Method for designing feeding mode of enzyme preparation in enzymolysis process through computer, application and biomass conversion method

Abstract

The invention relates to renewable energy sources and bioengineering technologies, and discloses a method for designing an enzyme preparation feeding mode in an enzymolysis process through a computer, application and a biomass conversion method. The method for designing the feeding mode of the enzyme preparation comprises the following steps: respectively determining the activity of each enzyme component in the presence of an inhibitor in the enzymolysis process, and setting the concentration of the inhibitor to be a plurality of different simulation concentrations; obtaining a crystal structure of an enzyme component from the protein database, selecting a model of an inhibitor, and performing molecular dynamics simulation on the enzyme component when the concentration of the inhibitor is the simulated concentration; and combining the activity data of each enzyme component with a molecular dynamics simulation result, and proposing a material supplementing time period and a material supplementing amount of each enzyme component. According to the design method, the adding strategy of the enzyme preparation during biomass enzymolysis can be optimized, the tolerance of the enzyme preparation to an inhibitor formed in the pretreatment process is improved, the yield of monosaccharide is increased, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to renewable energy and bioengineering technologies, and particularly to a method for designing the feeding mode of enzyme preparations during enzymatic hydrolysis by computer, its application, and a method for biomass conversion. Background Art

[0002] In today's society, the increasing demand for energy and concerns about environmental and social problems caused by the use of fossil fuels have made the search for new forms of energy one of the focuses of social attention. As a large-scale renewable aromatic resource, lignocellulosic biomass has great potential in the sustainable production of chemicals, fuels, and materials.

[0003] In industrial biorefining, biomass pretreatment remains the most commonly used method to improve its enzymatic hydrolysis efficiency. Acid and alkali pretreatment methods are particularly favored because they can effectively decompose the lignocellulose structure and promote the access of enzymes to carbohydrates. However, these pretreatment processes often produce various toxic inhibitors, such as phenols, acetic acid, and furfural, which can seriously affect the downstream enzymatic hydrolysis and fermentation processes. Therefore, finding sustainable and cost-effective solutions to mitigate the toxicity generated during pretreatment remains a severe challenge for the industrial application of biomass conversion technologies. In practice, the industry usually tends to tolerate the presence of inhibitors or adopt pretreatment methods that produce fewer inhibitors, rather than using expensive detoxification steps in the actual production process.

[0004] Developing a method that can obtain a general enzyme preparation feeding strategy without detoxification steps and can be widely applied to various enzymatic hydrolysis systems is highly desirable. Summary of the Invention

[0005] The object of the present invention is to overcome the problem that toxic inhibitors are easily generated during the biomass enzymatic hydrolysis pretreatment process in the prior art, and to provide a method for designing the feeding mode of enzyme preparations during enzymatic hydrolysis by computer, its application, and a method for biomass conversion. This design method can optimize the addition strategy of enzyme preparations during biomass enzymatic hydrolysis, improve the tolerance of enzyme preparations to inhibitors formed during the pretreatment process, increase the yield of monosaccharides, reduce production costs, and thus improve the economic benefits of industrial production and subsequent application processes.

[0006] To achieve the above object, in the first aspect of the present invention, there is provided a method for designing the feeding mode of enzyme preparations during enzymatic hydrolysis by computer. The enzyme preparation contains multiple enzyme components, and the method includes:

[0007] (1) Under the condition of the presence of inhibitors in the enzymatic hydrolysis process, measure the activities of each of the enzyme components respectively, where the concentrations of the inhibitors are set to multiple different simulated concentrations;

[0008] (2) Obtain the crystal structure of the enzyme component from the protein database, select the model of the inhibitor, and perform molecular dynamics simulation of the enzyme component when the concentration of the inhibitor is the simulated concentration;

[0009] (3) Combine the activity data of each enzyme component obtained in step (1) with the molecular dynamics simulation results obtained in step (2), and propose the feeding time period and feeding amount of each enzyme component.

[0010] The second aspect of the present invention provides the application of the feeding method of the enzyme preparation in the enzymatic hydrolysis and / or bioconversion of biomass obtained by the method as described above.

[0011] The third aspect of the present invention provides a method for the conversion of biomass, which includes: performing acid pretreatment or alkali pretreatment on biomass to form a pretreatment solution; subjecting the pretreatment solution to enzymatic hydrolysis, and during the enzymatic hydrolysis process, according to the feeding method of the enzyme preparation obtained by the method as described above, adding the corresponding enzyme component during the feeding time period.

[0012] Through the above technical solutions, the beneficial effects of the present invention are as follows: The method provided by the present invention is based on computer-aided molecular dynamics simulation, reveals the molecular mechanism of the inhibition of the enzyme components in the enzyme preparation by the generation of inhibitors during the enzymatic hydrolysis process, and combines the influence of inhibitors on the activity of enzyme components during the enzymatic hydrolysis process. From multiple perspectives, a guiding strategy for feeding the enzyme preparation during the enzymatic hydrolysis process is proposed, and the design of enzyme components resistant to inhibitors is carried out to obtain the feeding method of the enzyme preparation during the enzymatic hydrolysis process. When applied to the enzymatic hydrolysis or conversion process of biomass, it not only saves time and effort, effectively reduces the addition amount of the enzyme preparation during the enzymatic hydrolysis process, but also improves the monosaccharide yield, reduces the production cost, and further improves the economic benefits of industrial production and subsequent processes such as ethanol production. Description of the Drawings

[0013] Figure 1 It is a graph showing the relationship between the residual activity of each enzyme component in Example 1 and three inhibitors (syringaldehyde, vanillin, 4-hydroxycinnamic acid) at different concentrations. Among them, a-e correspond to exoglucanase I (CBH I), endoglucanase (EG), β-glucosidase (BG), xylanase (XYN), and lytic polysaccharide monooxygenase (LPMO) respectively;

[0014] Figure 2 It is the surface solvation phenomenon of exoglucanase I (CBH I), endoglucanase (EG), β-glucosidase (BG), xylanase (XYN), and lytic polysaccharide monooxygenase (LPMO) in Example 1 in inhibitors (syringaldehyde, vanillin, 4-hydroxycinnamic acid) at different concentrations. Among them, a is the co-solvent system of water and the inhibitor, and b is the water solvent system;

[0015] Figure 3 It is a solvation phenomenon diagram of the active sites of exocellulase I (CBH I), endocellulase (EG), β-glucosidase (BG), xylanase (XYN) and lytic polysaccharide monooxygenase (LPMO) in Example 1 in inhibitors (syringaldehyde, vanillin, 4-hydroxycinnamic acid) at different concentrations;

[0016] Figure 4 It is a protein interaction diagram of LPMO with exocellulase I (CBH I), endocellulase (EG), β-glucosidase (BG), and xylanase (XYN) in Example 1. Detailed implementation manners

[0017] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0018] The first aspect of the present invention provides a method for designing the feeding mode of enzyme preparations during enzymatic hydrolysis by a computer. The enzyme preparation contains multiple enzyme components, and the method includes:

[0019] (1) Under the condition of the presence of inhibitors in the enzymatic hydrolysis process, the activities of each of the enzyme components are measured respectively, where the concentration of the inhibitor is set to multiple different simulated concentrations;

[0020] (2) Obtain the crystal structure of the enzyme component from the protein database, select the model of the inhibitor, and perform molecular dynamics simulation of the enzyme component when the concentration of the inhibitor is the simulated concentration;

[0021] (3) Combine the activity data of each of the enzyme components obtained in step (1) with the molecular dynamics simulation results obtained in step (2), and propose the feeding time period and feeding amount of each of the enzyme components.

[0022] In the process of researching the enzymatic hydrolysis of biomass, the inventors of the present invention found that based on computer-aided molecular dynamics simulations, the molecular mechanism of the inhibition of the enzyme components in the enzyme preparation by the inhibitors generated during the enzymatic hydrolysis process was revealed. Combining the effect of the inhibitors on the activity of the enzyme components during the enzymatic hydrolysis process, guiding strategies for feeding the enzyme preparation during the enzymatic hydrolysis process were proposed from multiple perspectives, and the design of enzyme components resistant to inhibitors was carried out to obtain the feeding method of the enzyme preparation during the enzymatic hydrolysis process. Thus, in the case of the generation of inhibitors by acid hydrolysis or alkali hydrolysis, the enzyme activity of the enzyme preparation can be restored as much as possible. When applied to the biomass enzymatic hydrolysis or conversion process, not only the yield and conversion efficiency of monosaccharides are improved, but also the addition amount of the enzyme preparation during the enzymatic hydrolysis process is effectively reduced, saving time and effort and reducing production costs, thereby improving the economic benefits of industrial production and subsequent processes such as ethanol production.

