Novel tool enzyme combination for improving biodiesel conversion rate and application

Abstract

The invention discloses a novel tool enzyme combination for improving the biodiesel conversion rate and application, relates to the technical field of tool enzymes, and aims to solve the technical problems that existing lipase is high in monovalence, long in production period and large in energy consumption. S1.1, sample collection: collecting microbial samples from ecological environments such as soil, water bodies and plant rhizospheres in different regions in the world, and ensuring diversity; s1.2, primary screening: carrying out primary screening by utilizing an agar culture medium containing triglyceride, and observing the growth condition of microorganisms and the degradation capability of the microorganisms on the triglyceride; and S1.3, secondary screening: the microorganisms obtained through primary screening are inoculated into a reaction system containing standard triglyceride and a non-natural substrate, the standard triglyceride is soybean oil triglyceride, the non-natural substrate is a fatty acid methyl ester mixture, and culture is conducted under the conditions that the temperature ranges from 20 DEG C to 45 DEG C and the pH ranges from 6.2 to 9.2. The method has the advantage of carrying out directed evolution on the lipase.

Description

Technical Field

[0001] The present invention relates to the technical field of tool enzymes, and more specifically to a novel tool enzyme combination and application for improving biodiesel conversion rate. Background Art

[0002] The demand for fuel in industrial society continues to rise, while the reserves of non-renewable fossil fuels are decreasing, and the adverse consequences of large carbon emissions from fossil fuels have become apparent. Finding a better and cleaner fuel is necessary for environmental protection and relieving the pressure on fossil fuels. Biodiesel is a green and safe fuel that can be made from edible and inedible vegetable oils, animal fats, waste cooking oils and many other oils of poor quality.

[0003] Currently, the commercialized biodiesel production methods include chemical transesterification, enzyme catalysis, and whole-cell catalysis. The chemical transesterification method requires strong acid, alkali, high temperature and other conditions. The production conditions are harsh, the energy consumption is high, and an excess of methanol is required to participate in the reaction. The essence of whole-cell catalysis is to use intracellular or extracellular enzymes to complete the catalysis. Cell lysis is inevitable during the catalytic process, and intracellular substances are released into the reaction system, which brings inconvenience to the subsequent product extraction. In comparison, enzymatic catalysis has mild catalytic conditions, high catalytic efficiency, high process controllability, and low cost. It is the most ideal production method, and this method is also the current mainstream production method.

[0004] At present, there are still some disadvantages in the enzymatic production of biodiesel, such as the high unit price of lipase, long production cycle, and high energy consumption. Therefore, it is imperative to develop more advanced enzymes for biodiesel production. In view of this, we propose a new tool enzyme combination and application to improve the conversion rate of biodiesel. Summary of the invention

[0005] The purpose of the present invention is to provide a new type of tool enzyme combination and application for improving the conversion rate of biodiesel, so as to solve the technical problems of high unit price, long production cycle and high energy consumption of existing lipase.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a new tool enzyme combination and application for improving the biodiesel conversion rate, comprising the following steps:

[0007] S1. Extensive screening of enzymes;

[0008] S1.1. Sample collection:

[0009] Collect microbial samples from soil, water, plant rhizosphere and other ecological environments in different regions around the world to ensure diversity;

[0010] S1.2, initial screening:

[0011] A preliminary screening was conducted using agar medium containing triglycerides to observe the growth of microorganisms and their ability to degrade triglycerides;

[0012] S1.3, re-screening:

[0013] The microorganisms obtained in the initial screening are inoculated into a reaction system containing a standard triglyceride and a non-natural substrate, wherein the standard triglyceride is soybean oil triglyceride and the non-natural substrate is a fatty acid methyl ester mixture, and cultured at a temperature of 20°C to 45°C and a pH of 6.2 to 9.2, and the types and quantities of the products after the reaction and the substrate conversion rate are determined to screen out candidate enzyme sources with potential high-efficiency catalytic ability;

[0014] S1.4, Evaluation and Optimization:

[0015] Further evaluate the enzymatic properties of candidate enzyme sources, including thermal stability, pH stability, substrate specificity, etc., and select the optimal strain;

