CN116640757A - Construction method and application of immobilized enzyme system based on artificial antibody-antigen - Google Patents
Construction method and application of immobilized enzyme system based on artificial antibody-antigen Download PDFInfo
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 37
- 238000010276 construction Methods 0.000 title claims abstract description 14
- 239000000427 antigen Substances 0.000 title claims abstract description 13
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 claims abstract description 87
- 102000004190 Enzymes Human genes 0.000 claims abstract description 78
- 108090000790 Enzymes Proteins 0.000 claims abstract description 78
- 239000000463 material Substances 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000002262 Schiff base Substances 0.000 claims abstract description 13
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims abstract description 12
- 150000004753 Schiff bases Chemical class 0.000 claims abstract description 10
- 229940088598 enzyme Drugs 0.000 claims description 89
- 230000000694 effects Effects 0.000 claims description 58
- 238000006243 chemical reaction Methods 0.000 claims description 32
- 102000004882 Lipase Human genes 0.000 claims description 28
- 108090001060 Lipase Proteins 0.000 claims description 28
- 239000004367 Lipase Substances 0.000 claims description 28
- 235000019421 lipase Nutrition 0.000 claims description 28
- 229910004298 SiO 2 Inorganic materials 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 26
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 102000004139 alpha-Amylases Human genes 0.000 claims description 24
- 108090000637 alpha-Amylases Proteins 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 24
- 229940024171 alpha-amylase Drugs 0.000 claims description 23
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Natural products CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 19
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims description 19
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 19
- 238000002156 mixing Methods 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 10
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 9
- WMGVPDQNPUQRND-UHFFFAOYSA-N (2-methylphenyl)acetonitrile Chemical compound CC1=CC=CC=C1CC#N WMGVPDQNPUQRND-UHFFFAOYSA-N 0.000 claims description 8
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 8
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000002105 nanoparticle Substances 0.000 claims description 8
- QLNOVKKVHFRGMA-UHFFFAOYSA-N trimethoxy(propyl)silane Chemical group [CH2]CC[Si](OC)(OC)OC QLNOVKKVHFRGMA-UHFFFAOYSA-N 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 230000003100 immobilizing effect Effects 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- CDXSJGDDABYYJV-UHFFFAOYSA-N acetic acid;ethanol Chemical compound CCO.CC(O)=O CDXSJGDDABYYJV-UHFFFAOYSA-N 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 230000035484 reaction time Effects 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000006116 polymerization reaction Methods 0.000 claims description 4
- 239000004115 Sodium Silicate Substances 0.000 claims description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052911 sodium silicate Inorganic materials 0.000 claims description 2
- 230000000052 comparative effect Effects 0.000 description 12
- 238000011084 recovery Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- QUKGYYKBILRGFE-UHFFFAOYSA-N benzyl acetate Chemical compound CC(=O)OCC1=CC=CC=C1 QUKGYYKBILRGFE-UHFFFAOYSA-N 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- HSHNITRMYYLLCV-UHFFFAOYSA-N 4-methylumbelliferone Chemical compound C1=C(O)C=CC2=C1OC(=O)C=C2C HSHNITRMYYLLCV-UHFFFAOYSA-N 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 239000004382 Amylase Substances 0.000 description 3
- 102000013142 Amylases Human genes 0.000 description 3
- 108010065511 Amylases Proteins 0.000 description 3
- 235000019418 amylase Nutrition 0.000 description 3
- 229940007550 benzyl acetate Drugs 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- -1 catechol Schiff base Chemical class 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- ZMXYNJXDULEQCK-UHFFFAOYSA-N 2-amino-p-cresol Chemical compound CC1=CC=C(O)C(N)=C1 ZMXYNJXDULEQCK-UHFFFAOYSA-N 0.000 description 2
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 235000019445 benzyl alcohol Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920000344 molecularly imprinted polymer Polymers 0.000 description 2
- 238000011056 performance test Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- SYDNSSSQVSOXTN-UHFFFAOYSA-N 2-nitro-p-cresol Chemical compound CC1=CC=C(O)C([N+]([O-])=O)=C1 SYDNSSSQVSOXTN-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000222175 Diutina rugosa Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 241000712062 Patricia Species 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 241001417524 Pomacanthidae Species 0.000 description 1
- QOSMNYMQXIVWKY-UHFFFAOYSA-N Propyl levulinate Chemical compound CCCOC(=O)CCC(C)=O QOSMNYMQXIVWKY-UHFFFAOYSA-N 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000613130 Tima Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- PZJJKWKADRNWSW-UHFFFAOYSA-N trimethoxysilicon Chemical compound CO[Si](OC)OC PZJJKWKADRNWSW-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the technical field of immobilized enzymes, and particularly relates to a construction method and application of an immobilized enzyme system based on an artificial antibody-antigen. The method comprises the following steps: (1) By Fe 3 O 4 As a magnetic core, the magnetic core is modified by tetraethoxysilane; (2) grafting double bonds on the surface thereof by using MPS; (3) Constructing an artificial antibody material by taking catechol as a template molecule; (4) Preparing a catechol-enzyme complex by reacting an enzyme molecule with catechol through Schiff base; (5) And adding the catechol-enzyme complex solution into an artificial antibody material to immobilize the enzyme. The method is simple and convenient, can reduce the cost, improve the stability of the enzyme and has higher immobilization rate while realizing the specific directional immobilization of the enzyme.