[0023] According to the present invention, preferably, the method for measuring the activity of the enzyme components in step (1) includes: under the conditions of different substances and / or different simulated concentrations of the inhibitors, mixing a solution containing the enzyme components, a solution containing the corresponding substrate of the enzyme components, and a buffer in the same proportion to obtain a mixed solution, performing an enzymatic hydrolysis reaction on the mixed solution for the same time, and then measuring the absorbance value of the product of the enzymatic hydrolysis reaction. That is, in the presence of inhibitors and other conditions remaining unchanged, the changes in the activity of each enzyme component with different specific substances and / or concentrations of the inhibitors are investigated, which can further reflect the inhibitory effects of different inhibitors and their different concentrations on the activity of each enzyme component.

[0024] According to the present invention, the inhibitor can be any substance that inhibits the downstream enzymatic hydrolysis and fermentation processes generated during the acid hydrolysis or alkali hydrolysis pretreatment of biomass, such as phenolic substances, acetic acid, and furfural, preferably phenolic substances. More preferably, the inhibitor is selected from at least one of syringaldehyde, vanillin, and 4-hydroxycinnamic acid; syringaldehyde, vanillin, and 4-hydroxycinnamic acid may be generated simultaneously, or only one or two of them may be generated.

[0025] According to the present invention, the enzyme preparation is a hydrolytic enzyme preparation and may contain multiple conventional hydrolytic enzymes. Preferably, the multiple enzyme components are exoglucanase I, endoglucanase, β-glucosidase, xylanase, and lytic polysaccharide monooxygenase.

[0026] According to the present invention, preferably, the substrate corresponding to exoglucanase I is 4-nitrophenyl-β-D-cellobioside, the substrate corresponding to endoglucanase is carboxymethyl cellulose, the substrate corresponding to β-glucosidase is 4-nitrophenyl-β-D-glucopyranoside, the substrate corresponding to xylanase is xylan, and the substrate corresponding to lytic polysaccharide monooxygenase is horseradish peroxidase.

[0027] According to the present invention, preferably, the buffer solution is a citric acid buffer solution, and more preferably, the concentration of the citric acid buffer solution is 0.05 - 0.1 M.

[0028] According to the present invention, the concentration of the inhibitor can be set according to the concentration of the inhibitor obtained during different pretreatment processes for different biomasses. Preferably, the simulated concentration of the inhibitor in the mixture is 0.05 - 0.2 g / L, specifically, it can be 0.05 g / L, 0.11 g / L, 0.2 g / L, 0.5 g / L, or any value between the aforementioned two values; the concentration of the enzyme component in the mixture is 0.05 - 0.5 g / L, and a fixed concentration of the enzyme component can be selected to investigate the inhibitory effect of the inhibitor on the activity.

[0029] According to the present invention, conditions such as the temperature, time, and wavelength for measuring absorbance of the enzymatic hydrolysis reaction are determined according to different enzyme components and corresponding substrates. To improve the efficiency of the enzymatic hydrolysis reaction, preferably, the conditions of the enzymatic hydrolysis reaction include: the temperature is 20 - 60 °C, specifically, it can be 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, or any value between the aforementioned two values; the time is 20 - 120 min, specifically, it can be 20 min, 40 min, 60 min, 80 min, 100 min, 120 min, or any value between the aforementioned two values; the wavelength for measuring the absorbance value is 400 - 600 nm.

[0030] Exemplarily, the method for measuring the activity of exoglucanase I is to mix an exoglucanase I solution, a 4-nitrophenyl-β-D-cellobioside solution with a concentration of 5 - 50 mmol / L, and a citric acid buffer solution with a concentration of 0.05 - 0.1 M, and add an inhibitor corresponding to the simulated concentration (syringaldehyde, vanillin, or 4-hydroxycinnamic acid). After mixing, react at 20 - 60 °C for 20 - 60 min, add Na2CO3 with a concentration of 0.5 - 2 mol / L and water, and measure the absorbance value at 300 - 500 nm.

[0031] Exemplarily, the method for measuring the activity of endoglucanase is to mix an endoglucanase solution, a carboxymethyl cellulose solution with a concentration of 0.2 - 2 wt%, and a citric acid buffer solution with a concentration of 0.05 - 0.1 M, and add an inhibitor corresponding to the simulated concentration (syringaldehyde, vanillin, or 4-hydroxycinnamic acid). After mixing, react at 20 - 60 °C for 20 - 60 min, add 3,5-dinitrosalicylic acid (DNS) and react in boiling water for 2 - 10 min, then add water, and measure the absorbance value at 400 - 600 nm.

[0032] Exemplarily, the method for measuring β-glucosidase activity is to mix a β-glucosidase solution, a 4-nitrophenyl β-D-glucopyranoside solution with a concentration of 5 - 100 mmol / L, and a citrate buffer solution with a concentration of 0.05 - 0.1 M, and add an inhibitor corresponding to the simulated concentration (syringaldehyde, vanillin or 4-hydroxycinnamic acid). After mixing, react at 20 - 60 °C for 60 - 120 min, add Na2CO3 with a concentration of 0.5 - 2 mol / L and water, and measure the absorbance value at 300 - 500 nm.

[0033] Exemplarily, the method for measuring xylanase activity is to mix xylanase, xylan with a concentration of 1 - 5 wt%, and a citrate buffer solution with a concentration of 0.05 - 0.1 M, and add an inhibitor corresponding to the simulated concentration (syringaldehyde, vanillin or 4-hydroxycinnamic acid). After mixing, react at 20 - 60 °C for 20 - 60 min, add 3,5-dinitrosalicylic acid (DNS) and react in boiling water for 2 - 10 min, then add water, and measure the absorbance value at 400 - 600 nm.

[0034] Exemplarily, the method for measuring the activity of lytic polysaccharide monooxygenase is to mix lytic polysaccharide monooxygenase, horseradish peroxidase with a concentration of 20 - 50 U / mL, and Amplex Red with a concentration of 50 - 200 μM, and add an inhibitor corresponding to the simulated concentration (syringaldehyde, vanillin or 4-hydroxycinnamic acid). Add 0.5 - 2 mM ascorbic acid as an electron donor, and monitor at an excitation wavelength of 500 - 600 and an emission wavelength at 20 - 40 °C for 0.5 - 2 h.

[0035] According to the present invention, preferably, the process of the molecular dynamics simulation in step (2) includes: obtaining the crystal structure of the enzyme component from the Protein Data Bank, selecting the model of the inhibitor, simulating the basic solvent system and the co-solvent system using GROMOS software, performing MD simulation analysis on each enzyme component to obtain the energy and structure data of each enzyme component, and the action behavior of the co-solvent on the active site of the enzyme component; wherein, the co-solvent contains the inhibitor and the basic solvent. Further preferably, the basic solvent is water, and the concentration of the inhibitor in the co-solvent is 0.05 - 0.2 g / L.

[0036] Exemplarily, when the inhibitors are syringaldehyde, vanillin, and 4-hydroxycinnamic acid, three models are taken from the ATB and the optimized GROMOS96(54a7) force field parameters. The simulation system is filled with water molecules in a pure water system (i.e., the basic solvent system), and syringaldehyde, vanillin, and 4-hydroxycinnamic acid molecules are filled in a cosolvent system with three inhibitor concentrations of 0.05 - 0.2 g / L. The steepest descent method is used for energy minimization, and then, with position constraints on the hydrolase, equilibration is carried out in the NPT ensemble for 100 ps. Three independent computational simulations are performed with different initial atomic velocities, and coordinates, energies, and velocities are collected every 500 ps for later analysis.

[0037] According to the present invention, preferably, the process of combining the activity data of each of the enzyme components with the results of the molecular dynamics simulation in step (3) includes:

[0038] (a) Obtaining a specific fingerprint spectrum of the enzyme component in the cosolvent by analyzing the overall structural properties, overall solvation phenomenon, solvation phenomenon of the active site, free energy landscape, and enzyme-enzyme interaction of the enzyme component;

[0039] (b) Designing the enzyme activity recovery, surface desolvation, de-inhibition of the active site, and de-inhibition of enzyme interaction of each enzyme component respectively according to the actual enzyme activity change and specific fingerprint spectrum of the enzyme component, and proposing a feeding strategy for each enzyme component;

[0040] More preferably, the feeding strategy includes: residual activity guidance strategy, overall hydration guidance strategy, active site inhibition guidance strategy, and protein interaction guidance strategy.