[0016] S2, Isolation and purification of candidate enzymes;

[0017] Cell disruption: Ultrasonic waves are used to disrupt microbial cells and release lipase in the cells;

[0018] Initial separation: Remove cell debris and most impurities by centrifugation to obtain a crude enzyme solution containing lipase;

[0019] Multi-stage chromatography: Use ion exchange chromatography, gel filtration chromatography and affinity chromatography to purify the crude enzyme solution step by step to remove impurities and improve enzyme purity. During the purification process, strictly control the temperature, pH value and ionic strength to avoid enzyme activity loss;

[0020] Enzyme activity and purity detection: Enzyme activity determination method and protein purity detection technology are used to detect the activity and purity of the purified lipase to ensure that it meets the requirements of subsequent applications. The protein purity detection technology is SDS-PAGE electrophoresis;

[0021] S3, directed evolution and combination of enzymes;

[0022] Gene cloning and sequencing: Extract DNA from the purified lipase, perform gene cloning and sequencing, and obtain complete gene sequence information;

[0023] Directed evolution strategy design: Based on gene sequence information and enzyme property evaluation results, design directed evolution strategies such as site-directed mutagenesis and gene recombination to improve the catalytic efficiency, stability and substrate selectivity of the enzyme;

[0024] Mutant construction and screening: A mutant library is constructed through genetic engineering, and screening is performed under high temperature, high alcohol concentration, and the presence of non-natural substrates to select mutants with excellent performance;

[0025] Enzyme combination optimization: The selected mutants are subjected to combination tests, and the catalytic performance of the combined enzymes under different reaction conditions is measured to determine the optimal combination ratio and form a new tool enzyme combination with synergistic effects;

[0026] Preferably, in S1, a microfluidic chip is used for single cell culture and screening, each microchamber independently cultures a microbial cell, and cells with efficient catalytic ability are quickly identified by fluorescent labeling;

[0027] Combined with automated liquid handling systems and robotic operations, automatic sample loading, reaction, detection and data analysis can be achieved, significantly improving screening throughput and accuracy.

[0028] Preferably, in S3, deep sequencing technology is used to obtain gene sequence information of the mutant library, and bioinformatics analysis is combined to predict the effect of mutations on enzyme structure and function;

[0029] The three-dimensional structure of the target lipase is analyzed by cryo-electron microscopy to guide the design of site-directed mutagenesis and ensure the rationality and effectiveness of the mutation site.

[0030] Preferably, in S2, based on the amino acid sequence of the target lipase, short peptides or small molecule compounds that bind to it with high affinity are screened by computer simulation;

[0031] The screened ligands are chemically synthesized or genetically expressed, and their binding specificity and purification efficiency are verified through affinity purification experiments.

[0032] Preferably, in said S3, a disulfide bond stabilizing motif is introduced into the lipase gene to enhance the interaction and stability within the enzyme molecule;

[0033] Through gene fusion technology, lipase is connected to the stabilizing immunoglobulin Fc segment protein to form a fusion protein, thereby improving the enzyme's tolerance under extreme conditions.

[0034] A method for applying a novel tool enzyme combination for improving biodiesel conversion rate comprises the following steps:

[0035] S1, raw material pretreatment;

[0036] S1.1. Grease collection and classification:

[0037] Collect waste cooking grease, waste cooking oil and other waste animal and plant fats, classify them and remove impurities;

[0038] S1.2, degumming treatment:

[0039] Treat the oil with a degumming agent such as phosphate or citric acid at a temperature of 70°C-80°C to remove phospholipid impurities;

[0040] S1.3, deacidification treatment:

[0041] Adjust the pH value of oil to 4.5-5.0, use sodium hydroxide to neutralize and remove free fatty acids;

[0042] S1.4, Decolorization and deodorization:

[0043] Use activated carbon adsorbent to remove pigments and odor substances from grease;

[0044] S1.5, Dehydration treatment:

[0045] The water in the oil is removed by molecular sieve dehydration to reach the water content required for the reaction;

[0046] S2, enzyme combination addition and reaction condition optimization;

[0047] S2.1. Enzyme combination preparation:

