CN116202997A - Method for characterizing interaction of lignin and cellulase - Google Patents
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Abstract
The application discloses a method for characterizing interaction of lignin and cellulose, which comprises the steps of firstly preparing lignin solution, then dripping the lignin solution on a gold chip of a surface plasma resonance technology, and preparing a lignin film by spin coating; and (3) introducing cellulase into an SPR instrument, and monitoring the dynamic adsorption process between the lignin component and the cellulase in real time to obtain the interaction result of lignin and the cellulase. According to the method for representing interaction between lignin and cellulose, the SPR is utilized to evaluate the adsorption and dissociation processes between the cellulose and the lignin, so that the binding force and the desorption force of the interaction between the cellulose and the lignin can be effectively obtained, the action mechanism between the cellulose and the lignin can be studied in more detail, a solid theoretical basis is laid for the next scientific research, and the method has good practicability.
Description
Technical Field
The application belongs to the technical field of pretreatment of plant fiber raw materials, relates to a cellulose saccharification technology, and in particular relates to a method for representing interaction between lignin and cellulase.
Background
The preparation of bioethanol and bio-based chemicals from cellulosic waste by biorefinery has become a research hotspot in the fields of agriculture, forestry and industrial biotechnology in recent years. The preparation of bioethanol and bio-based chemicals from lignocellulosic resources mainly comprises the steps of raw material pretreatment, cellulose saccharification, sugar liquor fermentation, product extraction and the like. Among them, cellulose saccharification is a key technology for preparing bioethanol and bio-based chemicals from lignocellulose, and pretreatment is a primary step for realizing efficient cellulose saccharification efficiency. Dilute acid pretreatment is a common pretreatment method for biorefinery of lignocellulose and has been widely used for pretreatment of lignocellulosic feedstocks. However, researches show that the dilute acid pretreatment mainly used for degrading and dissolving hemicellulose has little effect on improving the saccharification efficiency of wood and bamboo raw materials, and the hemicellulose degradation greatly increases the porosity and specific surface area of the materials and improves the accessibility of cellulose by cellulose, but the saccharification rate of the pretreated materials is still lower. This is mainly lignin remaining in the material after pretreatment and pseudo lignin (carbohydrates in lignocellulose, which degrade during pretreatment to form globular substances formed by dehydration condensation and aromatization of glucose and xylose, called "pseudo lignin") formed during pretreatment, and has ineffective adsorption to cellulase, thereby reducing cellulase hydrolysis.
At present, research on interaction mechanisms of pseudo lignin and residual lignin generated in the dilute acid pretreatment process and cellulase is an important concern for biorefinery of lignocellulose. It is generally considered that the hydrophobic property, the chargeability, the functional group and other physical and chemical properties of lignin enable lignin molecules and cellulose molecules to be mutually adsorbed with a certain acting force, and the adsorbed cellulose and lignin are firmly combined, so that free cellulose in a hydrolysis system is reduced, and the cellulose saccharification efficiency is reduced.
The existing method for researching interaction between lignin and cellulase mainly comprises the steps of adding enzyme into lignin solution, measuring the difference value between the concentration of the enzyme in supernatant fluid after adsorption and the initial concentration of the enzyme, and obtaining the adsorption result of the lignin and the enzyme through beginning-to-end change. However, the conventional method cannot obtain the whole dynamic process and binding force of adsorption and desorption at a microscopic angle, and cannot effectively analyze the interaction mechanism of lignin and cellulose.
Disclosure of Invention
Aiming at the defects in the prior art, the technical problem to be solved by the application is to provide a method for representing interaction between lignin and cellulose, and the adsorption and dissociation processes between the cellulose and the lignin are evaluated by utilizing surface plasmon resonance (surface plasmon resonance, SPR) to obtain the interaction binding force and the interaction desorption force between the cellulose and the lignin.
In order to solve the technical problems, the technical scheme adopted by the application is as follows:
a method for representing interaction of lignin and cellulase comprises the steps of firstly preparing lignin solution, then dripping the lignin solution on a surface plasmon resonance technology gold chip, and preparing a lignin film by spin coating; and (3) introducing cellulase into an SPR instrument, and monitoring the dynamic adsorption process between the lignin component and the cellulase in real time to obtain the interaction result of lignin and the cellulase.