Description
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to a construction method and application of an immobilized enzyme system based on an artificial antibody-antigen.
Background
The enzyme as a catalyst has mild reaction, no pollution and catalytic effectHigh rate. However, free enzymes have the disadvantages of sensitivity to the external environment, poor stability and easy inactivation. The immobilization of the enzyme on the matrix material can remedy this disadvantage (Cesar Mateo, jose Palomo, gloria Fernandez-Lorente, et al im. Development of enzyme activity, stability and selectivity via immobilization techniques [ J)]Enzyme an d Microbial Technology,2007, 40:1451-1463). Conventional immobilized enzyme methods include physical adsorption, entrapment encapsulation (Mari a Fernz-Fernandez, sanrom Angeles, moldes diego. Recent dev elopments and applications of immobilized laccase [ J)]Biotechnology Advances,2013, 31:1808-1825) and covalent crosslinking (J-shaped Lio casar dos Santos, patri cia Daniela Mijone, gisel e F.sub. tima Morais Nunes, et al, component attachment of Candida rugosa lipase on chemically modified hybrid matrix of polysiloxane-polyvinyl alcohol with different activating compounds [ J ]]Journal of Chromatography B,2007, 61:229-236). The traditional immobilized enzyme method improves the stability of the enzyme, realizes the recycling of the enzyme and realizes the quantitative immobilization of the enzyme. However, the traditional immobilized enzyme method has the defects of low specificity, poor biocompatibility, poor repeated use effect and the like. To solve the above problems, natural molecules have been introduced into immobilized enzyme systems to solve the problem of specific recognition, inspired by DNA duplex base pairing, antigen-antibody recognition, etc. However, the natural antibodies or the biological molecular materials such as DNA chains have the defects of high cost, poor stability and difficult preservationRe nberg,Kae Sato,Kazuma Mawatari,et al.Serial DNA immobilization in micro-an d extended nanospace channels[J].Lab on a Chip,2009,9:1517–1523；Yun Liu,Huixiang Wang,Jingyu Huang,Jie Yang,Baohong Liu,Pengyuan Yang,Microchip-based ELISA strategy for the detection of low-level disease biomarker in serum,An alytica Chimica Acta,650(2009)77–82)。
Disclosure of Invention
The invention aims to provide a construction method and application of an immobilized enzyme system based on an artificial antibody-antigen, wherein the enzyme is subjected to covalent modification as an antigen, and specific recognition is carried out between the immobilized enzyme and an artificial antibody material, so that the specific directional immobilization of the enzyme is realized, the cost is reduced, the stability of the enzyme is improved, and the enzyme carrying amount is high.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in one aspect, the invention provides a method for constructing an immobilized enzyme system based on an artificial antibody-antigen, comprising the following steps:
(1) Fe is added to 3 O 4 Dispersing the nano particles in an ethanol-water mixed solvent system, and uniformly mixing with tetraethoxysilane in an alkaline environment to obtain Fe 3 O 4 @SiO 2 ；
(2) Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in toluene, adding triethylamine and alpha-methacrylic acid 3- (trimethoxy silicon based) propyl ester (MPS), and reflux mixing under nitrogen protection to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Using catechol as template molecule, and adding Fe prepared in the step (2) 3 O 4 @SiO 2 Carrying out polymerization reaction on the@MPS, catechol, alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in a toluene-acetonitrile mixed solution, and eluting the catechol by using an eluent to prepare an artificial antibody material;
(4) Performing Schiff base reaction on enzyme molecules and catechol in a buffer solution to obtain a catechol-enzyme complex solution;
(5) Adding the catechol-enzyme complex solution obtained in the step (4) into the artificial antibody material prepared in the step (3), uniformly mixing, and immobilizing the enzyme.
