A method of assessing selectivity of lipases towards fatty acids in enzymatic reactions

Abstract

The application discloses a kind of methods for evaluating the selectivity of lipase to fatty acid in enzymatic esterification reaction, including searching or constructing the 3D structure of lipase and fatty acid, idealizing the 3D structure of receptor lipase and ligand fatty acid and pretreating before docking, simulating docking by molecular docking software, obtaining the docking complex structure of substrate-enzyme, processing docking results using docking visualization software, and comprehensively analyzing the docking binding energy and hydrogen bond formation of different lipases and different fatty acids, to analyze the selectivity of lipase to fatty acid. The application uses molecular docking method to evaluate the selectivity of lipase to fatty acid in enzymatic reaction, significantly solves the problem of large experimental investment in lipase screening, greatly improves the efficiency of lipase screening and evaluation, and greatly shortens the screening time of the most suitable lipase in industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lipase application, and particularly relates to a method for evaluating the fatty acid selectivity of lipase in an enzymatic reaction. BACKGROUND

[0002] Lipase (EC 3.1.1.3), also known as glycerol ester hydrolase, is an important industrial hydrolytic enzyme, which can be used to catalyze esterification, transesterification, esterolysis and other reactions, and is widely used in the industrial production of food and medicine. Lipase can offset the activation energy required for the reaction through the binding energy generated by the combination with the substrate, thereby reducing the reaction energy barrier and improving the reaction efficiency. There are various types of lipases, and different lipases have great differences in positional selectivity and substrate selectivity. Therefore, in production, the best specific lipase needs to be selected according to the specific situation, and the unsuitable enzyme not only cannot efficiently catalyze the reaction, but also produces adverse by-products. However, the current screening of lipase is mainly carried out through single factor parallel experiment, which is reliable in result, but consumes a large amount of experimental time and wastes expensive enzyme raw materials, and is not an ideal enzyme screening method.

[0003] Molecular docking is a computational chemistry method that can simulate intermolecular interactions based on structure and predict the binding ability between receptors and ligands. This technology has been widely used in drug development and target prediction research. With the development of molecular docking software, the accuracy of docking has been continuously improved, and the binding energy algorithm has become increasingly accurate, which has become an important way to evaluate the binding ability between receptors and ligands. In enzymatic reactions, the catalytic mechanism of lipase is complex, and the catalytic triad in the enzyme catalyzes the reaction of the substrate by inducing the formation of a tetrahedral transition state. This process can be abstracted and simplified as a typical binding model of receptor and ligand, and the docking situation can be simulated using molecular docking model, so that the most suitable lipase can be screened based on structure and intermolecular forces without actual experiment. Therefore, using molecular docking to evaluate the fatty acid selectivity of lipase has the advantages of less experimental demand, high docking efficiency and reliable docking results, and is a breakthrough method for screening the most suitable lipase and evaluating the fatty acid selectivity of lipase. However, due to the short development time of molecular docking, the high learning cost of docking software, the unknown parameters of simulated lipase, and the barriers with related industries such as lipase, there is currently no related research on using molecular docking to evaluate the fatty acid selectivity of lipase. SUMMARY

[0004] This section aims to summarize some aspects of the embodiments of the application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the application.

[0005] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0006] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a method for evaluating the selectivity of lipase to fatty acid in an enzymatic reaction.

[0007] To solve the above technical problems, the present application provides the following technical solutions: comprising,

[0008] According to the protein source, amino acid sequence and element composition, the 3D structures of lipase and fatty acid are searched or constructed, and the structure rationality is verified;

[0009] The 3D structures of lipase and fatty acid are respectively idealized and pretreated before docking;

[0010] Through molecular docking software, semi-flexible docking is performed with lipase as the receptor and fatty acid as the ligand;

[0011] The docking results are processed using docking visualization software, and the docking binding energy, hydrogen bond formation and hydrophobic interaction of different lipases and different fatty acids are comprehensively analyzed to accurately evaluate the selectivity of lipase to fatty acid in the enzymatic reaction.

[0012] As a preferred scheme of the method for evaluating the selectivity of lipase to fatty acid in the enzymatic reaction according to the present application, wherein: the lipase is a lipase with known amino acid sequence or known structure.