The method for characterizing the interaction of lignin and cellulase comprises the following steps:
1) Preparing a pretreatment material;
2) Preparing Surface Lignin (SL);
3) Preparing residual lignin (MWL);
4) Detecting a dynamic adsorption process between lignin and cellulase;
preparing 0.5% of surface lignin DMSO solution and residual lignin DMSO solution, then dripping 100 mu L of solution on a surface plasmon resonance gold chip by using a flat-mouth injector, standing for 1min, spin-coating at a speed of 5kr/min by using a spin coater to prepare a lignin film, vacuum-drying at 40 ℃ for 4 hours, cleaning in deionized water to ensure complete removal of DMSO, and vacuum-drying at 40 ℃ for 12 hours;
before introducing the cellulase protein solution into the SPR instrument, introducing 0.05M sodium citrate buffer solution at a flow rate of 0.1mL/min to obtain a stable baseline; after stabilization, the solutions with the cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L were introduced for 4 minutes at a flow rate of 0.1mL/min, and the adsorption behavior of lignin and cellulase was observed; then, introducing 0.05M sodium citrate buffer solution to obtain a dissociation curve of dissociative cellulase on the lignin membrane; processing the SPR curve by adopting SPR Navi Data Viewer software, and analyzing the kinetic constants by adopting a washer software;
5) And obtaining a detection result.
In step 1), the preparation of the pretreated material is: pretreating bamboo scraps by using a 1% (g/mL) sulfuric acid solution, and washing residues to be neutral by using distilled water to obtain pretreated materials.
In step 2), the preparation of surface lignin is: extracting the pretreated material by adopting different solvents respectively to prepare surface lignin with different characteristics; the solvent is as follows: the volume ratio is 96:4, 4-dioxane/water solution, 95% alcohol and tetrahydrofuran.
The method for characterizing the interaction between lignin and cellulase comprises the following extraction processes: adding the pretreated material into a conical flask, adding a solvent according to the solid-to-liquid ratio of 1:10, and extracting for 1 day at room temperature under magnetic stirring at the rotating speed of 150 r/min; after the completion, carrying out solid-liquid separation, and adding fresh organic extraction solution again into the extracted solid to continue extraction; the above process is repeated three times, so that the surface lignin is extracted to the greatest extent; the three supernatants were collected and mixed together and the extraction solvent was removed by rotary evaporation and vacuum freeze drying to give lignin solids.
In step 3), the preparation of residual lignin is: taking the absolute dry lignin solid to a planetary ball mill, and effectively grinding for 6 hours at the rotating speed of 600 r/min.
In step 5), the detection result is: the adsorption capacity of different lignin components and cellulase is not obviously different, and the SL component ratio is MWL componentDissociation is easier to occur, and affinity K of SL to cellulase D The value is 39.4-52.6nM, and the MWL is K with cellulase D The value is 6.8-24.7nM.
The method for characterizing the interaction between lignin and cellulose is characterized in that the lignin is pseudo lignin.
The method for characterizing the interaction of lignin and cellulase comprises the following steps:
1) Preparing holocellulose;
2) Preparing a pretreatment material;
3) Preparation of pseudo lignin: extracting the pretreated material by adopting different solvents respectively to prepare pseudo lignin with different characteristics; the solvent is as follows: 1, 4-dioxane/water solution, 95% alcohol and tetrahydrofuran with the volume ratio of 96:4;
4) Dynamic adsorption process detection between pseudo lignin and cellulase
Preparing 0.5% pseudo lignin DMSO solution, then dripping 100 mu L of the solution on a surface plasmon resonance gold chip by using a flat-mouth injector, standing for 1min, spin-coating at a speed of 5kr/min by using a spin-coating instrument to prepare a pseudo lignin film, vacuum-drying at 40 ℃ for 4h, cleaning with deionized water to ensure complete removal of DMSO, and vacuum-drying at 40 ℃ for 12 h;
before introducing the cellulase protein solution into the SPR instrument, introducing 0.05M sodium citrate buffer solution at a flow rate of 0.1mL/min to obtain a stable baseline; after stabilization, the solutions with the cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L are introduced for 4 minutes at a flow rate of 0.1mL/min, and the adsorption behavior of pseudo lignin and cellulase is observed; then, introducing 0.05M sodium citrate buffer solution to obtain a dissociation curve of dissociative cellulase on the lignin membrane; processing the SPR curve by adopting SPR Navi Data Viewer software, and analyzing the kinetic constants by adopting a washer software;
5) Obtaining a detection result: the pseudo lignin obtained at higher temperature is more likely to dissociate from the cellulase.
