal effect
CN109536479A (en) * 2018-12-05 2019-03-29 清华大学 A kind of crosslinking immobilized bi-enzyme-surfactant complex and preparation method thereof
CN110862981A (en) * 2019-11-26 2020-03-06 南京工业大学 Method for immobilizing lipase by using nano material
CN115232784A (en) * 2022-07-25 2022-10-25 复旦大学附属中山医院 Medicine for inducing myocardial cell volume overload and preparation method of volume overload model of myocardial cell in vitro
CN115232784B (en) * 2022-07-25 2024-06-07 复旦大学附属中山医院 Medicament for inducing myocardial cell capacity overload and preparation method of capacity overload model of myocardial cells in vitro

Similar Documents

Publication Publication Date Title
CN107164358A (en) A kind of preparation method and applications of dopamine and its derivative Quick cross-linking surfactant enzyme nano-composite catalyst
Moreira et al. Lipase from Rhizomucor miehei immobilized on magnetic nanoparticles: performance in fatty acid ethyl ester (FAEE) optimized production by the Taguchi method
Feng et al. Enhancement of the catalytic activity and stability of immobilized aminoacylase using modified magnetic Fe3O4 nanoparticles
Ramakrishna et al. Controlling enzyme function through immobilisation on graphene, graphene derivatives and other two dimensional nanomaterials
Seenuvasan et al. Magnetic nanoparticles: a versatile carrier for enzymes in bio‐processing sectors
Zhou et al. Improved enzymatic activity by oriented immobilization on graphene oxide with tunable surface heterogeneity
CN104894094A (en) Enzyme immobilization method and enzyme preparation
WO2018223664A1 (en) Method for preparing lipase having high esterification activity using surfactant
Zhuang et al. Immobilization of lipase onto dopamine functionalized magnetic nanoparticles
CN105624128B (en) Immobilized monoamine oxidase and application thereof in synthesis of chiral azabicyclo compound
CN103756992B (en) A kind of chocolate microbacterium magnetic cell and its preparation method and application
CN106582810A (en) Preparation method of graphene immobilized enzyme catalyst
Yang et al. Enhanced reusability and activity: DNA directed immobilization of enzyme on polydopamine modified magnetic nanoparticles
CN103695409A (en) Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion
Li et al. Degradation of phthalic acid esters (PAEs) by an enzyme mimic and its application in the degradation of intracellular DEHP
CN106939305A (en) A kind of preparation method of surfactant enzyme nano-composite catalyst and application
CN105063010A (en) Multi-enzyme system with immobilized polyethylenimine and metal coordination and method for preparing multi-enzyme system
KR101287362B1 (en) Branched polymer microspheres with silica shell
Park et al. Direct removal of harmful cyanobacterial species by adsorption process and their potential use as a lipid source
Ein Ali Afjeh et al. Characteristics of glucose oxidase immobilized on Magnetic Chitosan Nanoparticles
CN110028063A (en) A kind of affine fixation support of graphene oxide and the preparation method and application thereof
CN101353654B (en) Lipase gel particle and preparation thereof
CN107034206A (en) A kind of enzyme-agglutinin conjugate nano particle and preparation method thereof
CN105037596B (en) The preparation method of the superparamagnetic carrier of crosslinked fixed fat enzyme
Lu et al. Synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate by immobilized cells using amino acid-modified magnetic nanoparticles

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20170915