[0048] The novel tool enzyme combination is prepared into a reaction solution according to an optimized ratio for later use;

[0049] S2.2, establishment of reaction system: the pretreated oil and fat are mixed with methanol or other low-carbon alcohols in a certain proportion, and an enzyme combination reaction solution or an immobilized enzyme carrier is added to form a reaction system;

[0050] S2.3, Optimization of reaction conditions:

[0051] The reaction is carried out at a temperature of 40°C-50°C and a pressure of 0.1MPa-1.0MPa, and the reaction conditions are optimized by adjusting the stirring speed, supplementing methanol, and adjusting the pH value to increase the conversion rate and reaction rate;

[0052] S3, reaction process monitoring;

[0053] S3.1, Online monitoring:

[0054] Use online monitoring equipment to monitor the temperature, pressure, reactant concentration and product generation of the reaction system in real time;

[0055] S3.2, Timed Sampling Analysis:

[0056] Take samples regularly during the reaction, analyze the composition and property changes of the samples, and evaluate the reaction progress and effect;

[0057] S3.3, Condition adjustment:

[0058] According to the monitoring and analysis results, timely adjust the reaction conditions to ensure efficient reaction;

[0059] S4, product separation and purification;

[0060] The biodiesel and glycerol phases were initially separated by centrifugal separation technology;

[0061] Biodiesel is purified using distillation technology to remove unreacted alcohols and water;

[0062] Introducing molecular sieves to remove trace catalyst residues and pigments in biodiesel and improve product purity;

[0063] S5, storage;

[0064] Store biodiesel samples under different temperature and light conditions and regularly test changes in their physical and chemical properties;

[0065] The antioxidant capacity and stability of biodiesel were evaluated by simulating long-term storage conditions through accelerated aging tests.

[0066] Preferably, in said S1;

[0067] Use ultrasonic waves to treat oils and fats, destroy the macromolecular structure in the oils and fats through the cavitation effect, and promote the separation of impurities;

[0068] Microwave heating technology is used to accelerate the deacidification, decolorization and dehydration process while keeping the nutritional components of the oil intact;

[0069] Supercritical carbon dioxide is used as a solvent for extraction and evolution to remove harmful substances and odor components in oils and fats.

[0070] Preferably, in said S2.3;

[0071] Combine JMP experimental design and statistical analysis software to conduct multi-factor and multi-level experimental design, reduce the number of experiments and improve optimization efficiency;

[0072] The artificial neural network model is used to learn and train the reaction data, establish a nonlinear relationship model between reaction conditions and product conversion rate, and predict the optimal combination of reaction conditions.

[0073] Preferably, in said S3;

[0074] The concentration changes of each component in the reaction system are monitored in real time using online chromatography analysis technology;

[0075] Set up an alarm system to automatically adjust when reaction conditions deviate from the set range.

[0076] Preferably, in said S4;

[0077] Deeply bleach biodiesel products to improve product transparency and color stability;

[0078] Introducing supercritical extraction technology to further purify biodiesel and remove trace impurities and by-products;

[0079] The purified biodiesel was subjected to long-term storage stability test to evaluate its reliability in long-term use.

[0080] Compared with the prior art, the present invention has the following beneficial effects:

[0081] The present invention collects microbial samples extensively, performs preliminary screening of microorganisms with triglyceride agar medium, and inoculates the preliminarily screened microorganisms into a reaction system containing standard triglycerides and non-natural substrates for rescreening, thereby obtaining candidate enzyme sources with potential high-efficiency catalytic ability, and evaluates the properties of the candidate enzyme sources with thermal stability, pH stability and substrate specificity, obtains high-activity and high-purity lipase through separation and purification technology, obtains gene sequence information by extracting DNA from the lipase for cloning and sequencing, designs directed evolution strategies of site-directed mutagenesis and gene recombination, constructs a mutant library through genetic engineering means, and screens the genes generated by the mutations under conditions of high temperature, high alcohol concentration and the presence of non-natural substrates after expression, thereby obtaining gene-directed evolved lipase, thereby solving the problems of high unit price, long production cycle and high energy consumption of existing lipase. DETAILED DESCRIPTION

[0082] In order to facilitate those skilled in the art to understand the technical solution of the present invention, the technical solution of the present invention is further explained in conjunction with the specification.

