CN115466406A - PH-UCST response type lignin-based zwitterionic compound, preparation method thereof and method for recovering cellulase at room temperature - Google Patents
PH-UCST response type lignin-based zwitterionic compound, preparation method thereof and method for recovering cellulase at room temperature Download PDFInfo
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Abstract
The invention discloses a pH-UCST response type lignin-based zwitterionic compound, a preparation method thereof and a method for recovering cellulase at room temperature. The invention synthesizes a pH-UCST response type lignin-based zwitterionic compound by grafting UCST response type betaine segments on enzymatic hydrolysis lignin, and efficiently recovers cellulase 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, the pH of the system is reduced to 4.0-4.5 and is reduced to room temperature after dissolution or enzymatic hydrolysis, the pH-UCST response type lignin-based zwitterionic compound and the cellulase are precipitated and separated simultaneously, and the cellulase is recovered by a solid-liquid separation method. The method utilizes the pH-UCST response of the pH-UCST response type lignin-based zwitterionic compound to efficiently recover the cellulase in the solution at room temperature, and the recovery process is simple to operate and is green and environment-friendly.
Description
Technical Field
The invention belongs to the technical field of lignocellulose enzymolysis, and particularly relates to a pH-UCST response type lignin-based zwitterionic compound, a preparation method thereof and a method for recovering cellulose at room temperature.
Background
Cellulose and hemicellulose in lignocellulose such as crop straws and the like can be converted into carbohydrate through pretreatment and enzymolysis technology, and then 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. The lignin accounts for 15-35% of lignocellulose, and the 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 utilized efficiently.
In the enzymolysis process of lignocellulose, the cost of enzyme can be reduced by realizing the recovery and the cyclic utilization of the cellulase. For example, cellulase can be recovered by methods and techniques such as a method of adding fresh substrate to the solution, a membrane separation method, and an enzyme immobilization method (Biofuels, bioprod. Bioref., 2017.11. The cellulase is a multi-component enzyme, the affinities of different components of cellulase to a substrate are different, wherein the affinity of beta-glucosidase to the substrate cellulose is poor, and the beta-glucosidase cannot be effectively recovered by a method of adding a fresh substrate for re-adsorption. The membrane separation method and the enzyme immobilization method are not favorable for industrial popularization due to high technical difficulty, high cost and the like. Compared with the prior art, the method has the advantages that the environment-responsive auxiliary agent is added, the response performance of the auxiliary agent is utilized to recover the cellulase, the process operation is simple, and the enzyme recovery effect is good. For example, pH-responsive lignin amphoteric surfactant pH-LC can be synthesized by grafting sulfonate ions and quaternary ammonium ions on enzymatic lignin (EHL), the pH of the system is reduced, and the pH-LC can precipitate due to the fact that carboxylate is protonated and the hydrophilicity of molecules is reduced. Synthesizing pH-LC with isoelectric point (Ip) of 2.2 by adjusting the access amount of sulfonate and quaternary ammonium ions, adding 3.0g/L of pH-LC into a corn cob residue enzymolysis system (pH 5.0), adjusting the pH of the system to 3.2, and saving 50% of cellulase dosage by using the pH-LC (ACS Sustainable chem.Eng.,2018.6: 10679-10686). The proportion of the sulfonic acid group and the quaternary ammonium ion grafted into the EHL has obvious influence on the pH-LC response performance and the enzyme recovery performance, and the proportion of the sulfonic acid group and the quaternary ammonium ion needs to be accurately regulated in the pH-LC synthesis process, so that the synthesis process is complex. Meanwhile, the pH response sensitivity of the pH-LC is poor, so that the acid consumption is large in the enzyme recovery process. Furthermore, the inventors set a problem to utilize the UCST response properties of sulfobetaine compounds to achieve recovery and recycling of cellulases at room temperature (sustatin. Energy. Fuels,2020,5, 750-757; green chem.,2021, 23. When the temperature of the system is lower than UCST (cellulose ether temperature control) by adding a sulfobetaine polymer PSPE into a cellulase solution, the hydrophilicity of molecules is reduced due to association of positive and negative ions in the molecules, and the PSPE and the enzyme are mainly co-precipitated through hydrophobic effect. For example, by adding PSPE to a corn cob residue enzymatic digestion system (50 ℃) and cooling the system to room temperature, at least 50% of the enzyme is saved by PSPE (Green chem.,2021, 23. However, the binding force between the PSPE and cellulose is weak, so that the enzyme recovery performance of the auxiliary agent is poor. Therefore, the synthesis of the intelligent response type lignin surfactant with high sensitivity and high enzyme recovery performance has important significance for reducing the enzyme dosage.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a preparation method of a pH-UCST response type lignin-based zwitterionic compound.