[0013] As a preferred scheme of the method for evaluating the selectivity of lipase to fatty acid in the enzymatic reaction according to the present application, wherein: the fatty acid is a fatty acid with known structure.

[0014] As a preferred scheme of the method for evaluating the selectivity of lipase to fatty acid in the enzymatic reaction according to the present application, wherein: the enzymatic reaction includes one of enzymatic esterification reaction, enzymatic transesterification reaction, enzymatic alcoholysis reaction, and enzymatic hydrolysis reaction.

[0015] As a preferred scheme of the method for evaluating the selectivity of lipase to fatty acid in the enzymatic reaction according to the present application, wherein: the 3D structures of lipase and fatty acid are searched and constructed, wherein the search database includes one or more of PDB, PubChem, UniProt database, and the construction software includes one of Chem3D, YASARA, and Chimera.

[0016] As a preferred scheme of the method for evaluating the selectivity of lipase to fatty acid in the enzymatic reaction according to the present application, wherein: the verification software for verifying the structure rationality includes one or more of PROCHECK, Verify 3D and QMEAN.

[0017] As a preferred solution of the method for evaluating the selectivity of lipase to fatty acid in enzymatic reaction, wherein: the processing mode of the idealization processing and the pre-docking processing includes one or more of removing water molecules, adding polar hydrogen atoms, calculating electrons, and energy minimization, and the processing software used includes one of AutodockTools, Chimera, and GROMACS.

[0018] As a preferred solution of the method for evaluating the selectivity of lipase to fatty acid in enzymatic reaction, wherein: the docking operation of the semi-flexible docking includes one or more of setting a receptor and a ligand, setting a ligand torsion bond, setting a docking box, and running docking, and the number of times of the semi-flexible docking is 1-200.

[0019] As a preferred solution of the method for evaluating the selectivity of lipase to fatty acid in enzymatic reaction, wherein: the software of the molecular docking includes one of Gold, Flex X, AutoDock, and Autodock Vina.

[0020] As a preferred solution of the method for evaluating the selectivity of lipase to fatty acid in enzymatic reaction, wherein: the docking visualization software includes one or more of PyMOL, LigPlus, and AutoDock.

[0021] The present application has the following beneficial effects:

[0022] The present application uses the molecular docking method to evaluate the selectivity of lipase to fatty acid in enzymatic reaction, breaks through the traditional method of judging the selectivity of lipase only through actual experiments and experience in the past, uses structure-based computer simulation to significantly improve the efficiency of evaluating the selectivity of lipase, greatly shortens the time of screening lipase, and strengthens the reliability of lipase screening. It is a major breakthrough in the screening of the best enzymatic reaction lipase in industrial production and scientific research, and lays a foundation for further research of molecular docking in the field of lipase screening and research. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the description examples.

[0024] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, therefore the present application is not limited to the specific embodiments disclosed below.

[0025] Second, the "one embodiment" or "an embodiment" described herein refers to a particular feature, structure, or characteristic described in one or more implementations. The appearances of "in one embodiment" or "in an embodiment" in various places in the specification are not necessarily all referring to the same embodiment nor are they necessarily all referring to a single, alternative embodiment planarly exclusive of the other embodiments.

[0026] In the present application, the content of triglyceride in the actual experimental reaction product is detected by HPLC-RID. Before HPLC-RID analysis, 20 mg of the reaction product is weighed, 1 mL of mobile phase is added to dissolve the sample, and then the sample is analyzed by HPLC-RID after passing through a 0.22 μm organic filter membrane;

[0027] Chromatographic conditions: silica gel column (4.6 mm x 250 mm x 5 μm, Phenomenex, USA), column temperature 30°C, mobile phase flow rate 1.0 mL / min, sample mass concentration 20 mg / mL, injection volume 20 μL, mobile phase n-hexane / isopropanol / formic acid (15:1:0.003, v / v / v). Each component is qualitatively analyzed by using a standard sample, and quantitatively analyzed by using the area normalization method.

[0028] Unless otherwise specified, the software and materials used in the examples are commercially available.