The principle of the invention is as follows: by Fe 3 O 4 For the magnetic core, the magnetic core is modified by tetraethoxysilane, and a layer of SiO is coated on the surface of the magnetic core 2 Then, grafting double bonds on the surface of the material by using a silane coupling agent MPS, and constructing a catechol molecularly imprinted polymer, namely an artificial antibody material, by using catechol as an antigen template molecule; subsequent selection of template molecules with "antigenCatechol with similar structure is formed into catechol-enzyme complex by combining aldehyde group of para-position of phenolic hydroxyl and amino group of enzyme molecule through Schiff base reaction; and then the compound is connected to an artificial antibody material by utilizing the specific recognition between the phenolic hydroxyl end and the catechol molecularly imprinted polymer, so as to realize the immobilization of the enzyme.
In the above technical solution, further, in step (1), the Fe 3 O 4 The particle size of the nano particles is 100-400nm;
in the ethanol-water mixed solvent system, the volume ratio of ethanol to water is 4:1, a step of;
the alkaline environment is provided by ammonia water, and the volume ratio of the ammonia water to the tetraethoxysilane is 2:1, a step of;
the Fe is 3 O 4 The molar ratio of the sodium silicate to the tetraethoxysilane is 1:5-1:10.
in the above technical solution, in step (2), further, the volume ratio of triethylamine to 3- (trimethoxysilyl) propyl α -Methacrylate (MPS) is 1:1-1:3, the Fe 3 O 4 @SiO 2 The ratio of the mass of the alpha-methacrylic acid 3- (trimethoxysilyl) propyl ester (MPS) to the volume of the mixture is 0.1-0.5g:1mL.
In the above technical scheme, further, the volume ratio of toluene to acetonitrile in the toluene-acetonitrile mixed solution is 1:1-1:5.
in the above technical scheme, in step (3), the molar mass of catechol and Fe 3 O 4 @Si O 2 The mass ratio of @ MPS is 1-3mmol:1g, the molar ratio of catechol to alpha-methacrylic acid (MAA) being 1:2-1:6, the molar ratio of catechol to Ethylene Glycol Dimethacrylate (EGDMA) is 1:5-1:20, a step of;
the polymerization reaction temperature is 60-80 ℃ and the time is 12-24 hours;
the eluent is acetic acid and the volume ratio of ethanol is 9:1 in acetic acid-ethanol mixture.
In the above technical solution, in step (4), the buffer solution is a phosphate buffer solution of 0.01M; the enzyme molecule is any one of lipase, alpha-amylase and alpha-glucosidase;
the mass ratio of the catechol to the lipase is 1:50-1:250, wherein the mass ratio of the catechol to the alpha-amylase is 1:10-1:40, wherein the ratio of the activity of the alpha-glucosidase to the mass of the catechol is 200-1500U:1 μg;
the reaction temperature of the reaction of the catechol and the enzyme molecule Schiff base is 20-60 ℃, the reaction pH value is 6-8, and the reaction time is 1-6h.
In the above technical solution, in step (4), further, the reaction parameters of the catechol and the enzyme molecule schiff base need to be optimized according to different types of enzymes:
the optimal conditions for the reaction of lipase and catechol Schiff base are as follows: the reaction temperature is preferably 40-52 ℃, and the pH value is preferably 7.2-7.6;
the optimal conditions for the reaction of the alpha-amylase and the catechol Schiff base are as follows: the reaction temperature is preferably 20-45 ℃, and the pH value is preferably 6.5-7.6.
The optimal conditions for the reaction of the alpha-glucosidase and the catechol Schiff base are as follows: the reaction temperature is preferably 35-55 ℃, and the pH value is preferably 6.5-7.6.
In the above technical scheme, in step (5), the dosage relationship between the artificial antibody polymer and the catechol-enzyme complex is as follows: adding 5-20mg artificial antibody material into 1mL catechol-enzyme complex;
the mixing speed is 100-300rpm;
the immobilization time of the enzyme is 1-24h, and the immobilization temperature is 10-60 ℃.
In another aspect, the invention provides the use of the above construction method in the field of biocatalysis.