[0041] Exemplarily, when syringaldehyde, vanillin, and 4-hydroxycinnamic acid are used as inhibitors, the process of combining the activity data of each enzyme component with the results of the molecular dynamics simulation can be:

[0042] (a) Combining the actual activity inhibition results of different enzyme components in three inhibitor environments obtained in the experiment, and the analysis of overall structural properties, overall solvation phenomenon, solvation phenomenon of the active site, free energy landscape, and enzyme-enzyme interaction obtained in the molecular dynamics simulation, to obtain a specific fingerprint spectrum of each hydrolase in the cosolvent;

[0043] (b) Performing specific design methods on them according to the specific fingerprint spectrum of each hydrolase in the cosolvent; according to the actual activity inhibition results of the hydrolase in three inhibitor environments, selecting the precise activity change as the design source of the compounding scheme, and proposing Strategy 1: residual activity guidance strategy;

[0044] Based on the overall solvation phenomenon on the surface of the hydrolase and the solvation phenomenon at the active site, the precise change in the number of water molecules was selected as the source for designing the compounding scheme, and Strategy 2: Overall Hydration Guidance Strategy and Strategy 3: Active Site Inhibition Guidance Strategy were proposed;

[0045] According to the activation and inhibition effects of the enzyme component (lytic polysaccharide monooxygenase) on the other four enzyme components (exocellulase I, endocellulase, β-glucosidase, xylanase), the precise synergy was used as the source for designing the compounding scheme, and Strategy 4: Protein Interaction Guidance Strategy was proposed.

[0046] According to the present invention, when enzyme preparations are required during the enzymatic hydrolysis process, the method provided by the present invention for designing the feeding method of enzyme preparations during the enzymatic hydrolysis process by computer can be adopted to form corresponding enzyme preparation feeding strategies to improve the tolerance of enzyme preparations. For example, in the process of producing ethanol by enzymatic hydrolysis fermentation of biomass raw materials such as corn stover, corn cob, and sorghum stalk.

[0047] The second aspect of the present invention provides the application of the feeding method of enzyme preparations during the enzymatic hydrolysis process obtained by the method as described above in biomass enzymatic hydrolysis and / or biomass fermentation conversion.

[0048] In the present invention, the biomass can be any biomass raw material containing cellulose. Preferably, the biomass is lignocellulose, and more preferably at least one of corn stover, corn cob, and sorghum stalk.

[0049] In the present invention, the biomass fermentation conversion can be a process of using the monosaccharides obtained by enzymatic hydrolysis of biomass as a culture substrate to ferment any production strain, so as to obtain a large amount of required products through the growth and proliferation of the production strain. Preferably, the product of the biomass fermentation conversion is ethanol, and correspondingly, the production strain uses ethanol-fermenting bacteria.

[0050] The third aspect of the present invention provides a method for converting biomass, which includes: performing acid pretreatment or alkali pretreatment on the biomass to form a pretreatment solution; enzymatically hydrolyzing the pretreatment solution, and adopting the feeding method of enzyme preparations obtained by the method as described above during the enzymatic hydrolysis process, and adding the corresponding enzyme components during the feeding time period.

[0051] According to the present invention, preferably, the multiple enzyme components are exocellulase I, endocellulase, β-glucosidase, xylanase, and lytic polysaccharide monooxygenase; the feeding time periods are 10 - 15 h, 20 - 28 h, 32 - 40 h, and 44 - 52 h respectively.

[0052] According to the present invention, preferably, the enzyme preparation feeding method includes: for every 1g of the dry weight of the biomass, when the enzymatic hydrolysis is carried out for 10-15h, 0.1-2.5mg of exocellulase I, 0-0.5mg of endocellulase, 0-2.5mg of β-glucosidase, 0-2.5mg of xylanase, and 0.03-0.5mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth;

[0053] After the enzymolysis is carried out for 20-28 hours, 0.1-2.5 mg of exocellulase I, 0-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-2.5 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth;

[0054] After the enzymolysis is carried out for 32-40 hours, 0.1-2.5 mg of exocellulase I, 0-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-1.75 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth;

[0055] After the enzymolysis is carried out for 44-52 hours, 0.1-2.5 mg of exocellulase I, 0.15-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-2.5 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth.

[0056] According to the present invention, preferably, relative to every 1g of the biomass, the initial enzyme dosage of the enzymatic hydrolysis is 0.6-2.5mg of exocellulase I, 0.6-2.5mg of endocellulase, 0.6-2.5mg of β-glucosidase, 0.6-2.5mg of xylanase, and 0.6-2.5mg of polysaccharide-dissolving monooxygenase.

[0057] According to the present invention, preferably, the conditions of the enzymolysis include: the temperature is 30-50 ° C, specifically 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, or any value between the two aforementioned values; the stirring rate is 200-300 rpm, specifically 200 rpm, 220 rpm, 240 rpm, 260 rpm, 280 rpm, 300 rpm, or any value between the two aforementioned values; the time is 48-72h, specifically 48h, 52h, 56h, 60h, 64h, 68h, 72h, or any value between the two aforementioned values. Under this preferred embodiment, it is beneficial to further improve the efficiency of biomass enzymolysis, cooperate with the enzyme preparation feeding method, obtain higher monosaccharide yield, and better economic benefits.

[0058] In the present invention, the initial pH of the enzymatic hydrolysis is 4-5, and the treatment solution obtained by acid pretreatment or alkali pretreatment can be adjusted by using a pH regulator.

[0059] According to the present invention, preferably, the biomass is lignocellulose, more preferably at least one of corn stalks, corn cobs and sorghum stalks.

[0060] According to the present invention, preferably, the solid content of the biomass is 5-20 wt%; the pretreatment conditions include: temperature of 100-300°C, time of 10-30 min. The biomass is mixed with water to form a raw material with a solid content of 5-20 wt%, and the acid pretreatment or alkali pretreatment is performed.

[0061] In the present invention, the acid pretreatment is carried out with an acid, preferably sulfuric acid, and the alkali pretreatment is carried out with an alkali, preferably sodium hydroxide and / or potassium hydroxide. Further preferably, the amount of acid or alkali added is 1-10 wt % relative to the biomass raw material having a solid content of 5-20 wt %.

[0062] According to the present invention, preferably, the conditions of the acid pretreatment or alkali pretreatment include: a temperature of 100-300°C and a time of 10-30 minutes. Under this preferred embodiment, it is beneficial to further improve the efficiency of biomass enzymatic hydrolysis, cooperate with the enzyme preparation feeding method, obtain a higher monosaccharide yield, and better economic benefits.

[0063] According to the present invention, preferably, the transformation method further comprises: using the enzymatic hydrolyzate obtained by enzymatic hydrolysis as a culture medium to ferment the production strain.

[0064] According to the present invention, preferably, the production strain is an ethanol fermentation bacteria, more preferably Saccharomyces cerevisiae.

[0065] According to the present invention, preferably, the fermentation conditions include: inoculation amount of 0.3-0.5g dry cell weight / kg biomass dry weight; temperature of 25-35°C, specifically 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or any value between the two aforementioned values; speed of 120-180rpm, specifically 120rpm, 130rpm, 150rpm, 160rpm, 170rpm, 180rpm, or any value between the two aforementioned values; time of 60-96h, specifically 60h, 66h, 72h, 78h, 84h, 90h, 96h, or any value between the two aforementioned values. Under this preferred embodiment, it is beneficial to further improve the utilization rate of biomass enzymatic hydrolysis products, increase the yield of ethanol, and make the overall economic benefits of ethanol production process better.

[0066] The present invention will be described in detail below through examples.

[0067] In the following examples, the enzyme activity parameters were determined by a microplate reader, and the glucose, xylose, and ethanol contents were measured by liquid chromatography; the corn stover raw material was sourced from a farm in Hebei.

[0068] In the following examples, exoglucanase I (CBH I) was purchased from Sigma Aldrich, endoglucanase (EG) and β-glucosidase (BG) were both purchased from Megazyme, and the protein contents of CBH I, EG, and BG were 2.97, 0.33, and 5.05 mg / mL, respectively; xylanase (XYN) was purchased from Megazyme, and the product specification was 8000 Units.

[0069] Lytic polysaccharide monooxygenase (LPMO) was derived from self-expression in the laboratory, and the product specification was 3 mg / mL.

