CN115466406B - PH-UCST responsive lignin-based zwitterionic compound and preparation method thereof and method for recycling cellulase at room temperature - Google Patents
PH-UCST responsive lignin-based zwitterionic compound and preparation method thereof and method for recycling cellulase at room temperature Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention discloses a pH-UCST responsive lignin-based zwitterionic compound and a preparation method thereof and a method for recycling cellulase at room temperature. According to the invention, a UCST response type betaine fragment is grafted on enzymatic hydrolysis lignin to synthesize a pH-UCST response type lignin-based zwitterionic compound, and cellulase is efficiently recovered at room temperature by utilizing the response performance of the pH-UCST response type lignin-based zwitterionic compound, specifically, the lignin-based zwitterionic compound is added into a cellulase solution or an enzymatic hydrolysis system, after dissolution or enzymatic hydrolysis, the pH of the system is reduced to 4.0-4.5 and cooled to room temperature, so that the pH-UCST response type lignin-based zwitterionic compound and the cellulase are simultaneously precipitated and separated out, and the cellulase is recovered by a solid-liquid separation method. The method utilizes the pH-UCST of the pH-UCST responsive lignin-based zwitterionic compound to efficiently recycle the cellulase in the solution at room temperature, and the recycling process is simple to operate and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of lignocellulose enzymolysis, and particularly relates to a pH-UCST responsive lignin-based zwitterionic compound and a preparation method thereof and a method for recovering cellulase at room temperature.
Background
Cellulose and hemicellulose in lignocellulose such as crop straws and the like can be converted into saccharide compounds through pretreatment and enzymolysis technology, and biofuel and other chemicals are prepared through a biological method or a chemical method, but the technical bottleneck problems of low enzymolysis efficiency and high enzymolysis cost still exist at present. Lignin accounts for 15-35% of lignocellulose, and lignin remained in the existing biorefinery process including cellulosic ethanol is mainly used for combustion, so that the lignin is difficult to be recycled and efficiently utilized.
In the enzymolysis process of lignocellulose, the enzyme cost can be reduced by realizing the recovery and the recycling of cellulase. For example, cellulases (Biofuels, bioprod. Bioref., 2017.11:150-167) can be recovered by methods and techniques such as fresh substrate addition, membrane separation, and enzyme immobilization. Cellulase is a multicomponent enzyme and the affinity of the different components of cellulase to the substrate is different, wherein the affinity of the beta-glucosidase to the substrate cellulose is poor and the beta-glucosidase cannot be recovered effectively by adding fresh substrate for re-adsorption. And the membrane separation method and the enzyme immobilization method are not beneficial to industrialized popularization due to the reasons of high technical difficulty, high cost and the like. In contrast, by adding the environment response type auxiliary agent, the cellulase is recovered by utilizing the response performance of the auxiliary agent, the process is simple to operate, and the enzyme recovery effect is good. For example, pH responsive lignin amphoteric surfactant pH-LC can be synthesized by grafting sulfonate and quaternary ammonium ions on Enzymatic Hydrolysis Lignin (EHL), the pH of the system is lowered, and as carboxylate is protonated, the molecular hydrophilicity is reduced, and the pH-LC precipitates. The isoelectric point (Ip) is synthesized to be pH-LC of 2.2 by adjusting the access amount of sulfonate and quaternary ammonium ions, 3.0g/L of pH-LC is added into a corncob residue enzymolysis system (pH 5.0), the pH of the system is adjusted to be 3.2, and the use amount of cellulase (ACS Sustainable chem. Eng., 2018.6:10679-10686) can be saved by 50% by utilizing the pH-LC. The ratio of sulfonate ions to quaternary ammonium ions which are connected in the EHL can have obvious influence on pH-LC response performance and enzyme recovery performance, the ratio of sulfonate ions to quaternary ammonium ions needs to be accurately regulated in the pH-LC synthesis process, and the synthesis process is complex. Meanwhile, the pH response sensitivity of the pH-LC is poor, so that the acid consumption in the enzyme recovery process is relatively large. Furthermore, the inventors' subject group utilized UCST response properties of sulfobetaines to achieve recovery and recycling of cellulases at room temperature (Sustin. Energ. Fuels,2020,5:750-757; green Chem.,2021, 23:2738-2746). When the system temperature is lower than UCST, the molecular hydrophilicity is reduced due to the association of positive and negative ions in the molecule, and PSPE and enzyme are co-precipitated mainly through hydrophobic effect. For example, PSPE is added into an enzymolysis system (50 ℃) of corncob residues, the system is cooled to room temperature, and at least 50% of enzyme can be saved by using the PSPE (Green chem.,2021, 23:2738-2746). However, the enzyme recovery performance of the auxiliary agent is poor due to the weak binding force between PSPE and cellulose. Therefore, the synthesis of the intelligent response lignin surfactant with high sensitivity and high enzyme recovery performance has important significance for reducing the enzyme dosage.