[0029] Example 1

[0030] The present embodiment provides a method for evaluating the selectivity of lipase to fatty acid in an enzymatic reaction, which can be used for all lipases with known amino acid sequences or known structures, and known structures of fatty acids in enzymatic esterification, enzymatic transesterification, enzymatic alcoholysis, and enzymatic hydrolysis. The specific steps are as follows:

[0031] S1: Search or construct 3D structures of lipase and fatty acid with high accuracy according to the protein source, amino acid sequence, and element composition, and verify the structural rationality;

[0032] The searched database can be one or more of PDB, PubChem, and UniProt database, the software used for constructing 3D structure can be one of Chem3D, YASARA, and Chimera, and the verification software used for verifying the structural rationality can be one or more of PROCHECK, Verify 3D, and QMEAN.

[0033] S2: Respectively, idealize the 3D structure of lipase and fatty acid and perform pre-processing before docking;

[0034] Specifically, the processing includes one or more of removing water molecules, adding polar hydrogen atoms, calculating electrons, and energy minimization, and the processing software can be one of AutodockTools, Chimera, and GROMACS.

[0035] S3: performing semi-flexible docking by taking the lipase as a receptor and the fatty acid as a ligand through a molecular docking software;

[0036] Specifically, the docking operation includes one or more of setting the receptor and the ligand, setting the torsion bond of the ligand, setting the docking box, and running the docking, the number of docking is 1-200 times, and the docking software can be one of Gold, Flex X, AutoDock, and AutodockVina.

[0037] S4: processing the docking result by using a docking visualization software;

[0038] Specifically, the visualization software can be one of PyMOL, LigPlus, and AutoDock, and the docking binding energy, hydrogen bond formation, and hydrophobic interaction of different lipases and different fatty acids are comprehensively analyzed to realize accurate evaluation of the selectivity of the lipase to the fatty acid in the enzymatic reaction.

[0039] Embodiment 2

[0040] To verify the effectiveness of the scheme, the selectivity of different lipases to branched fatty acids in the enzymatic esterification reaction is evaluated to screen the most suitable lipase for the production of branched fatty acid glycerides by the enzymatic esterification reaction, specifically as follows:

[0041] 1) According to the protein source, amino acid sequence, and element composition, the 3D structures of the required lipases (Lipozyme 435, Lipozyme RMIM, and NS40086) and fatty acids (iso-C15:0 and anteiso-C15:0) are searched from PDB, PubChem, and UniProt databases;

[0042] 2) The ligand and receptor structures are introduced into AutodockTools, the lipase model is processed by removing water, adding polar hydrogen atoms, and calculating electrons, and the torsion bond of the fatty acid model is set, and the two are taken as the initial structure for docking;

[0043] 3) In AutodockTools, the lipase structure is set as rigid, the fatty acid molecule is set as flexible, the catalytic triad of the enzyme is taken as the center, and the size is set as 30A x 30A x 30A; The network grid is used as a docking space, 10 docking results are generated each time, the maximum energy difference between the best and worst binding results is 4 kcal / mol, 10 docking operations are performed each time, the results of successful docking of glycerides and the active site of the lipase catalytic triad are selected from the docking results, and the average value of the binding energy is taken to evaluate the selectivity of the lipase to different fatty acids;

[0044] 4) 3D and 2D rendering, data processing and geometric analysis of the molecular docking results are performed using PyMOL respectively, the interaction between fatty acids and amino acid residues in the active site of the lipase is visualized, the binding energy of different fatty acids and the lipase residues is comprehensively analyzed, the selectivity of the lipase to fatty acids is clarified, and the binding energy of three different lipases to three fatty acids is shown in Table 1;

[0045] Table 1

[0046]

[0047] It can be seen that the docking results of Lipozyme 435 and the two branched fatty acids in the three enzymes have smaller binding energy, which indicates that the enzyme can release more energy when it binds to the two branched fatty acids, thereby reducing the activation energy required for the reaction and accelerating the esterification reaction rate of branched fatty acids and glycerol.