[0083] The preparation method of a novel tool enzyme combination for improving biodiesel conversion rate comprises the following steps:

[0084] S1. Extensive screening of enzymes;

[0085] S1.1. Sample collection:

[0086] Collect microbial samples from soil, water, plant rhizosphere and other ecological environments in different regions around the world to ensure diversity;

[0087] S1.2, initial screening:

[0088] A preliminary screening was conducted using agar medium containing triglycerides to observe the growth of microorganisms and their ability to degrade triglycerides;

[0089] S1.3, re-screening:

[0090] The microorganisms obtained in the initial screening are inoculated into a reaction system containing a standard triglyceride and a non-natural substrate, wherein the standard triglyceride is soybean oil triglyceride and the non-natural substrate is a fatty acid methyl ester mixture, and cultured at a temperature of 20°C to 45°C and a pH of 6.2 to 9.2, and the types and quantities of the products after the reaction and the substrate conversion rate are determined to screen out candidate enzyme sources with potential high-efficiency catalytic ability;

[0091] S1.4, Evaluation and Optimization:

[0092] Further evaluate the enzymatic properties of candidate enzyme sources, including thermal stability, pH stability, substrate specificity, etc., and select the optimal strain;

[0093] S2, Isolation and purification of candidate enzymes;

[0094] Cell disruption: Ultrasonic waves are used to disrupt microbial cells and release lipase in the cells;

[0095] Initial separation: Remove cell debris and most impurities by centrifugation to obtain a crude enzyme solution containing lipase;

[0096] Multi-stage chromatography: Use ion exchange chromatography, gel filtration chromatography and affinity chromatography to purify the crude enzyme solution step by step to remove impurities and improve enzyme purity. During the purification process, strictly control the temperature, pH value and ionic strength to avoid enzyme activity loss;

[0097] Enzyme activity and purity detection: Enzyme activity determination method and protein purity detection technology are used to detect the activity and purity of the purified lipase to ensure that it meets the requirements of subsequent applications. The protein purity detection technology is SDS-PAGE electrophoresis;

[0098] S3, directed evolution and combination of enzymes;

[0099] Gene cloning and sequencing: Extract DNA from the purified lipase, perform gene cloning and sequencing, and obtain complete gene sequence information;

[0100] Directed evolution strategy design: Based on gene sequence information and enzyme property evaluation results, design directed evolution strategies such as site-directed mutagenesis and gene recombination to improve the catalytic efficiency, stability and substrate selectivity of the enzyme;

[0101] Mutant construction and screening: A mutant library is constructed through genetic engineering, and screening is performed under high temperature, high alcohol concentration, and the presence of non-natural substrates to select mutants with excellent performance;

[0102] Enzyme combination optimization: The selected mutants are subjected to combination tests, and the catalytic performance of the combined enzymes under different reaction conditions is measured to determine the optimal combination ratio and form a new tool enzyme combination with synergistic effects;

[0103] In S1, microfluidic chips are used for single-cell culture and screening. Each microchamber independently cultures a microbial cell. Cells with efficient catalytic ability are quickly identified by fluorescent labeling. Combined with an automated liquid handling system and robot operation, automatic sample loading, reaction, detection and data analysis are achieved, significantly improving screening throughput and accuracy. In S3, deep sequencing technology is used to obtain gene sequence information of the mutant library, combined with bioinformatics analysis to predict the effect of mutations on enzyme structure and function, and the three-dimensional structure of the target lipase is analyzed by cryo-electron microscopy to guide the design of site-directed mutations to ensure the rationality and effectiveness of the mutation site. In S2, based on the amino acid sequence of the target lipase, short peptides or small molecule compounds that bind to it with high affinity are screened by computer simulation; the screened ligands are chemically synthesized or genetically engineered for expression, and their binding specificity and purification efficiency are verified by affinity purification experiments. In S3, a disulfide bond stabilizing motif is introduced into the lipase gene to enhance the interaction and stability within the enzyme molecule; the lipase is connected to the immunoglobulin Fc segment protein with a stabilizing effect through gene fusion technology to form a fusion protein to improve the tolerance of the enzyme under extreme conditions.