Disclosure of Invention
In order to solve the defects and shortcomings of the prior art, the primary purpose of the invention is to provide a preparation method of a pH-UCST responsive lignin-based zwitterionic compound.
It is another object of the present invention to provide a pH-UCST responsive lignin-based zwitterionic compound prepared by the above method.
It is still another object of the present invention to provide a method for recovering cellulase at room temperature using the above-mentioned pH-UCST responsive lignin-based zwitterionic compound.
According to the invention, the lignin-based zwitterionic compound with pH-UCST response performance is synthesized by grafting the latent UCST response betaine fragment on the enzymatic hydrolysis lignin, and the cellulase is efficiently recovered at room temperature by utilizing the response performance of the lignin-based zwitterionic compound. Compared with pH-LC, betaine ions are easy to associate into salt at low temperature, the molecular hydrophilicity is reduced, the response performance of the auxiliary agent is improved, the consumption of acid in the enzyme recovery process is saved, and the binding capacity of the auxiliary agent and the enzyme is improved. In addition, the synthesis process does not need to regulate and control the access proportion of the positive ions and the negative ions, and the synthesis process is simple. Not only can realize the full-component resource utilization of lignocellulose, but also has important significance for reducing the production cost of biorefinery and cellulosic ethanol.
According to the invention, a series of pH-UCST response lignin-based amphoteric ion compounds are synthesized, the influence of factors such as a molecular structure, a grafting amount, a concentration, a buffer solution pH and an ionic strength on the pH-UCST response performance is examined, and on the basis, the influence rules of the factors such as the zwitterionic compound structure, the grafting amount, the concentration, the buffer solution pH and the ionic strength on the cellulase recovery performance under different pH values and temperatures are further studied, so that the efficient recovery of the cellulase in the lignocellulose enzymolysis system at room temperature is realized by adding the pH-UCST response lignin-based amphoteric ion compounds.
The invention aims at realizing the following technical scheme:
a preparation method of a pH-UCST responsive lignin-based zwitterionic compound comprises the following steps:
(1) Reacting N, N-dimethylethanolamine and 3-bromopropane sodium sulfonate at 50-80 ℃ for 3-12 hours to obtain isethionic betaine, and then placing the isethionic betaine in thionyl chloride and reacting at 0-60 ℃ for 3-6 hours to obtain a sulfobetaine fragment;
acidifying a phosphorylcholine calcium salt tetrahydrate by oxalic acid at room temperature, then obtaining a phosphoric acid betaine sodium salt by sodium salt replacement, and carrying out halogenation reaction on the phosphoric acid betaine sodium salt and epichlorohydrin at 70-90 ℃ for 6-12 h under an acidic condition to obtain a phosphoric acid ester betaine fragment;
(2) Under alkaline conditions, the pH-UCST responsive lignin-based zwitterionic compound is obtained by grafting sulfobetaine fragments and/or phosphate betaine fragments on lignin phenolic hydroxyl sites.
