CN113481255B - Enzymolysis method of cellulose-containing raw material - Google Patents
Enzymolysis method of cellulose-containing raw material Download PDFInfo
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- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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Abstract
The invention relates to the technical fields of renewable energy sources and bioengineering, and discloses an enzymolysis method of a cellulose-containing raw material. The enzymolysis method of the cellulose-containing raw material comprises the following steps: carrying out enzymolysis on a raw material containing cellulose in the presence of modified lignin to obtain an enzymolysis product; the preparation method of the modified lignin comprises the following steps: mixing lignin raw materials with p-toluenesulfonic acid for pretreatment to obtain pretreated lignin, and mixing the pretreated lignin with a modifier for modification. The modified lignin is used as a catalytic auxiliary agent, so that the conformation of the cellulase can be improved, the activity of the cellulase is increased, and the enzymolysis efficiency of cellulose-containing raw materials is enhanced.
Description
Technical Field
The invention relates to the technical fields of renewable energy sources and bioengineering, in particular to an enzymolysis method of a cellulose-containing raw material.
Background
The lignocellulose materials such as straw and the like are continuously degraded to obtain fuel ethanol, and the fuel ethanol and the common gasoline are mixed according to a certain proportion, so that the fuel ethanol can be used for automobiles without influencing the running performance of the automobiles. The renewable ethanol gasoline is taken as a clean fuel and is the focus of research in the current new energy field. The lignocellulose resources in China are rich, the renewable resources are also effective means for relieving the energy crisis, the method has important significance for relieving the environmental pollution, the reasonable utilization of agricultural waste straws and the like can be promoted, and the method accords with the sustainable development strategy and the development direction of renewable energy in China.
At present, although the method for producing ethanol by hydrolyzing agricultural wastes such as straw and the like through cellulase is widely accepted and adopted on a large scale, the lignocellulose mainly comprises cellulose, hemicellulose and lignin, has stable properties, has low enzymolysis saccharification efficiency and relatively high cost, and therefore, the wider application of the lignocellulose is limited. In the process of hydrolyzing lignocellulose by using cellulase, the enzymolysis efficiency is influenced by various factors such as hydrolysis resistance of a substrate, hydrolysis activity of the cellulase, hydrolysis process conditions and the like, so that the reduction of the production cost of lignocellulose ethanol is limited.
Adsorption of cellulose substrate and cellulase is a precondition for cellulose hydrolysis, which is only hydrolyzed by cellulose when the cellulase is adsorbed on cellulose. Therefore, improving the adsorption performance between cellulase and cellulose plays an important role in enhancing the enzymatic hydrolysis of cellulose.
Disclosure of Invention
The invention aims to solve the problems of low cellulose hydrolysis efficiency and high cost in the prior art, and provides an enzymolysis method for a raw material containing cellulose, which can improve the hydrolysis efficiency of cellulose and the yield of bioethanol.
In order to achieve the above object, the present invention provides a method for enzymatic hydrolysis of a cellulose-containing raw material, comprising: carrying out enzymolysis on a raw material containing cellulose in the presence of modified lignin to obtain an enzymolysis product; the preparation method of the modified lignin comprises the following steps: mixing lignin raw materials with p-toluenesulfonic acid for pretreatment to obtain pretreated lignin, and mixing the pretreated lignin with a modifier for modification.
Preferably, the para-toluenesulfonic acid is mixed with the lignin feedstock in the form of a preheated para-toluenesulfonic acid-water solution; the concentration of the p-toluenesulfonic acid in the p-toluenesulfonic acid-water solution is 50-70 mass%, the preheating temperature is 70-90 ℃, and the pretreatment conditions at least meet the following conditions: the temperature is 60-90deg.C, and the time is 10-30min.
Preferably, the lignin raw material is straw and/or wheat straw; the preparation method of the modified lignin further comprises the following steps: and carrying out solid-liquid separation I on the pretreatment feed liquid obtained by pretreatment to obtain pretreatment liquid, mixing the pretreatment liquid with water, carrying out solid-liquid separation II to obtain crude lignin, washing the crude lignin to be neutral, and drying to obtain the pretreatment lignin.
Preferably, the volume ratio of the pretreatment liquid to water is 1:5-20, wherein the mixing time of the pretreatment liquid and water is 1-5min, and the conditions of solid-liquid separation II at least meet the following conditions: the rotating speed is 8000-10000rpm, and the time is 8-15min.
