CN107177645B - Method for promoting lignocellulose enzymolysis and cooling and recovering cellulase by using amphoteric surfactant - Google Patents
Method for promoting lignocellulose enzymolysis and cooling and recovering cellulase by using amphoteric surfactant Download PDFInfo
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Abstract
The invention discloses a method for promoting lignocellulose enzymolysis and cooling and recovering cellulose by using an amphoteric surfactant. Adding lignocellulose into a buffer solution, adding an amphoteric surfactant and cellulase, heating for reaction to obtain a saccharification hydrolysate of the lignocellulose, performing solid-liquid separation to obtain an enzymolysis liquid, and reducing the temperature of the enzymolysis liquid to simultaneously precipitate the amphoteric surfactant and the cellulase for recycling. The method of the invention firstly provides a method for recovering the cellulase by simple temperature reduction by utilizing the characteristic that the amphoteric surfactant has critical solution temperature. The method disclosed by the invention not only can effectively improve the enzymolysis efficiency of the lignocellulose and recover certain cellulase, but also is simple to operate, does not need additional equipment, is green and environment-friendly, and the recovered cellulase keeps high activity.
Description
Technical Field
The invention relates to the technical field of lignocellulose enzymolysis, in particular to a method for promoting lignocellulose enzymolysis and recovering cellulose by using an amphoteric surfactant.
Background
The lignocellulose biorefinery fuel ethanol is one of effective and feasible technologies for replacing gasoline. In the refining process, whether the cellulase can carry out high-efficiency enzymolysis on the lignocellulose substrate is a key technical bottleneck. Meanwhile, the cellulase has the characteristics of low activity, high consumption, high price and the like, and thus the industrialization of the cellulosic ethanol is directly hindered. The realization of the high-efficiency recycling of the cellulase is an important way for reducing the cost of the bioethanol. At present, the cellulase recycling technology is used for recycling by ultrafiltration membrane recycling and fresh substrate re-adsorption. Immobilization of cellulases is also an effective way to increase enzyme stability and reduce cellulase costs.
Researchers have immobilized cellulase on silica chips, silica, glass beads, calcium alginate gel beads and magnetic nanoparticles. Because the loaded cellulase is insoluble in water, the loaded cellulase has better hydrolysis capacity on soluble carboxymethyl cellulose, but has lower enzymolysis efficiency on real lignocellulose substrates. In order to ensure that the cellulase has high hydrolysis efficiency and is convenient to recover, intelligent macromolecules with environmental response (such as temperature and pH) become the object of research, because acid and alkali are consumed in the process of adjusting the pH, polymers with temperature response are adopted to recover the cellulase, the cellulase is more environment-friendly, some nonionic polymers such as polyisopropylacrylamide have sensitive temperature response properties, the cellulase and the nonionic polymers are bonded together by covalent bonds, and the modified cellulase can have the properties of high-temperature precipitation and low-temperature dissolution.
Two types of copolymers with the Lowest Critical Solution Temperature (LCST) are synthesized by copolymerization of methacrylamide having amide groups with N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). A series of polymers with LCST in the range of 20.9-60.5 ℃ were synthesized experimentally. Endoglucanase produced by thermophilic bacteria Pyrococcushorikoshii was transaminated with pyridoxal-5-phosphate to produce a ketone-containing protein, and the transaminated cellulase was then co-polymerized with an amide bond-containing polymer to synthesize a bioconjugate. Endoglucanases retain more than 60% activity after two recycles and produce more soluble sugars than the unmodified enzyme alone (Journal of the American Chemical Society,2013,135: 293-.
A thermo-responsive Polymer (PNMN) formed by polymerization of N-isopropyl methacrylamide (nimaa) with methyl acrylate and N- (hydroxymethyl) acrylamide is used with cellulase to form a bioconjugate. The LCST of the Polymer (PNMN) was adjusted to 51.6 ℃ by small molecule quenching, at which point the recovery was 98.5% and cellulase was covalently bound to PNMN via 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to synthesize a bioconjugate (PNMN-C) that maintained 85.2% of its original activity after 5 repeated cycles of hydrolysis (Journal of Molecular Catalysis B: Enzymatic,2016,128: 39-45.).