Preferably, the molar ratio of the N, N-dimethylethanolamine to the 3-bromopropane sodium sulfonate in the step (1) is 1-1.5: 1.
preferably, the solvent for the reaction of N, N-dimethylethanolamine and sodium 3-bromopropanesulfonate in the step (1) is at least one of methanol, ethyl acetate, acetone, acetonitrile, N-dimethylformamide and N, N-dimethylacetamide.
Preferably, the molar ratio of oxalic acid, phosphorylcholine calcium salt tetrahydrate and sodium salt in step (1) is 1.2:1:0.5 to 1.6, wherein the sodium salt used for sodium salt substitution is at least one of anhydrous sodium carbonate and anhydrous sodium bicarbonate.
Preferably, the molar ratio of the epichlorohydrin to the sodium salt of the phosphoric acid betaine in the step (1) is 1 to 1.5:1.
preferably, the acid in the acidic condition in the step (1) is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, formic acid and acetic acid, wherein the molar ratio of the acid to the sodium salt of phosphoric acid betaine is 0.1-0.5: 1.2.
preferably, the pH of the alkaline condition of step (2) is 10 to 12.
Preferably, the ratio of the sulfobetaine fragment and/or the phosphate betaine fragment to the amount of phenolic hydroxyl species in lignin of step (2) is from 1 to 4:1.
preferably, the reaction temperature of the step (2) by grafting the sulfobetaine fragment and/or the phosphate betaine fragment on the lignin phenolic hydroxyl site is 60-90 ℃ for 3-9 hours; the reaction uses water as a solvent, and the mass fraction of lignin in the reaction system is 20%.
Preferably, the lignin in the step (2) is at least one of alkali lignin, kraft lignin, lignin sulfonate, enzymatic lignin and organic solvent lignin.
The structural formulas of the sulfobetaine and the phosphobetaine fragments are respectively as follows
The pH-UCST responsive lignin-based zwitterionic compound prepared by the method.
A method for recycling cellulase by utilizing the pH-UCST responsive lignin-based zwitterionic compound at room temperature comprises the following steps:
adding the pH-UCST response lignin-based zwitterionic compound into a cellulase solution, dissolving at 40-60 ℃, or adding the pH-UCST response lignin-based zwitterionic compound into a lignocellulose enzymolysis system, and carrying out enzymolysis for 24-72 h at 40-60 ℃; then the pH is reduced as required, and the temperature is reduced to the room temperature, so that the pH-UCST responsive lignin-based zwitterionic compound and the cellulase are simultaneously precipitated and separated out, and the cellulase is recovered by a solid-liquid separation method.
Preferably, the pH of the cellulase solution or the lignocellulose enzymolysis system is 4-6, the ionic strength is 10-200 mmol/L, the concentration of cellulase protein is 40-2500 mg/L, and the cellulase is derived from Trichoderma viride and Aspergillus niger.
More preferably, the cellulase solution lignocellulose enzymolysis system has a pH of 5 and an ionic strength of 50mmol/L.
Preferably, if the pH of the system is higher than 4.5, the pH of the system is adjusted to 3.0-4.5 after enzymolysis and the temperature is reduced to room temperature; more preferably, the pH is adjusted to 4.0 to 4.5, and most preferably 4.0.
Preferably, the mass ratio of the pH-UCST responsive lignin-based zwitterionic compound to the cellulase is 1-50:1; more preferably 30:1.
preferably, the room temperature is 15-30 ℃.
Preferably, the cooling method is a natural cooling method or cooling by a refrigeration device.
Preferably, the solid-liquid separation method is at least one of natural sedimentation, decantation, filtration and centrifugation.
