CN107227305B - Complex immobilization method of acetylcholinesterase - Google Patents
Complex immobilization method of acetylcholinesterase Download PDFInfo
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Abstract
The invention discloses a compound immobilization method of acetylcholinesterase, belonging to the field of enzyme immobilization. The composite immobilization method of the invention comprises the following steps: (1) using molecular simulation software or a module to carry out molecular docking scoring on the small molecules and the region I and the region II on the acetylcholinesterase molecules respectively; (2) obtaining a total score of the small molecules through calculation; (3) connecting the preferential small molecules or the good small molecules with the surface of the immobilized carrier, and then incubating with acetylcholinesterase to obtain the composite immobilized acetylcholinesterase. The invention is based on the three-dimensional structure information of enzyme protein molecules in a breakthrough manner, and designs an immobilized carrier and an immobilized mode on a molecular level by analyzing the structure and property difference of different areas on acetylcholinesterase molecules, so that the conformation of the enzyme is optimal, the activity of the enzyme is retained to the maximum extent, the stability of the immobilized enzyme is greatly improved, the immobilized amount of the enzyme is increased, the enzyme can be repeatedly used, and the application range is wider.
Description
Technical Field
The invention belongs to the field of enzyme immobilization, and particularly relates to a compound immobilization method of acetylcholinesterase.
Background
Acetylcholinesterase (AChE, EC 3.1.1.7) is a large class of cholinesterase, widely exists in human bodies, animals, insects and the like, can specifically hydrolyze acetylcholine into choline and acetic acid, and is an important enzyme related to nerve conduction. The acetylcholinesterase can be specifically inhibited by organophosphorus and carbamate pesticides, so that the acetylcholinesterase is widely applied to a plurality of fields such as pesticide detection, environmental detection and the like.
Enzyme immobilization technology (Enzyme immobilization) refers to a class of technologies that bind or confine enzymes in a certain area, but retain their catalytic properties and can be recovered and reused. Compared with free enzyme, the immobilized enzyme has the characteristics of high stability, easy separation, reusability and the like, and is widely applied to the fields of industry, agriculture, medicines, foods and the like.
Currently, conventional immobilization methods of common enzymes, such as adsorption and cross-linking, are frequently used for immobilization of acetylcholinesterase (AChE). In the immobilization process of acetylcholinesterase (AChE), researches are mostly focused on finding a mild immobilization material and an immobilization method with good affinity, and structural characteristics and regional differences of acetylcholinesterase (AChE) protein molecules are not considered, so that the structure of part of enzymes in the immobilization process of acetylcholinesterase (AChE) is changed, and the activity is greatly reduced. This seriously affects the use of immobilized enzymes and becomes a bottleneck for the practical application of immobilized acetylcholinesterase (AChE).
The structural basis of acetylcholinesterase is protein, and the amino acid sequence and the three-dimensional crystal structure information of the molecule are completely resolved. Meanwhile, the research on the regional structure function relationship of acetylcholinesterase is complete. Research shows that different regions on acetylcholinesterase molecules have different physicochemical properties (such as hydrophobicity, chargeability and the like), and there are significant regional functional differences.
In order to improve the performance of immobilized acetylcholinesterase (AChE) in the prior art, it is important to develop an enzyme immobilization method which is designed at a molecular level and specially aims at the difference of acetylcholinesterase regions.
Disclosure of Invention
The invention provides a compound immobilization method of acetylcholinesterase based on a molecular docking screening technology, aiming at solving the problem of activity reduction caused by structural change in the immobilization process of acetylcholinesterase in the prior art. The specific technical scheme is as follows:
a complex immobilization method of acetylcholinesterase comprises the following steps:
(1) and (3) zone scoring: constructing three-dimensional structure data of acetylcholinesterase, and performing molecular docking scoring on the small molecules and a region I and a region II on the acetylcholinesterase molecules respectively by using molecular docking software or a module to obtain a region I score and a region II score of the small molecules;
(2) evaluation classification: calculating the total score of the small molecules in the step (1), and classifying the small molecules into preferential small molecules, good small molecules and eliminated small molecules according to the total score;
(3) enzyme immobilization: and (3) connecting the preferential small molecules or the good small molecules in the step (2) with the surface of an immobilized carrier, and then incubating with acetylcholinesterase to obtain the composite immobilized acetylcholinesterase.