[0048] 5) The accuracy of the evaluation of the selectivity of the lipase to fatty acids by molecular docking is verified by comparing the catalytic experimental results of the lipase in the actual experiment, and the specific experimental steps are as follows:

[0049] 2g of fatty acids is weighed in a sandwich enzyme reactor, glycerol (glycerol:fatty acid = 3:1, mol / mol) is added, a magnetic stirrer is used for stirring, the system temperature is allowed to reach 70℃, and then 5wt% of the three lipases (Lipozyme 435, Lipozyme RMIM and NS40086) are added, the product is taken out after 12h of reaction, 4000rpm centrifugation is performed for 10min to remove the lipase, and the content of branched fatty acid triglycerides obtained by the catalysis of the three enzymes is shown in Table 2.

[0050] Table 2

[0051] Lipase type Lipozyme 435 NS40086 Lipozyme RM IM Branched fatty acid triglycerides (%) 44.8 35.5 39.7

[0052] By comparing the results in Table 1 and Table 2, it can be shown that the selectivity of the three enzymes to anteiso-C15:0 and iso-C15:0, two branched fatty acids, is Lipozyme 435>Lipozyme RM IM>NS40086 from large to small, Lipozyme 435 is the most suitable lipase for the preparation of branched fatty acid triglycerides by enzymatic esterification reaction among the three enzymes, and the effectiveness of the method is proved.

[0053] Example 3

[0054] This example is to screen the optimal lipase for the production of polyunsaturated fatty acid glycerides by enzymatic alcoholysis reaction. The difference from Example 1 is only that the lipase species in step 1) is immobilized lipase Eversa Transform 2.0, PS "Amano" SD and AK "Amano", the fatty acid species is adjusted to EPA (C20:5 n-3) and DHA (C22:5 n-3), and the docking of the above three enzymes with fatty acids is simulated by molecular docking, and the resulting binding energy is shown in Table 3.

[0055] Table 3

[0056]

[0057] The fatty acid sample in step 5) is adjusted to fish oil sample, the glycerol is adjusted to 60 wt% ethanol aqueous solution, the reaction temperature is adjusted to 40°C, the lipase species is adjusted to immobilized Eversa Transform 2.0, PS "Amano" SD and AK "Amano", and the polyunsaturated fatty acid glyceride content is shown in Table 4.

[0058] Table 4

[0059] Lipase type Eversa Transform 2.0 PS "Amano" SD AK "Amano" Polyunsaturated fatty acid glycerides (%) 32.9 25.7 28.2

[0060] It can be seen that the selectivity of the three enzymes on EPA and DHA, two polyunsaturated fatty acids, is Eversa Transform 2.0 > AK "Amano" > PS "Amano" SD, and Eversa Transform 2.0 is the optimal lipase for the production of polyunsaturated fatty acid glycerides by enzymatic alcoholysis reaction among the three enzymes, which is consistent with the actual experimental results (Table 4).

[0061] Example 4

[0062] This example is to screen the optimal lipase for the production of conjugated linoleic acid glycerides by enzymatic interesterification reaction. The difference from Example 1 is only that the lipase species in step 1) is adjusted to Lipozyme RM IM, Lipozyme TL IM and Novozyme 435, the fatty acid species is adjusted to 9c,11t-CLA and 10t,12c-CLA, and the docking of the above three enzymes with fatty acids is simulated by molecular docking, and the resulting binding energy is shown in Table 5.

[0063] Table 5

[0064]

[0065] The fatty acid sample in step 5) is adjusted to be conjugated linoleic acid methyl ester, the glycerol is adjusted to be soybean oil, and the lipase species is adjusted to be Lipozyme RM IM, Lipozyme TL IM and Novozyme 435, to obtain the content of conjugated linoleic acid triglyceride as shown in Table 6;

[0066] Table 6

[0067] Lipase type Lipozyme RM IM Lipozyme TL IM Novozyme 435 Conjugated linoleic acid triglycerides (%) 45.7 40.6 50.1

[0068] It can be seen that the selectivity of the three enzymes to conjugated linoleic acid is Novozyme 435>Lipozyme RM IM>Lipozyme TL IM, and Novozyme 435 is the most suitable lipase for the preparation of conjugated linoleic acid glyceride by enzymatic transesterification reaction among the three enzymes, which is consistent with the actual experimental results (Table 6).