The mechanism of the invention is as follows:
the response mechanism of the pH-UCST lignin-based zwitterionic compound is as follows, and under the condition of higher pH (the pH is more than or equal to 4.75), the molecular hydrophilicity is increased and the solubility of the zwitterionic compound is increased due to the increase of the ionization degree of COOH; under the condition of meta-acid (pH is less than or equal to 4.75), COO is used - The degree of protonation increases, the molecular hydrophilicity decreases, the solubility of the zwitterionic compound decreases, and the lignin-based zwitterionic compound has a pH response. When the temperature is higher than UCST, the molecular hydrophilicity is increased and the solubility of the zwitterionic compound is increased because the betaine is in an ionization state; at a temperature below UCST, the intermolecular association between betaine ions reduces the molecular hydrophilicity, the solubility of the zwitterionic compound reduces, and the lignin-based zwitterionic compound has UCST response.
The mechanism of recovering enzyme by using the pH-UCST type lignin-based zwitterionic compound is as follows, the pH-UCST type lignin-based zwitterionic compound can be dissolved under the enzymolysis condition (pH is 5.0 and 40-60 ℃), the pH of the system is reduced to 4.0-4.5 after enzymolysis, the system is cooled to room temperature, and the pH-UCST type lignin-based zwitterionic compound and the enzyme are coprecipitated as carboxylate radicals are protonated and positive and negative ions are associated in betaine to form salt.
Compared with the prior art, the invention has the following advantages:
(1) Compared with the prior work of recovering the cellulase by using a pH response lignin-based auxiliary agent (grafting a cationic group or polyethylene glycol onto lignin), the method has the advantages that the pH-UCST response lignin-based zwitterionic compound is used for recovering the cellulase, the enzyme recovery rate is high, only a small amount of acid and alkali are needed in the recovery process, and the method is environment-friendly;
(2) Compared with the prior work of recycling cellulase by using octadecylsulfopropyl betaine (SB 3-18) and sulfobetaine polymer (PSPE), the method has the advantages that the pH-UCST responsive lignin-based zwitterionic compound is utilized to recycle the cellulase, the enzyme recovery rate is high, and the added value of industrial lignin is improved;
(3) The invention has simple operation for recycling the cellulase, and can recycle the cellulase by adjusting the temperature of a recycling system without additional equipment.
Drawings
FIG. 1 is a FT-IR infrared spectrum characterization of pH-UCST responsive lignin-based sulfobetaine LSB-400 of example 3 (400 is the percentage of sulfobetaine fragment to the amount of phenolic hydroxyl species in lignin during synthesis).
FIG. 2 is a graph of the pH-UCST response of 3g/L LSB-100 of example 2 in an aqueous phase system.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
In the following examples, reagents used in addition to the pH-UCST lignin-based zwitterionic compounds were all commercially available. The cellulase in the examples is Cellic CTec2 which is currently widely used. The methods for measuring the enzymatic activity of cellulase filters in the examples can be found in the literature (Methods in Enzymology, 1988.160:87-112.).
The comparative example relates to sulfobetaine polymers (PSPE-1) which are prepared in the laboratory, specific synthetic process reference methods (Green chem.,2021, 23:2738-2746). The comparative example involved quaternary ammonium lignosulfonate (pH-LC) being a laboratory homemade, specific synthetic process reference method (ACS stable chem.eng., 2018.6:10679-10686).
Example 1
The specific synthesis method of the pH-UCST lignin-based sulfobetaine is as follows:
sulfobetaine fragment synthesis: adding N, N-dimethylethanolamine (1.2 eq) into an N, N-dimethylacetamide solution system of 3-bromopropane sodium sulfonate (1.0 eq) at 70 ℃, stirring the system at constant temperature for 6 hours, and obtaining an intermediate isethionic betaine after the reaction is finished; the mixture is stirred and reacted in thionyl chloride at constant temperature (the reaction temperature is 0 ℃) for 3 hours to synthesize the intermediate sulfobetaine fragment.