Further, the acetylcholinesterase is human acetylcholinesterase, or human acetylcholinesterase with similar functions and more than 85% of similarity with the amino acid sequence of the acetylcholinesterase.
Furthermore, the amino acid sequence of the human acetylcholinesterase is shown in SEQ ID No. 1.
The three-dimensional structure data of the acetylcholinesterase in the step (1) come from public data or are constructed by INSIGHTii and Modeller software.
In the step (1), the small molecule is a molecule or a functional group which is positioned at the tail end of the immobilized carrier and can interact with acetylcholinesterase molecules, and the molecular weight of the small molecule is 50-8000 Da.
Further, the small molecule is not limited to the following species: antibiotics, pesticides, veterinary or human pharmaceuticals.
Further, the small molecule may specifically be an organophosphate, a steroid, a tetracycline, an amide or a low molecular weight polymer.
The region I in step (1) is a region on the acetylcholinesterase molecule consisting of amino acids VAL330, VAL331, LYS332, ASP333, GLU334, GLY335, SER336, ARG395, GLU396, SER399, ASP400, GLY403, ASP404, VAL408, VAL429, GLU431, TRP442, MET443, GLY444, TYR510, LEU524 and ARG 525.
The region II in the step (1) is a region on the acetylcholinesterase molecule, which is composed of the amino acids GLN71, TYR72, ASP74, GLY82, THR83, TRP86, ASN87, PRO88, GLY120, GLY121, GLY122, TYR124, SER125, GLY126, ALA127, LEU130, TYR133, GLN202, SER203, ALA204, SER229, GLY230, TRP236, TRP286, VAL294, PHE295, ARG296, PHE297, TYR337, PHE338, TYR341, VAL407, TRP439, MET443, PRO446, HIS447, GLY448, TYR449 and ILE 451.
The molecular docking software or module in the step (1) is Dock, 3D-DOCK, AutoDock, Surflex, Glide, Gold, FlexX, Z-DOCK, FTDOCK, Molegro Virtual Docker or Affinity molecular simulation software, module or program.
And (2) the molecular docking scoring in the step (1) is to calculate the binding constant (logKa) of the small molecule and the acetylcholinesterase region site according to the molecular docking.
The method for calculating the total score of the small molecules in the step (2) comprises the following steps: if the score of the area II of the small molecule is more than or equal to zero, the total score of the small molecule is equal to the score of the area I minus the score of the area II; if the small molecule's region II score is less than zero, then the total score for the small molecule is equal to its region I score.
The method for classifying the small molecules in the step (2) comprises the following steps: if the total score of the small molecules is more than 7.0, the small molecules are preferential small molecules, and are preferentially considered and selected when the acetylcholinesterase is immobilized; if the total score of the small molecules is more than or equal to 4.5 and less than or equal to 7.0, the small molecules are good-class small molecules and can be considered and selected when the acetylcholinesterase is immobilized; if the total score of the small molecules is less than 4.5, the small molecules are eliminated small molecules and are not considered and selected when the acetylcholinesterase is immobilized.
The immobilized carrier in the step (3) is a polymer, an electrode, a mesoporous material, a nano material, an inorganic material or a composite material.
In the step (3), the connection between the small molecules and the surface of the immobilized carrier is direct, indirect or compound chemical connection or physical connection.
The invention has the beneficial effects that:
1. the invention is based on the three-dimensional structure information of enzyme protein molecules, establishes a multi-site evaluation method for small molecule screening by analyzing the structure and property difference of different areas on acetylcholinesterase molecules, designs an immobilized carrier and an immobilization mode on a molecular level, enables the enzyme conformation to be optimal, and retains the activity of the enzyme to the maximum extent.
2. The stability of the immobilized acetylcholinesterase is greatly improved, the immobilized acetylcholinesterase can be repeatedly used for many times, and the application range of the immobilized acetylcholinesterase is wider.
3. The immobilized amount of the acetylcholinesterase is increased, and the method can be widely applied to industrial production.
Detailed Description
The following examples facilitate a better understanding of the invention, but are not intended to limit the invention thereto.