Another object of the present invention is to provide a pH-UCST-responsive lignin-based zwitterionic compound prepared by the above method.
Still another object of the present invention is to provide the above method for recovering cellulase at room temperature using a pH-UCST responsive lignin-based zwitterionic compound.
According to the invention, a potential UCST response type betaine fragment is grafted on enzymatic hydrolysis lignin to synthesize a lignin-based zwitterionic compound with pH-UCST response performance, and cellulase is efficiently recovered at room temperature by utilizing the response performance. Compared with pH-LC, betaine ions are easy to associate into salts at low temperature, the molecular hydrophilicity is reduced, the response performance of the aid is improved, the acid consumption in the enzyme recovery process is reduced, and the binding capacity of the aid and the enzyme is improved. In addition, the synthesis process does not need to regulate and control the access proportion of positive ions and negative ions, and is simple. Not only can realize the resource utilization of the whole components of the lignocellulose, but also has important significance for reducing the production cost of biorefinery and cellulosic ethanol.
The invention synthesizes a series of pH-UCST response type lignin-based zwitterionic compounds, inspects the influence of factors such as molecular structure, grafting amount, concentration, buffer solution pH and ionic strength on the pH-UCST response performance, further researches the influence rule of factors such as the zwitterionic compound structure, grafting amount, concentration, buffer solution pH and ionic strength on the cellulase recovery performance of the zwitterionic compound at different pH and temperature, and realizes the efficient recovery of cellulase in a lignocellulose enzymolysis system at room temperature by adding the pH-UCST response type lignin-based zwitterionic compounds.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a pH-UCST response type lignin-based zwitterionic compound comprises the following steps:
(1) Reacting N, N-dimethylethanolamine and 3-bromopropylsodium sulfonate at 50-80 ℃ for 3-12 hours to obtain hydroxyethyl betaine, placing the hydroxyethyl betaine in thionyl chloride, and reacting at 0-60 ℃ for 3-6 hours to obtain a sulfobetaine fragment;
acidifying a choline phosphate calcium salt tetrahydrate compound by oxalic acid at room temperature, then replacing sodium salt to obtain betaine phosphate sodium salt, and performing halogenation reaction on the betaine phosphate sodium salt and epoxy chloropropane at 70-90 ℃ for 6-12 h under an acidic condition to obtain a phosphate betaine fragment;
(2) Under alkaline conditions, a pH-UCST response type lignin-based zwitterionic compound is obtained by grafting a sulfobetaine fragment and/or a phosphate betaine fragment on a lignin phenolic hydroxyl site.
Preferably, the molar ratio of the N, N-dimethylethanolamine to the sodium 3-bromopropylsulfonate in the step (1) is 1 to 1.5:1.
preferably, the solvent for the reaction of the N, N-dimethylethanolamine and the sodium 3-bromopropylsulfonate in the step (1) is at least one of methanol, ethyl acetate, acetone, acetonitrile, N-dimethylformamide and N, N-dimethylacetamide.
Preferably, the mole ratio of the oxalic acid, the calcium phosphocholine salt tetrahydrate and the sodium salt in the step (1) is 1.2:1: 0.5-1.6, wherein the sodium salt used for sodium salt replacement is at least one of anhydrous sodium carbonate and anhydrous sodium bicarbonate.