The invention has the following beneficial effects:
1. the method is simple and convenient, can reduce the cost and improve the stability of the enzyme while realizing the specific directional fixation of the enzyme, has higher immobilization rate, and the immobilization rate reaches more than 60 percent, breaks through the defects that the traditional immobilized enzyme material can only realize quantitative fixation, and the biological molecular material is used for directional immobilization of the enzyme and has high price, and creates conditions for simple, convenient and practical specific directional immobilization.
2. The invention utilizes the artificial antibody material to fix the enzyme, thereby not only having good immobilization effect, but also having higher enzyme activity;
3. the invention uses Fe 3 O 4 The immobilized enzyme is easy to separate from the solution system for the magnetic core, and can be reused.
Drawings
FIG. 1 is a schematic diagram showing the construction process of the artificial antibody-antigen immobilized enzyme system of the present invention.
Detailed Description
The technical solution of the present invention will be further described by the following specific embodiments, and it should be apparent to those skilled in the art that the examples are only for aiding in understanding the present invention and should not be construed as limiting the present invention in any way.
Example 1
In this embodiment, the construction of the immobilized enzyme system for lipase is carried out, and the construction method comprises the following steps:
(1) 0.6g of Fe 3 O 4 The volume ratio of the nano particles dispersed in 200mL of ethanol to water is 4:1, 8mL of ammonia water and 4mL of tetraethoxysilane are added into an ethanol-water mixed solvent system, and evenly mixed for 6 hours at 35 ℃ to obtain Fe 3 O 4 @SiO 2 ；
(2) Taking 0.5g of Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in 50mL toluene, adding 1mL triethylamine and 2mL 3- (trimethoxysilyl) propyl alpha-Methacrylate (MPS), and reflux mixing under nitrogen protection to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Taking 0.2g of Fe prepared in the step (2) 3 O 4 @SiO 2 @MPS with 0.25mmol catechol, 0.5mmol alpha-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azobisisobutyronitrile at 60℃in a volume ratio of 4:1 in a toluene-acetonitrile mixed solvent system for 24 hours, and then using acetic acid and ethanol with the volume ratio of 9:1, eluting catechol from the acetic acid-ethanol mixed solution to obtain an artificial antibody material;
(4) Performing Schiff base reaction on lipase and catechol in phosphate buffer solution with concentration of 0.01M to obtain catechol-enzyme complex solution; the reaction conditions are as follows: the concentration of the catechol solution is 25 mug/mL, the concentration of the alpha-amylase is 4mg/mL, the reaction temperature is 50 ℃, the pH value is 7.5, and the reaction time is 4 hours;
(5) Adding 1mL of the catechol-enzyme complex solution obtained in the step (4) into 5mg of the artificial antibody material obtained in the step (3), uniformly mixing by using a uniformly mixing instrument with the rotating speed of 300rpm, and immobilizing the enzyme for 11 hours at the immobilization temperature of 20 ℃ to obtain the immobilized lipase.
Example 2
In this example, the construction of an immobilized enzyme system for α -amylase is performed by the following steps:
(1) 0.6g of Fe 3 O 4 The volume ratio of the nano particles dispersed in 200mL of ethanol to water is 4:1, 8mL of ammonia water and 4mL of tetraethoxysilane are added into an ethanol-water mixed solvent system, and evenly mixed for 6 hours at 35 ℃ to obtain Fe 3 O 4 @SiO 2 ；
(2) Taking 0.5g of Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in 50mL toluene, adding 1mL triethylamine and 2mL 3- (trimethoxysilyl) propyl alpha-Methacrylate (MPS), and reflux mixing under nitrogen protection to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Taking 0.2g of Fe prepared in the step (2) 3 O 4 @SiO 2 @MPS with 0.25mmol catechol, 0.5mmol alpha-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azobisisobutyronitrile at 60℃in a volume ratio of 4:1 in a toluene-acetonitrile mixed solvent system for 24 hours, and then using acetic acid and ethanol with the volume ratio of 9:1, eluting catechol from the acetic acid-ethanol mixed solution to obtain an artificial antibody material;
(4) Carrying out Schiff base reaction on alpha-amylase and catechol in phosphate buffer solution with the concentration of 0.01M to obtain catechol-enzyme complex solution; the reaction conditions are as follows: the concentration of the catechol solution is 30 mug/mL, the concentration of the alpha-amylase is 1mg/mL, the reaction temperature is 36 ℃, the pH value is 6.8, and the reaction time is 5 hours;
(5) Adding 1mL of the catechol-enzyme complex solution obtained in the step (4) into 5mg of the artificial antibody material obtained in the step (3), uniformly mixing by using a uniformly mixing instrument with the rotating speed of 300rpm, and immobilizing the enzyme for 11h at the immobilization temperature of 20 ℃ to obtain the immobilized lipase.