However, the temperature-sensitive macromolecule-loaded cellulase still faces some problems: 1. the cellulase is recovered at a higher temperature, and is easy to inactivate; 2. the cellulase is easy to inactivate when the cellulase is grafted on a polymer, 3, the cellulase is a multi-component enzyme system, the grafting reaction and the recovery rate are different, and the recovery affects the activity of the enzyme system.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the method for recovering the cellulase by promoting the enzymolysis of the lignocellulose by using the amphoteric surfactant and reducing the temperature, which has the advantages of low energy consumption, environmental protection and capability of recovering the cellulase by only reducing the temperature of enzymolysis liquid naturally or by refrigeration equipment.
The amphoteric surfactant needs to have proper critical solution temperature (UCST) so that the amphoteric surfactant can be completely dissolved in buffer solution at 40-60 ℃ under the enzymolysis temperature, and can be precipitated from enzymolysis liquid by a cooling method after enzymolysis is finished.
The purpose of the invention is realized by the following scheme:
the method for promoting lignocellulose enzymolysis and cooling to recover cellulose by using the amphoteric surfactant comprises the following steps: adding lignocellulose into a buffer solution, adding an amphoteric surfactant and cellulase, heating to 40-60 ℃, reacting for 24-96 h to obtain a saccharification hydrolysate of the lignocellulose, performing solid-liquid separation to obtain an enzymolysis liquid, and reducing the temperature of the enzymolysis liquid to simultaneously precipitate the amphoteric surfactant and the cellulase for recycling; the UCST of the amphoteric surfactant is 0-40 ℃.
To further achieve the object of the present invention, preferably, the amphoteric surfactant is at least one of an amino acid type amphoteric surfactant, a betaine type amphoteric surfactant, a sulfobetaine type amphoteric surfactant, and a phosphate betaine amphoteric surfactant.
Preferably, the amphoteric surfactant is C8H18(CH3)2N+(CH2)OSO3 ﹣、C12H26(CH2)2N+CH2PO4 ﹣、C18H38(CH3)2N+O﹣、C14H30(CH3)2N+(CH2)3SO3 ﹣、C16H34(CH3)2N+(CH2)3SO3 ﹣And C8H17P(CH3)2At least one of O.
Preferably, the method for obtaining the enzymatic liquid by solid-liquid separation is a natural settling method, a decantation method, a filtration method, a centrifugation method or a combination of these methods.
Preferably, the method for reducing the temperature of the enzymolysis liquid is a natural cooling method or cooling by a refrigeration device.
Preferably, the buffer solution is an acetic acid-sodium acetate buffer solution, a citric acid-sodium citrate buffer solution or a phosphate buffer solution with pH of 4.5-6.2 and ionic strength of 5-200 mmol/L.
Preferably, the lignocellulose is at least one of pine, eucalyptus, poplar, ash, sea buckthorn, bur, fir, birch, corncob, corn stover, wheat straw, bagasse, straw, rice hull, edible fungus matrix and peanut shell.
Preferably, the amount of the buffer solution is 5-50 times of the mass of the lignocellulose.
Preferably, the mass ratio of the amphoteric surfactant to the lignocellulose is 10-50: 100.
preferably, the dosage of the cellulase is 3-30 FPU/g calculated by the content of glucan in the lignocellulose.