Preferably, the modification process comprises: and mixing the pretreated lignin with alkali liquor and the modifier, performing modification reaction to obtain modification feed liquid, and diluting, regulating pH value and performing solid-liquid separation III on the modification feed liquid to obtain modified solid.
Preferably, the alkali in the alkaline solution is used in an amount of 0.07-3g and the modifier in an amount of 0.017-0.48g relative to 1g of the pretreated lignin.
Preferably, the modifier contains carboxylate and quaternary ammonium salt, and the mass ratio of the carboxylate to the quaternary ammonium salt is 1:0.25-14;
preferably, the quaternary ammonium salt is mixed with the pretreatment lignin, the lye in the form of a quaternary ammonium salt-water solution having a concentration of 50-70 mass%.
Preferably, the preparation method of the cellulose-containing raw material comprises the following steps: mixing straw and/or wheat straw with p-toluenesulfonic acid-water solution, incubating, filtering to obtain a filtered solid, and washing the filtered solid to be neutral; the mass ratio of the straw and/or wheat straw to the p-toluenesulfonic acid-water solution is 1:20-40 parts; the mass fraction of the p-toluenesulfonic acid in the p-toluenesulfonic acid-water solution is 50-70 mass percent, and the temperature of the p-toluenesulfonic acid-water solution is 70-90 ℃; the hatching conditions at least meet the following conditions: the temperature is 60-90deg.C, and the time is 10-30min.
Preferably, the enzymolysis process comprises: and mixing the cellulose-containing raw material with buffer solution and cellulase, performing enzymolysis reaction, and performing solid-liquid separation IV to obtain the enzymolysis product.
Preferably, the buffer is selected from at least one of citric acid-water solution, sodium citrate-water solution and sodium acetate-water solution;
the amount of the buffer solution is 19-99mL, the amount of the cellulase is 5-10FPU and the amount of the modified lignin is 10-200mg relative to 1g of dry weight of the cellulose-containing raw material;
the conditions of the enzymolysis reaction at least meet the following conditions: the temperature is 30-60 ℃, the rotating speed is 150-200rpm, and the time is 60-84h.
Through the technical scheme, the invention has the beneficial effects that:
according to the enzymolysis method for the cellulose-containing raw material, the lignin is modified by using the p-toluenesulfonic acid and the modifier, and the modified lignin is used as a catalytic auxiliary agent in the enzymolysis process of the cellulose-containing raw material, so that the hydrogen bond interaction and the hydrophobic interaction between the modified lignin and cellulose molecules can be reduced, and the adsorption quantity of the cellulose molecules is reduced; the modified lignin can be combined with cellulase to form a compound with lower free energy of combination, and the conformation of the cellulase is improved, so that the activity of the cellulase is increased, and the enzymolysis efficiency of cellulose-containing raw materials is enhanced;
in the enzymolysis method of the cellulose-containing raw material, after the straws and/or the wheat straw are pretreated by the p-toluenesulfonic acid, the pretreatment liquid obtained by solid-liquid separation can be further modified to form modified lignin, the pretreated solid is used as the cellulose-containing raw material, and further enzymolysis is carried out to obtain the reducing sugar, so that the utilization rate of the straws and/or the wheat straw can be improved.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The invention provides an enzymolysis method of a cellulose-containing raw material, which comprises the following steps: carrying out enzymolysis on a raw material containing cellulose in the presence of modified lignin to obtain an enzymolysis product; the preparation method of the modified lignin comprises the following steps: mixing lignin raw materials with p-toluenesulfonic acid for pretreatment to obtain pretreated lignin, and mixing the pretreated lignin with a modifier for modification.
According to the invention, the lignin modification process mainly comprises the steps of pretreating p-toluenesulfonic acid to obtain pretreated lignin (mainly p-toluenesulfonic acid), and modifying the treated lignin by a modifier to form carboxylated and quaternized modified lignin; the modified lignin is used as a catalytic auxiliary agent in the enzymolysis process of the cellulose-containing raw material, and the modified lignin can reduce the hydrogen bond interaction and the hydrophobic interaction between the modified lignin and cellulose molecules, so that the adsorption quantity of the modified lignin to the cellulose molecules is reduced; and can combine with cellulase to form a complex with lower free energy of combination, and the modified lignin improves the conformation of the cellulase so as to increase the activity of the cellulase.