Lignin-based sulfobetaine synthesis (LSB-x): 100g of an aqueous EHL solution (EHL 20% by mass) was prepared at pH 10 and a number of sulfobetaine fragments were added. The system is reacted for 3 hours at 90 ℃ and is purified by an acid precipitation method to obtain a product LSB-x (x percent is the percentage of the quantity of phenolic hydroxyl substances in sulfobetaine fragments and enzymolysis lignin, wherein x=100 and 400).
Synthesis of phosphate betaine fragment: oxalic acid (1.2 eq) was added to an aqueous solution of phosphocholine calcium salt (1.0 eq) at room temperature, stirred at constant temperature for 3h, after the reaction was completed, the precipitate was removed by liquid-solid separation and the supernatant was collected. Further adding sodium bicarbonate (1.6 eq) into the supernatant, and stirring at room temperature for 0.5h at constant temperature to obtain an intermediate sodium phosphate betaine; further, it was reacted with epichlorohydrin (1.5 eq) at 90℃under acidic conditions for 9 hours to synthesize an intermediate sulfobetaine fragment, wherein the acid in the acidic conditions was hydrochloric acid (0.3 eq).
Lignin-based phosphate betaine synthesis (LPB-x): 100g of an aqueous EHL solution (EHL 20% by mass) was prepared at pH 10 and several phosphobetaine fragments were added. The system was reacted at 90 ℃ for 3 hours and purified by acid precipitation to obtain the product LPB-x (x% is the percentage of the amount of phenolic hydroxyl species in the phosphate betaine fragment and the enzymatically hydrolyzed lignin, where x=100 and 400).
Example 2 (recovery of enzyme in buffer solution)
Adding 30 mass parts of LSB-100 into 1 mass part of cellulase solution with the protein concentration of 100mg/L (prepared by 50mmol/L acetic acid-sodium acetate buffer solution) at 50 ℃, regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution, cooling the system to the room temperature of 25 ℃ to precipitate and separate out the LSB-100 and the cellulase, and separating solid and liquid by a centrifugal machine, wherein the solid phase is the recovered LSB-100 and the cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 3 (recovery of enzyme in buffer solution)
Adding 30 mass parts of LSB-400 into 1 mass part of solution with the cellulase protein concentration of 100mg/L (pH=5.0, 50mmol/L of acetic acid-sodium acetate buffer solution is prepared), regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution, cooling the system to the room temperature of 25 ℃ to precipitate and separate out the LSB-400 and the cellulase, and separating solid and liquid by a centrifugal machine, wherein the solid phase is the recovered LSB-400 and the cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 4 (recovery of enzyme in buffer solution)
Adding 1 mass fraction of LSB-100 into 1 mass fraction solution (pH=5.0, 50mmol/L acetic acid-sodium acetate buffer solution) with cellulase protein concentration of 100mg/L at 50deg.C, adjusting pH of the system to 4.0 with dilute hydrochloric acid solution, cooling to room temperature of 25deg.C to precipitate LSB-100 and cellulase, separating solid and liquid by a centrifuge, and collecting solid phase as recovered LSB-100 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 5 (recovery of enzyme in buffer solution)
Adding 30 mass parts of LSB-100 into 1 mass part of solution with the cellulase protein concentration of 100mg/L (pH=5.0, 50mmol/L of acetic acid-sodium acetate buffer solution is prepared), regulating the pH of the system to 4.5 by using a dilute hydrochloric acid solution, cooling the system to the room temperature of 25 ℃ to precipitate and separate out the LSB-100 and the cellulase, and separating solid and liquid by a centrifugal machine, wherein the solid phase is the recovered LSB-100 and the cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 6 (recovery of enzymes in a Lignocellulase System)
Adding 30 mass fractions of LSB-100 into a corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the concentration of cellulase protein of 100mg/L and 1 mass fraction at 50 ℃, regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate and separate out the LSB-100 and the cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered LSB-100 and the cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 7 (recovery of enzymes in a Lignocellulase System)
