CN109609487A - A kind of biological enzyme of graft polymers and preparation method thereof and fixing means - Google Patents
A kind of biological enzyme of graft polymers and preparation method thereof and fixing means Download PDFInfo
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- CN109609487A CN109609487A CN201811406148.XA CN201811406148A CN109609487A CN 109609487 A CN109609487 A CN 109609487A CN 201811406148 A CN201811406148 A CN 201811406148A CN 109609487 A CN109609487 A CN 109609487A
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Abstract
The invention discloses a kind of biological enzyme of graft polymers, connect biological enzyme and polymer with double aryl hydrazone bonds that succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generates by bridging agent 4- formoxyl succinimido benzoic ether S-4FB.The invention also discloses the preparation method of the biological enzyme of above-mentioned graft polymers and fixing means.The present invention selects synthesising macromolecule copolymer as immobilization carrier, compares other carriers, and enzyme is more compact in conjunction with carrier, and mechanical strength also has been significantly improved；The performance of enzyme is more stable after fixed simultaneously, can repeatedly use, be catalyzed chain reaction.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of biological enzyme of graft polymers, the present invention relates to above-mentioned
The preparation method and fixing means of biological enzyme.
Background technique
The technique for fixing of enzyme is that biological enzyme is fixed on surfaces of carrier materials or inside by physically or chemically effect.Enzyme quilt
After fixation, structural stability is improved, is separated convenient for recycling, being easy to product, can operate continuously in fixed bed, catalysis substrate connects
Lock reactor greatly reduces production cost.Therefore, enzyme immobilizatio study and apply industrial production, chemical analysis, medicine and
The various fields such as environmental project are all concerned, and are had become in the world across one of multi-disciplinary forward position focus research field.
Taking it by and large enzyme immobilizatio method has physics and two kinds of chemical method.Physical method immobilised enzymes, such as Chinese section
Publication " a kind of controlled pore carrier-free immobilized lipase and preparation method thereof the " (application of Guangzhou Energy Research Institute, institute
Number CN201510358156, publication date 2015.09.16, publication number CN104911162A), Chinese Academy of Agricultural Sciences oil crops
Publication " a kind of using pollen as immobilised enzymes of carrier and preparation method thereof " (application number of research institute
CN201610556371, publication date 2016.11.16, publication number CN106119239A), Shanghai base oneself upon Biotechnology Co., Ltd
Publication " a kind of fixed enzyme vector and its preparation method and application " (application number CN201410831584, publication date
2015.04.22, publication number CN104531668A) etc., although easy to operate, enzyme activity loss is small, and recycling rate of waterused is low, leads
Corresponding cost is caused to improve；Chemical method immobilised enzymes, such as Academy of Military Medicine, PLA's radiation and radiation medicine
Publication " a kind of preparation and its application of immobilization proteinase reagent " CN 103882002A (application number of research institute
CN201410062221, publication date 2014.06.25, publication number CN103882002A), Chinese Academy of Agricultural Sciences oil crops grind
Study carefully publication " a kind of immobilised enzymes, fixed enzyme vector and preparation method thereof " (application number CN201610964102, it is public
Open a day 2017.03.29, publication number CN106542568A), patent " a kind of preparation of immobilised enzymes disclosed in Anhui Polytechnic University
Method " 104404021 A (application number CN201410619223, publication date 2015.03.11, publication number CN104404021A) of CN
It is bigger than physical method Deng the loss of, enzyme activity, but after immobilization, immobilization after a number of uses still keeps enzyme activity, meets large-scale production
It needs, wherein the use of polymer being fixed biological enzyme, catalysis chain reaction, realizing the simple and clean approach such as continuous operation.
Fixation techniques for enzyme very abundant and diversification can generally be selected accordingly by actual needs, from most
Initial absorption method, crosslinking and covalent coupling method till now.However, being specifically included there are still many deficiencies: carrier material
That expects is expensive, is less likely to be applied to industrial bio conversion；Immobilization operating process is complicated；Used carrier is unsuitable for aqueous
Environment；Mechanical strength and filterability drop；A possibility that being catalyzed chain reaction, utilization rate are low.
Summary of the invention
The object of the present invention is to provide a kind of biological enzyme of graft polymers, fixed when solving conventional method immobilized enzyme to carry
The dispersion of body material, brittleness is big, recycling number is limited, cannot be catalyzed the problem of chain reaction.
It is a further object of the present invention to provide the preparation methods of the biological enzyme of above-mentioned graft polymers.
Third object of the present invention is to provide the fixing means of the biological enzyme of above-mentioned graft polymers.
The technical scheme adopted by the invention is that a kind of biological enzyme of graft polymers, passes through bridging agent 4- formoxyl amber
Amber imide benzoic ether S-4FB and succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generate double
Aryl hydrazone bond connects biological enzyme and polymer.
Feature of the present invention also resides in,
Polymer surfaces have the functional group that covalent cross-linking is easily carried out with enzyme.