[0104] A method for applying a novel enzyme combination for improving biodiesel conversion rate comprises the following steps:

[0105] S1, raw material pretreatment;

[0106] S1.1. Grease collection and classification:

[0107] Collect waste cooking grease, waste cooking oil and other waste animal and plant fats, classify them and remove impurities;

[0108] S1.2, degumming treatment:

[0109] Treat the oil with a degumming agent such as phosphate or citric acid at a temperature of 70°C-80°C to remove phospholipid impurities;

[0110] S1.3, deacidification treatment:

[0111] Adjust the pH value of oil to 4.5-5.0, use sodium hydroxide to neutralize and remove free fatty acids;

[0112] S1.4, Decolorization and deodorization:

[0113] Use activated carbon adsorbent to remove pigments and odor substances from grease;

[0114] S1.5, Dehydration treatment:

[0115] The water in the oil is removed by molecular sieve dehydration to reach the water content required for the reaction;

[0116] S2, enzyme combination addition and reaction condition optimization;

[0117] S2.1. Enzyme combination preparation:

[0118] The novel tool enzyme combination is prepared into a reaction solution according to an optimized ratio for later use;

[0119] S2.2, establishment of reaction system: the pretreated oil and fat are mixed with methanol or other low-carbon alcohols in a certain proportion, and an enzyme combination reaction solution or an immobilized enzyme carrier is added to form a reaction system;

[0120] S2.3, Optimization of reaction conditions:

[0121] The reaction is carried out at a temperature of 40°C-50°C and a pressure of 0.1MPa-1.0MPa, and the reaction conditions are optimized by adjusting the stirring speed, supplementing methanol, and adjusting the pH value to increase the conversion rate and reaction rate;

[0122] S3, reaction process monitoring;

[0123] S3.1, Online monitoring:

[0124] Use online monitoring equipment to monitor the temperature, pressure, reactant concentration and product generation of the reaction system in real time;

[0125] S3.2, Timed Sampling Analysis:

[0126] Take samples regularly during the reaction, analyze the composition and property changes of the samples, and evaluate the reaction progress and effect;

[0127] S3.3, Condition adjustment:

[0128] According to the monitoring and analysis results, timely adjust the reaction conditions to ensure efficient reaction;

[0129] S4, product separation and purification;

[0130] The biodiesel and glycerol phases were initially separated by centrifugal separation technology;

[0131] Biodiesel is purified using distillation technology to remove unreacted alcohols and water;

[0132] Introducing molecular sieves to remove trace catalyst residues and pigments in biodiesel and improve product purity;

[0133] S5, storage;

[0134] Store biodiesel samples under different temperature and light conditions and regularly test changes in their physical and chemical properties;

[0135] The antioxidant capacity and stability of biodiesel were evaluated by simulating long-term storage conditions through accelerated aging tests.

[0136] In S1;

[0137] Use ultrasonic waves to treat oils and fats, destroy the macromolecular structure in the oils and fats through the cavitation effect, and promote the separation of impurities;

[0138] Microwave heating technology is used to accelerate the deacidification, decolorization and dehydration process while keeping the nutritional components of the oil intact;

[0139] Supercritical carbon dioxide is used as a solvent for extraction and evolution to remove harmful substances and odor components in oils and fats.

[0140] S2.3;

[0141] Combine JMP experimental design and statistical analysis software to carry out multi-factor and multi-level experimental design, reduce the number of experiments and improve optimization efficiency; use artificial neural network model to learn and train reaction data, establish a nonlinear relationship model between reaction conditions and product conversion rate, and predict the optimal combination of reaction conditions.

[0142] In S3;

[0143] The concentration changes of each component in the reaction system are monitored in real time using online chromatography analysis technology;

[0144] Set up an alarm system to automatically adjust when reaction conditions deviate from the set range.