Example 3
In this embodiment, the construction of the immobilized enzyme system for α -glucosidase is performed by the following steps:
(1) Will be 0.6g Fe 3 O 4 The volume ratio of the nano particles dispersed in 200mL of ethanol to water is 4:1, 8mL of ammonia water and 4mL of tetraethoxysilane are added into an ethanol-water mixed solvent system, and evenly mixed for 6 hours at 35 ℃ to obtain Fe 3 O 4 @SiO 2 ；
(2) Taking 0.5g of Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in 50mL toluene, adding 1mL triethylamine and 2mL 3- (trimethoxysilyl) propyl alpha-Methacrylate (MPS), and reflux mixing under nitrogen protection to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Taking 0.2g of Fe prepared in the step (2) 3 O 4 @SiO 2 @MPS with 0.25mmol catechol, 0.5mmol alpha-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azobisisobutyronitrile at 60℃in a volume ratio of 4:1 in a toluene-acetonitrile mixed solvent system for 24 hours, and then using acetic acid and ethanol with the volume ratio of 9:1, eluting catechol from the acetic acid-ethanol mixed solution to obtain an artificial antibody material;
(4) Carrying out Schiff base reaction on alpha-glucosidase and catechol in phosphate buffer solution with the concentration of 0.01M to obtain catechol-enzyme complex solution; the reaction conditions are as follows: the concentration of the catechol solution is 28 mug/mL, the volume ratio of the alpha-glucosidase to the catechol solution is 2.5%, the reaction temperature is 45 ℃, the pH value is 7.3, and the reaction time is 5 hours;
(5) Adding 1mL of the catechol-enzyme complex solution obtained in the step (4) into 5mg of the artificial antibody material obtained in the step (3), uniformly mixing by using a uniformly mixing instrument with the rotating speed of 300rpm, and immobilizing the enzyme for 11h at the immobilization temperature of 20 ℃ to obtain the immobilized lipase.
Comparative example 1
The difference from example 1 is that the matrix material is a non-artificial antibody material, and the other processes are completely identical to example 1;
the preparation method of the non-artificial antibody material comprises the following steps:
(1) 0.6g of Fe 3 O 4 The volume ratio of the nano particles dispersed in 200mL of ethanol to water is 4:1, 8mL of ammonia water and 4mL of tetraethoxysilane are added into an ethanol-water mixed solvent system, and evenly mixed for 6 hours at 35 ℃ to obtain Fe 3 O 4 @SiO 2 ；
(2) Taking 0.5g of Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in 50mL toluene, adding 1mL triethylamine and 2mL 3- (trimethoxysilyl) propyl alpha-Methacrylate (MPS), and reflux mixing under nitrogen protection to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Taking 0.2g of Fe prepared in the step (2) 3 O 4 @SiO 2 @MPS with 0.5mmol of alpha-methacrylic acid, 2.5mmol of ethylene glycol dimethacrylate, 20mg of azobisisobutyronitrile at 60℃in a volume ratio of 4:1 in a toluene-acetonitrile mixed solvent system for 24 hours, and then using acetic acid and ethanol with the volume ratio of 9:1, eluting catechol from the mixed solution of acetic acid and ethanol to obtain the non-artificial antibody material.
Comparative example 2
The difference from example 2 is that the non-artificial antibody material of comparative example 1 was used, unlike the matrix material, and the other procedures were exactly identical to example 2.
Comparative example 3
The difference from example 3 is that the non-artificial antibody material of comparative example 1 was used, unlike the matrix material, and the other procedures were completely identical to example 3.
Performance test:
the immobilized enzyme materials prepared in example 1, example 2, example 3 and comparative example 1, comparative example 2, comparative example 3 were subjected to performance test by the following methods:
(1) Enzyme loading amount:
the enzyme absorbance of the supernatant before and after immobilization is measured by a Coomassie brilliant blue method under ultraviolet 595nm, and is substituted into a BS A bovine serum albumin standard curve to determine the protein concentration before and after immobilization so as to calculate the immobilization rate and the immobilization amount.
And (3) a solid load rate formula: yield (%) = (C 0 -C t )/C 0 ×100％
The formula of the immobilization amount: immobilization amount= (C 0 -C t )×V×1000/M
Wherein C is 0 For protein concentration, C before immobilization t The concentration of protein after immobilization, V is the volume of solution used for immobilization, and M is the mass of matrix.