Another technical solution of the present invention is that a kind of preparation method of the biological enzyme of graft polymers is specifically pressed
Implement according to following steps:
Step 1, the synthesis of polymer precursor and bridging agent:
It weighs polymerization reaction monomer, initiator and catalyst, is added solvent, under nitrogen protection, be heated to 50-60 DEG C of progress
Polymerization reaction obtains polymer solids；Polymer solids are added in the mixed solution of water and ethylenediamine, heating is stirred at 70-85 DEG C
12-18h is mixed, the cooling centrifugation of product is cleaned repeatedly with deionized water, is dried in vacuo, contained-NH2Polymer precursor；
In anhydrous n,N-Dimethylformamide DMF, 4- carboxyl benzaldehyde and n-hydroxysuccinimide NHS and 1- is added
(3- dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride EDCl is reacted, and product is through extraction, washing, drying, dense
It contracts, be recrystallized to give bridging agent 4- formoxyl succinimido benzoic ether S-4FB；
6- hydrazinonicotinic acid is dissolved in anhydrous n,N-Dimethylformamide DMF, triethylamine, acetone, room temperature are sequentially added
After being stirred to react, be added n-hydroxysuccinimide, EDCl stirring, the reaction was continued, product through extraction, washing, drying, concentration,
Succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic is obtained after purification；
Step 2, modification biological enzyme and polymer-modified synthesis:
The S-4FB modified biological enzyme obtained using step 1, obtains modified biological enzyme；
The S-HyNic obtained using step 1 with contain-NH2Polybenzazole precursor precursor reactant, obtain modified polymer；
Step 3, the synthesis of graft polymers biological enzyme:
By the modified biological enzyme of step 2 and modified polymer reaction to get the biological enzyme for arriving graft polymers.
The features of the present invention also characterized in that
The molar ratio of polymerization reaction monomer, initiator and catalyst is 1:1:10-1000, polymerization reaction monomer in step 1
Mass volume ratio with solvent is 1g/1-3ml, the volume ratio 1:1-1.5 of water and ethylenediamine.
Initiator is azodiisobutyronitrile in step 1, and catalyst is sodium hydrogensulfite, and solvent is tetrahydrofuran.
The molar ratio of 4- carboxyl benzaldehyde, NHS and EDCl is 1:0.8-1.5:0.8-1.5,4- carboxyl benzaldehyde in step 1
Molar ratio with anhydrous DMF is 1:50-60.
6- hydrazinonicotinic acid in step 1, acetone, n-hydroxysuccinimide and EDCL molar ratio be 1:1-1.4:1.1-
1.5:1.1-1.3.
The specific synthesis process of modification biological enzyme in step 2 are as follows: biological enzyme is dissolved in phosphate buffer solution, by step
1 obtained S-4FB is dissolved in DMF, then the phosphate buffer solution of biological enzyme is added in the DMF solution of S-4FB under room temperature
React 2-3h, the ratio that biological enzyme molecule and -4FB group are controlled in reaction process is greater than 1:9, ultrafiltration to get；
Polymer-modified specific synthesis process in step 2 are as follows:-NH will be contained obtained in step 12Polybenzazole precursor
Body is dissolved in MOPS buffer, and the S-HyNic normal-temperature reaction 2-3h being dissolved in DMF, control-NH in reaction process2Base
Group and the ratio of-HyNic group less than 1:2, ultrafiltration to get.
Control-HyNic group and -4FB group ratio are 20-23:1 in step 3 reaction process.
Third technical solution of the present invention is a kind of fixing means of the biological enzyme of graft polymers, will be clear
Clean carrier surface is soaked with MOPS buffer, then removes MOPS buffer with suction pipe, carrier is placed in centrifuge tube, add
Enter the phosphate buffer of the biological enzyme of graft polymers, impregnates, to realize the fixation of biological enzyme；Remove extra biological enzyme grafting
Polymer solution washs carrier surface with MOPS buffer, and being eventually adding MOPS buffer soaks conventional carrier surface completely
Not yet, stored refrigerated in refrigerator.
The invention has the advantages that the present invention uses polymer precursor and biological enzyme presoma, then with efficient
Aldehyde hydrazine reaction one kettle way realizes the structure composition regulation of biological enzyme graft polymers, greatly expands biological enzyme graft polymers
The designability of structure composition and its route of synthesis.The present invention selects synthesising macromolecule copolymer as immobilization carrier, compares it
His carrier, enzyme is more compact in conjunction with carrier, and mechanical strength also has been significantly improved；The performance of enzyme is more steady after fixed simultaneously
It is fixed, it can repeatedly use, be catalyzed chain reaction.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram of the biological enzyme of graft polymers of the present invention；
Fig. 2 is in the fixed HRP enzymatic activity measurement figure of conventional surface；
Fig. 3 is that HRP enzymatic activity measurement figure is fixed in glass tube；
Fig. 4 is modified HRP enzyme stability measurement figure.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of biological enzyme of graft polymers of the present invention, passes through bridging agent 4- formoxyl succinimido benzoic ether
(S-4FB) the double aryl hydrazone bonds generated with succinimido -6- diazanyl-niacinamide acetone hydrazone (S-HyNic) reaction connect life
Object enzyme and polymer.Its structure generates BAH key as shown in Figure 1, reacting by the bridging agent group on polymer and enzyme, will
Polymer chain is connected with enzyme molecule, and BAH key is double aryl hydrazone bonds that aldehyde hydrazine reaction generates.
The surface of used polymer has functional group abundant (such as hydroxyl, sulfydryl, carboxyl, amino), easily and enzyme
Covalent cross-linking is carried out, the fixation of enzyme is carried out, such as polyketone, polyvinyl alcohol, amino-polyethyleneglycols sulfydryl, sulfhydryl modified is gathered in oneself
Ester, carboxyl butyronitrile, carboxyl terminated vinyl, polyaniline, polyacrylamide.The biological enzyme being applicable in includes HRP enzyme, protease, surpasses
Superoxide dismutase, lipase, glycosidase, ketoreductase, restriction endonuclease, pancreatin etc..