According to the invention, the p-toluenesulfonic acid is mixed with the lignin feedstock in the form of a preheated aqueous p-toluenesulfonic acid solution; the concentration of the p-toluenesulfonic acid in the p-toluenesulfonic acid-water solution is 50-70 mass%, and the preheating temperature is 70-90 ℃, specifically, may be 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, or any value between the two values. The inventors have found that in this preferred embodiment, it is advantageous to increase the efficiency of the pretreatment of lignin with p-toluene sulfonic acid.
According to the invention, the pretreatment conditions at least satisfy: the temperature is 60-90deg.C, specifically 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, or any value between the above two values; the time is 10-30min, specifically 10min, 15min, 20min, 25min, 30min, or any value between the above two values.
According to the invention, the lignin feedstock may be any lignin-containing feedstock material or lignin-containing solution. Preferably, the lignin raw material is straw and/or wheat straw; specifically, the straw can be corn straw, rape straw, cotton straw, wheat straw and rice straw.
Because the lignin content in the straw and/or wheat straw is low, after the straw and/or wheat straw is pretreated, the pretreated straw and/or wheat straw can be directly used for modification to obtain modified lignin; the pretreatment lignin is preferably subjected to effective separation to increase the purity of the lignin, which is beneficial to further modification of the pretreatment lignin. Illustratively, the method of preparing the modified lignin further comprises: and carrying out solid-liquid separation I on the pretreatment feed liquid obtained by pretreatment to obtain pretreatment liquid, mixing the pretreatment liquid with water, carrying out solid-liquid separation II to obtain crude lignin (solid), washing the crude lignin to be neutral, and drying to obtain the pretreatment lignin so as to realize effective separation of the pretreatment lignin.
According to the present invention, the water may be any form of water, such as deionized water, purified water, distilled water, preferably deionized water, which refers to purified water from which impurities in the form of ions are removed, which may be prepared by a conventional preparation method; drying may be carried out using any one or more conventional drying processes and equipment, for example freeze drying, microwave drying or vacuum heat drying.
Further preferably, the volume ratio of the pretreatment liquid to water is 1:5-20, specifically can be 1: 5. 1: 10. 1: 15. 1:20, or any value between the two values; the pretreatment liquid is mixed with water for 1-5min, specifically 1min, 2min, 3min, 4min, 5min, or any value between the two values. In the present invention, any conventional separation process and apparatus may be used for the solid-liquid separation I and the solid-liquid separation II, for example, filtration, suction filtration or centrifugation. The solid-liquid separation I is preferably filtration, and the solid-liquid separation II is preferably centrifugation; the conditions of the solid-liquid separation II at least meet the following conditions: the rotation speed is 8000-10000rpm, specifically 8000rpm, 8500rpm, 9000rpm, 9500rpm, 10000rpm, or any value between the above two values; the time is 8-15min, and can be specifically 8min, 10min, 12min, 14min, 15min, or any value between the above two values. The inventors found that in this preferred embodiment, it is advantageous to increase the purity of the pre-modified lignin, and thus the promotion of cellulase hydrolysis.
According to the invention, the modification process comprises: and mixing the pretreated lignin with alkali liquor and the modifier, performing modification reaction to obtain modification feed liquid, and diluting, regulating pH value and performing solid-liquid separation III on the modification feed liquid to obtain modified solid. The dilution can be performed by deionized water, and the pH adjustment specifically means that the pH of the diluted modified feed liquid is adjusted by using acid liquor (such as hydrochloric acid solution and sulfuric acid solution) until the amount of lignin precipitation is not increased.
In the invention, any conventional separation process and equipment can be adopted for the solid-liquid separation III, for example, filtration, suction filtration or centrifugation is adopted, and centrifugation is preferred; the alkali liquor can be any alkaline solution, for example, naOH solution with the mass fraction of 5-15% can be used; the pH can be adjusted by conventional adjustment methods, such as adding alkali liquor or acid liquor, or adding deionized water for dilution, etc.
Preferably, the alkali in the alkaline solution is used in an amount of 0.07-3g and the modifier in an amount of 0.017-0.48g relative to 1g of the pretreated lignin.