[0069] Example 5

[0070] This example is used to screen the most suitable lipase for preparing α-linolenic acid by enzymatic hydrolysis of flaxseed oil. The difference from Example 1 is only that the lipase species in step 1) is adjusted to be Lipase-AY, Lipozyme RM IM, Lipozyme TL IM and Novozym 435; the fatty acid species is adjusted to be α-linolenic acid (C18:3 n-3); glycidyl oleate, glycidyl palmitate and glycidyl linoleate are constructed by Chem3D, and the docking of the above four enzymes with α-linolenic acid is simulated by molecular docking, and the binding energy is shown in Table 7.

[0071] Table 7

[0072]

[0073] The fatty acid sample in step 5) is adjusted to be α-linolenic acid glyceride sample, and the glycerol is adjusted to be pH=6 phosphate buffer, and the lipase species is adjusted to be Lipase-AY, Lipozyme RM IM, Lipozyme TL IM and Novozym 435, to obtain the content of α-linolenic acid as shown in Table 8;

[0074] Table 8

[0075] Lipase type Lipase-AY Lipozyme RM IM Lipozyme TL IM Novozym 435 Alpha-linolenic acid (%) 73.5 48.1 57.2 53.6

[0076] It can be seen that the selectivity of the four enzymes to alpha-linolenic acid is Lipase-AY>Lipozyme TL Novozym435>Lipozyme RM IM, respectively, and Lipase-AY is the most suitable lipase for the enzymatic hydrolysis reaction of preparing alpha-linolenic acid from linseed oil among the four enzymes, which is consistent with the actual experimental results (Table 8).

[0077] Comparative Example 1

[0078] The difference between the present comparative example and Example 2 is that the operation of calculating electrons in the pre-treatment of molecular docking in step 2) is removed, and the docking space network grid size in step 3) is adjusted to The rest of the step conditions are the same as those in Example 2, and the binding energies of three different lipases to three fatty acids are shown in Table 9;

[0079] Table 9

[0080]

[0081] The results in Table 9 show that the binding energies obtained by adjusting the molecular docking process parameters in the present comparative example are all positive numbers, which have no actual reference significance, indicating that the pre-treatment (water removal, hydrogen addition, calculation of electrons, etc.) and the docking parameter setting (docking box, flexibility or rigidity, etc.) of the lipase in the present method have a significant influence on the docking results, and improper treatment cannot obtain effective binding energy results.

[0082] In summary, the present application uses the molecular docking method to evaluate the fatty acid selectivity of lipase in the enzymatic reaction, which significantly solves the problems of large experimental demand and high detection cost in the screening experiment of lipase, breaks through the limitation that a large number of actual experiments are necessary for screening lipase in the traditional lipase industry, greatly improves the screening efficiency of lipase in the enzymatic reaction industry, greatly shortens the screening time of lipase, significantly improves the accuracy of lipase screening, and is a major breakthrough in solving the complex, time-consuming and costly problems of lipase screening process. It lays a foundation for further research of molecular docking in the field of evaluation of lipase selectivity.

[0083] The present application uses the molecular docking method, takes lipase as the receptor and takes fatty acid as the ligand, simulates the binding process of lipase and fatty acid substrate based on structure, water transport interaction and hydrogen bond, etc., and calculates the binding energy of lipase and substrate to evaluate the catalytic activity of lipase, which successfully applies molecular docking to the exploration of fatty acid selectivity of lipase for the first time, and opens up a new method for the research of fatty acid selectivity of lipase.

[0084] The method of screening the optimal lipase for enzymatic reaction by molecular docking is effective to obtain the optimal lipase result, which is highly consistent with the result of actual experiment screening lipase, and has no loss required by actual experiment, and is a high-efficiency and accurate method of screening and evaluating lipase.

[0085] The molecular docking effectively quantifies the fatty acid selectivity of lipase in the form of accurate and representative binding energy, clearly and intuitively shows the selectivity difference of lipase for different fatty acids, and is conducive to researchers and relevant personnel to screen and evaluate the fatty acid selectivity of lipase in detail and efficiently.

[0086] The molecular docking screening the fatty acid selectivity of lipase in enzymatic reaction has wide application range, is suitable for esterification reaction, ester exchange reaction, alcoholysis reaction, hydrolysis reaction, glycerolysis reaction and almost all enzymatic reactions catalyzed by lipase, and can be widely promoted and applied.