A kind of preparation method of the biological enzyme of graft polymers, is specifically implemented according to the following steps:
Step 1, the synthesis of polymer precursor and bridging agent:
The synthesis of 1.1 polymer precursors:
According to molar ratio 1:1:10-1000, weigh respectively polymerization reaction monomer, initiator azodiisobutyronitrile (AIBN) and
Catalyst sodium hydrogensulfite (NaHSO3)；Weighed all reagents are added in reaction vessel, then solvent is added into container
Tetrahydrofuran (THF) (mass volume ratio of polymerization reaction monomer and tetrahydrofuran is 1g/1-3ml)；Container is sealed and is passed through
Nitrogen removes the oxygen in container；Reaction vessel is placed in magnetic stirring apparatus and is heated, is made in reaction vessel at 50-60 DEG C
Reaction monomers sufficiently reacted with initiator, catalyst, generate polymer；Polymer is purified with n-hexane, filtering, and 50-60 DEG C
It is dry, obtain pure polymer solids.By polymer solids be added to water and ethylenediamine (water and ethylenediamine volume ratio 1:1-1.5,
In mixed solution of the mass volume ratio of polymer solids and water and ethylenediamine mixed liquor for 0.02-0.03g/ml), 70-85 DEG C
Lower heating stirring 12-18h, the cooling centrifugation of product are cleaned repeatedly with deionized water, are dried in vacuo, are contained-NH2Polymer
Presoma.
The synthesis of 1.2 bridging agent 4- formoxyl succinimido benzoic ether S-4FB:
In anhydrous n,N-Dimethylformamide (DMF), 4- carboxyl benzaldehyde and n-hydroxysuccinimide (NHS) and 1-
Chloroform water extraction is added in (3- dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride (EDCl) reaction, saturated salt is washed
Wash (water, chloroform, salt water consumption are isometric with DMF), anhydrous magnesium sulfate dry 2-4h, concentrated by rotary evaporation, recrystallization (hexane: second
Acetoacetic ester=1:1), obtain S-4FB.Wherein the molar ratio of 4- carboxyl benzaldehyde, NHS and EDCl are 1:0.8-1.5:0.8-1.5,
The molar ratio of 4- carboxyl benzaldehyde and anhydrous DMF is 1:50-60.
1.3 bridging agent succinimido -6- diazanyls-niacinamide acetone hydrazone S-HyNic synthesis:
6- chlorine apellagrin is dissolved in hydrazine hydrate, is condensed back 6-8h, concentrated by rotary evaporation；The said goods are dissolved in water again again
In, to be acidified to pH be that faintly acid (4-5) is settled out hydrochloride for the dilute hydrochloric acid that is 20%-35% through mass fraction；Ethanol washing, again
It crystallizes (ethyl alcohol: water=2:1), obtains 6- hydrazinonicotinic acid, the molar ratio 1:15-20 of 6- chlorine apellagrin and hydrazine hydrate；In anhydrous DMF and
In triethylamine (volume ratio 8:1-5), 6- hydrazinonicotinic acid is successively reacted with acetone, n-hydroxysuccinimide and EDCl, and chlorine is added
It is imitative wash (water, chloroform, salt water consumption are isometric with DMF) with water extraction, saturated sodium-chloride, anhydrous magnesium sulfate dries 2-4h,
Concentrated by rotary evaporation, column chromatography DCM:MeOH (50:1) purifying, obtain S-HyNic.6- hydrazinonicotinic acid, acetone, N- hydroxysuccinimidyl acyl are sub-
The molar ratio of amine and EDCL are 1:1-1.4:1.1-1.5:1.1-1.3.
Step 2, the synthesis of modification biological enzyme:
Biological enzyme is dissolved in phosphate buffer solution (PB1,0.1M dibastic sodium phosphate, 0.15M NaCl, PH=7.2), it will
The S-4FB that step 1.2 obtains is dissolved in DMF, and room temperature in the DMF solution of S-4FB is added in the phosphate buffer solution of biological enzyme
Lower reaction 2-3h, the ratio that biological enzyme molecule and -4FB group are controlled in reaction process are greater than 1:9, obtain 4FB through multiple ultrafiltration
The biological enzyme of modification.
Step 3, polymer-modified synthesis:
- NH will be contained obtained in step 1.12Polymer precursor be dissolved in MOPS buffer, it is micro- with being dissolved in
Measure the S-HyNic normal-temperature reaction 2-3h in DMF, control-NH in reaction process2The ratio of group and-HyNic group is less than 1:2,
The modified polymer of HyNic is obtained through ultrafiltration.
Step 4, the synthesis of graft polymers biological enzyme:
By the polymer for the HyNic modification that the biological enzyme of 4FB that step 2 obtains modification and step 3 obtain in reacting 1-2h
Or so, control-HyNic group and -4FB group ratio are 20-23:1 in reaction process, and repeated ultrafiltration purification is grafted
The biological enzyme of polymer.
A kind of fixing means of the biological enzyme of graft polymers, specifically: by clean conventional carrier surface (glass, paper
Open, cotton, timber, glass tube) it is soaked with MOPS buffer (MopsB1,0.1M MOPS, 0.15M NaCl, PH=7.6), so
Remove MOPS buffer with suction pipe afterwards, carrier is placed in centrifuge tube, the phosphoric acid buffer of the biological enzyme of graft polymers is added
Liquid (PB1,0.1M dibastic sodium phosphate, 0.15M NaCl, PH=7.2) and impregnate 1 hour after, to realize the fixation of biological enzyme.It removes
Extra biological enzyme graft polymer solution (can be reserved for recycle), secures the conventional carrier surface MOPS of biological enzyme
Buffer washs 3 times, and MOPS buffer, which is added, is totally submerged conventional carrier surface, stored refrigerated in refrigerator.