In the following examples, acetylcholinesterase (AChE) was selected and used in the form of three-dimensional crystal structure of human acetylcholinesterase molecule (three-dimensional crystal structure is derived from Protein Data Bank) with PDB number 1F8Uhttp://www.rcsb.org/pdb)。
Based on the three-dimensional structure information of the acetylcholinesterase protein molecules, an immobilized carrier and an immobilization mode are designed on a molecular level by analyzing the structure and property difference of different regions on the acetylcholinesterase molecules. The three-dimensional structure of acetylcholinesterase can be constructed by using software such as INSIGHTii and modeler, and the three-dimensional structure data of the enzyme protein disclosed in the existing database can also be used.
Region I is a region of the acetylcholinesterase molecule consisting of amino acids VAL330, VAL331, LYS332, ASP333, GLU334, GLY335, SER336, ARG395, GLU396, SER399, ASP400, GLY403, ASP404, VAL408, VAL429, GLU431, TRP442, MET443, GLY444, TYR510, LEU524 and ARG 525.
Region II is a region of the acetylcholinesterase molecule consisting of amino acids GLN71, TYR72, ASP74, GLY82, THR83, TRP86, ASN87, PRO88, GLY120, GLY121, GLY122, TYR124, SER125, GLY126, ALA127, LEU130, TYR133, GLN202, SER203, ALA204, SER229, GLY230, TRP236, TRP286, VAL294, PHE295, ARG296, PHE297, TYR337, PHE338, TYR341, VAL407, TRP439, MET443, PRO446, HIS447, TYR 448, TYR449 and ILE 451.
The small molecule in the present invention refers to a molecule or functional group located at the end of the immobilization carrier, capable of interacting with the surface of acetylcholinesterase molecule, and used for linking the enzyme protein to the immobilization carrier, such as but not limited to the following species: antibiotics, pesticides, veterinary drugs, and human drugs, specifically organophosphorus compounds, steroid compounds, tetracyclines, amides, and low molecular weight polymers.
The activity detection method of the composite immobilized acetylcholinesterase prepared by the invention comprises the following steps:
reaction System preparation (final volume 0.2ml)
0.1mol/L, pH 8.0 phosphate buffer solution 100. mu.L
0.75mmol/L substrate (thioacetylcholine iodide) 50. mu.L
Enzyme source (protein content is adjusted to 40-80 mug/mL) or immobilized enzyme with equal enzyme amount of 50 muL
After the system is prepared, the reaction is carried out for 5min at 37 ℃, then 1.8ml of DTNB-phosphate-ethanol reagent is added, the colorimetric determination is carried out at the wavelength of 412nm, a blank tube which adjusts the transmittance to be 100% is added with a color developing agent, and then enzyme liquid which is equal to the amount of the determination tube is added, so as to eliminate the influence of the enzyme on the light absorption.
Example 1
The acetylcholinesterase complex immobilization method specifically comprises the following steps:
(1) and (3) carrying out small molecule screening of the region I and the region II by using a Suflex-Dock molecular docking module. The candidate small molecules are derived from a common organic small molecule library (small molecule compound database in Purchasable range in Zinc database, http:// Zinc. The results of the molecular docking showed that the molecule numbered ZINC18060741(Cas60-09-3) scored 8.723 for domain I and-0.897 for domain II with acetylcholinesterase.
(2) Since its region II score is less than zero, the total score of the ZINC18060741 molecule is 8.723, belonging to the preferred class of small molecules, which are preferentially used for immobilization of acetylcholinesterase.
(3) 15ml of a 20% volume fraction solution of terephthaldehyde in ethanol was mixed with 2g of amino silica gel, stirred at 30 ℃ for 30min and filtered through a sand core funnel. Adding the silica gel into 30ml of ethanol, dropwise adding 20ml of 5% by mass ZINC18060741 ethanol solution, and stirring and reacting at 40 ℃ for 60 min. And (3) carrying out suction filtration, washing for 3 times by using 20ml of ethanol solution, and washing for 3 times by using 20ml of water to prepare the immobilized carrier. 1g of the immobilized carrier was dispersed in 30ml of 0.1M Tris-HCl buffer solution (pH 7.4), 1ml of 30U 0.1M Tris-HCl solution of acetylcholinesterase was added, the reaction was stirred at 0 ℃ for 15min, and the mixture was filtered and washed 3 times with 0.1M Tris-HCl buffer solution (pH 7.4) at 20 ℃ to prepare immobilized acetylcholinesterase complex.