The mechanism of the invention is as follows: because common amphoteric surfactants have critical solution temperature (UCST), when the temperature is higher than the UCST, the amphoteric surfactants are dissolved in buffer solution, so that ineffective adsorption of cellulase on lignin is reduced, and enzymolysis of lignocellulose is promoted; when the temperature is lower than UCST, the amphoteric surfactant is separated out, and the cellulase in the solution is precipitated together when the amphoteric surfactant and the cellulase have certain hydrophobic effect. The amphoteric surfactant needs to have proper critical solution temperature (UCST) so that the amphoteric surfactant can be completely dissolved in buffer solution at the enzymolysis temperature (40-60 ℃), and can be conveniently precipitated from enzymolysis liquid by a cooling method after enzymolysis is finished.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the method takes the amphoteric surfactant as an enzymolysis auxiliary agent, has no inhibition effect on the enzymolysis of pure cellulose, and can improve the enzymolysis saccharification yield of lignocellulose by 13.7-72.1%.
(2) The method has simple operation for recovering the cellulase, and the cellulase can be recovered only by reducing the temperature of the enzymolysis liquid naturally or by refrigeration equipment.
(3) Compared with the method for fixing the cellulase on the nonionic polymer and recovering the cellulase by raising the temperature, which is generally researched at present, the method can avoid the high-temperature inactivation of the cellulase and the inactivation of the immobilization reaction.
(4) The invention does not need additional equipment in the operation process, has low energy consumption and is environment-friendly.
Drawings
FIG. 1 shows the temperature dependence of the solubility of SB3-16 in pH4.8(50mM) sodium acetate buffer.
FIG. 2 is a process flow diagram of the process of accelerating the hydrolysis of lignocellulose by amphoteric surfactants and recovering cellulase by lowering the temperature, and examples 1-6 are all operated according to the process flow diagram, but the operation is not supplemented with amphoteric surfactants and cellulase.
Detailed Description
For a better understanding of the present invention, the present invention is further described below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. The reagents used in the following examples are commercially available. The microcrystalline cellulose type in the examples was PH101 (from Sigma Aldrich), fiberThe cellulase is currently widely used Cellic CTec2, and the substrate comprises dilute acid pretreated eucalyptus (eucalyptus-DA) and acid sulfite treated pine (pine-SpORL); the amphoteric surfactant used is SB3-16, C8APSO4、C12PPS and C18DAO. The glucose concentration in the hydrolysate was determined by means of a biosensing analyzer (SBA-40E, institute of bioscience, Shandong province).
Example 1
As shown in fig. 2, 100 parts by mass of microcrystalline cellulose is taken, added to 5000 parts by mass of acetic acid-sodium acetate buffer solution with pH of 4.8 and ionic strength of 50mmol/L, 50 parts by mass of SB3-16 is added, 10FPU/g of cellulase in terms of mass of microcrystalline cellulose is added, reaction is performed at 50 ℃ for 48 hours, after the reaction is completed, enzymolysis liquid is obtained by centrifugal separation, the temperature of the enzymolysis liquid is reduced to 0 ℃, centrifugal separation is performed when a large amount of precipitates appear in the solution, the obtained solid is added to a sample with the same initial enzymolysis conditions (substrate and buffer) for enzymolysis again for 48 hours (cellulase and amphoteric surfactant are not supplemented), glucose content of two times of enzymolysis is measured by a biosensor analyzer, and statistical results are shown in table 1.
Example 2
As shown in fig. 2, 100 parts by mass of eucalyptus-DA is taken, added to 5000 parts by mass of citric acid-sodium citrate buffer solution with pH of 5.0 and ionic strength of 25mmol/L, 50 parts by mass of SB3-16 is added, 5FPU/g of cellulase calculated by glucan in the substrate is added, reaction is performed at 50 ℃ for 48 hours, after the reaction is completed, an enzymolysis liquid is obtained by centrifugal separation, the temperature of the enzymolysis liquid is reduced to 0 ℃, centrifugal separation is performed when a large amount of precipitates appear in the solution, the obtained solid is added to a sample with the same initial enzymolysis conditions (substrate and buffer) for enzymolysis again for 48 hours (cellulase and amphoteric surfactant are not supplemented), glucose content of enzymolysis for two times is measured by a biosensor analyzer, and statistical results are shown in table 1.