(2) The enzyme activity is defined as the amount of enzyme consumed to hydrolyze 1. Mu. Mol of substrate per minute or to produce 1. Mu. Mol of product is defined as 1U.
(3) Reusability of enzyme
The activity of the enzyme measured for the first time was defined as 100%, and after the immobilized enzyme was separated from the solution system by a magnet, the activity was measured repeatedly, and the ratio of the repeated measurement to the first measurement was compared.
(4) Comparison of temperature adaptability of immobilized enzyme to free enzyme
The activities of the immobilized enzyme and the free enzyme at different temperatures were determined and the best definition was chosen as 100% respectively and the remaining temperature activities were compared with them.
(5) Comparison of pH adaptability of immobilized enzyme to free enzyme
The activities of the immobilized and free enzymes at different pH conditions were determined and the best definition was chosen as 100% respectively and the remaining pH activities were compared.
The performance measurements were as follows:
the results of the immobilized amounts, the enzyme activities, and the repeated enzyme activities of examples 1 to 3 and comparative examples 1 to 3 are shown in Table 1.
TABLE 1
The immobilization amount of the artificial antibody-antigen immobilized enzyme system of example 1 to lipase was 13.22mg protein /g supp ort Initial enzyme Activity is 104.66U/g support The immobilization rate is 73.58 percent, and the enzyme activity is 104.30U/g when the catalyst is repeatedly used support The method comprises the steps of carrying out a first treatment on the surface of the The immobilized amount of the non-artificial antibody material of comparative example 1 to lipase was 0.77mg protein /g support Compared with the immobilized lipase of the artificial antibody material, the immobilized lipase has a quite different effect, has a certain initial activity of 75.87U/g suppor t But only 5.17U/g remains when repeatedly used support 。
The immobilization amount of the alpha-amylase of example 2 was 14.78mg protein /g support Initial enzyme Activity is 199.33u/g support The immobilization rate is 68.29 percent, and the enzyme activity is 199.33U/g when the catalyst is repeatedly used support The method comprises the steps of carrying out a first treatment on the surface of the The non-artificial antibody material of comparative example 2 had an immobilization of 0.20mg for alpha-amylase protein /g support Having a volume of 114.30U/g support The activity recovery effect was rapidly decreased at the same time as the initial enzyme activity of (A) was re-used, and only 29.74U/g was left support 。
The immobilization amount of the α -glucosidase of example 3 was 25.09mg protein /g support Initial enzyme Activity is 143.25U/g support The immobilization rate is 62.84 percent, and the enzyme activity is 137.70U/g when the catalyst is repeatedly used support The method comprises the steps of carrying out a first treatment on the surface of the The immobilization amount of the non-artificial antibody material of comparative example 2 to alpha-glucosidase was 5.91mg protein /g support The initial enzyme activity is 70.29U/g support The enzyme activity only remains 29.46U/g when the kit is repeatedly used support 。
Meanwhile, the immobilized lipase, alpha-amylase and alpha-glucosidase materials prepared in the embodiments 1-3 of the invention have the initial enzyme activities of 62.79%, 47.48% and 39.85% respectively when being repeatedly used for 15 times, and the fact proves that the immobilized enzyme by using the artificial antibody materials has good immobilization effect, higher enzyme activity and higher stability when being repeatedly used compared with the non-artificial antibody.
The optimal enzymatic reaction conditions of the enzymes may be changed after the enzymes are immobilized, and the initial activities of the free enzymes and the immobilized enzymes are defined as 100%, respectively, and the experimental results show that the optimal enzymatic reaction temperatures of the three immobilized enzymes are changed compared with the free enzymes. The activity recovery effect of the immobilized alpha-amylase in example 2 is higher than that of the free amylase at high temperature or low temperature, the relative activity of the immobilized alpha-amylase at 25 ℃ is 77.21%, and the free amylase is 67.68%; the relative activity of immobilized alpha-amylase at 50℃was 95.31% and the activity of free amylase was 79.76%. The activity recovery effect of the immobilized alpha-glucosidase in example 3 was similar to that of the free enzyme at low temperature, but the activity was 47.82% and 38.51% of that of the immobilized alpha-glucosidase at 80℃in comparison with the free enzyme at high temperature. In example 1, the activity of the immobilized lipase was not as high as that of the free enzyme at low temperature, but at high temperature, the activity of the immobilized lipase was 93.72% at 50℃and only 87.35% of the free lipase was left. In combination, when the enzyme is immobilized with artificial antibody material, the adaptability of the enzyme to higher extreme temperatures is improved.