The recovery rate of the enzyme activity of the complex immobilized acetylcholinesterase measured by the acetylcholinesterase activity measuring method is 79.8%. The compound immobilized enzyme can be repeatedly used for 10 times, and the activity of the compound immobilized enzyme can be kept above 76.2% compared with the activity of the compound immobilized enzyme used for the first time. The compound immobilized enzyme is stored for 10 days at the temperature of 18 ℃ below zero and 0.1M tris-HCl solution, and the activity of the compound immobilized enzyme can be kept over 93.0 percent compared with the activity of the compound immobilized enzyme used in the first day.
Example 2
The acetylcholinesterase complex immobilization method specifically comprises the following steps:
(1) and (3) carrying out small molecule screening of the region I and the region II by using an AutoDock molecular docking module. The candidate small molecules are derived from a common organic small molecule library (small molecule compound database in Purchasable range in Zinc database, http:// Zinc. Molecular docking results showed that the molecule numbered ZINC05273799(Cas108608-63-5) scored 7.923 for acetylcholinesterase and 0.897 for region II.
(2) Since its zone II score is greater than zero, the total score of the ZINC05273799 molecule is 7.026, belonging to the preferential class of small molecules, preferentially used for the immobilization of acetylcholinesterase.
(3) Adding 5g of quartz filter membrane into 3mol/L hydrochloric acid, stirring and reacting at 100 ℃ for 8h, filtering, and drying and activating at 120 ℃ for 12 h. After activation, 5g of the quartz filter membrane were added with 100ml of anhydrous toluene and 5ml of 3-aminopropyltriethoxysilane, refluxed at 110 ℃ for 24 hours, filtered, and washed 3 times with 20ml of toluene. Vacuum drying at room temperature for 6h to obtain the amino quartz filter membrane. 20ml of terephthalic dialdehyde ethanol solution with the volume fraction of 20 percent is mixed with 3g of amino quartz filter membrane, the mixture is stirred and reacted for 30min at the temperature of 30 ℃, the quartz filter membrane is added with 30ml of ethanol, 20ml of ZINC05273799 ethanol solution with the mass fraction of 5 percent is dripped, and the mixture is stirred and reacted for 90min at the temperature of 40 ℃. And (3) carrying out suction filtration, washing for 3 times by using 20ml of ethanol solution, and washing for 3 times by using 20ml of water to prepare the immobilized carrier. The immobilized acetylcholinesterase complex is prepared by placing 1g of the immobilized carrier in 30ml of 0.1M Tris-HCl buffer solution (pH 7.4), adding 1ml of 25U of 0.1M Tris-HCl solution of acetylcholinesterase, reacting at 0 deg.C with stirring for 15min, filtering, and washing 3 times with 0 deg.C 20ml of 0.1M Tris-HCl buffer solution (pH 7.4).
The recovery rate of the enzyme activity of the complex immobilized acetylcholinesterase measured by the acetylcholinesterase activity measuring method is 81.7%. The compound immobilized enzyme can be repeatedly used for 10 times, and the activity of the compound immobilized enzyme can be maintained by more than 78.1% compared with the activity of the compound immobilized enzyme used for the first time. The compound immobilized enzyme is stored for 10 days at the temperature of 18 ℃ below zero and 0.1M tris-HCl solution, and the activity of the compound immobilized enzyme can be kept above 95.6 percent compared with the activity of the compound immobilized enzyme used in the first day.
Example 3
The acetylcholinesterase complex immobilization method specifically comprises the following steps:
(1) and (3) carrying out small molecule screening of the region I and the region II by using a Suflex-Dock molecule docking module. The candidate small molecules are derived from a common organic small molecule library (small molecule compound database in Purchasable range in Zinc database, http:// Zinc. The results of molecular docking showed that the molecule numbered ZINC04272010(Cas538-41-0) scored 7.142 for acetylcholinesterase and 1.067 for domain I and domain II.
(2) Since the score of the region II is larger than zero, the total score of the ZINC04272010 molecule is 6.075, and the ZINC04272010 molecule belongs to a good class of small molecules and can be used for immobilization of acetylcholinesterase.