[0087] It should be noted that the above examples are only used to illustrate the technical solutions of the present application, not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method of assessing the selectivity of a lipase towards fatty acids in an enzymatic reaction, characterized in that: The method is used for screening the optimum lipase for producing branched fatty acid glyceride by evaluating the selectivity of different lipases to branched fatty acids in the production of enzymatic esterification reaction, and the steps are as follows: S1: searching the 3D structure of the required lipase and fatty acid from the PDB, PubChem and UniProt databases according to the protein source, amino acid sequence and element composition; S2: importing the above ligand and receptor structure into the software for idealization processing and pre-processing of docking, processing the lipase model by removing water, adding polar hydrogen atoms and calculating electrons, and setting the torsional bond of the fatty acid model, and taking the two as the starting structure for docking; S3: setting the lipase structure as rigid and the fatty acid molecule as flexible in AutodockTools, setting the network grid with a size of 40Åx40Åx40Å as the docking space with the catalytic triad of the enzyme as the center, generating 10 docking results each time, and the maximum energy difference between the best and worst binding results is 4 kcal / mol, and each docking is run for 1-200 times, and the results of successful docking of glyceride and the active site of the catalytic triad of the lipase are selected, and the average binding energy is taken to evaluate the selectivity of the lipase to different fatty acids; S4: using docking visualization software to perform 3D and 2D rendering, data processing and geometric analysis on the molecular docking results, visualizing the interaction between the fatty acid and the amino acid residues in the active site of the lipase, and comprehensively analyzing the binding energy and other indicators of different fatty acids and lipase residues; S5: verifying the accuracy of the molecular docking in evaluating the selectivity of the lipase to fatty acids by comparing the catalytic experimental results of the lipase in the actual experiment, and the specific experimental steps are as follows: The fatty acid is placed in a sandwich enzyme reactor, glycerol is added according to a molar ratio of 3:1, a magnetic stirrer is used for stirring, the system temperature is allowed to reach 70℃, 5 wt% of the lipase to be tested is added, the product is taken out after 12 h of reaction, and the lipase is removed by centrifugation at 4000 rpm for 10 min; The content of triglyceride in the reaction product is detected by HPLC-RID, and before HPLC-RID analysis, 20 mg of the reaction product is weighed, 1 mL of mobile phase is added to dissolve the sample, and after passing through a 0.22 μm organic filter membrane, the sample is analyzed by HPLC-RID; The chromatographic conditions are as follows: 4.6 mmx250 mmx5 μm, silica gel column, column temperature is 30℃, flow rate of mobile phase is 1.0 mL / min, sample mass concentration is 20 mg / mL, injection volume is 20 μL, mobile phase is n-hexane / isopropyl alcohol / formic acid with a volume ratio of 15:1:0.003, each component is qualitatively analyzed by using standard samples, and is quantitatively analyzed by using area normalization method, and finally the content of branched fatty acid glyceride catalyzed by different enzymes is obtained, and the selectivity of the lipase to different fatty acids is screened.

2. The method of assessing selectivity of a lipase towards fatty acids in an enzymatic reaction according to claim 1, characterized in that: The lipase is a lipase with known amino acid sequence or known structure, including Lipozyme 435, Lipozyme RMIM, NS40086.

3. The method of assessing selectivity of a lipase towards fatty acids in an enzymatic reaction according to claim 1, wherein: The fatty acids are structurally known fatty acids, including iso-C15:0 and anteiso-C15:

0.

4. The method of assessing selectivity of a lipase towards a fatty acid in an enzymatic reaction according to any one of claims 1 to 3, wherein: The enzymatic reaction includes one of an enzymatic esterification reaction, an enzymatic transesterification reaction, an enzymatic alcoholysis reaction, and an enzymatic hydrolysis reaction.

5. The method of assessing selectivity of a lipase towards fatty acids in an enzymatic reaction as claimed in claim 1, wherein: The software for idealization processing and pre-docking processing includes one of Autodock Tools, Chimera, and GROMACS.

6. The method of assessing selectivity of a lipase towards fatty acids in an enzymatic reaction as claimed in claim 1, wherein: The docking visualization software includes one or several of PyMOL, LigPlus, and AutoDock.

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