According to the invention, the modifier may be selected from substances capable of carboxylating and quaternizing the pretreated lignin, for example, the modifier is configured to contain carboxylate and quaternary ammonium salts. The carboxylate refers to salts formed by carboxylic acid and contains carboxylate anions, and concretely, the carboxylate can be at least one of sodium chloroacetate, potassium chloroacetate, sodium 3-chloropropionate and sodium 2-chloropropionate; the quaternary ammonium salt refers to a compound formed by substituting four hydrogen atoms in ammonium ions with hydrocarbon groups, and specifically can be 3-chloro-2-hydroxypropyl trimethyl ammonium chloride. Illustratively, the modifier comprises sodium chloroacetate and 3-chloro-2-hydroxypropyl trimethylammonium chloride.
Preferably, the mass ratio of the carboxylate to the quaternary ammonium salt is 1:0.25-14. The inventor finds that under the preferred specific embodiment, the stability of the modified lignin is improved, and the promotion effect on cellulose enzymolysis is improved.
In the invention, carboxylate and quaternary ammonium salt can be respectively mixed with pretreated lignin and alkali liquor in the form of solid or liquid solution. Illustratively, the carboxylate salt is mixed as a solid with the pretreated lignin, lye at a temperature of 60-90 ℃ and stirred until it is completely dissolved.
Preferably, the quaternary ammonium salt is mixed with the pretreatment lignin, the lye in the form of a quaternary ammonium salt-water solution having a concentration of 50-70 mass%.
According to the present invention, the cellulose-containing raw material may be any cellulose-containing raw material substance or cellulose-containing solution. The cellulose-containing raw material can be the existing agricultural residues such as crop straw and agricultural herbaceous plants including corn straw, corncob, grass, paper, barley straw, wheat straw, paper and the like; or forest residue such as hardwood, cork, nut shell, leaf, cotton seed wadding, willow branch, oat hull, etc., and energy crops such as switchgrass, and energy grass; the cellulose-containing raw material may be directly used as the agricultural residue, or the agricultural residue may be subjected to pretreatment. Preferably, the preparation method of the cellulose-containing raw material comprises the following steps: mixing straw and/or wheat straw with p-toluenesulfonic acid-water solution, incubating, filtering to obtain a filtered solid, and washing the filtered solid to neutrality.
At this time, the straw and/or wheat straw can be used as a lignin raw material while being used as a raw material for preparing cellulose; specifically, mixing straw and/or wheat straw with p-toluenesulfonic acid-water solution, incubating (preprocessing) to obtain a preprocessing liquid, carrying out solid-liquid separation on the preprocessing liquid to obtain a preprocessing liquid and a preprocessing solid (filtering solid), washing the preprocessing solid to be neutral to obtain a cellulose-containing raw material, mixing the preprocessing liquid with deionized water, carrying out solid-liquid separation II to obtain crude lignin, washing the crude lignin to be neutral, and drying to obtain the preprocessed lignin. The inventors have found that in this preferred embodiment, it is advantageous to increase the utilization of straw and/or wheat straw.
According to the invention, after the straws and/or the wheat straws are crushed, the crushed straws and/or the wheat straws are mixed with the p-toluenesulfonic acid-water solution, so that the effect of pretreatment of lignin and the like by the p-toluenesulfonic acid can be optimized; the incubation (pretreatment) process may be terminated with deionized water.
Preferably, the mass ratio of the straw and/or wheat straw to the p-toluenesulfonic acid-water solution is 1:20-40 parts; the mass fraction of the p-toluenesulfonic acid in the p-toluenesulfonic acid-water solution is 50-70 mass percent, the temperature of the p-toluenesulfonic acid-water solution is 70-90 ℃, and the p-toluenesulfonic acid-water solution can be 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃ or any value between the two values; the hatching conditions at least meet the following conditions: the temperature is 60-90deg.C, specifically 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, or any value between the above two values; the time is 10-30min, specifically 10min, 15min, 20min, 25min, 30min, or any value between the above two values.
Preferably, the enzymolysis process comprises: and mixing the cellulose-containing raw material with buffer solution and cellulase, performing enzymolysis reaction, and performing solid-liquid separation IV to obtain the enzymolysis product.
In the invention, any conventional separation process and equipment can be adopted for the solid-liquid separation IV, for example, filtration, suction filtration or centrifugation can be adopted, and filtration is preferable; the cellulase may be a conventional cellulase material, for example, beta-glucosidase.