(3) 2g of amino-modified magnetic oxidized carbon nanoparticles (200-400nm) were used. Mixing 20ml of a terephthaldehyde ethanol solution with the volume fraction of 20% with 2g of amino-modified magnetic carbon oxide nano-particles (200-. The immobilized carrier was prepared by magnetic separation and washing 3 times with 20ml of ethanol solution and 3 times with 20ml of water. The immobilized acetylcholinesterase complex is prepared by adding 1g of the immobilized carrier into 30ml of 0.1M Tris-HCl buffer solution (pH 7.4), adding 1ml of 25U of 0.1M Tris-HCl solution of acetylcholinesterase, reacting at 0 ℃ for 15min with stirring, filtering, and washing 3 times with 0 ℃ 20ml of 0.1M Tris-HCl buffer solution (pH 7.4).
The recovery rate of the enzyme activity of the composite immobilized acetylcholinesterase measured by the acetylcholinesterase activity measuring method is 65.8%. The compound immobilized enzyme can be repeatedly used for 10 times, and the activity of the compound immobilized enzyme can be maintained by more than 63.2 percent compared with the activity of the compound immobilized enzyme used for the first time. The compound immobilized enzyme is stored for 10 days at the temperature of 18 ℃ below zero and 0.1M tris-HCl solution, and the activity of the compound immobilized enzyme can be kept more than 90.6 percent compared with the activity of the compound immobilized enzyme used in the first day.
SEQUENCE LISTING
<110> university of agriculture in China
<120> acetylcholinesterase composite immobilization method
<130>2017
<160>1
<170>PatentIn version 3.3
<210>1
<211>583
<212>PRT
<213> human (Homo sapiens)
<400>1
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Leu Arg Gly Ile Arg Leu Lys Thr Pro Gly Gly Pro Val Ser Ala Phe
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Leu Gly Ile Pro Phe Ala Glu Pro Pro Met Gly Pro Arg Arg Phe Leu
35 40 45
Pro Pro Glu Pro Lys Gln Pro Trp Ser Gly Val Val Asp Ala Thr Thr
50 55 60
Phe Gln Ser Val Cys Tyr Gln Tyr Val Asp Thr Leu Tyr Pro Gly Phe
65 70 75 80
Glu Gly Thr Glu Met Trp Asn Pro Asn Arg Glu Leu Ser Glu Asp Cys
85 90 95
Leu Tyr Leu Asn Val Trp Thr Pro Tyr Pro Arg Pro Thr Ser Pro Thr
100 105 110
Pro Val Leu Val Trp Ile Tyr Gly Gly Gly Phe Tyr Ser Gly Ala Ser
115 120 125
Ser Leu Asp Val Tyr Asp Gly Arg Phe Leu Val Gln Ala Glu Arg Thr
130 135 140
Val Leu Val Ser Met Asn Tyr Arg Val Gly Ala Phe Gly Phe Leu Ala
145 150 155 160
Leu Pro Gly Ser Arg Glu Ala Pro Gly Asn Val Gly Leu Leu Asp Gln
165 170 175
Arg Leu Ala Leu Gln Trp Val Gln Glu Asn Val Ala Ala Phe Gly Gly
180 185 190
Asp Pro Thr Ser Val Thr Leu Phe Gly Gln Ser Ala Gly Ala Ala Ser
195 200 205
Val Gly Met His Leu Leu Ser Pro Pro Ser Arg Gly Leu Phe His Arg
210 215 220
Ala Val Leu Gln Ser Gly Ala Pro Asn Gly Pro Trp Ala Thr Val Gly
225 230 235 240
Met Gly Glu Ala Arg Arg Arg Ala Thr Gln Leu Ala His Leu Val Gly
245 250 255
Cys Pro Pro Gly Gly Thr Gly Gly Asn Asp Thr Glu Leu Val Ala Cys
260 265 270
Leu Arg Thr Arg Pro Ala Gln Val Leu Val Asn His Glu Trp His Val
275 280 285
Leu Pro Gln Glu Ser Val Phe Arg Phe Ser Phe Val Pro Val Val Asp
290 295 300
Gly AspPhe Leu Ser Asp Thr Pro Glu Ala Leu Ile Asn Ala Gly Asp
305 310 315 320
Phe His Gly Leu Gln Val Leu Val Gly Val Val Lys Asp Glu Gly Ser
325 330 335
Tyr Phe Leu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp Asn Glu Ser
340 345 350
Leu Ile Ser Arg Ala Glu Phe Leu Ala Gly Val Arg Val Gly Val Pro
355 360 365
Gln Val Ser Asp Leu Ala Ala Glu Ala Val Val Leu His Tyr Thr Asp
370 375 380
Trp Leu His Pro Glu Asp Pro Ala Arg Leu Arg Glu Ala Leu Ser Asp
385 390 395 400
Val Val Gly Asp His Asn Val Val Cys Pro Val Ala Gln Leu Ala Gly
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Arg Leu Ala Ala Gln Gly Ala Arg Val Tyr Ala Tyr Val Phe Glu His
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Arg Ala Ser Thr Leu Ser Trp Pro Leu Trp Met Gly Val Pro His Gly
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Cys Ala Phe Trp Asn Arg Phe Leu Pro Lys Leu Leu Ser Ala Thr Asp
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Thr Leu Asp Glu Ala Glu Arg Gln Trp Lys Ala Glu Phe His Arg Trp
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Ser Ser Tyr Met Val His Trp Lys Asn Gln Phe Asp His Tyr Ser Lys