[0070] Saccharomyces cerevisiae was purchased from Angel Yeast Co., Ltd., and other raw materials and reagents were all conventional commercially available products.

[0071] In the following examples, the absorbance value was measured by pipetting 200 μL of the supernatant into a microplate and using a microplate reader. The ethanol concentration was measured by centrifuging the sample at 10000 rcf for 10 min to obtain the supernatant, diluting it 10 times, and then immediately performing sterile filtration through a 0.22 μm filter and analyzing it by high performance liquid chromatography.

[0072] Example 1

[0073] 1. The enzyme components of the enzyme preparation include five hydrolytic enzyme components: exoglucanase I (CBH I), endoglucanase (EG), β-glucosidase (BG), xylanase (XYN), and lytic polysaccharide monooxygenase (LPMO). The activities of each enzyme component were measured separately under the condition of the presence of inhibitors (syringaldehyde, vanillin, 4-hydroxycinnamic acid) generated during the enzymatic hydrolysis process.

[0074] (1) Determination of the activity of exoglucanase I (CBH I): Mix 0.1 mL of exoglucanase I solution (CBH I-Tr), 0.7 mL of 4-nitrophenyl-β-D-cellobioside solution with a concentration of 10 mmol / L, and 0.1 mL of citric acid buffer solution with a concentration of 0.05 M, and add inhibitors (syringaldehyde, vanillin or 4-hydroxycinnamic acid) respectively to obtain a mixed solution, so that the concentrations of inhibitors in the mixed solution are 0.2 g / L of syringaldehyde, 0.5 g / L of syringaldehyde, 0.2 g / L of vanillin, 0.5 g / L of vanillin, 0.05 g / L of 4-hydroxycinnamic acid, and 0.11 g / L of 4-hydroxycinnamic acid; then react the 6 kinds of mixed solutions at 50 °C for 40 min, add 2 mL of Na2CO3 with a concentration of 1 mol / L and 10 mL of water, and measure the absorbance at 400 nm. The results are shown in Figure 1 ;

[0075] (2) Determination of the activity of endoglucanase (EG): Mix 0.1 mL of endoglucanase solution, 0.14 mL of carboxymethyl cellulose solution with a concentration of 1 wt%, and 0.1 mL of citric acid buffer solution with a concentration of 0.05 M, and add inhibitors (syringaldehyde, vanillin or 4-hydroxycinnamic acid) respectively to obtain a mixed solution, so that the concentrations of inhibitors in the mixed solution are 0.2 g / L of syringaldehyde, 0.5 g / L of syringaldehyde, 0.2 g / L of vanillin, 0.5 g / L of vanillin, 0.05 g / L of 4-hydroxycinnamic acid, and 0.11 g / L of 4-hydroxycinnamic acid; then react the 6 kinds of mixed solutions at 50 °C for 40 min, add 0.6 mL of 3,5-dinitrosalicylic acid (DNS) and react in boiling water for 5 min, then add 2 mL of water, and measure the absorbance at 550 nm. The results are shown in Figure 1 ;

[0076] (3) Determination of the activity of β-glucosidase (BG): Mix 0.1 mL of β-glucosidase solution, 0.7 mL of 4-nitrophenyl β-D-glucopyranoside solution with a concentration of 5 mmol / L, and 0.1 mL of citric acid buffer solution with a concentration of 0.05 M, and add inhibitors (syringaldehyde, vanillin or 4-hydroxycinnamic acid) respectively to obtain a mixed solution, so that the concentrations of inhibitors in the mixed solution are 0.2 g / L of syringaldehyde, 0.5 g / L of syringaldehyde, 0.2 g / L of vanillin, 0.5 g / L of vanillin, 0.05 g / L of 4-hydroxycinnamic acid, and 0.11 g / L of 4-hydroxycinnamic acid; then react the 6 kinds of mixed solutions at 50 °C for 120 min, add 2 mL of Na2CO3 with a concentration of 1 mol / L and 10 mL of water, and measure the absorbance at 400 nm. The results are shown in Figure 1 ;

[0077] (4) Determination of xylanase (XYN) activity: Mix 0.1 mL of xylanase solution, 0.14 mL of xylan solution with a concentration of 1 wt%, and 0.1 mL of citrate buffer with a concentration of 0.05 M, and add inhibitors (syringaldehyde, vanillin or 4-hydroxycinnamic acid) respectively to obtain a mixed solution, so that the concentrations of inhibitors in the mixed solution are syringaldehyde 0.2 g / L, syringaldehyde 0.5 g / L, vanillin 0.2 g / L, vanillin 0.5 g / L, 4-hydroxycinnamic acid 0.05 g / L, 4-hydroxycinnamic acid 0.11 g / L; then react the 6 kinds of mixed solutions at 50 °C for 40 min, add 0.6 mL of 3,5-dinitrosalicylic acid (DNS) and react in boiling water for 5 min, then add 2 mL of water, and measure the absorbance at 550 nm. The results are shown in Figure 1 ;

[0078] (5) Determination of lytic polysaccharide monooxygenase (LPMO) activity: Mix 0.1 mL of lytic polysaccharide monooxygenase solution, 0.05 mL of horseradish peroxidase with a concentration of 30 U / mL, and 0.05 mL of Amplex Red with a concentration of 100 μM, and add inhibitors (syringaldehyde, vanillin or 4-hydroxycinnamic acid) respectively to obtain a mixed solution, so that the concentrations of inhibitors in the mixed solution are syringaldehyde 0.2 g / L, syringaldehyde 0.5 g / L, vanillin 0.2 g / L, vanillin 0.5 g / L, 4-hydroxycinnamic acid 0.05 g / L, 4-hydroxycinnamic acid 0.11 g / L; then continuously monitor the fluorescence intensity of the 6 kinds of mixed solutions at 30 °C, an excitation wavelength of 560 nm and an emission wavelength of 590 nm for 1 hour. The results are shown in Figure 1 。

[0079] 2. Obtain the crystal structures of each enzyme component from the protein database, select the model of the inhibitor, and perform molecular dynamics simulations of the enzyme component at the simulated concentration of the inhibitor;

[0080] (5) The crystal structures of CBH I, EG, BG, XYN and LPMO were obtained from the protein database, and MD simulations were performed using Gromacs2022.5. The GROMOS96 (54a7) force field was used to simulate the hydrolase in syringaldehyde, vanillin, and 4-hydroxycinnamic acid solvents. The hydrolase crystal structure was placed in a cubic box of SPC / E water with a minimum distance of 1.2 nm. According to the experimental conditions, the simulation system was filled with water molecules in the pure water system, and filled with syringaldehyde, vanillin, 4-hydroxycinnamic acid in the inhibitor system with concentrations of 0.05-0.2 g / L. For the cinnamic acid molecule, in order to avoid the most unfavorable interaction, the steepest descent method was first used for energy minimization. Then, NVT and NPT equilibrium were performed for 100 ps respectively with position restraints on the hydrolase. To avoid errors, three independent molecular dynamics (MD) simulations were performed with different starting atomic velocities (100 ns at 298 K, 1 bar, with a time step of 1 fs). Coordinates, energy, and velocity were collected every 500 ps for MD analysis. All analyses were calculated using the GROMACS simulation software package tool.

[0081] Among them, SDF (Spatial Distribution Function) is a function used to describe the spatial distribution of other molecules or atoms around a reference molecule. In molecular dynamics simulations, SDF is often used to study the three-dimensional distribution of solvents (such as water) around solutes (such as proteins or ions). Commonly used analysis tools include GROMACS's own gmxspatial or VMD (Visual Molecular Dynamics Tool) combined with script analysis. Areas with high SDF values ​​indicate high solvent density, that is, solvent molecules are more stable or tend to aggregate; areas with low SDF values ​​indicate fewer solvent molecules.

[0082] Hydration Shell refers to the layer of solvent (water) molecules around the solute molecules that interact directly or indirectly with them. The range of the hydration shell is usually defined by the distance from the solvent molecules to the solute surface (for example, the first shell is The second shell is ). The formation of hydration shell reflects the interaction between solute and solvent; its analysis tool is gmx mindist in GROMACS, and the analysis steps are:

[0083] a. Calculate the radial distribution function (RDF), that is, the distribution of solvent molecules relative to the solute;

[0084] b. Determine the range of the hydration shell according to the peak position of the RDF graph. For example, the distance range of the first peak of the RDF can be defined as the first hydration shell;

[0085] c. The water molecules can be directly defined with a distance of 0.35 nm. The radii of different shells and their integrals can be used to calculate the number of water molecules.