According to the present invention, the buffer is at least one selected from the group consisting of a citric acid-water solution, a sodium citrate-water solution and a sodium acetate-water solution, and for example, a citric acid-water solution having a concentration of 50mM and a pH of 4.8 is used as the buffer. The inventors have found that in this preferred embodiment, it is advantageous to increase the reaction efficiency of cellulase hydrolysis.
According to the invention, the buffer is used in an amount of 19-99mL, the cellulase is used in an amount of 5-10FPU, and the modified lignin is used in an amount of 10-200mg relative to 1g of dry weight of the cellulose-containing feedstock; the conditions of the enzymolysis reaction at least meet the following conditions: the temperature is 30-60deg.C, specifically 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, or any value between the above two values; the rotation speed is 150-200rpm, and can be 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the two values; the time is 60-84h, and can be specifically 60h, 65h, 70h, 75h, 80h, 84h, or any value between the two values.
As a relatively preferred embodiment of the enzymatic hydrolysis method of a cellulose-containing feedstock in the present invention, it comprises the steps of:
s1, mixing straws and/or wheat straws with p-toluenesulfonic acid-water solution, incubating (preprocessing) to obtain preprocessing feed liquid, carrying out solid-liquid separation on the preprocessing feed liquid to obtain preprocessing liquid and preprocessing solids (filtering solids), washing the preprocessing solids to be neutral to obtain cellulose-containing raw materials, mixing the preprocessing liquid with deionized water, carrying out solid-liquid separation II to obtain crude lignin, washing the crude lignin to be neutral, and drying to obtain the preprocessing lignin;
s2, mixing the pretreated lignin obtained in the step S1 with alkali liquor and a modifier, and then carrying out modification reaction to obtain a modified feed liquid, diluting the modified feed liquid by deionized water, adjusting pH, and carrying out solid-liquid separation III to obtain modified solid, namely modified lignin;
s3, mixing the cellulose-containing raw material obtained in the step S1 with the citric acid buffer solution, the cellulase and the modified lignin obtained in the step S2, performing enzymolysis reaction, and performing solid-liquid separation IV to obtain the enzymolysis product.
The present invention will be described in detail by examples.
In the following examples, the content of reducing sugar in the hydrolysate was measured by DNS method; corn stalk is purchased from the Lianyuangang city of Jiangsu province, and other raw materials and reagents are conventional commercial products.
Example 1
S1, mixing 65% of p-toluenesulfonic acid-water solution with the preheating temperature of 70 ℃ and crushed corn stalks according to a mass ratio of 1:40, after mixing, incubating for 20min at 70 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, washing the pretreatment solid with deionized water to neutrality to obtain cellulose-containing raw material, and mixing the pretreatment liquid and deionized water according to a volume ratio of 1:10, after mixing for 3min, centrifuging for 10min at a rotation speed of 10000rpm to obtain crude lignin, washing the crude lignin to be neutral by deionized water, and freeze-drying to obtain pretreated lignin;
s2, mixing 0.5g of the pretreated lignin obtained in the step S1 with 10mL of NaOH solution with the mass fraction of 15%, adding 0.05g of sodium chloroacetate, stirring at the temperature of 80 ℃ until the lignin is completely dissolved, slowly adding 0.1mL of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution with the mass fraction of 65%, carrying out modification reaction to obtain modification feed liquid, diluting the modification feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modification solid, namely modified lignin;
s3, mixing 2g of the cellulose-containing raw material obtained in the step S1 with 98mL of citric acid buffer solution (50 mM, pH 4.8), adding 8FPU of beta-glucosidase and 100mg of modified lignin obtained in the step S2, performing enzymolysis for 72 hours at the temperature of 50 ℃ and the rotating speed of 180rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 84.48%.