Preferably, the molar ratio of the epichlorohydrin to the betaine sodium phosphate in the step (1) is 1-1.5: 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 betaine sodium phosphate salt is 0.1-0.5: 1.2.
preferably, the pH of the alkaline condition in step (2) is 10 to 12.
Preferably, the ratio of the sulfobetaine fragment and/or the phosphate betaine fragment in the step (2) to the amount of phenolic hydroxyl substances in the lignin is 1-4: 1.
preferably, the reaction temperature for grafting the sulfobetaine fragment and/or the phosphate betaine fragment on the lignin phenolic hydroxyl site in the step (2) is 60-90 ℃, and the reaction time is 3-9 h; the reaction uses water as a solvent, and the mass fraction of lignin in a reaction system is 20%.
Preferably, the lignin in step (2) is at least one of alkali lignin, kraft lignin, lignosulfonate, enzymatic lignin and organosolv lignin.
The structural formulas of the sulfobetaine and the phosphobetaine are respectively as follows
The pH-UCST response type lignin-based zwitterionic compound prepared by the method.
A method for recovering cellulase at room temperature by using the pH-UCST response type lignin-based zwitterionic compound comprises the following specific steps:
adding the pH-UCST response type lignin-based zwitterionic compound into a cellulase solution, and dissolving at 40-60 ℃, or adding the pH-UCST response type lignin-based zwitterionic compound into a lignocellulose enzymolysis system, and carrying out enzymolysis for 24-72 h at 40-60 ℃; and then reducing the pH value as required, cooling to room temperature to simultaneously precipitate and separate out the pH-UCST response type lignin-based zwitterionic compound and the cellulase, and recovering the cellulase by a solid-liquid separation method.
Preferably, the pH value of the cellulase solution or the lignocellulose enzymolysis system is 4-6, the ionic strength is 10-200 mmol/L, the concentration of the cellulase protein is 40-2500 mg/L, and the cellulase is derived from Trichoderma reesei fungus and Aspergillus niger fungus.
More preferably, the cellulase solution lignocellulose enzymolysis system has pH of 5 and ionic strength of 50mmol/L.
Preferably, if the pH of the system is higher than 4.5, the pH of the system needs to be 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, most preferably 4.0.
Preferably, the mass ratio of the pH-UCST response type lignin-based zwitterionic compound to the cellulase is 1-50; more preferably 30:1.
preferably, the room temperature is 15 to 30 ℃.
Preferably, the cooling method is a natural cooling method or cooling by refrigeration equipment.
Preferably, the solid-liquid separation method is at least one of a natural settling method, a decantation method, a filtration method, and a centrifugation method.
The mechanism of the invention is as follows:
the pH-UCST type lignin-based zwitterionic compound has the following response mechanism, and under the condition of higher pH (the pH is more than or equal to 4.75), due to the fact that the ionization degree of COOH is increased, the hydrophilicity of molecules is increased, and the solubility of the zwitterionic compound is increased; under the condition of meta-acid (pH is less than or equal to 4.75), due to COO - The protonation degree is increased, the molecular hydrophilicity is reduced, the solubility of the zwitterionic compound is reduced, and the lignin-based zwitterionic compound has pH response. When the temperature is higher than UCST, the betaine is in an ionized state, the hydrophilicity of molecules is increased, and the solubility of zwitterionic compounds is increased; when the temperature is lower than UCST, the intramolecular betaine ion association reduces the molecule hydrophilicity, the solubility of the zwitterion compound reduces, and the lignin-based zwitterion compound has UCST response.