The present invention uses polymer precursor and biological enzyme presoma, is then realized and is given birth to efficient aldehyde hydrazine reaction one kettle way
The structure composition of object enzyme graft polymers regulates and controls, greatly expand the structure composition of biological enzyme graft polymers designability and
Its route of synthesis.
The present invention selects synthesising macromolecule copolymer as immobilization carrier, compares other carriers, and enzyme is tighter in conjunction with carrier
It gathers, mechanical strength also has been significantly improved；The performance of enzyme is more stable after fixed simultaneously, can repeatedly use, be catalyzed
Chain reaction.
The biological enzyme of graft polymers of the present invention not only generates double aryl hydrazone bonds, connection by two kinds of bridging agent groups
Biological enzyme and polymer；The functional group (such as amino, metal ion or anionic group) on carrier is by target enzyme simultaneously
Affinity interaction (electrostatic interaction and chelation), the physical absorption of enzyme can be remarkably promoted；Epoxy group, ammonia on polymer
The functional group of base and substrate surface is chemically crosslinked, and enzyme is promoted to be chemically bonded on carrier；Furthermore the list that the present invention selects
Body contains epoxy group, the carrier containing epoxy group, the epoxy group and protein macromolecule chain on surface after synthetic polymer
On a variety of groups (amino, sulfydryl, phenolic hydroxyl group etc.) be easy to react, keep the fixation of enzyme more stable.
Below by taking glycidyl methacrylate (GMA) monomer as an example, the present invention is specifically described.
Embodiment 1
Step 1: weigh respectively azodiisobutyronitrile, sodium hydrogensulfite, glycidyl methacrylate (GMA) 0.05g,
0.03g, 4g (molar ratio 1:1:100)；Weighed all reagents are added in reaction vessel, then solvent four is added into container
Hydrogen furans 5mL.Container is sealed to and is passed through nitrogen, removes oxygen 30min；60 DEG C of heating stirring 30min, the reaction in reaction vessel
Monomer is sufficiently reacted with initiator, catalyst, and product is taken out；Polymer is added drop-wise to dropwise in 30mL n-hexane, it is to be generated
N-hexane is filtered in flocculent deposit, and solvent volatilization obtains pure polymer.After 60 DEG C of freeze-day with constant temperature 10h, PGMA solid is obtained.
The above-mentioned PGMA solid of 2g is added in the mixed solution of 50mL water and 75mL ethylenediamine, 12h, the cooling centrifugation of product are stirred at 80 DEG C
It is cleaned repeatedly with deionized water, is dried in vacuo spare, product PGMA-NH2It indicates.
In 30mL anhydrous DMF, 4- carboxyl benzaldehyde 1.00g is added, NHS 0.843g, EDCl is added under room temperature
1.507g being stirred overnight.30mL chloroform CHCl is added in 50mL round-bottomed flask3It is extracted twice with water, compound organic phase saturation chlorine
Change sodium solution washing.Magnesium sulfate dry in right amount is added and is filtered to remove magnesium sulfate overnight.Filtered organic phase is rotated, is obtained
Crude product.Crude product n-hexane after drying: ethyl acetate=1:1 (5mL:5mL) recrystallization.It is needle-shaped to be cooled to room temperature appearance
Crystal after 2-3 hours, is put into refrigerator (4 DEG C), takes out after crystal is smooth, 48 hours dry at 50 DEG C, obtains S-4FB.
1.901g 6- chlorine apellagrin is dissolved into 10mL hydrazine hydrate, 6h is condensed back, vacuum concentration drying is re-dissolved in
It is acidified in 35%HCL solution, adjustment PH is 5, and 6- hydrazinonicotinic acid hydrochloride can be obtained in precipitation to be precipitated, uses ethyl alcohol after filtering
Washing, recrystallization (ethyl alcohol: water=2:1), obtain 1.245g6- hydrazinonicotinic acid.Above-mentioned made 6- hydrazinonicotinic acid 1.083g is taken to be dissolved in
It in 20mL anhydrous DMF, is added 2.5mL triethylamine (excess), addition 0.5mL acetone in Xiang Shangshu solution, room temperature magnetic agitation 4h,
Then addition 1.023g n-hydroxysuccinimide 0.778g, EDCl 1.023g are stirred overnight, and the extraction of 30mL chloroform is added, pure
After change, crude product is purified (methylene chloride: methanol=50:1) with column chromatography again, obtains 0.937g S-HyNic.
Step 2: measuring 2.5mL from 82.3 μM of HRP (1eq) stostes and be dissolved in phosphate buffer solution (PB1,0.2M
NaCl, PH=7.2), it mixes with the DMF solution of the S-4FB (7.5eq) of 72mL 20mM, is obtained after reacting at room temperature 3h through multiple ultrafiltration
The HRP, i.e. HRP-4FB modified to 4FB.
Step 3: by 8.73 μm of ol PGMA-NH in step 12(NH2, 1eq) be dissolved in MOPS buffer (MopsB1,
0.1M MOPS, 0.15M NaCl, PH=7.6) in, it is anti-with the S-HyNic (20eq) for 3.49 μm of ol being dissolved in micro DMF
2h is answered, MES buffer solution (MESB1,0.1M MES, 0.15NaCl, PH=4.7) is used through ultrafiltration, obtains polymer P GMA-
HyNic。
Step 4: by 958nmol (22eq) PGMA-HyNic in (1eq) HRP-4FB of 44nmol in step 2 and step 3 in room
Temperature reaction 5h, repeated ultrafiltration purification obtain biological enzyme graft polymers.