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Gln Asp Arg Cys Ser Asp Leu
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Claims (6)
1. A method for immobilizing acetylcholinesterase by compounding is characterized by comprising the following steps:
(1) constructing three-dimensional structure data of acetylcholinesterase, and performing molecular docking scoring on the small molecules and a region I and a region II on the acetylcholinesterase molecules respectively by using molecular docking software or a module to obtain a region I score and a region II score of the small molecules;
the region I is a region consisting of amino acids VAL330, VAL331, LYS332, ASP333, GLU334, GLY335, SER336, ARG395, GLU396, SER399, ASP400, GLY403, ASP404, VAL408, VAL429, GLU431, TRP442, MET443, GLY444, TYR510, LEU524 and ARG525 on an acetylcholinesterase molecule;
the region II is a region on an acetylcholinesterase molecule and composed of amino acids GLN71, TYR72, ASP74, GLY82, THR83, TRP86, ASN87, PRO88, GLY120, GLY121, GLY122, TYR124, SER125, GLY126, ALA127, LEU130, TYR133, GLN202, SER203, ALA204, SER229, GLY230, TRP236, TRP286, VAL294, PHE295, ARG296, PHE297, TYR337, PHE338, TYR341, VAL407, TRP439, MET443, PRO446, HIS447, TYR 448, TYR449 and ILE 451;
the molecular docking scoring is to calculate the binding constant of the obtained small molecule and the acetylcholinesterase region site according to the molecular docking;
(2) calculating the total score of the small molecules in the step (1), and classifying the small molecules into preferential small molecules, good small molecules and eliminated small molecules according to the total score;
the method for calculating the total score of the small molecules comprises the following steps: if the score of the area II of the small molecule is more than or equal to zero, the total score of the small molecule is equal to the score of the area I minus the score of the area II; if the score of the region II of the small molecule is less than zero, the total score of the small molecule is equal to the score of the region I of the small molecule;
the method for classifying the small molecules comprises the following steps: if the total score of the small molecules is more than 7.0, the small molecules are priority small molecules; if the total score of the small molecules is more than or equal to 4.5 and less than or equal to 7.0, the small molecules are good class small molecules; if the total score of the small molecules is less than 4.5, the small molecules are eliminated small molecules;
(3) connecting the preferential small molecules or the good small molecules in the step (2) with the surface of an immobilized carrier, and then incubating with acetylcholinesterase to obtain composite immobilized acetylcholinesterase;
the connection between the small molecules and the surface of the immobilized carrier is direct, indirect or compound chemical connection or physical connection.
2. The composite immobilization method according to claim 1, wherein the acetylcholinesterase is human acetylcholinesterase.
3. The composite immobilization method according to claim 1, wherein the three-dimensional structure data of acetylcholinesterase in step (1) is obtained from public data or is constructed by INSIGHTii and Modeller software.
4. The composite immobilization method according to claim 1, wherein the small molecule in step (1) is a molecule or a functional group that is located at the end of the immobilization carrier and is capable of interacting with acetylcholinesterase molecules, and has a molecular weight of 50-8000 Da.
5. The composite immobilization method of claim 1, wherein the molecular docking software or module in step (1) is Dock, 3D-DOCK, AutoDock, Surflex, Glide, Gold, FlexX, Z-DOCK, FTDOCK, MolegroVirtual Docker, or Affinity molecular simulation software, module, or program.
6. The composite immobilization method according to claim 1, wherein the immobilization support in step (3) is a polymer, an electrode, a mesoporous material, a nanomaterial, an inorganic material, or a composite material.
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