The mechanism for recovering the enzyme by utilizing 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 is 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 because carboxylic acid radicals are protonated and positive and negative ions in betaine are associated to form salt.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) Compared with the work of recovering the cellulase by using the pH-UCST response type lignin-based auxiliary agent (cationic groups or polyethylene glycol are grafted to lignin) in the earlier stage of the subject group of the inventor, the cellulase is recovered by using the pH-UCST response type lignin-based zwitterionic compound, the enzyme recovery rate is high, only a small amount of acid and alkali are needed in the recovery process, and the method is green and environment-friendly;
(2) Compared with the work of recovering cellulase by using octadecyl sulfopropyl betaine (SB 3-18) and sulfobetaine polymer (PSPE) in the earlier stage of the subject group of the inventor, the cellulase is recovered by using the pH-UCST response type lignin-based zwitterionic compound, the enzyme recovery rate is high, and the additional value of industrial lignin is improved;
(3) The method for recovering the cellulase is simple to operate, and the cellulase can be recovered by adjusting the temperature of a recovery system without additional equipment.
Drawings
FIG. 1 is a FT-IR spectrum characterization of pH-UCST responsive lignosulfoniobetaine LSB-400 (400 is the amount of phenolics hydroxyl species in lignin as a percentage of the sulfonate betaine fragment during synthesis) from example 3.
FIG. 2 is a graph of the pH-UCST response of 3g/L LSB-100 of example 2 in an aqueous system.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
In the following examples, reagents other than the lignin-based zwitterionic compound of the pH-UCST type are commercially available. The cellulase in the examples is currently widely used Cellic CTec2. In the examples, the cellulase-containing enzyme activity of filter paper was measured by the method described in reference (Methods in Enzymology,1988.160: 87-112).
The comparative example relates to a sulfobetaine polymer (PSPE-1) self-made by the laboratory, a specific synthetic process reference method (Green chem.,2021,23, 2738-2746. The comparative example relates to the synthesis of quaternary ammonium lignosulphonate (pH-LC) as self-made in the laboratory, a specific synthetic process reference method (ACS sustaineble chem. Eng., 2018.6.
Example 1
The specific synthesis method of the pH-UCST type lignin-based sulfobetaine comprises the following steps:
and (3) synthesizing a sulfobetaine fragment: adding N, N-dimethylethanolamine (1.2 eq) into an N, N-dimethylacetamide solution system of 3-bromopropylsodium sulfonate (1.0 eq) at 70 ℃, stirring the system at constant temperature for 6 hours, and obtaining an intermediate hydroxyethyl sulfoacid betaine after the reaction is finished; further stirring and reacting the intermediate product in thionyl chloride at constant temperature (the reaction temperature is 0 ℃) for 3 hours to synthesize an intermediate sulfobetaine fragment.
Ligno-based sulfobetaine Synthesis (LSB-x): 100g of EHL aqueous solution (EHL accounts for 20% of the solution mass) with the pH of 10 is prepared, and a plurality of sulfobetaine fragments are 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% is the percentage of the sulfobetaine fragment and the phenolic hydroxyl substance in the enzymatic hydrolysis lignin, wherein x =100 and 400).
Phosphate betaine fragment synthesis: adding oxalic acid (1.2 eq) into aqueous solution of choline phosphocholine calcium salt (1.0 eq) at room temperature, stirring at constant temperature for 3 hours, removing precipitate through liquid-solid separation after reaction is finished, and collecting supernatant. Further adding sodium bicarbonate (1.6 eq) into the supernatant, and stirring at constant temperature for 0.5h at room temperature to obtain an intermediate sodium betaine phosphate; further reacting the intermediate product with epichlorohydrin (1.5 eq) at 90 ℃ under an acidic condition for 9h to synthesize an intermediate sulfobetaine fragment, wherein the acid in the acidic condition is hydrochloric acid (0.3 eq).
Lignin-based phosphate betaine Synthesis (LPB-x): 100g of EHL aqueous solution (EHL accounts for 20% of the solution mass) with the pH of 10 is prepared, and a plurality of phosphate betaine fragments are added. The system is reacted for 3 hours at 90 ℃, and is purified by an acid precipitation method to obtain a product LPB-x (x% is the percentage of the phosphate betaine fragment and the phenolic hydroxyl substance in the enzymatic hydrolysis lignin, wherein x =100 and 400).