With ABTS/H2O2The stability of enzymatic activity and enzyme, enzymatic activity measurement process are measured for substrate are as follows: add into quartz cell
Enter 930 μ L ABTS-Phosphate buffer, 10 μ L H202Solution and 60 μ L about 1nM HRP phosphate buffered solutions；Quickly but sufficiently
After concussion, A is recorded at 25 DEG C414nm2 minutes (data spacing: 10s)；Each sample measurement is three times；Pass through measurement data
(A414nm) after linear recurrence divided by the time, obtain ABTS-Initial rate (the Δ A that free radical generates414nm/s).Enzyme stability is surveyed
Amount process are as follows: in the activity of different time intervals measurement HRP, finally obtain ABTS-Initial rate (the Δ that free radical generates
A414nm/ s) with the relationship of storage time.
Use the K of biological enzyme graft polymers substrate for enzymatic activity prepared by the feed ratioMFor 1.15 ± 0.1 (mM)；Reaction
Constant Kcat is 17.5 ± 0.1 (s-1)；I.e. catalytic efficiency is 15.2 ± 0.1.
The fixation of biological enzyme: clean conventional surface (glass, paper, cotton, timber) MOPS buffer is soaked, after
Remove MOPS buffer with suction pipe, carrier be placed in centrifuge tube, and the buffer that biological enzyme graft polymers is added is added,
After impregnating 1 hour, extra biological enzyme graft polymers is removed from centrifuge tube, and washed coverslip 3 times with MOPS buffer,
MOPS buffer, which is added, to be totally submerged glass cover-slip to be saved in 4 DEG C of refrigerators.
Surface enzyme activity measurement: it the bioenzyme activity measurement of desorption: is sucked out with suction pipe slow in a certain amount of centrifuge tube
Fliud flushing (has absorption biological enzyme graft polymers glass cover-slip) in the centrifuge tube, be added in cuvette, substrate A BTS/ is added
H2O2, UV/vis spectrum 2 minutes for recording the measurement mixture with 15 seconds intervals prepare each sample and measure three times, pass through
Product ABTS is obtained divided by the time after the measurement linear recurrence of the data obtained-The initial rate of generation.Adsorb the activity of biological enzyme
Measurement: removing the buffer in centrifuge tube, and substrate A BTS/H is added2O2With buffer, it is gently inverted centrifuge tube per minute, 15 points
Zhong Hou takes out substrate solution from reaction tube, with UV/vis spectrum record product to ABTS-Absorbance, washed with buffer
Centrifuge tube, and at 4 DEG C until further using.As shown in the curve that 1:1:100 is represented in Fig. 2, in the enzyme that conventional surface is fixed,
Its ABTS-Absorbance at 414nm is floated 2.0 or so.
Enzymatic activity measurement in managing: catalysis substrate is flowed with certain speed, and the glass-micropipe both ends for flowing through absorption biological enzyme connect
Meet rubber tube, substrate A BTS/H2O2The glass-micropipe of attaching organism enzyme graft polymers is reacted by catalysis, the product ABTS of reaction-?
It is recorded in cuvette through real-time UV/vis spectrum.It is fixed in glass tube as shown in the curve that 1:1:100 is represented in Fig. 3
Enzyme, ABTS-Absorbance at 414nm is floated 10 or so.
The activity stabilized Journal of Sex Research of biological enzyme graft polymers on the adsorbent material: load there is into biological enzyme graft polymers
Adsorbent material store the different time, study its bioactivity with storage using identical method is measured with bioenzyme activity
Deposit the stability of time.As shown in the curve that 1:1:100 is represented in Fig. 4, which stores at 4 DEG C
After 30 days, which is still able to maintain its activity, and relative activity is up to 1.0.
Embodiment 2
Referring to embodiment 1, weigh polymerization reaction azodiisobutyronitrile, sodium hydrogensulfite, monomer methacrylic acid shrink it is sweet
Grease (GMA) 0.008g, 0.005g, 2g (1:1:300), remaining condition are constant.It is grafted using biological enzyme prepared by the feed ratio
The K of polymer substrate for enzymatic activityMFor 0.93 ± 0.1 (mM)；Reaction constant KcatFor 19.0 ± 0.1 (s-1)；I.e. catalytic efficiency is
20.4±0.1.As shown in the curve that 1:1:300 is represented in Fig. 2, fixed enzyme, ABTS on conventional surface-At 414nm
Absorbance 1.5 or so float.As shown in the curve that 1:1:300 is represented in Fig. 3, fixed enzyme, ABTS in glass tube-
Absorbance at 414nm is floated 4 or so.As shown in the curve that 1:1:300 is represented in Fig. 4, the biological enzyme graft polymers
After solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is up to 0.95.
Embodiment 3
Referring to embodiment 1, weigh polymerization reaction azodiisobutyronitrile, sodium hydrogensulfite, monomer methacrylic acid shrink it is sweet
Grease (GMA) 0.05g, 0.03g, 2g (1:1:500), remaining condition is constant, is grafted using biological enzyme prepared by the feed ratio poly-
Close the K of object substrate for enzymatic activityMFor 1.43 ± 0.1 (mM)；Reaction constant KcatFor 28.5 ± 0.1 (s-1)；I.e. catalytic efficiency is 19.9
±0.1.As shown in the curve that 1:1:500 is represented in Fig. 2, fixed enzyme, ABTS on conventional surface-Suction at 414nm
Luminosity floats 0.4 or so.As shown in the curve that 1:1:500 is represented in Fig. 3, fixed enzyme, ABTS in glass tube-?
Absorbance at 414nm is floated 2 or so.As shown in the curve that 1:1:500 is represented in Fig. 4, the biological enzyme graft polymers is molten
After liquid stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is up to 0.85.