[0145] S4 in;

[0146] Deeply bleach biodiesel products to improve product transparency and color stability;

[0147] Introducing supercritical extraction technology to further purify biodiesel and remove trace impurities and by-products;

[0148] The purified biodiesel was subjected to long-term storage stability test to evaluate its reliability in long-term use.

[0149] The embodiments of the present invention disclose preferred embodiments, but are not limited thereto. A person skilled in the art can easily understand the spirit of the present invention based on the above embodiments and make different extensions and changes. However, as long as they do not deviate from the spirit of the present invention, they are all within the protection scope of the present invention.

Claims

1. A method for preparing a novel enzyme combination for improving biodiesel conversion rate, characterized in that: The following steps are involved: S1. Extensive screening of enzymes; S1.

1. Sample collection: Collect microbial samples from soil, water, plant rhizosphere and other ecological environments in different regions around the world to ensure diversity; S1.2, initial screening: A preliminary screening was conducted using agar medium containing triglycerides to observe the growth of microorganisms and their ability to degrade triglycerides; S1.3, re-screening: The microorganisms obtained in the initial screening are inoculated into a reaction system containing a standard triglyceride and a non-natural substrate, wherein the standard triglyceride is soybean oil triglyceride and the non-natural substrate is a fatty acid methyl ester mixture, and cultured at a temperature of 20°C to 45°C and a pH of 6.2 to 9.2, and the types and quantities of the products after the reaction and the substrate conversion rate are determined to screen out candidate enzyme sources with potential high-efficiency catalytic ability; S1.4, Evaluation and Optimization: Further evaluate the enzymatic properties of candidate enzyme sources, including thermal stability, pH stability, substrate specificity, etc., and select the optimal strain; S2, Isolation and purification of candidate enzymes; Cell disruption: Ultrasonic waves are used to disrupt microbial cells and release lipase in the cells; Initial separation: Remove cell debris and most impurities by centrifugation to obtain a crude enzyme solution containing lipase; Multi-stage chromatography: Use ion exchange chromatography, gel filtration chromatography and affinity chromatography to purify the crude enzyme solution step by step to remove impurities and improve enzyme purity. During the purification process, strictly control the temperature, pH value and ionic strength to avoid enzyme activity loss; Enzyme activity and purity detection: Enzyme activity determination method and protein purity detection technology are used to detect the activity and purity of the purified lipase to ensure that it meets the requirements of subsequent applications. The protein purity detection technology is SDS-PAGE electrophoresis; S3, directed evolution and combination of enzymes; Gene cloning and sequencing: Extract DNA from the purified lipase, perform gene cloning and sequencing, and obtain complete gene sequence information; Directed evolution strategy design: Based on gene sequence information and enzyme property evaluation results, design directed evolution strategies such as site-directed mutagenesis and gene recombination to improve the catalytic efficiency, stability and substrate selectivity of the enzyme; Mutant construction and screening: A mutant library is constructed through genetic engineering, and screening is performed under high temperature, high alcohol concentration, and the presence of non-natural substrates to select mutants with excellent performance; Enzyme combination optimization: The screened mutants are subjected to combination tests, and the catalytic performance of the combined enzymes under different reaction conditions is measured to determine the optimal combination ratio and form a new tool enzyme combination with synergistic effects.

2. The method for preparing a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In S1, a microfluidic chip is used for single cell culture and screening, each microchamber independently cultures a microbial cell, and cells with efficient catalytic ability are quickly identified by fluorescent labeling; Combined with automated liquid handling systems and robotic operations, automatic sample loading, reaction, detection and data analysis can be achieved, significantly improving screening throughput and accuracy.

3. The method for preparing a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In S3, deep sequencing technology is used to obtain gene sequence information of the mutant library, and bioinformatics analysis is combined to predict the effect of mutations on enzyme structure and function; The three-dimensional structure of the target lipase is analyzed by cryo-electron microscopy to guide the design of site-directed mutagenesis and ensure the rationality and effectiveness of the mutation site.

4. The method for preparing a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In S2, based on the amino acid sequence of the target lipase, short peptides or small molecule compounds that bind to it with high affinity are screened by computer simulation; The screened ligands are chemically synthesized or genetically expressed, and their binding specificity and purification efficiency are verified through affinity purification experiments.