Example 2 (recovery of enzyme in buffer solution)
Adding 30 parts by mass of LSB-100 into 1 part by mass of cellulase solution with the protein concentration of 100mg/L (pH =5.0, 50mmol/L acetic acid-sodium acetate buffer solution is prepared) at 50 ℃, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution, reducing the temperature of the system to 25 ℃, precipitating the LSB-100 and the cellulase, and separating solid and liquid through a centrifugal machine, wherein the solid phase is the recovered LSB-100 and the cellulase. The relative filter paper enzyme activity of the recovered cellulase was 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 (pH =5.0, 50mmol/L acetic acid-sodium acetate buffer solution) with cellulase protein concentration of 100mg/L at 50 ℃, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution, reducing the temperature of the system to 25 ℃, precipitating and separating the LSB-400 and the cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered LSB-400 and the cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Example 4 (recovery of enzyme in buffer solution)
Adding 1 mass part of LSB-100 into 1 mass part of solution with cellulase protein concentration of 100mg/L (pH =5.0, 50mmol/L acetic acid-sodium acetate buffer solution is prepared) at 50 ℃, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution, reducing the temperature of the system to room temperature of 25 ℃, precipitating LSB-100 and cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is recovered LSB-100 and cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Example 5 (recovery of enzyme in buffer solution)
Adding 30 parts by mass of LSB-100 into 1 part by mass of solution with the cellulase protein concentration of 100mg/L (pH =5.0, prepared by 50mmol/L of acetic acid-sodium acetate buffer solution) at 50 ℃, adjusting the pH of the system to 4.5 by using dilute hydrochloric acid solution, reducing the temperature of the system to 25 ℃, precipitating the LSB-100 and the cellulase, and separating solid and liquid through a centrifugal machine, wherein the solid phase is the recovered LSB-100 and the cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Example 6 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 parts by mass of LSB-100 into a 1 part by mass of corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the cellulase protein concentration of 100mg/L at 50 ℃, adjusting the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis is carried out for 48 hours, cooling the system to the room temperature of 25 ℃, precipitating and separating 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 enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Example 7 (enzyme recovery in lignocellulosic hydrolysis System)
Adding 1 mass part of LSB-100 into a 1 mass part of corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the cellulase protein concentration of 100mg/L at 50 ℃, adjusting the pH of the system to 4.0 by using a dilute hydrochloric acid solution after enzymolysis is carried out for 48 hours, cooling the system to the room temperature of 25 ℃, precipitating and separating 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 enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Example 8 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 parts by mass of LPB-100 into a 1 part by mass corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with cellulase protein concentration of 100mg/L at 50 ℃, after 48 hours of enzymolysis, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution, cooling the system to room temperature of 25 ℃, precipitating the LPB-100 and the cellulase, and separating solid and liquid by a centrifuge, wherein the solid phase is the recovered LPB-100 and the cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in Table 1
Example 9 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 parts by mass of LPB-400 into a 1 part by mass corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with cellulase protein concentration of 100mg/L at 50 ℃, after 48 hours of enzymolysis, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution, cooling the system to room temperature of 25 ℃, precipitating the LPB-400 and the cellulase, and separating solid and liquid through a centrifugal machine, wherein the solid phase is the recovered LPB-400 and the cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in Table 1
Comparative example 1 (recovery of enzyme in buffer solution)