Embodiment 4
Referring to embodiment 1, weigh polymerization reaction azodiisobutyronitrile, sodium hydrogensulfite, monomer methacrylic acid shrink it is sweet
Grease (GMA) 0.008g, 0.005g, 2g (1:1:50), remaining condition is constant, is grafted using biological enzyme prepared by the feed ratio poly-
Close the K of object substrate for enzymatic activityMFor 1.72 ± 0.1 (mM)；Reaction constant KcatFor 15 ± 0.1 (s-1)；I.e. catalytic efficiency be 8.72 ±
0.1.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is reachable
1.0 left and right.Fixed enzyme, ABTS on conventional surface-Absorbance at 414nm is floated 2.4 or so.In glass tube
Fixed enzyme, ABTS-Absorbance at 414nm is floated 1.1 or so.
Embodiment 5
Referring to embodiment 1, weigh polymerization reaction azodiisobutyronitrile, sodium hydrogensulfite, monomer methacrylic acid shrink it is sweet
Grease (GMA) 0.003g, 0.002g, 2g (1:1:50), remaining condition is constant, is grafted using biological enzyme prepared by the feed ratio poly-
Close the K of object substrate for enzymatic activityMFor 1.56 ± 0.1 (mM)；Reaction constant KcatFor 29 ± 0.1 (s-1)；I.e. catalytic efficiency is 18.58
±0.1.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is reachable
0.7 or so.Fixed enzyme, ABTS on conventional surface-Absorbance at 414nm is floated 1 or so.It is solid in glass tube
Fixed enzyme, ABTS-Absorbance at 414nm is floated 0.25 or so.
Embodiment 6
Referring to embodiment 1, change horseradish peroxidase is superoxide dismutase, remaining condition is constant, resulting life
The K of object enzyme graft polymers catalysis substrateMFor 1.25 ± 0.1 (mM)；Reaction constant KcatFor 23.6 ± 0.1 (s-1)；That is catalysis effect
Rate is 18.8 ± 0.1.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, relatively
Activity is up to 0.74.Fixed enzyme, ABTS on conventional surface-Absorbance at 414nm is floated 0.3 or so.In glass
Fixed enzyme, ABTS in glass pipe-Absorbance at 414nm is floated 1.5 or so.
Embodiment 7
Referring to embodiment 1, change horseradish peroxidase is protease, remaining condition is constant, resulting biological enzyme grafting
The K of polymer catalyzing substrateMFor 1.52 ± 0.1 (mM)；Reaction constant KcatFor 30.0 ± 0.1 (s-1)；I.e. catalytic efficiency is 19.7
±0.1.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is reachable
0.84.Fixed enzyme, ABTS on conventional surface-Absorbance at 414nm is floated 0.4 or so.It is solid in glass tube
Fixed enzyme, ABTS-Absorbance at 414nm is floated 1.8 or so.
Embodiment 8
Referring to embodiment 1, change horseradish peroxidase is lipase, remaining condition is constant, resulting biological enzyme grafting
The K of polymer catalyzing substrateMFor 1.18 ± 0.1 (mM)；Reaction constant KcatFor 16.4 ± 0.1 (s-1)；I.e. catalytic efficiency is 13.9
±0.1.After the biological enzyme graft polymer solution stores 30 days at 4 DEG C, which is still able to maintain its activity, and relative activity is reachable
0.54.Fixed enzyme, ABTS on conventional surface-Absorbance at 414nm is floated 0.2 or so.It is solid in glass tube
Fixed enzyme, ABTS-Absorbance at 414nm is floated 1.0 or so.
Embodiment 9
Step 1: weigh respectively azodiisobutyronitrile, sodium hydrogensulfite, glycidyl methacrylate (GMA) (mole
Than 1:1:10)；Weighed all reagents are added in reaction vessel, then solvents tetrahydrofurane (GMA and four is added into container
The mass volume ratio of hydrogen furans is 1g/ml).Container is sealed to and is passed through nitrogen, removes oxygen 30min；50 DEG C of heating stirrings
30min, the reaction monomers in reaction vessel sufficiently react with initiator, catalyst, product are taken out；Polymer is added dropwise dropwise
Into n-hexane, n-hexane is filtered in flocculent deposit to be generated, and solvent volatilization obtains pure polymer.50 DEG C of freeze-day with constant temperature
After 10h, PGMA solid is obtained.Above-mentioned PGMA solid is added in the mixed solution of water and ethylenediamine (volume ratio 1:1), is stirred at 70 DEG C
18h is mixed, the cooling centrifugation of product is cleaned repeatedly with deionized water, is dried in vacuo spare, product PGMA-NH2It indicates.
In anhydrous DMF, 4- carboxyl benzaldehyde is added, is added NHS, EDCl reaction under room temperature, 4- carboxyl benzaldehyde, NHS,
EDCl molar ratio 1:1.5:1.5,4- carboxyl benzaldehyde, anhydrous DMF molar ratio are 1:50.Chloroform CHCl is added in round-bottomed flask3With
Water is extracted twice, and compound organic phase is washed with saturated sodium chloride solution.The dry 2h of appropriate anhydrous magnesium sulfate is added, is filtered to remove sulphur
Sour magnesium.Filtered organic phase is rotated, crude product is obtained.Crude product volume ratio n-hexane after drying: ethyl acetate=1:1 weight
Crystallization.It is cooled to room temperature and acicular crystal occurs, after 2-3 hours, be put into refrigerator (4 DEG C), taken out after crystal is smooth, at 50 DEG C
It is 48 hours dry, obtain S-4FB.