Example 2
S1, mixing p-toluenesulfonic acid-water solution with the preheating temperature of 80 ℃ and the mass fraction of 50% with crushed corn stalks according to the mass ratio of 1:20, incubating for 10min at 90 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, washing the pretreatment solid with deionized water to neutrality to obtain cellulose-containing raw material, and mixing the pretreatment liquid and deionized water according to a volume ratio of 1:20, after mixing for 5min, centrifuging for 15min at a rotating speed of 8000rpm to obtain crude lignin, washing the crude lignin to be neutral by deionized water, and freeze-drying to obtain pretreated lignin;
s2, mixing 10g of the pretreated lignin obtained in the step S1 with 10mL of NaOH solution with the mass fraction of 15%, adding 0.136g of sodium chloroacetate, stirring at the temperature of 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.068mL of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution with the mass fraction of 50%, carrying out modification reaction to obtain modified feed liquid, diluting the modified feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modified solid, namely modified lignin;
s3, mixing 1g of the cellulose-containing raw material obtained in the step S1 with 99mL of citric acid buffer solution (50 mM, pH 4.8), adding 5FPU of beta-glucosidase and 10mg of modified lignin obtained in the step S2, performing enzymolysis for 84 hours at the temperature of 60 ℃ and the rotating speed of 150rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 83.19%.
Example 3
S1, mixing 70% of p-toluenesulfonic acid-water solution with the preheating temperature of 90 ℃ and crushed corn stalks according to a mass ratio of 1:30, incubating for 30min at 60 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, washing the pretreatment solid with deionized water to neutrality to obtain cellulose-containing raw material, and mixing the pretreatment liquid and deionized water according to a volume ratio of 1:5, after mixing for 1min, centrifuging for 8min at the rotating speed of 9000rpm to obtain crude lignin, washing the crude lignin to be neutral by deionized water, and freeze-drying to obtain pretreated lignin;
s2, mixing 1g of the pretreated lignin obtained in the step S1 with 5mL of 10% NaOH solution by mass fraction, adding 0.032g of sodium chloroacetate, stirring at 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.64mL of 70% 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution by mass fraction, carrying out modification reaction to obtain modified feed liquid, diluting the modified feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modified solid, namely modified lignin;
s3, mixing 5g of the cellulose-containing raw material obtained in the step S1 with 95mL of citric acid buffer solution (50 mM, pH 4.8), adding 10FPU of beta-glucosidase and 200mg of modified lignin obtained in the step S2, performing enzymolysis for 60 hours at the temperature of 30 ℃ and the rotating speed of 200rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 86.73%.
Example 4
The enzymatic hydrolysis of cellulose-containing raw material was carried out in the same manner as in example 3, except that sodium chloroacetate in step S2 was replaced with ammonium citrate and 3-chloro-2-hydroxypropyl trimethylammonium chloride was replaced with tetrabutylammonium bromide.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 80.16%.
Example 5
Enzymolysis of the cellulose-containing raw material was performed in accordance with the method of example 3, except that step 2 was replaced with: adding 0.032g of 1-chloropropionic acid sodium into 1g of the pretreated lignin obtained in the step S1, stirring at the temperature of 80 ℃ until the lignin is completely dissolved, slowly adding 0.64mL of 70% 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution, carrying out modification reaction to obtain modification feed liquid, diluting the modification feed liquid with deionized water, adjusting the pH value of the solution, centrifuging and removing supernatant to obtain modified solid, namely the modified lignin.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 81.75%.
Example 6
Enzymolysis of the cellulose-containing raw material was performed in accordance with the method of example 3, except that step 2 was replaced with: mixing 1g of the pretreated lignin obtained in the step S1 with 5mL of NaOH solution with the mass fraction of 10%, adding 0.032g of sodium chloroacetate, stirring at the temperature of 80 ℃ until the sodium chloroacetate is completely dissolved, carrying out modification reaction to obtain modified feed liquid, diluting the modified feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modified solid, namely modified lignin.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 80.92%.
Example 7
Enzymolysis of cellulose-containing raw materials was performed in the same manner as in example 3 except that in step S1, the 70% by mass aqueous solution of p-toluenesulfonic acid at a preheating temperature of 90℃was replaced with the 70% by mass aqueous solution of p-toluenesulfonic acid at room temperature (25 ℃).
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 82.01%.
Example 8
Enzymolysis of the cellulose-containing raw material was performed in accordance with the method of example 3, except that step S2 was replaced with: mixing 1g of the pretreated lignin obtained in the step S1 with 30mL of NaOH solution with the mass fraction of 15%, adding 0.32g of sodium chloroacetate, stirring at the temperature of 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.64mL of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution with the mass fraction of 70%, carrying out modification reaction to obtain modification feed liquid, diluting the modification feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modification solid, namely the modified lignin.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 83.27%.