Adding 1 mass fraction LSB-100 into 1 mass fraction corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with cellulase protein concentration of 100mg/L at 50deg.C, regulating pH to 4.0 with dilute hydrochloric acid solution after enzymolysis for 48 hr, cooling to room temperature of 25deg.C to precipitate LSB-100 and cellulase, separating solid and liquid by centrifuge, and collecting solid phase which is recovered LSB-100 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Example 8 (recovery of enzymes from a Lignocellulase System)
Adding 30 mass fractions of LPB-100 into a corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with 1 mass fraction of cellulase protein concentration of 100mg/L at 50 ℃, regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate LPB-100 and cellulase, separating solid and liquid by a centrifuge, and obtaining the solid phase which is the recovered LPB-100 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1
Example 9 (recovery of enzymes from a Lignocellulase System)
Adding 30 mass fractions of LPB-400 into a corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with 1 mass fraction of cellulase protein concentration of 100mg/L at 50 ℃, regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate LPB-400 and cellulase, separating solid and liquid by a centrifuge, and obtaining the solid phase which is the recovered LPB-400 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1
Comparative example 1 (recovery of enzyme in buffer solution)
At 50 ℃, 30 mass fractions of PSPE-1 (Mw=198.5 kDa) are added into a 1 mass fraction cellulase solution (pH=5.0, 50mmol/L acetic acid-sodium acetate buffer is prepared, 50 ℃) with the protein concentration of 100mg/L, the system temperature is reduced to room temperature of 25 ℃, PSPE-1 and cellulase are precipitated and separated out, and solid-liquid is separated by a centrifuge, and the solid phase is the recovered PSPE-1 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Comparative example 2 (recovery of enzymes in a Lignocellulase System)
Adding 30 mass fractions of PSPE-1 (Mw=198.5 kDa) into a 1 mass fraction corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with a cellulase protein concentration of 100mg/L at 50 ℃, carrying out enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate and separate PSPE-1 and cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered PSPE-1 and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Comparative example 3 (recovery of enzymes in a lignocellulose enzymatic hydrolysis System)
Adding 30 mass fractions of pH-LC into a corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with 1 mass fraction of cellulase protein concentration of 100mg/L at 50 ℃, regulating the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate and separate out the pH-LC and the cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered pH-LC and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
Comparative example 4 (recovery of enzymes in a lignocellulose enzymatic hydrolysis System)
Adding 30 mass fractions of pH-LC into a corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with 1 mass fraction of cellulase protein concentration of 100mg/L at 50 ℃, regulating the pH of the system to 3.0 by using a dilute hydrochloric acid solution after enzymolysis for 48 hours, cooling the system to room temperature of 25 ℃ to precipitate and separate out the pH-LC and the cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered pH-LC and cellulase. The relative filter paper activities of the recovered cellulases were directly measured and calculated, and the results are shown in Table 1.
TABLE 1 relative Filter paper enzyme Activity of pH-UCST responsive lignin-based sulfobetaines recovery cellulases
From Table 1, it can be seen that the pH-UCST responsive lignin-based zwitterionic compound can effectively recover the cellulase in the solution, the recovered cellulase keeps high filter paper enzyme activity, and the enzyme recovery effect is significantly better than that of the polymers PSPE and pH-LC. In comparative example 4, pH-LC was added to the corncob residue enzymatic hydrolysis system, and after the enzymatic hydrolysis was completed, the pH of the enzymatic hydrolysate was reduced to 3.0 to recover a large amount of enzyme. Compared with pH-LC, the enzyme recovery process consumes a great amount of acid and alkali in an ineffective way, and the process is more environment-friendly.