6- chlorine apellagrin is dissolved into hydrazine hydrate according to molar ratio 1:15, is condensed back 6h, is concentrated in vacuo dry be redissolved
It is acidified in 20%HCl solution, adjustment pH is 4, and 6- hydrazinonicotinic acid hydrochloride can be obtained in precipitation to be precipitated, uses second after filtering
Alcohol washing, recrystallization (ethyl alcohol: water=2:1), obtain 6- hydrazinonicotinic acid.Above-mentioned made 6- hydrazinonicotinic acid is taken to be dissolved in anhydrous DMF,
It is added triethylamine (excess), acetone is added in Xiang Shangshu solution, it is sub- that N- hydroxysuccinimidyl acyl is then added in room temperature magnetic agitation 4h
Amine, EDCl are stirred overnight, 6- hydrazinonicotinic acid, acetone, n-hydroxysuccinimide and EDCL molar ratio 1:1:1.5:1.3, nothing
Chloroform extraction is added in water DMF and triethylamine volume ratio 8:5, after purification, anhydrous magnesium sulfate dry 2-4h, concentrated by rotary evaporation, crude product
Purified (methylene chloride: methanol=50:1) with column chromatography again, obtains S-HyNic.
Step 2: measured from HRP stoste it is a certain amount of be dissolved in phosphate buffer solution (PB1,0.2M NaCl, PH=7.2),
It is mixed with the DMF solution of S-4FB, reacts at room temperature 2h, the ratio that biological enzyme molecule and -4FB group are controlled in reaction process is 1:
10, the HRP, i.e. HRP-4FB of 4FB modification are obtained through multiple ultrafiltration.
Step 3: by PGMA-NH in step 12(MopsB1,0.1M MOPS, 0.15M are dissolved in MOPS buffer
NaCl, PH=7.6) in, 3h is reacted with the S-HyNic being dissolved in micro DMF, control-NH in reaction process2Group with-
The ratio of HyNic group is 1:1, uses MES buffer solution (MESB1,0.1M MES, 0.15NaCl, PH=4.7) through ultrafiltration,
Obtain polymer P GMA-HyNic.
Step 4: will be in step 2: HRP-4FB and PGMA-HyNic in step 3 be controlled in room temperature reaction 2h, reaction process
System-HyNic group and -4FB group ratio are 20:1, and repeated ultrafiltration purification obtains biological enzyme graft polymers.
Embodiment 10
Step 1: weigh respectively azodiisobutyronitrile, sodium hydrogensulfite, glycidyl methacrylate (GMA) (mole
Than 1:1:1000)；Weighed all reagents are added in reaction vessel, then into container be added solvents tetrahydrofurane (GMA with
The mass volume ratio of tetrahydrofuran is 1g/3ml).Container is sealed to and is passed through nitrogen, removes oxygen 30min；55 DEG C of heating stirrings
30min, the reaction monomers in reaction vessel sufficiently react with initiator, catalyst, product are taken out；Polymer is added dropwise dropwise
Into n-hexane, n-hexane is filtered in flocculent deposit to be generated, and solvent volatilization obtains pure polymer.55 DEG C of freeze-day with constant temperature
After 11h, PGMA solid is obtained.Above-mentioned PGMA solid is added in the mixed solution of water and ethylenediamine (volume ratio 1:1.2), at 85 DEG C
15h is stirred, the cooling centrifugation of product is cleaned repeatedly with deionized water, is dried in vacuo spare, product PGMA-NH2It indicates.
In anhydrous DMF, 4- carboxyl benzaldehyde is added, is added NHS, EDCl reaction under room temperature, 4- carboxyl benzaldehyde, NHS,
EDCl molar ratio 1:0.8:0.8,4- carboxyl benzaldehyde, anhydrous DMF molar ratio are 1:55.Chloroform CHCl is added in round-bottomed flask3With
Water is extracted twice, and compound organic phase is washed with saturated sodium chloride solution.The dry 2h of appropriate anhydrous magnesium sulfate is added, is filtered to remove sulphur
Sour magnesium.Filtered organic phase is rotated, crude product is obtained.Crude product volume ratio n-hexane after drying: ethyl acetate=1:1 weight
Crystallization.It is cooled to room temperature and acicular crystal occurs, after 2-3 hours, be put into refrigerator (4 DEG C), taken out after crystal is smooth, at 50 DEG C
It is 48 hours dry, obtain S-4FB.
6- chlorine apellagrin is dissolved into hydrazine hydrate according to molar ratio 1:20, is condensed back 7h, is concentrated in vacuo dry be redissolved
It is acidified in 30%HCl solution, adjustment pH is 4, and 6- hydrazinonicotinic acid hydrochloride can be obtained in precipitation to be precipitated, uses second after filtering
Alcohol washing, recrystallization (ethyl alcohol: water=2:1), obtain 6- hydrazinonicotinic acid.Above-mentioned made 6- hydrazinonicotinic acid is taken to be dissolved in anhydrous DMF,
It is added triethylamine (excess), acetone is added in Xiang Shangshu solution, it is sub- that N- hydroxysuccinimidyl acyl is then added in room temperature magnetic agitation 3h
Amine, EDCl are stirred overnight, 6- hydrazinonicotinic acid, acetone, n-hydroxysuccinimide and EDCL molar ratio 1:1.2:1.2:1.2,
Chloroform extraction is added in anhydrous DMF and triethylamine volume ratio 8:3, after purification, anhydrous magnesium sulfate dry 2-4h, concentrated by rotary evaporation, thick production
Object is purified (methylene chloride: methanol=50:1) with column chromatography again, obtains S-HyNic.
Step 2: measured from HRP stoste it is a certain amount of be dissolved in phosphate buffer solution (PB1,0.2M NaCl, PH=7.2),
It is mixed with the DMF solution of S-4FB, reacts at room temperature 2h, the ratio that biological enzyme molecule and -4FB group are controlled in reaction process is 1:
15, the HRP, i.e. HRP-4FB of 4FB modification are obtained through multiple ultrafiltration.