5. The method for preparing a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In said S3, a disulfide bond stabilizing motif is introduced into the lipase gene to enhance the interaction and stability within the enzyme molecule; Through gene fusion technology, lipase is connected to the stabilizing immunoglobulin Fc segment protein to form a fusion protein, thereby improving the enzyme's tolerance under extreme conditions.

6. An application method of a new type of enzyme combination for improving biodiesel conversion rate, characterized in that: The following steps are involved: S1, raw material pretreatment; S1.

1. Grease collection and classification: Collect waste cooking grease, waste cooking oil and other waste animal and plant fats, classify them and remove impurities; S1.2, degumming treatment: Treat the oil with a degumming agent such as phosphate or citric acid at a temperature of 70°C-80°C to remove phospholipid impurities; S1.3, deacidification treatment: Adjust the pH value of oil to 4.5-5.0, use sodium hydroxide to neutralize and remove free fatty acids; S1.4, Decolorization and deodorization: Use activated carbon adsorbent to remove pigments and odor substances from grease; S1.5, Dehydration treatment: The water in the oil is removed by molecular sieve dehydration to reach the water content required for the reaction; S2, enzyme combination addition and reaction condition optimization; S2.

1. Enzyme combination preparation: The novel tool enzyme combination is prepared into a reaction solution according to an optimized ratio for later use; S2.2, establishment of reaction system: the pretreated oil and fat are mixed with methanol or other low-carbon alcohols in a certain proportion, and an enzyme combination reaction solution or an immobilized enzyme carrier is added to form a reaction system; S2.3, Optimization of reaction conditions: The reaction is carried out at a temperature of 40°C-50°C and a pressure of 0.1MPa-1.0MPa, and the reaction conditions are optimized by adjusting the stirring speed, supplementing methanol, and adjusting the pH value to increase the conversion rate and reaction rate; S3, reaction process monitoring; S3.1, Online monitoring: Use online monitoring equipment to monitor the temperature, pressure, reactant concentration and product generation of the reaction system in real time; S3.2, Timed Sampling Analysis: Take samples regularly during the reaction, analyze the composition and property changes of the samples, and evaluate the reaction progress and effect; S3.3, Condition adjustment: According to the monitoring and analysis results, timely adjust the reaction conditions to ensure efficient reaction; S4, product separation and purification; The biodiesel and glycerol phases were initially separated by centrifugal separation technology; Biodiesel is purified using distillation technology to remove unreacted alcohols and water; Introducing molecular sieves to remove trace catalyst residues and pigments in biodiesel and improve product purity; S5, storage; Store biodiesel samples under different temperature and light conditions and regularly test changes in their physical and chemical properties; The antioxidant capacity and stability of biodiesel were evaluated by simulating long-term storage conditions through accelerated aging tests.

7. The application method of a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In said S1; Use ultrasonic waves to treat oils and fats, destroy the macromolecular structure in the oils and fats through the cavitation effect, and promote the separation of impurities; Microwave heating technology is used to accelerate the deacidification, decolorization and dehydration process while keeping the nutritional components of the oil intact; Supercritical carbon dioxide is used as a solvent for extraction and evolution to remove harmful substances and odor components in oils and fats.

8. The application method of the novel enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In said S2.3; Combine JMP experimental design and statistical analysis software to conduct multi-factor and multi-level experimental design, reduce the number of experiments and improve optimization efficiency; The artificial neural network model is used to learn and train the reaction data, establish a nonlinear relationship model between reaction conditions and product conversion rate, and predict the optimal combination of reaction conditions.

9. The application method of a novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In said S3; The concentration changes of each component in the reaction system are monitored in real time using online chromatography analysis technology; Set up an alarm system to automatically adjust when reaction conditions deviate from the set range.

10. The application method of the novel tool enzyme combination for improving biodiesel conversion rate according to claim 1, characterized in that: In said S4; Deeply bleach biodiesel products to improve product transparency and color stability; Introducing supercritical extraction technology to further purify biodiesel and remove trace impurities and by-products; The purified biodiesel was subjected to long-term storage stability test to evaluate its reliability in long-term use.