Example 9
Enzymolysis of the cellulose-containing raw material was performed in accordance with the method of example 3, except that step S3 was replaced with: after 5g of the cellulose-containing raw material obtained in step S1 was mixed with 80mL of a citric acid buffer (50 mM, pH 4.8), 12FPU of beta-glucosidase and 300mg of the modified lignin obtained in step S2 were added, and the mixture was subjected to enzymolysis at 30℃and 200rpm for 60 hours, and after the completion of the enzymolysis, the mixture was filtered to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 79.82%.
Example 10
S1, mixing 70% of p-toluenesulfonic acid-water solution with the preheating temperature of 90 ℃ and crushed corn stalks according to a mass ratio of 1:30, incubating for 30min at 60 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, and mixing the pretreatment liquid and the deionized water according to a volume ratio of 1:5, after mixing for 1min, centrifuging for 8min at the rotating speed of 9000rpm to obtain crude lignin, washing the crude lignin to be neutral by deionized water, and freeze-drying to obtain pretreated lignin;
s2, mixing 1g of the pretreated lignin obtained in the step S1 with 5mL of 10% NaOH solution by mass fraction, adding 0.032g of sodium chloroacetate, stirring at 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.64mL of 70% 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution by mass fraction, carrying out modification reaction to obtain modified feed liquid, diluting the modified feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modified solid, namely modified lignin;
s3, mixing a raw material solution containing 5g of cellulose with 95mL of citric acid buffer solution (50 mM, pH 4.8), adding 10FPU of beta-glucosidase and 200mg of modified lignin obtained in the step S2, performing enzymolysis for 60 hours at the temperature of 30 ℃ and the rotating speed of 200rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 80.59%.
Example 11
S1, mixing lignin with 70% of p-toluenesulfonic acid-water solution with a preheating temperature of 90 ℃ in a mass ratio of 1:30, incubating for 30min at 60 ℃, adding deionized water to terminate the reaction to obtain a pretreatment liquid, and mixing the pretreatment liquid with deionized water according to a volume ratio of 1:5, after mixing for 1min, centrifuging for 8min at the rotating speed of 9000rpm to obtain crude lignin, washing the crude lignin to be neutral by deionized water, and freeze-drying to obtain pretreated lignin;
s2, mixing 1g of the pretreated lignin obtained in the step S1 with 5mL of 10% NaOH solution by mass fraction, adding 0.032g of sodium chloroacetate, stirring at 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.64mL of 70% 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution by mass fraction, carrying out modification reaction to obtain modified feed liquid, diluting the modified feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modified solid, namely modified lignin;
s3, mixing a raw material solution containing 5g of cellulose with 95mL of citric acid buffer solution (50 mM, pH 4.8), adding 10FPU of beta-glucosidase and 200mg of modified lignin obtained in the step S2, performing enzymolysis for 60 hours at the temperature of 30 ℃ and the rotating speed of 200rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 81.55%.
Comparative example 1
S1, mixing 70% of p-toluenesulfonic acid-water solution with the preheating temperature of 90 ℃ and crushed corn stalks according to a mass ratio of 1:30, after mixing, incubating for 30min at the temperature of 60 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, and washing the pretreatment solid with deionized water to neutrality to obtain cellulose-containing raw material;
s2, mixing 5g of the cellulose-containing raw material obtained in the step S1 with 95mL of citric acid buffer solution (50 mM, pH 4.8), adding 10FPU of beta-glucosidase, performing enzymolysis for 60 hours at the temperature of 30 ℃ and the rotating speed of 200rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this comparative example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 78.64%.