Step 3: by PGMA-NH in step 12(MopsB1,0.1M MOPS, 0.15M are dissolved in MOPS buffer
NaCl, PH=7.6) in, 3h is reacted with the S-HyNic being dissolved in micro DMF, control-NH in reaction process2Group with-
The ratio of HyNic group is 1:0.5, uses MES buffer solution (MESB1,0.1M MES, 0.15NaCl, PH=through ultrafiltration
4.7) polymer P GMA-HyNic, is obtained.
Step 4: by the control-in room temperature reaction 2h, reaction process of PGMA-HyNic in HRP-4FB in step 2 and step 3
HyNic group and -4FB group ratio are 23:1, and repeated ultrafiltration purification obtains biological enzyme graft polymers.
Claims (10)
1. a kind of biological enzyme of graft polymers, which is characterized in that pass through bridging agent 4- formoxyl succinimide yl benzoic acid
Ester S-4FB connects biology with double aryl hydrazone bonds that succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic reaction generates
Enzyme and polymer.
2. a kind of biological enzyme of graft polymers according to claim 1, which is characterized in that the polymer surfaces have
The functional group of covalent cross-linking is easily carried out with enzyme.
3. a kind of preparation method of the biological enzyme of graft polymers, which is characterized in that be specifically implemented according to the following steps:
Step 1, the synthesis of polymer precursor and bridging agent:
It weighs polymerization reaction monomer, initiator and catalyst, is added solvent, under nitrogen protection, be heated to 50-60 DEG C and polymerize
Reaction obtains polymer solids；Polymer solids are added in the mixed solution of water and ethylenediamine, heating stirring at 70-85 DEG C
12-18h, the cooling centrifugation of product, is cleaned repeatedly with deionized water, is dried in vacuo, is contained-NH2Polymer precursor；
In anhydrous n,N-Dimethylformamide DMF, 4- carboxyl benzaldehyde and n-hydroxysuccinimide NHS and 1- (3- is added
Dimethylaminopropyl) -3- ethyl-carbodiimide hydrochloride EDCl reacted, and product is through extraction, washing, drying, concentration, again
Crystallization obtains bridging agent 4- formoxyl succinimido benzoic ether S-4FB；
6- hydrazinonicotinic acid is dissolved in anhydrous n,N-Dimethylformamide DMF, triethylamine, acetone is sequentially added, is stirred at room temperature
After reaction, n-hydroxysuccinimide, EDCl stirring is added, the reaction was continued, and product is through extraction, washing, drying, concentration, purifying
After obtain succinimido -6- diazanyl-niacinamide acetone hydrazone S-HyNic；
Step 2, modification biological enzyme and polymer-modified synthesis:
The S-4FB modified biological enzyme obtained using step 1, obtains modified biological enzyme；
The S-HyNic obtained using step 1 with contain-NH2Polybenzazole precursor precursor reactant, obtain modified polymer；
Step 3, the synthesis of graft polymers biological enzyme:
By the modified biological enzyme of step 2 and modified polymer reaction to get the biological enzyme for arriving graft polymers.
4. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that polymerize in the step 1 anti-
The molar ratio for answering monomer, initiator and catalyst is 1:1:10-1000, the mass volume ratio of polymerization reaction monomer and tetrahydrofuran
For 1g/1-3ml, the volume ratio 1:1-1.5 of water and ethylenediamine.
5. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that initiator in the step 1
For azodiisobutyronitrile, catalyst is sodium hydrogensulfite, and solvent is tetrahydrofuran.
6. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that 4- carboxyl in the step 1
The molar ratio of benzaldehyde, NHS and EDCl is 1:0.8-1.5:0.8-1.5, and the molar ratio of 4- carboxyl benzaldehyde and anhydrous DMF is 1:
50-60。
7. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that 6- diazanyl in the step 1
Niacin, acetone, n-hydroxysuccinimide and EDCL molar ratio be 1:1-1.4:1.1-1.5:1.1-1.3.
8. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that modified life in the step 2
The specific synthesis process of object enzyme are as follows: biological enzyme is dissolved in phosphate buffer solution, the S-4FB that step 1 is obtained is dissolved in DMF
In, then the phosphate buffer solution of biological enzyme is added in the DMF solution of S-4FB and reacts 2-3h under room temperature, controlled in reaction process
The ratio for making biological enzyme molecule and -4FB group is greater than 1:9, ultrafiltration to get；
Polymer-modified specific synthesis process in step 2 are as follows:-NH will be contained obtained in step 12Polymer precursor it is molten
Solution is in MOPS buffer, and the S-HyNic normal-temperature reaction 2-3h being dissolved in DMF, control-NH in reaction process2Group with-
The ratio of HyNic group be less than 1:2, ultrafiltration to get.
9. a kind of biological enzyme of graft polymers according to claim 3, which is characterized in that step 3 reaction process
Middle control-HyNic group and -4FB group ratio are 20-23:1.
10. a kind of fixing means of the biological enzyme of graft polymers according to claim 1 or 2, which is characterized in that will be clear
Clean carrier surface is soaked with MOPS buffer, then removes MOPS buffer with suction pipe, carrier is placed in centrifuge tube, and
The phosphate buffer of the biological enzyme of graft polymers is added, impregnates, to realize the fixation of biological enzyme；Extra biological enzyme is removed to connect
Branch polymer solution, carrier surface is washed with MOPS buffer, being eventually adding MOPS buffer keeps conventional carrier surface complete
Submergence, it is stored refrigerated in refrigerator.
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