Adding 30 mass parts of PSPE-1 (Mw =198.5 kDa) into 1 mass part of cellulase solution with the protein concentration of 100mg/L (pH =5.0, 50mmol/L acetic acid-sodium acetate buffer solution preparation, 50 ℃) at 50 ℃, cooling the system temperature to room temperature of 25 ℃, precipitating the PSPE-1 and the cellulase, and separating solid and liquid through a centrifuge, wherein the solid phase is the recovered PSPE-1 and cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Comparative example 2 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 mass parts of PSPE-1 (Mw =198.5 kDa) into a 1 mass part of corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the cellulase protein concentration of 100mg/L at 50 ℃, cooling the system temperature to 25 ℃ after enzymolysis is carried out for 48 hours, precipitating and separating out the PSPE-1 and the cellulase, and separating solid and liquid through a centrifugal machine, wherein the solid phase is the recovered PSPE-1 and cellulase. The relative filter paper enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Comparative example 3 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 parts by mass of pH-LC into a 1 part by mass of corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the cellulase protein concentration of 100mg/L at 50 ℃, adjusting the pH of the system to 4.0 by using dilute hydrochloric acid solution after enzymolysis is carried out for 48 hours, cooling the system to room temperature of 25 ℃, precipitating and separating 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 enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
Comparative example 4 (enzyme recovery in lignocellulose enzymolysis System)
Adding 30 parts by mass of pH-LC into a 1 part by mass of corncob residue enzymolysis system (pH 5.0, 50mmol/L,50 ℃) with the cellulase protein concentration of 100mg/L at 50 ℃, adjusting the pH of the system to 3.0 by using a dilute hydrochloric acid solution after enzymolysis is carried out for 48 hours, cooling the system to the room temperature of 25 ℃, precipitating and separating 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 enzyme activity of the recovered cellulase was directly measured and calculated, and the results are shown in table 1.
TABLE 1 relative filter paper enzyme activity of pH-UCST responsive ligno-sulfobetaine to cellulase recovery
As can be seen from the table 1, the pH-UCST response type 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 obviously superior to that of polymer PSPE and pH-LC. In comparative example 4, pH-LC was added to the corn cob residue enzymolysis system, and after the enzymolysis was completed, the pH of the enzymolysis solution was adjusted to 3.0 to recover more enzymes. Compared with pH-LC, a large amount of acid and alkali are consumed in the enzyme recovery process, and the process is more green and environment-friendly.
LSB-400 lignin phenolic hydroxyl group 1374cm in FIG. 1 compared with FT-IR infrared spectrum of EHL -1 The intensity of the stretching vibration peak is obviously reduced, which shows that the relative content of phenolic hydroxyl in LSB-400 is reduced, and 1329cm in LSB-400 -1 C-N telescopic vibration absorber with quaternary ammonium saltAnd (4) collecting peaks, which indicates that the sulfobetaine fragment is successfully grafted to the phenolic hydroxyl site of the lignin.
In FIG. 2, LSB-100 is completely dissolved at pH5.0 at 50 ℃, the pH of the system is adjusted to be low, the system is cooled to room temperature, and the hydrophilicity of the molecule is reduced due to protonation of the carboxylate, so that the system becomes turbid. Adjusting the pH value of the system to 4.0, and reducing the dissolution percentage of the LSB-100 to 30.7% at room temperature; the pH value of the system is adjusted to 2.0, the dissolution percentage of LSB-100 is respectively reduced to 1.8 percent at room temperature, and 98.2 percent of LSB-100 can be recycled. The results show that LSB-100 has sensitive pH-UCST responsiveness.
Compared with the work of recovering the cellulase in the solution by using the pH response lignin sulfonic acid quaternary ammonium salt in the earlier stage of the subject group of the inventor, the method for recovering the cellulase by using the pH-UCST response type lignin-based zwitterionic compound has the advantages that the enzyme recovery effect is good, a large amount of acid and alkali are not required to be consumed in the enzyme recovery process, and the method is green and environment-friendly; in addition, compared with the work of recovering the cellulase by using octadecyl sulfopropyl betaine (SB 3-18) and sulfobetaine polymer (PSPE) at the earlier stage of the subject group of the inventor, the cellulase is recovered by using the pH-UCST response type lignin-based zwitterionic compound, the enzyme recovery rate is high, and the additional value of industrial lignin can be improved; the optimized product can recover more than 98% of the filter paper enzyme activity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of a pH-UCST response type lignin-based zwitterionic compound is characterized by comprising the following steps:
(1) Reacting N, N-dimethylethanolamine and 3-bromopropyl sodium sulfonate at 50-80 ℃ for 3-12 h to obtain hydroxyethyl betaine sulfonate, placing the hydroxyethyl betaine sulfonate in thionyl chloride, and reacting at 0-60 ℃ for 3-6 h to obtain a betaine sulfonate fragment;
acidifying a choline phosphate calcium salt tetrahydrate compound by oxalic acid at room temperature, then replacing sodium salt to obtain betaine phosphate sodium salt, and performing halogenation reaction on the betaine phosphate sodium salt and epoxy chloropropane at 70-90 ℃ for 6-12 h under an acidic condition to obtain a phosphate betaine fragment;
(2) Under alkaline conditions, a pH-UCST response type lignin-based zwitterionic compound is obtained by grafting a sulfobetaine fragment and/or a phosphate betaine fragment on a lignin phenolic hydroxyl site.