LSB-400 lignin phenolic hydroxyl 1374cm compared to FT-IR Infrared Spectrometry for EHL in FIG. 1 -1 The intensity of the stretching vibration peak at the position is obviously reduced, which shows that the relative content of phenolic hydroxyl groups in LSB-400 is reduced, and the content of phenolic hydroxyl groups in LSB-400 is 1329cm -1 The quaternary ammonium salt C-N telescopic vibration absorption peak appears, which shows that the sulfobetaine fragment is successfully connected into the lignin phenolic hydroxyl site.
In FIG. 2 LSB-100 is completely dissolved at 50℃at pH5.0, the system pH is lowered and the system is cooled to room temperature, the hydrophilicity of the molecule is reduced as the carboxylate is protonated, and the system becomes cloudy. Regulating the pH value of the system to 4.0, and reducing the LSB-100 dissolution percentage to 30.7% at room temperature; and regulating the pH value of the system to 2.0, and respectively reducing the dissolution percentage of LSB-100 to 1.8 percent and 98.2 percent at room temperature so as to recycle the LSB-100. The results indicate that LSB-100 has a sensitive pH-UCST responsiveness.
Compared with the prior work of recovering the cellulose in the solution by using the pH responsive lignin sulfonic acid quaternary ammonium salt, the invention utilizes the pH-UCST responsive lignin-based amphoteric ion compound to recover the cellulose, has good enzyme recovery effect, does not need to consume a large amount of acid and alkali in the enzyme recovery process, and is green and environment-friendly; in addition, compared with the prior work of recycling cellulase by using octadecylsulfopropyl betaine (SB 3-18) and sulfobetaine polymer (PSPE), the method has the advantages that the pH-UCST responsive lignin-based zwitterionic compound is utilized to recycle the cellulase, the enzyme recovery rate is high, and the added value of industrial lignin can be improved; the optimized product can recover more than 98% of the filter paper enzyme activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (11)
1. A method for recycling cellulase at room temperature by utilizing pH-UCST responsive lignin-based zwitterionic compound is characterized by comprising the following steps:
adding a pH-UCST responsive lignin-based zwitterionic compound into a cellulase solution, dissolving at 40-60 ℃, then regulating the pH of the system to 4.0-4.5, cooling to room temperature, simultaneously precipitating and separating the pH-UCST responsive lignin-based zwitterionic compound and the cellulase, and recovering the cellulase by a solid-liquid separation method; the preparation method of the pH-UCST responsive lignin-based zwitterionic compound comprises the following steps:
(1) Reacting N, N-dimethylethanolamine and 3-bromopropane sodium sulfonate at 50-80 ℃ for 3-12 hours to obtain isethionic betaine, and then placing the isethionic betaine in thionyl chloride and reacting at 0-60 ℃ for 3-6 hours to obtain a sulfobetaine fragment;
acidifying a phosphorylcholine calcium salt tetrahydrate by oxalic acid at room temperature, then obtaining a phosphoric acid betaine sodium salt by sodium salt replacement, and carrying out halogenation reaction on the phosphoric acid betaine sodium salt and epichlorohydrin at 70-90 ℃ for 6-12 h under an acidic condition to obtain a phosphoric acid ester betaine fragment;
(2) Under alkaline conditions, the pH-UCST responsive lignin-based zwitterionic compound is obtained by grafting sulfobetaine fragments and/or phosphate betaine fragments on lignin phenolic hydroxyl sites.