Example 3
As shown in FIG. 2, 250 parts by mass of pine-SPORL was added to 5000 parts by mass of a phosphate buffer solution having a pH of 5.5 and an ionic strength of 100mmol/L,adding 100 parts by mass of C12PPS, 10FPU/g of cellulase counted by glucan in a substrate is added, the reaction is carried out for 72 hours at the temperature of 50 ℃, after the reaction is finished, enzymolysis liquid is obtained through centrifugal separation, the temperature of the enzymolysis liquid is reduced to 0 ℃, centrifugal separation is carried out when a large amount of precipitates appear in the solution, the obtained solid is added into a sample with the same initial enzymolysis conditions (the substrate and a buffer solution) for enzymolysis for 72 hours again (the cellulase and the amphoteric surfactant are not supplemented), the glucose content of the two times of enzymolysis is measured through a biosensing analyzer, and the statistical result is shown in table 1.
Example 4
As shown in FIG. 2, 100 parts by mass of microcrystalline cellulose was added to 5000 parts by mass of an acetic acid-sodium acetate buffer solution having a pH of 4.8 and an ionic strength of 50mmol/L, and 50 parts by mass of C was added8APSO4Adding 10FPU/g of cellulase based on the mass of microcrystalline cellulose, reacting at 45 ℃ for 48 hours, after the reaction is finished, carrying out centrifugal separation to obtain enzymolysis liquid, reducing the temperature of the enzymolysis liquid to 0 ℃, carrying out centrifugal separation when a large amount of precipitates appear in the solution, adding the obtained solid into a sample with the same initial enzymolysis conditions (substrate and buffer) for carrying out enzymolysis for 48 hours again (cellulose and amphoteric surfactant are not supplemented), and measuring the glucose content of the two times of enzymolysis by using a biosensing analyzer, wherein the statistical result is shown in Table 1.
Example 5
As shown in figure 2, 100 parts by mass of eucalyptus-DA is added into 5000 parts by mass of citric acid-sodium citrate buffer solution with pH of 6.0 and ionic strength of 25mmol/L, and 50 parts by mass of C is added8APSO4Adding 20FPU/g of cellulase counted by glucan in a substrate, reacting for 48 hours at the temperature of 52 ℃, after the reaction is finished, carrying out centrifugal separation to obtain enzymolysis liquid, reducing the temperature of the enzymolysis liquid to 5 ℃, carrying out centrifugal separation when a large amount of precipitates appear in the solution, adding the obtained solid into a sample with the same initial enzymolysis conditions (the substrate and the buffer) for carrying out enzymolysis for 48 hours again (the cellulase and the amphoteric surfactant are not supplemented), measuring the glucose content of the two times of enzymolysis through a biosensing analyzer, and calculating the result as shown in Table 1.
Example 6
As shown in FIG. 2, 250 parts by mass of pine-SPORL was added to 5000 parts by mass of phosphate buffer solution having pH of 5.5 and ionic strength of 100mmol/L, and 100 parts by mass of C was added18DAO, adding 10FPU/g of cellulase counted by glucan in a substrate, reacting at the temperature of 50 ℃ for 72 hours, after the reaction is finished, carrying out centrifugal separation to obtain enzymolysis liquid, reducing the temperature of the enzymolysis liquid to 0 ℃, carrying out centrifugal separation when a large amount of precipitates appear in the solution, adding the obtained solid into a sample with the same initial enzymolysis conditions (the substrate and a buffer solution) for carrying out enzymolysis for 72 hours again (cellulose and amphoteric surfactant are not supplemented), measuring the glucose content of the enzymolysis twice through a biosensing analyzer, and obtaining the statistical result shown in Table 1.
Corresponding blank comparative examples are made in the above examples, and the blank refers to a sample which is subjected to enzymolysis for the same time without adding an amphoteric surfactant under the same enzymolysis conditions. The recovery rate (r) of the cellulase protein is calculated by comparing the ultraviolet absorption peak at 278nm of the cellulase before and after recovery:
h0is the ultraviolet absorbance at 278nm of the cellulase solution before recovery, h1The ultraviolet absorbance of the cellulase solution left after recovery at 278 nm.