[0086] The number of water molecules at the active site refers to the number of water molecules that are relatively close to the molecular active site (usually defined by key residues) during the simulation; these water molecules may directly participate in chemical reactions, provide stability, or form indirect interactions with the active site through hydrogen bonds. The analysis tool is gmx mindist in GROMACS, and the analysis steps are as follows:

[0087] a. Define the key atoms or residues of the active site;

[0088] b. Set a distance threshold (e.g., ), and select the water molecules within this distance range;

[0089] c. Count the number of water molecules changing with time;

[0090] d. Output the number of water molecules near the active site in each frame.

[0091] The Solvent-Residue Contact Frequency refers to the frequency of interaction between solvent molecules and residues of proteins or other macromolecules in molecular dynamics (MD) simulations. This is an important indicator for evaluating the strength of the interaction between solute and solvent and its stability, and has important applications especially in the fields of protein-solvent interaction, enzyme catalysis, drug design, etc.

[0092] The contact frequency refers to the number of contacts between solvent molecules and the target residue within a certain time range. Usually, the contact is defined as the distance between the solvent molecule and the residue being less than a set threshold (such as or ); the level of the contact frequency can reflect the stability and distribution of solvent molecules around the target residue. GROMACS provides a variety of tools to calculate the contact frequency between solvent and residue, and the most commonly used ones are tools such as gmx make_ndx (generate groups) and gmx mindist (calculate the shortest distance).

[0093] The contact frequency map can obtain the contact situation changing with time by analyzing the contact frequencies between different residues and water molecules or other solvent molecules.

[0094] 3. Combine the activity data of each of the enzyme components with the results of the molecular dynamics simulations to propose the feeding time periods and feeding amounts for each of the enzyme components.

[0095] (6) Combining steps (1) - (4), in the presence of phenolic inhibitors, all hydrolases showed varying degrees of activity decline; among them, syringaldehyde had a strong inhibitory effect on CBH I and EG, and CBH I completely lost its activity after 12 hours ( Figure 1 as shown in a - b); in contrast, vanillin and 4 - hydroxycinnamic acid had only a slight inhibitory effect on most hydrolases, but BG was very sensitive to all three inhibitors and had lost its activity after 12 hours ( Figure 1 as shown in c); XYN and LPMO showed a similar pattern, with their activities gradually decreasing after 24 hours of hydrolysis in all inhibitor environments ( Figure 1 as shown in d - e). Based on the actual activity inhibition results of different hydrolases at different concentrations of different inhibitors, the precise activity changes were selected as the design source for the compounding scheme, and Strategy 1: Residual Activity Guidance Strategy was proposed.

[0096] Combining the overall structural properties obtained from the molecular dynamics simulations, the overall solvation phenomenon (such as Figure 2 shown), the solvation phenomenon of the active site (such as Figure 3 shown), and the enzyme - enzyme interaction analysis (such as Figure 4 shown), the specific fingerprint spectra of each hydrolase in the co - solvent were obtained. The results of the molecular dynamics simulations showed that as the inhibitor concentration increased, the number of hydration layers on the surfaces of the five hydrolases gradually decreased, while the number of inhibitor layers increased correspondingly; the frequency of residues contacting inhibitors or water was also concentration - dependent ( Figure 2 as shown in a - b), indicating that as the inhibitor concentration increased, water molecules were removed from the enzyme surface by the inhibitor, ultimately affecting the enzyme activity. Therefore, the precise change in the number of water molecules was selected as the design source for the compounding scheme, and Strategy 2: Overall Hydration Guidance Strategy was proposed.

[0097] The active site is the central region of enzyme catalysis. Conducting molecular dynamics simulation studies on this region, the results are as Figure 3 shown in a. As the inhibitor molecule concentration increased, a continuous decrease in the number of water molecules at the active sites of all five hydrolases was observed. In addition, it was also observed that inhibitor molecules gradually entered the active pockets of XYN and LPMO ( Figure 3 as shown in b), and strongly interacted with the active sites, effectively replacing water molecules ( Figure 3As shown in Figures c-d, this indicates that the inhibitor can directly block the active sites of XYN and LPMO, preventing the substrate from entering and interfering with the normal function of the enzyme. In contrast, this phenomenon was not observed in CBH I, EG, or BG. Therefore, based on the change in the number of water molecules around the enzyme active site as the design origin of the compounding scheme, Strategy 3: Active Site Inhibition Guidance Strategy was proposed.

[0098] The hydrolase (lytic polysaccharide monooxygenase LPMO) has activation and inhibition effects on four other hydrolases (exocellulase I CBH I, endocellulase EG, β-glucosidase BG, xylanase XYN). As Figure 4 shown in Figures c-d, LPMOs have significant synergy with BG (degree of synergy DS increases from 1 to 2.4) and XYN (degree of synergy DS increases from 1 to 1.23); on the contrary, LPMO strongly inhibits CBH I, and as the LPMO dosage increases, the DS value decreases from 1.0 to 0.44 ( Figure 4 as shown in Figure a); the DS value in the EG-LPMO system remains unchanged, indicating a lack of synergy between EG and LPMO ( Figure 4 as shown in Figure b). Based on the above results, using precise synergy as the design origin of the compounding scheme, Strategy 4: Protein Interaction Guidance Strategy was proposed.

[0099] That is, in actual research, first determine that under conventional conditions, the addition amounts of the four hydrolases CBH I, EG, BG, and XYN are 2.5 mg / g of dry corn stover and 0.6 mg / g of dry corn stover; then, according to the above residual activity data or simulation results, calculate the percentage reduction of the selected factors (for example, residual activity, overall hydration, degree of hydration layer of the active center, degree of protein interaction), and define the latter reduction as the amount of enzyme activity reduction. In order to theoretically restore 100% of the enzyme activity, supplement the corresponding amount of enzyme every 12 h. The specific feeding method of the enzyme preparation finally obtained is:

[0100] Relative to every 1 g of the dry biomass, during the enzymatic hydrolysis for 10 - 15 h, add 0.1 - 2.5 mg of exocellulase I, 0 - 0.5 mg of endocellulase, 0 - 2.5 mg of β-glucosidase, 0 - 2.5 mg of xylanase, and 0.03 - 0.5 mg of lytic polysaccharide monooxygenase to the fermentation broth;

[0101] During the enzymatic hydrolysis for 20 - 28 h, add 0.1 - 2.5 mg of exocellulase I, 0 - 0.5 mg of endocellulase, 0 - 2.5 mg of β-glucosidase, 0 - 2.5 mg of xylanase, and 0.03 - 0.5 mg of lytic polysaccharide monooxygenase to the fermentation broth;

[0102] When the enzymatic hydrolysis proceeds for 32 - 40 h, add 0.1 - 2.5 mg of exoglucanase I, 0 - 0.5 mg of endoglucanase, 0 - 2.5 mg of β-glucosidase, 0 - 1.75 mg of xylanase, and 0.03 - 0.5 mg of lytic polysaccharide monooxygenase to the fermentation broth;

[0103] When the enzymatic hydrolysis proceeds for 44 - 52 h, add 0.1 - 2.5 mg of exoglucanase I, 0.15 - 0.5 mg of endoglucanase, 0 - 2.5 mg of β-glucosidase, 0 - 2.5 mg of xylanase, and 0.03 - 0.5 mg of lytic polysaccharide monooxygenase to the fermentation broth.

[0104] Example 2

[0105] (1) Use sulfuric acid as a pretreatment reagent (the addition amount is 2% by weight of the corn straw raw material), and perform acid pretreatment on the corn straw raw material with a solid content of 10% by weight at 121 °C for 20 min, and then adjust the pH to 4.7 to obtain an acid pretreatment solution;

[0106] (2) Use sodium hydroxide as a pretreatment reagent (the addition amount is 2% by weight of the corn straw raw material), and perform acid pretreatment on the corn straw raw material with a solid content of 10% by weight at 121 °C for 20 min, and then adjust the pH to 4.7 to obtain an alkali pretreatment solution;

[0107] (3) Add 2.5 mg / g of CBH I, 2.5 mg / g of EG, 2.5 mg / g of BG, 2.5 mg / g of XYN, and 0.6 mg / g of LPMO to the acid pretreatment solution and the alkali pretreatment solution respectively, based on the dry weight of the corn straw; perform enzymatic hydrolysis at 50 °C with a stirring rate of 250 rpm for 60 h;

[0108] During the enzymatic hydrolysis of the acid pretreatment solution, the following enzyme preparations are fed:

[0109] When the enzymatic hydrolysis reaches 12 h, based on every 1 g of the dry weight of the corn straw, add 2.5 mg of CBH I, 0 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO as a feed supplement;

[0110] When the enzymatic hydrolysis reaches 24 h, based on every 1 g of the dry weight of the corn straw, add 2.5 mg of CBH I, 0 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO as a feed supplement;

[0111] At 36 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0.18 mg of EG, 2.5 mg of BG, 1.6 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0112] At 48 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0.35 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0113] Finally, the enzymatic hydrolysate of the acid-pretreated solution was obtained.