2. A method for recycling cellulase at room temperature by utilizing pH-UCST responsive lignin-based zwitterionic compound is characterized by comprising the following steps:
adding a pH-UCST responsive lignin-based zwitterionic compound into a lignocellulose enzymolysis system, and carrying out enzymolysis for 24-72 h at 40-60 ℃; then regulating the pH of the system to 4.0-4.5, cooling to room temperature, simultaneously precipitating and separating out the pH-UCST responsive lignin-based zwitterionic compound and the cellulase, and recovering the cellulase by a solid-liquid separation method;
the preparation method of the pH-UCST responsive lignin-based zwitterionic compound comprises the following steps:
(1) Reacting N, N-dimethylethanolamine and 3-bromopropane sodium sulfonate at 50-80 ℃ for 3-12 hours to obtain isethionic betaine, and then placing the isethionic betaine in thionyl chloride and reacting at 0-60 ℃ for 3-6 hours to obtain a sulfobetaine fragment;
acidifying a phosphorylcholine calcium salt tetrahydrate by oxalic acid at room temperature, then obtaining a phosphoric acid betaine sodium salt by sodium salt replacement, and carrying out halogenation reaction on the phosphoric acid betaine sodium salt and epichlorohydrin at 70-90 ℃ for 6-12 h under an acidic condition to obtain a phosphoric acid ester betaine fragment;
(2) Under alkaline conditions, the pH-UCST responsive lignin-based zwitterionic compound is obtained by grafting sulfobetaine fragments and/or phosphate betaine fragments on lignin phenolic hydroxyl sites.
3. The method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1 or 2, wherein the molar ratio of N, N-dimethylethanolamine to sodium 3-bromopropanesulfonate in step (1) is 1 to 1.5:1, a step of;
the molar ratio of oxalic acid, the phosphorylcholine calcium salt tetrahydrate and the sodium salt in the step (1) is 1.2:1:0.5 to 1.6, wherein the sodium salt used for sodium salt replacement is at least one of anhydrous sodium carbonate and anhydrous sodium bicarbonate;
the molar ratio of the epichlorohydrin to the sodium salt of the phosphoric acid betaine in the step (1) is 1 to 1.5:1, a step of;
the acid in the acidic condition in the step (1) is at least one of hydrochloric acid, sulfuric acid, phosphoric acid, formic acid and acetic acid, wherein the molar ratio of the acid to the sodium salt of phosphoric acid betaine is 0.1-0.5: 1.2;
the ratio of the sulfobetaine fragment and/or the phosphate betaine fragment to the amount of phenolic hydroxyl substances in lignin in the step (2) is 1-4: 1, a step of;
and (2) grafting a sulfobetaine fragment and/or a phosphate betaine fragment on the phenolic hydroxyl site of lignin, wherein the reaction temperature is 60-90 ℃ and the time is 3-9 h.
4. The method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1 or 2, wherein the pH of the alkaline conditions of step (2) is from 10 to 12;
the lignin in the step (2) is at least one of alkali lignin, kraft lignin, lignosulfonate, enzymatic lignin and organic solvent lignin.
5. The method for recovering cellulase at room temperature by using a pH-UCST responsive lignin-based zwitterionic compound according to claim 1, wherein the pH of the cellulase solution is 4-6, the ionic strength is 10-200 mmol/L, and the concentration of cellulase protein is 40-2500 mg/L.
6. The method for recovering cellulase at room temperature by using a pH-UCST responsive lignin-based zwitterionic compound according to claim 2, wherein the pH of the lignocellulose enzymolysis system is 4-6, the ionic strength is 10-200 mmol/L, and the concentration of cellulase protein is 40-2500 mg/L.
7. The method for recovering cellulase at room temperature using a pH-UCST responsive lignin-based zwitterionic compound of claim 5 wherein the cellulase solution has a pH of 5 and an ionic strength of 50mmol/L.
8. The method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound of claim 6 wherein said lignocellulosic enzymatic hydrolysate has a pH of 5 and an ionic strength of 50mmol/L.
9. The method for recovering cellulase at room temperature by using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1 or 2, wherein the mass ratio of the pH-UCST-responsive lignin-based zwitterionic compound to cellulase is 1-50:1.
10. The method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1 or 2, wherein the mass ratio of the pH-UCST-responsive lignin-based zwitterionic compound to cellulase is 30:1.
11. a method for recovering cellulase at room temperature using a pH-UCST responsive lignin-based zwitterionic compound according to claim 1 or 2, wherein the cellulase is derived from trichoderma reesei fungus and aspergillus niger fungus; the room temperature is 15-30 ℃.
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