As can be seen from Table 1, the amphoteric surfactant can effectively promote the enzymolysis of lignocellulose, and can recover certain cellulase, and at the same time, the amphoteric surfactant can be recycled in the process.
FIG. 1 shows the solubility of SB3-16 in pH4.8(50mM) sodium acetate buffer as a function of temperature, illustrating the feasibility of recovering cellulase by lowering the temperature.
FIG. 2 is a process flow diagram of the process of accelerating lignocellulose enzymolysis by the amphoteric surfactant and recovering the cellulase through temperature reduction, and the process does not involve a complex process, does not need additional equipment, and is low in energy consumption and environment-friendly.
TABLE 1 cases of accelerating lignocellulose enzymolysis and recovering cellulase by amphoteric surfactant
The embodiments of the present invention are not limited to the above-described 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 they are included in the scope of the present invention.
Claims (9)
1. The method for promoting lignocellulose enzymolysis and cooling and recovering cellulose by using the amphoteric surfactant is characterized by comprising the following steps of: adding lignocellulose into a buffer solution, adding an amphoteric surfactant and cellulase, heating to 40-60 ℃, reacting for 24-96 h to obtain a saccharification hydrolysate of the lignocellulose, performing solid-liquid separation to obtain an enzymolysis liquid, and reducing the temperature of the enzymolysis liquid to simultaneously precipitate the amphoteric surfactant and the cellulase for recycling; the UCST of the amphoteric surfactant is 0-40 ℃;
the amphoteric surfactant is sulfobetaine amphoteric surfactant, phosphate betaine amphoteric surfactant and C18At least one of DAOs.
2. The method according to claim 1, wherein the amphoteric surfactant is SB3-16, C8APSO4And C12At least one of PPS.
3. The method according to claim 1, wherein the method for obtaining the enzymatic liquid by solid-liquid separation is natural sedimentation, decantation, filtration, centrifugation or a combination thereof.
4. The method of claim 1, wherein the temperature of the enzymatic liquid is lowered by natural cooling or by refrigeration.
5. The method according to claim 1, wherein the buffer solution is an acetic acid-sodium acetate buffer, a citric acid-sodium citrate buffer or a phosphate buffer with pH = 4.5-6.2 and an ionic strength of 5-200 mmol/L.
6. The method of claim 1, wherein the lignocellulose is at least one of pine, eucalyptus, poplar, ash, sea buckthorn, cypress, cedar, birch, corncob, corn stover, wheat straw, bagasse, rice straw, rice hulls, edible fungus substrate, and peanut hulls.
7. The method according to claim 1, wherein the amount of the buffer is 5 to 50 times the mass of the lignocellulose.
8. The method according to claim 1, wherein the mass ratio of the amphoteric surfactant to the lignocellulose is 10-50: 100.
9. the method according to claim 1, wherein the amount of the cellulase is 3 to 30FPU/g in terms of glucan content in lignocellulose.
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| CN111100888B (en) * | 2018-10-26 | 2022-06-07 | 中国石油化工股份有限公司 | Method for promoting lignocellulose enzymolysis and saccharification |
| CN112522347A (en) * | 2020-12-24 | 2021-03-19 | 浙江华康药业股份有限公司 | Method for improving cellulase enzymolysis saccharification efficiency under high-solid enzymolysis system |
| CN113151225B (en) * | 2021-04-09 | 2023-06-20 | 华南理工大学 | Polymer modified beta-glucosidase, preparation thereof and application thereof in lignocellulose enzymolysis |
| CN115466406B (en) * | 2021-06-11 | 2023-08-22 | 华南理工大学 | PH-UCST responsive lignin-based zwitterionic compound and preparation method thereof and method for recycling cellulase at room temperature |
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