[0114] During the enzymatic hydrolysis of the alkali-pretreated solution, the following enzyme preparations were added by feeding:

[0115] At 12 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0.2 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0116] At 24 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0.4 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0117] At 36 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0 mg of EG, 2.5 mg of BG, 1.75 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0118] At 48 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 2.5 mg of CBH I, 0.15 mg of EG, 2.5 mg of BG, 2.5 mg of XYN, and 0.5 mg of LPMO were added by feeding;

[0119] Finally, the enzymatic hydrolysate of the alkali-pretreated solution was obtained.

[0120] The yields of glucose in the enzymatic hydrolysates of the acid-pretreated solution and the alkali-pretreated solution were measured to be 17.73 g / L and 22.97 g / L respectively, and the yields of xylose were 13.35 g / L and 11.91 g / L respectively.

[0121] Example 3

[0122] (1) Corn stover raw materials with a solid content of 20% by weight were pretreated with sulfuric acid (the addition amount was 8% by weight of the corn stover raw materials) at a temperature of 260 °C for 10 min, and then the pH was adjusted to 4.7 to obtain an acid-pretreated solution;

[0123] (2) The corn stover raw material with a solid content of 20% by weight was pretreated with sodium hydroxide as a pretreatment reagent (the addition amount was 8% by weight of the corn stover raw material) at a temperature of 260 °C for 10 min, and then the pH was adjusted to 4.7 to obtain the alkali pretreatment solution;

[0124] (3) 1.5 mg / g of CBH I, 1.5 mg / g of EG, 1.5 mg / g of BG, 1.5 mg / g of XYN, and 1.5 mg / g of LPMO were respectively added to the acid pretreatment solution and the alkali pretreatment solution based on the dry weight of the corn stover; The enzymatic hydrolysis was carried out at a temperature of 40 °C and a stirring rate of 200 rpm for 72 h;

[0125] During the enzymatic hydrolysis of the acid pretreatment solution, the following enzyme preparation feeding was carried out:

[0126] At 10 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.2 mg of CBH I, 0 mg of EG, 0 mg of BG, 0 mg of XYN, and 0.03 mg of LPMO were added as supplementary feed;

[0127] At 20 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.2 mg of CBH I, 0 mg of EG, 0 mg of BG, 0 mg of XYN, and 0.03 mg of LPMO were added as supplementary feed;

[0128] At 32 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.2 mg of CBH I, 0 mg of EG, 0 mg of BG, 0 mg of XYN, and 0.03 mg of LPMO were added as supplementary feed;

[0129] At 44 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.2 mg of CBH I, 0 mg of EG, 0 mg of BG, 0 mg of XYN, and 0.03 mg of LPMO were added as supplementary feed;

[0130] Finally, the enzymatic hydrolysate of the acid pretreatment solution was obtained.

[0131] During the enzymatic hydrolysis of the alkali pretreatment solution, the following enzyme preparation feeding was carried out:

[0132] At 10 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.1 mg of CBH I, 0.2 mg of EG, 0.23 mg of BG, 0.22 mg of XYN, and 0.11 mg of LPMO were added as supplementary feed;

[0133] At 20 h of enzymatic hydrolysis, relative to every 1 g of the dry weight of the corn stover, 0.1 mg of CBH I, 0.2 mg of EG, 0.23 mg of BG, 0.22 mg of XYN, and 0.11 mg of LPMO were added as supplementary feed;

[0134] At 32 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.2 mg of EG, 0.23 mg of BG, 0.22 mg of XYN, and 0.11 mg of LPMO were added by feeding;

[0135] At 44 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.2 mg of EG, 0.23 mg of BG, 0.22 mg of XYN, and 0.11 mg of LPMO were added by feeding;

[0136] Finally, the enzymatic hydrolysate of the alkali pretreatment solution was obtained.

[0137] The yields of glucose in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured to be 16.67 g / L and 23.01 g / L, respectively, and the yields of xylose were 13.48 g / L and 11.69 g / L, respectively.

[0138] Example 4

[0139] (1) For the corn stover raw material with a solid content of 5 wt%, using sulfuric acid as the pretreatment reagent (the addition amount is 5 wt% of the corn stover raw material), after acid pretreatment at 180 °C for 25 min, the pH was adjusted to 4.7 to obtain the acid pretreatment solution;

[0140] (2) For the corn stover raw material with a solid content of 5 wt%, using sodium hydroxide as the pretreatment reagent (the addition amount is 5 wt% of the corn stover raw material), after acid pretreatment at 180 °C for 25 min, the pH was adjusted to 4.7 to obtain the alkali pretreatment solution;

[0141] (3) 1 mg / g of CBH I, 1 mg / g of EG, 1 mg / g of BG, 1 mg / g of XYN, and 2.5 mg / g of LPMO were added to the acid pretreatment solution and the alkali pretreatment solution respectively; enzymatic hydrolysis was carried out at 30 °C with a stirring rate of 300 rpm for 50 h;

[0142] During the enzymatic hydrolysis of the acid pretreatment solution, the following enzyme preparation feeding was carried out:

[0143] At 15 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO were added by feeding;

[0144] At 28 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO were added as supplements;

[0145] At 40 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO were added as supplements;

[0146] At 52 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.1 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO were added as supplements;

[0147] Finally, the enzymatic hydrolysate of the acid-pretreated solution was obtained.

[0148] During the enzymatic hydrolysis of the alkali-pretreated solution, the following enzyme preparations were added as supplements:

[0149] At 15 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.3 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO were added as supplements;

[0150] At 28 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.3 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO were added as supplements;

[0151] At 40 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.3 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO were added as supplements;

[0152] At 52 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.3 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO were added as supplements;

[0153] Finally, the enzymatic hydrolysate of the alkali-pretreated solution was obtained.

[0154] The yields of glucose in the enzymatic hydrolysates of the acid-pretreated solution and the alkali-pretreated solution were measured to be 18.21 g / L and 24.05 g / L, respectively, and the yields of xylose were 13.64 g / L and 12.3 g / L, respectively.

[0155] Example 5

[0156] Perform pretreatment and enzymatic hydrolysis according to the method of Example 2, except that the feeding method of the enzyme preparation in step (3) is replaced with:

[0157] During the enzymatic hydrolysis of the acid pretreated solution, perform the following feeding of the enzyme preparation:

[0158] At 15 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 0.975 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO;

[0159] At 28 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 0.975 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO;

[0160] At 40 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 0.975 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO;

[0161] At 52 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 0.975 mg of CBH I, 0.18 mg of EG, 0.15 mg of BG, 0.3 mg of XYN, and 0.04 mg of LPMO;

[0162] Finally, obtain the enzymatic hydrolysate of the acid pretreated solution.

[0163] During the enzymatic hydrolysis of the alkali pretreated solution, perform the following feeding of the enzyme preparation:

[0164] At 15 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 1.07 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO;

[0165] At 28 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 1.07 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO;

[0166] At 40 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, supplement and add 1.07 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO;

[0167] At 52 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 1.07 mg of CBH I, 0.5 mg of EG, 0.4 mg of BG, 0.6 mg of XYN, and 0.095 mg of LPMO were added in fed-batch mode;

[0168] Finally, the enzymatic hydrolysate of the alkali-pretreated solution was obtained.

[0169] The yields of glucose in the enzymatic hydrolysates of the acid-pretreated solution and the alkali-pretreated solution were measured to be 18.59 g / L and 24.81 g / L respectively, and the yields of xylose were 13.49 g / L and 12.46 g / L respectively.

[0170] Using the enzymatic hydrolysates of the acid-pretreated solution and the alkali-pretreated solution obtained in Example 5 as fermentation raw materials, Saccharomyces cerevisiae was inoculated at an inoculation amount of 0.4 g of dry cell weight / kg of dry weight of corn stover, and fermented for 72 h at a temperature of 30 °C and a rotation speed of 150 rpm. The ethanol yield of this process was measured to be 96%.