And comparing the activity recovery effects of the immobilized lipase, the immobilized alpha-amylase and the immobilized alpha-glucosidase with the corresponding free enzymes under different pH values, wherein the optimal pH value of the enzymatic reaction of the alpha-glucosidase, the lipase and the alpha-amylase is changed after the immobilized lipase and the immobilized alpha-amylase are immobilized on an artificial antibody material. The immobilized lipase has more excellent activity recovery effect under alkaline conditions, and the recovery rate of the immobilized lipase activity is 77.27 +/-0.25% when the pH=10.5, and the activity of the free lipase is only 39.65+/-0.33%. Although the activity recovery effect of the immobilized α -glucosidase was not as good as that of the free enzyme under the acidic condition, the activity recovery rate thereof under the alkaline condition was higher than that of the free enzyme, and when ph=9, the activity recovery efficiency of the free enzyme was 64.35%, and the activity recovery effect of the immobilized enzyme was 72.72%. The immobilized alpha-amylase is superior to the free enzyme in both peracid and overbase conditions after immobilization, and the relative activity of the immobilized alpha-amylase is 82.37% at ph=10.5, and 69.46% in the free state. When ph=5.5, the recovery rate of the activity of the immobilized α -amylase was 89.55%, and the recovery rate of the activity of the enzyme in the free state was 81.04%, and the activity retention effect of the enzyme in the peracid-or overbase-facing environment was improved by immobilizing the enzyme with an artificial antibody material as a whole.
Application example 1
The immobilized lipase is used for catalytic synthesis of benzyl acetate, and the synthesis conditions are as follows: adding 500 mu L of vinyl acetate and 5 mu L of benzyl alcohol into 50mg of immobilized lipase material, reacting at 70 ℃ for 6 hours, and balancing the reaction; after evaporating the unreacted reactants at 70℃from the mixture after 6 hours of reaction, the yield of benzyl acetate as a reaction product was determined by liquid phase and found to be 88.72%. After separating the supernatant from the immobilized lipase material by a magnet, adding vinyl acetate and benzyl alcohol continuously, and continuously injecting the sample for 5 times, wherein the yield of the obtained benzyl acetate is 74.13% of that of the product obtained by primary catalysis. This demonstrates that the lipase immobilized by this method is excellent in not only stability in aqueous solution and recyclability but also stability in organic solvent.
Application example 2
The immobilized alpha-amylase is used for catalyzing and synthesizing o-amino-p-methylphenol, and the synthesis conditions are as follows: after adding the starch solution to the immobilized alpha-amylase material and activating for 10min at 37 ℃, the supernatant is sucked out to react with 2-nitro-4-methylphenol, the product quantity is basically stable when the reaction is carried out for 9 min at 100 ℃, and the liquid phase is used for detection at 275nm, so that the yield is 58.88%. And then, the continuous sample injection efficiency of the immobilized alpha-amylase material for catalyzing the reaction of the 2-amino-p-methylphenol is measured, and after 5 times of repeated use, the catalytic efficiency is 80.86 percent of that of the primary use.
Application example 3
The immobilized alpha-glucosidase is used for catalyzing and synthesizing 4-methylumbelliferone, and the synthesis conditions are as follows: the reaction was carried out at 37℃by adding 4-methylumbelliferone- α -D-pyran-glucoside solution to immobilized α -glucosidase material, and after 30min, the yield was measured at 254nm by high performance liquid chromatography, at which time the reaction yield was 77.54%. And then, measuring the continuous sample injection effect of the immobilized alpha-glucosidase for catalyzing the 4-methylumbelliferone-alpha-D-glucopyranoside to be converted into 4-methylumbelliferone. After 5 consecutive catalytic times, the yield was 81.12% of the initial catalytic.
Application examples 1-3 the test results are shown in table 2.
TABLE 2
The invention is mainly researched aiming at the feasibility of an artificial antibody-antigen immobilized enzyme system, after a single antibody immobilized enzyme system is successfully constructed, the performance of the single antibody immobilized enzyme system is measured, and compared with a common immobilized enzyme material, the immobilized effect and stability of the artificial antibody immobilized enzyme material are better, and finally the immobilized enzyme material is successfully applied.