2. The method for preparing a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1, wherein the molar ratio of the N, N-dimethylethanolamine to the sodium 3-bromopropylsulfonate in the step (1) is 1-1.5: 1;
the mole ratio of oxalic acid, the calcium choline phosphate 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 mol ratio of the epichlorohydrin to the sodium salt of the phosphoric acid betaine in the step (1) is 1-1.5: 1;
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 betaine sodium phosphate is 0.1-0.5: 1.2;
the ratio of the sulfobetaine fragment and/or the phosphate betaine fragment in the step (2) to the amount of phenolic hydroxyl substances in the lignin is 1-4: 1;
the reaction temperature of grafting sulfobetaine fragments or phosphate betaine fragments on the phenolic hydroxyl sites of the lignin is 60-90 ℃, and the reaction time is 3-9 h.
3. The method for preparing a pH-UCST-responsive lignin-based zwitterionic compound according to claim 1, wherein the alkaline condition in step (3) is a pH of 10 to 12;
the lignin in the step (3) is at least one of alkali lignin, sulfate lignin, lignosulfonate, enzymatic lignin and organic solvent lignin.
4. A pH-UCST responsive lignin-based zwitterionic compound produced by the method of any one of claims 1 to 3.
5. A method for recovering cellulase at room temperature using the pH-UCST-responsive lignin-based zwitterionic compound of claim 4, comprising:
adding the pH-UCST response type lignin-based zwitterionic compound into a cellulase solution, and dissolving at 40-60 ℃, or adding the pH-UCST response type lignin-based zwitterionic compound into a lignocellulose enzymolysis system, and carrying out enzymolysis at 40-60 ℃ for 24-72 h; and then reducing the pH value as required, cooling to room temperature to simultaneously precipitate and separate out the pH-UCST response type lignin-based zwitterionic compound and the cellulase, and recovering the cellulase by a solid-liquid separation method.
6. The method for recovering cellulase at room temperature by using the pH-UCST response type lignin-based zwitterionic compound as claimed in claim 5, wherein the pH of the cellulase solution or 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 by using the pH-UCST response type lignin-based zwitterionic compound according to claim 6, wherein the pH of the cellulase solution or the lignocellulose enzymolysis system is 5, and the ionic strength is 50mmol/L.
8. The method for recovering cellulase at room temperature by using the pH-UCST-responsive lignin-based zwitterionic compound according to claim 5, wherein the mass ratio of the pH-UCST-responsive lignin-based zwitterionic compound to the cellulase is 1-50.
9. The method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 8, wherein the mass ratio of the pH-UCST-responsive lignin-based zwitterionic compound to cellulase is 30:1.
10. the method for recovering cellulase at room temperature using a pH-UCST-responsive lignin-based zwitterionic compound according to claim 5, wherein the cellulase is derived from Trichoderma reesei fungi and Aspergillus niger fungi; if the pH value of the system is higher than 4.5, the pH value of the system needs to be adjusted to 3.0-4.5 and cooled to room temperature after dissolution or enzymolysis; the room temperature is 15-30 ℃.
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