[0171] The ethanol concentration in the fermentation sample was analyzed using an HPLC and a Biorad Aminex HPX-87H chromatographic column, and the ethanol productivity was calculated according to the following formula;

[0172] Ethanol productivity = ethanol concentration in the fermentation broth / (total sugar concentration in the enzymatic hydrolysate * 0.511) * 100%. The PMI of the process in Example 5 was evaluated according to the following formula, and its value was 90.84;

[0173] PMI = total input mass (kg) in the process / product mass (kg).

[0174] Example 6

[0175] Pretreatment and enzymatic hydrolysis were carried out according to the method of Example 2, except that steps (1) and (2) were replaced with:

[0176] (1) For the corn stover raw material with a solid content of 30% by weight, sulfuric acid was used as the pretreatment reagent (addition amount: 2% by weight of the corn stover raw material), and acid pretreatment was carried out at 121 °C for 20 min, and then the pH was adjusted to 4.7 to obtain the acid-pretreated solution;

[0177] (2) For the corn stover raw material with a solid content of 30% by weight, sodium hydroxide was used as the pretreatment reagent (addition amount: 2% by weight of the corn stover raw material), and acid pretreatment was carried out at 121 °C for 20 min, and then the pH was adjusted to 4.7 to obtain the alkali-pretreated solution.

[0178] The yields of glucose in the enzymatic hydrolysates of the acid-pretreated solution and the alkali-pretreated solution were measured, and the results are shown in Table 1.

[0179] Example 7

[0180] The pretreatment and enzymatic hydrolysis were carried out according to the method of Example 2, except that steps (1) and (2) were replaced with:

[0181] (1) For the corn stover raw material with a solid content of 10% by weight, sulfuric acid was used as the pretreatment reagent (the addition amount was 2% by weight of the corn stover raw material), and acid pretreatment was carried out at a temperature of 90 °C for 20 min. Then, the pH was adjusted to 4.7 to obtain the acid pretreatment solution.

[0182] (2) For the corn stover raw material with a solid content of 10% by weight, sodium hydroxide was used as the pretreatment reagent (the addition amount was 2% by weight of the corn stover raw material), and acid pretreatment was carried out at a temperature of 90 °C for 20 min. Then, the pH was adjusted to 4.7 to obtain the alkali pretreatment solution.

[0183] The glucose yields in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured, and the results are shown in Table 1.

[0184] Example 8

[0185] The pretreatment and enzymatic hydrolysis were carried out according to the method of Example 2, except that the initial enzyme addition amount in step (3) was replaced with:

[0186] 3 mg / g of CBH I, 3 mg / g of EG, 3 mg / g of BG, 3 mg / g of XYN, and 3 mg / g of LPMO based on the dry weight of corn stover were added to the acid pretreatment solution and the alkali pretreatment solution respectively. Enzymatic hydrolysis was carried out at a temperature of 50 °C and a stirring rate of 250 rpm for 60 h.

[0187] The glucose yields in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured, and the results are shown in Table 1.

[0188] Comparative Example 1

[0189] The pretreatment and enzymatic hydrolysis of corn stover were carried out according to the method of Example 2, except that step (3) was replaced with:

[0190] (3) The acid pretreatment solution and the alkali pretreatment solution were respectively washed with water for detoxification, washed about three times with clear water until the pH was neutral, the solid was placed in an oven to dry, and then dissolved in a 10% system to obtain the solution to be enzymatically hydrolyzed.

[0191] Add 12.5 mg / g of CBH I, 3.03 mg / g of EG, 12.5 mg / g of BG, 11.6 mg / g of XYN, and 2.6 mg / g of LPMO based on the dry weight of corn stover to the solution to be enzymatically hydrolyzed in the acid pretreatment solution at one time. Enzymatically hydrolyze for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatically hydrolyzed solution of the acid pretreatment solution.

[0192] Add 12.5 mg / g of CBH I, 32.5 mg / g of EG, 12.5 mg / g of BG, 11.75 mg / g of XYN, and 2.6 mg / g of LPMO based on the dry weight of corn stover to the solution to be enzymatically hydrolyzed in the alkali pretreatment solution at one time. Enzymatically hydrolyze for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatically hydrolyzed solution of the alkali pretreatment solution.

[0193] Measure that the glucose yields in the enzymatically hydrolyzed solutions of the acid pretreatment solution and the alkali pretreatment solution are 15.55 g / L and 20.51 g / L respectively, and the xylose yields are 12.71 g / L and 10.31 g / L respectively.

[0194] Use the enzymatically hydrolyzed solutions of the acid pretreatment solution and the alkali pretreatment solution obtained in Example 5 as fermentation raw materials. Inoculate Saccharomyces cerevisiae at an inoculation amount of 0.4 g of dry cell weight / kg of corn stover dry weight and ferment at a temperature of 30 °C and a rotation speed of 150 rpm for 72 h; measure the ethanol yield of this process to be 97% according to the method in Example 5.

[0195] Evaluate the PMI of the process of Comparative Example 1 according to the method in Example 5, and its value is 310.83.

[0196] Comparative Example 2

[0197] Perform the pretreatment and enzymatic hydrolysis of corn stover according to the method of Example 2, except that step (3) is replaced with:

[0198] (3) Add 3.3 mg / g of CBH I, 2.5 mg / g of EG, 2.5 mg / g of BG, 2.5 mg / g of XYN, and 0.72 mg / g of LPMO based on the dry weight of corn stover to the acid pretreatment solution; enzymatically hydrolyze for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatically hydrolyzed solution of the acid pretreatment solution.

[0199] Add CBH I at 2.9 mg / g of dry weight of corn stover, EG at 3.3 mg / g of dry weight of corn stover, BG at 3.42 mg / g of dry weight of corn stover, XYN at 3.29 mg / g of dry weight of corn stover, and LPMO at 1.04 mg / g of dry weight of corn stover to the alkali pretreatment solution; perform enzymatic hydrolysis for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatic hydrolysate of the alkali pretreatment solution.

[0200] The yields of glucose in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured to be 14.71 g / L and 19.96 g / L respectively, and the yields of xylose were 12.06 g / L and 9.10 g / L respectively.

[0201] Comparative Example 3

[0202] Perform the pretreatment and enzymatic hydrolysis of corn stover according to the method of Example 2, except that step (3) is replaced with:

[0203] (3) Add CBH I at 2.9 mg / g of dry weight of corn stover, EG at 3.22 mg / g of dry weight of corn stover, BG at 3.1 mg / g of dry weight of corn stover, XYN at 3.7 mg / g of dry weight of corn stover, and LPMO at 0.76 mg / g of dry weight of corn stover to the acid pretreatment solution; perform enzymatic hydrolysis for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatic hydrolysate of the acid pretreatment solution;

[0204] Add CBH I at 3.7 mg / g of dry weight of corn stover, EG at 4.5 mg / g of dry weight of corn stover, BG at 3.7 mg / g of dry weight of corn stover, XYN at 4.9 mg / g of dry weight of corn stover, and LPMO at 0.98 mg / g of dry weight of corn stover to the alkali pretreatment solution; perform enzymatic hydrolysis for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm to obtain the enzymatic hydrolysate of the alkali pretreatment solution.

[0205] The yields of glucose in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured to be 14.92 g / L and 20.90 g / L respectively, and the yields of xylose were 12.38 g / L and 9.19 g / L respectively.

[0206] Comparative Example 4

[0207] Perform the pretreatment and enzymatic hydrolysis of corn stover according to the method of Example 2, except that step (3) is replaced with:

[0208] (3) Add CBH I at 2.5 mg / g of dry corn stover weight, EG at 2.5 mg / g of dry corn stover weight, BG at 2.5 mg / g of dry corn stover weight, XYN at 2.5 mg / g of dry corn stover weight, and LPMO at 0.6 mg / g of dry corn stover weight to the acid pretreatment solution and the alkali pretreatment solution respectively; perform enzymatic hydrolysis for 60 h at a temperature of 50 °C and a stirring rate of 250 rpm;

[0209] During the enzymatic hydrolysis of the acid pretreatment solution, the following enzyme supplements are added:

[0210] At 8 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0211] At 16 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0212] At 30 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0.18 mg of EG, 2 mg of BG, 1.6 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0213] At 42 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0.35 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0214] Finally, obtain the enzymatic hydrolysate of the acid pretreatment solution.