The applicant states that the present invention illustrates the preparation and use of the artificial antibody-antigen immobilized enzyme system of the present invention by the above examples, but the present invention is not limited to the above steps, i.e., it is not meant to necessarily rely on the above process steps for implementation. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected materials and addition of auxiliary components, selection of specific modes, etc. fall within the scope of the invention and the scope of the disclosure.
Claims (8)
1. A construction method of an immobilized enzyme system based on an artificial antibody-antigen is characterized in that,
the method comprises the following steps:
(1) Fe is added to 3 O 4 Dispersing the nano particles in an ethanol-water mixed solvent system, and uniformly mixing with tetraethoxysilane in an alkaline environment to obtain Fe 3 O 4 @SiO 2 ；
(2) Fe prepared in the step (1) 3 O 4 @SiO 2 Dispersing in toluene, adding triethylamine, 3- (trimethoxysilyl) propyl alpha-methacrylate(MPS) and carrying out reflux mixing under the protection of nitrogen to obtain Fe 3 O 4 @SiO 2 @MPS；
(3) Using catechol as template molecule, and adding Fe prepared in the step (2) 3 O 4 @SiO 2 Carrying out polymerization reaction on the@MPS, catechol, alpha-methacrylic acid, ethylene glycol dimethacrylate and azodiisobutyronitrile in a toluene-acetonitrile mixed solution, and eluting the catechol by using an eluent to prepare an artificial antibody material;
(4) Performing Schiff base reaction on enzyme molecules and catechol in a buffer solution to obtain a catechol-enzyme complex solution;
(5) Adding the catechol-enzyme complex solution obtained in the step (4) into the artificial antibody material prepared in the step (3), uniformly mixing, and immobilizing the enzyme.
2. The method according to claim 1, wherein in the step (1), the Fe 3 O 4 The particle size of the nano particles is 100-400nm;
in the ethanol-water mixed solvent system, the volume ratio of ethanol to water is 4:1, a step of;
the alkaline environment is provided by ammonia water, and the volume ratio of the ammonia water to the tetraethoxysilane is 2:1, a step of;
the Fe is 3 O 4 The molar ratio of the sodium silicate to the tetraethoxysilane is 1:5-1:10.
3. the method of claim 1, wherein in step (2), the volume ratio of triethylamine to 3- (trimethoxysilyl) propyl α -methacrylate is 1:1-1:3, the Fe 3 O 4 @SiO 2 The ratio of the mass of the alpha-methacrylic acid 3- (trimethoxysilyl) propyl ester to the volume of the alpha-methacrylic acid 3- (trimethoxysilyl) propyl ester is 0.1-0.5g:1mL.
4. The construction method according to claim 1, wherein the volume ratio of toluene to acetonitrile in the toluene-acetonitrile mixed solution is 1:1-1:5.
5. according to the weightsThe method according to claim 1, wherein in the step (3), the catechol molar mass is equal to Fe 3 O 4 @SiO 2 The ratio of @ MPS mass is 1-3mmol:1g, the molar ratio of catechol to alpha-methacrylic acid being 1:2-1:6, the mol ratio of catechol to glycol dimethacrylate is 1:5-1:20, a step of;
the polymerization reaction temperature is 60-80 ℃ and the time is 12-24 hours;
the eluent is acetic acid and the volume ratio of ethanol is 9:1 in acetic acid-ethanol mixture.
6. The method of claim 1, wherein in step (4), the buffer solution is a 0.01M phosphate buffer solution;
the enzyme molecule is any one of lipase, alpha-amylase and alpha-glucosidase;
the mass ratio of the catechol to the lipase is 1:50-1:250;
the mass ratio of the catechol to the alpha-amylase is 1:10-1:40, a step of performing a;
the ratio of the activity of the alpha-glucosidase to the mass of the catechol is 200-1500U:1 μg;
the reaction temperature of the reaction of the catechol and the enzyme molecule Schiff base is 20-60 ℃, the reaction pH value is 6-8, and the reaction time is 1-6h.
7. The method of claim 1, wherein in step (5), the amount of the artificial antibody polymer and the amount of the catechol-enzyme complex are in the following relationship: adding 5-20mg artificial antibody material into 1mL catechol-enzyme complex;
the mixing speed is 100rpm-300rpm;
the immobilization time of the enzyme is 1-24h, and the immobilization temperature is 10-60 ℃.
8. Use of the construction method according to any one of claims 1 to 7 in the field of biocatalysis.
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