Comparative example 2
S1, mixing 70% of p-toluenesulfonic acid-water solution with the preheating temperature of 90 ℃ and crushed corn stalks according to a mass ratio of 1:30, after mixing, incubating for 30min at the temperature of 60 ℃, adding deionized water to terminate the reaction to obtain pretreatment feed liquid, filtering the pretreatment feed liquid to obtain pretreatment liquid and pretreatment solid, and washing the pretreatment solid with deionized water to neutrality to obtain cellulose-containing raw material;
s2, mixing 1g of lignin with 5mL of NaOH solution with the mass fraction of 10%, adding 0.032g of sodium chloroacetate, stirring at the temperature of 80 ℃ until the sodium chloroacetate is completely dissolved, slowly adding 0.64mL of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride-water solution with the mass fraction of 70%, carrying out modification reaction to obtain modification feed liquid, diluting the modification feed liquid with deionized water, adjusting the pH of the solution, centrifuging to remove supernatant, and obtaining modification solid, namely modified lignin;
s3, mixing 5g of the cellulose-containing raw material obtained in the step S1 with 95mL of citric acid buffer solution (50 mM, pH 4.8), adding 10FPU of beta-glucosidase and 200mg of modified lignin obtained in the step S2, performing enzymolysis for 60 hours at the temperature of 30 ℃ and the rotating speed of 200rpm, and filtering after the enzymolysis reaction is finished to obtain an enzymolysis product.
In this example, the content of reducing sugar in the enzymatic hydrolysate was measured, and the Enzymatic Hydrolysis Efficiency (EHE) of cellulose in the cellulose-containing raw material was calculated to be 77.39%.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (8)
1. A method for enzymatic hydrolysis of a cellulose-containing feedstock, the method comprising:
s1, mixing straw and/or wheat straw with p-toluenesulfonic acid-water solution for pretreatment to obtain pretreatment liquid, filtering the pretreatment liquid to obtain pretreatment liquid and pretreatment solid, washing the pretreatment solid to be neutral to obtain cellulose-containing raw material, mixing the pretreatment liquid with deionized water, centrifuging to obtain crude lignin, washing the crude lignin to be neutral, and drying to obtain pretreated lignin;
s2, mixing the pretreated lignin obtained in the step S1 with alkali liquor and a modifier, performing a modification reaction to obtain a modified feed liquid, and diluting, regulating pH and performing solid-liquid separation III on the modified feed liquid to obtain modified lignin; the modifier comprises carboxylate and quaternary ammonium salt;
and S3, carrying out enzymolysis on the cellulose-containing raw material obtained in the step S1 in the presence of the modified lignin obtained in the step S2 to obtain an enzymolysis product.
2. The enzymatic hydrolysis method according to claim 1, characterized in that the p-toluenesulfonic acid is mixed with the straw and/or wheat straw in the form of preheated aqueous p-toluenesulfonic acid;
the concentration of the p-toluenesulfonic acid in the p-toluenesulfonic acid-water solution is 50-70 mass%, the preheating temperature is 70-90 ℃, and the pretreatment conditions are as follows: the temperature is 60-90deg.C, and the time is 10-30min.
3. The enzymolysis method according to claim 1, wherein the volume ratio of the pretreatment liquid to deionized water is 1:5-20, wherein the mixing time of the pretreatment liquid and deionized water is 1-5min, and the centrifugation conditions are as follows: the rotating speed is 8000-10000rpm, and the time is 8-15min.
4. The enzymatic hydrolysis method according to claim 1, characterized in that the amount of alkali in the alkaline solution is 0.07-3g and the amount of modifier is 0.017-0.48g relative to 1g of the pretreated lignin.
5. The enzymatic hydrolysis method according to claim 1, characterized in that the mass ratio of the carboxylate to the quaternary ammonium salt is 1:0.25-14;
the quaternary ammonium salt is mixed with the pretreatment lignin and the alkali liquor in the form of a quaternary ammonium salt-water solution with the concentration of 50-70 mass%.
6. The enzymatic hydrolysis method according to any one of claims 1 to 3, characterized in that the mass ratio of the straw and/or wheat straw to the p-toluenesulfonic acid-water solution is 1:20-40.
7. A method according to any one of claims 1 to 3, wherein the enzymatic hydrolysis process comprises: and mixing the cellulose-containing raw material with buffer solution and cellulase, performing enzymolysis reaction, and performing solid-liquid separation IV to obtain the enzymolysis product.
8. The method according to claim 7, wherein the buffer is at least one selected from the group consisting of citric acid-aqueous solution, sodium citrate-aqueous solution and sodium acetate-aqueous solution;
the amount of the buffer solution is 19-99mL, the amount of the cellulase is 5-10FPU and the amount of the modified lignin is 10-200mg relative to 1g of dry weight of the cellulose-containing raw material;
the conditions of the enzymolysis reaction are as follows: the temperature is 30-60 ℃, the rotating speed is 150-200rpm, and the time is 60-84h.
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