[0215] During the enzymatic hydrolysis of the alkali pretreatment solution, the following enzyme supplements are added:

[0216] At 8 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0.2 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0217] At 16 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0.4 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0218] At 30 h of enzymatic hydrolysis, add 0.04 mg of CBH I, 0 mg of EG, 2 mg of BG, 1.75 mg of XYN, and 0.6 mg of LPMO per 1 g of dry corn stover weight as a supplement;

[0219] At 42 h of enzymatic hydrolysis, relative to every 1 g of dry weight of corn stover, 0.04 mg of CBH I, 0.15 mg of EG, 2 mg of BG, 2 mg of XYN, and 0.6 mg of LPMO were added by feeding.

[0220] Finally, the enzymatic hydrolysate of the alkali pretreatment solution was obtained.

[0221] The yields of glucose in the enzymatic hydrolysates of the acid pretreatment solution and the alkali pretreatment solution were measured to be 14.83 g / L and 20.43 g / L respectively, and the yields of xylose were 12.19 g / L and 9.73 g / L respectively.

[0222] Table 1

[0223]

[0224] From the above, it can be seen that Examples 2, 3, 4, and 5 were fermented by the feeding addition method of the enzyme preparation obtained by the method of the present invention. Under the acid and alkali pretreatment conditions, the glucose yield obtained in Example 5 was increased by 19.55% and 20.97% compared with Comparative Example 1 (one-time addition according to the residual activity guiding strategy); compared with Comparative Example 1, PMI in Example 5 was approximately reduced by 70%, greatly improving the economy of the process. The results of Examples 2, 3, 4, and 5 and Comparative Examples 1, 2, 3, and 4 show that the method of the present invention is based on computer simulation and uses the protein interaction guiding strategy to produce higher monosaccharide yields, while saving production costs and improving the economy of the entire ethanol process.

[0225] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for designing the feeding mode of enzyme preparation in enzymolysis process by computer, characterized in that: The enzyme preparation contains multiple enzyme components, and the method comprises: (1) measuring the activity of each of the enzyme components in the presence of an inhibitor of the enzymatic hydrolysis process, wherein the concentration of the inhibitor is set to a plurality of different simulated concentrations; (2) obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the inhibitor, and performing a molecular dynamics simulation of the enzyme component when the concentration of the inhibitor is a simulated concentration; (3) Combining the activity data of each enzyme component obtained in step (1) with the molecular dynamics simulation results obtained in step (2), and proposing a feeding time period and feeding amount for each enzyme component.

2. The method according to claim 1, characterized in that The method for determining the activity of the enzyme component in step (1) comprises: mixing a solution containing the enzyme component, a solution containing a substrate corresponding to the enzyme component and a buffer solution in the same proportion to obtain a mixed solution under the conditions of different inhibitor substances and / or different simulated concentrations, subjecting the mixed solution to an enzymatic hydrolysis reaction for the same time, and then determining the absorbance value of the product of the enzymatic hydrolysis reaction; Preferably, the simulated concentration of the inhibitor in the mixed solution is 0.05-0.2 g / L, and the concentration of the enzyme component is 0.05-0.5 g / L; Preferably, the conditions of the enzymatic hydrolysis reaction include: a temperature of 20-60° C. and a time of 20-120 min; and a wavelength of 400-600 nm for measuring the absorbance value.

3. The method according to claim 2, characterized in that The inhibitor is selected from at least one of syringaldehyde, vanillin and 4-hydroxycinnamic acid; Preferably, the plurality of enzyme components are exocellulase I, endocellulase, β-glucosidase, xylanase and polysaccharide-dissolving monooxygenase; Preferably, the substrate corresponding to the exocellulase I is 4-nitrophenyl-β-D-cellobioside, the substrate corresponding to the endocellulase is carboxymethyl cellulose, the substrate corresponding to the β-glucosidase is 4-nitrophenyl-β-D-pyranoglucoside, the substrate corresponding to the xylanase is xylan, and the substrate corresponding to the polysaccharide-dissolving monooxygenase is horseradish peroxidase; The buffer is a citric acid buffer.

4. The method according to any one of claims 1 to 3, characterized in that The molecular dynamics simulation process in step (2) includes: obtaining the crystal structure of the enzyme component from a protein database, selecting a model of the inhibitor, simulating a basic solvent system and a co-solvent system using GROMOS software, performing MD simulation analysis on each of the enzyme components, obtaining energy and structural data of each of the enzyme components, and the action behavior of the co-solvent on the active site of the enzyme component; wherein the co-solvent contains the inhibitor and a base solvent; Preferably, the base solvent is water, and the concentration of the inhibitor in the co-solvent is 0.05-0.2 g / L.

5. The method according to claim 4, characterized in that The process of combining the activity data of each enzyme component with the molecular dynamics simulation results in step (3) includes: (a) obtaining a specific fingerprint spectrum of the enzyme component in the co-solvent by analyzing the overall structural properties, overall solvation phenomena, solvation phenomena of active sites, free energy landscape, and enzyme-enzyme interactions of the enzyme component; (b) according to the actual enzyme activity change and the specific fingerprint spectrum of the enzyme component, respectively carry out the design of enzyme activity recovery, surface desolvation, active site deinhibition, and enzyme interaction deinhibition of the enzyme component, and propose a feeding strategy for each enzyme component; Preferably, the feeding strategies include: residual activity guidance strategy, overall hydration guidance strategy, active site inhibition guidance strategy, and protein interaction guidance strategy.

6. Application of the enzyme preparation feeding mode in the enzymatic hydrolysis process obtained by the method according to any one of claims 1 to 5 in biomass enzymatic hydrolysis and / or biomass fermentation conversion; Preferably, the biomass is lignocellulose, more preferably at least one of corn stalks, corn cobs and sorghum stalks; The product of the biomass fermentation conversion is ethanol.

7. A biomass conversion method, characterized in that: The conversion method comprises: subjecting the biomass to acid pretreatment or alkali pretreatment to form a pretreatment liquid; subjecting the pretreatment liquid to enzymatic hydrolysis, and during the enzymatic hydrolysis process, using the enzyme preparation obtained by the method described in any one of claims 1 to 5 as a feeding method, and adding the corresponding enzyme components during the feeding time period.

8. The conversion method according to claim 7, characterized in that The multiple enzyme components are exocellulase I, endocellulase, β-glucosidase, xylanase and polysaccharide-dissolving monooxygenase; The feeding time periods are 10-15h, 20-28h, 32-40h, and 44-52h respectively; Preferably, the enzyme preparation feeding method includes: for every 1g of the dry weight of the biomass, when the enzymolysis is performed for 10-15h, 0.1-2.5mg of exocellulase I, 0-0.5mg of endocellulase, 0-2.5mg of β-glucosidase, 0-2.5mg of xylanase, and 0.03-0.5mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth; After the enzymolysis is carried out for 20-28 hours, 0.1-2.5 mg of exocellulase I, 0-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-2.5 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth; After the enzymolysis is carried out for 32-40 hours, 0.1-2.5 mg of exocellulase I, 0-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-1.75 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth; After the enzymolysis is carried out for 44-52 hours, 0.1-2.5 mg of exocellulase I, 0.15-0.5 mg of endocellulase, 0-2.5 mg of β-glucosidase, 0-2.5 mg of xylanase, and 0.03-0.5 mg of polysaccharide-dissolving monooxygenase are added to the fermentation broth.

9. The conversion method according to claim 7 or 8, characterized in that Relative to 1g of the biomass, the initial enzyme dosage of the enzymatic hydrolysis is 0.6-2.5mg of exocellulase I, 0.6-2.5mg of endocellulase, 0.6-2.5mg of β-glucosidase, 0.6-2.5mg of xylanase, and 0.6-2.5mg of polysaccharide-dissolving monooxygenase; Preferably, the conditions for the enzymatic hydrolysis include: a temperature of 30-50°C, a stirring rate of 200-300 rpm, and a time of 48-72 h; Preferably, the biomass is lignocellulose, more preferably at least one of corn stalks, corn cobs and sorghum stalks; Preferably, the solid content of the biomass is 5-20% by weight; the pretreatment conditions include: temperature of 100-300° C., and time of 10-30 min.

10. The conversion method according to claim 7 or 8, characterized in that: The transformation method further comprises: using the enzymatic hydrolyzate obtained by enzymatic hydrolysis as a culture medium to ferment the production strain; Preferably, the production strain is an ethanol fermentation strain, more preferably Saccharomyces cerevisiae; Preferably, the fermentation conditions include: an inoculation amount of 0.3-0.5 g dry cell weight / kg biomass dry weight, a temperature of 25-35° C., a rotation speed of 120-180 rpm, and a time of 60-96 h.

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