Use of SPR for detecting interactions between lignin and cellulases.
The beneficial effects are that: compared with the prior art, the method for representing interaction between lignin and cellulose utilizes SPR to evaluate the adsorption and dissociation processes between cellulose and lignin, so as to obtain the interaction binding force and desorption force between the cellulose and lignin. The characterization result shows that:
1) The presence of a stronger interaction relationship between the cellulase and the MWL results in less free cellulase content in the enzymatic hydrolysis process system during enzymatic hydrolysis, and thus lower yields of enzymatic hydrolysis of the substrate.
2) Extraction of the SL component from the DAP-BR surface by three different organic solvents exposes more residual lignin component (MWL), resulting in more non-productive binding to the enzyme and reduced enzymatic hydrolysis performance of the substrate.
3) The false lignin prepared by the higher pretreatment temperature is easier to dissociate from the cellulose, which is beneficial to the separation of more enzymes adsorbed on the false lignin in a free state in an enzyme hydrolysis system after the enzyme is resolved, thereby improving the cellulose hydrolysis yield.
4) The pseudo lignin extracted by the ethanol has the strongest binding capacity with the cellulase, which is one of the reasons for the stronger effect of inhibiting the hydrolysis of microcrystalline cellulase.
It can be seen that SPR may have broader application in characterizing the interaction between cellulases and lignin.
Drawings
FIG. 1 is a graph of the results of real-time monitoring of adsorption and resolution kinetics of a cellulase solution on membranes of different lignin components; in the figure, (a) Dio-SL; (b) Eth-SL; (c) THF-SL; (d) DAP-MWL; (e) Dio-MWL; (f) Eth-MWL; (g) THF-MWL;
FIG. 2 is a graph showing the results of real-time monitoring of adsorption and desorption kinetics of a cellulase solution on different pseudo lignin component membranes; in the figure, (a) 160-Dio-PL; (b) 160-Eth-PL; (c) 160-THF-PL; (d) 170-Dio-PL; (e) 170-Eth-PL; (f) 170-THF-PL; (g) 180-Dio-PL; (h) 180-Eth-PL; (i) 180-THF-PL.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Example 1
1. Preparation of pretreated Material
1kg of absolute dried bamboo chips are transferred into a 15L high-temperature reactor, a sulfuric acid solution with the solid-to-liquid ratio of 1:6 is added into the reactor, and the reactor is reacted for 1h at 160 ℃. And after the completion, rapidly taking out and separating solid from liquid. The residue was washed with distilled water to neutrality to give a pretreated material, labeled DAP-BR (diluted acid pretreated bamboo residues). A part of the pretreated material is stored in a refrigerator at 4 ℃ for subsequent enzymatic hydrolysis, and the other part is naturally air-dried for lignin extraction.
2. Preparation of surface lignin
DAP-BR was extracted with different solvents, respectively, to prepare Surface Lignin (SL) with different characteristics. The solvent is as follows: 1, 4-Dioxa-ring/water (96:4, v/v) solution, ethanol/water (95:5, v/v) solution and tetrahydrofuran solvent (100%). The specific process is as follows:
80g of DAP-BR were weighed into conical flasks, 800mL of solvent were added in a solid-to-liquid ratio of 1:10, and extracted for 1 day with magnetic stirring at 150r/min at room temperature. After the completion of the solid-liquid separation, fresh organic extraction solution is added again to the extracted solid to continue the extraction. The above process is repeated three times, so as to ensure that the surface lignin is extracted to the greatest extent. The three supernatants were collected and mixed together and the extraction solvent was removed by rotary evaporation and vacuum freeze drying to give lignin solids.
Surface lignin obtained by extraction of DAP-BR from 1, 4-dioxane/water (96:4, v/v) solution, ethanol/water (95:5, v/v) solution and tetrahydrofuran solvent (100%) was labeled as Dio-SL, eth-SL and THF-SL, respectively. The solid material remaining after extraction was washed with a large amount of distilled water, air dried to ensure complete removal of the organic solvent, and collected in a self-sealing bag for use, labeled Dio-BR, eth-BR and THF-BR, respectively.
3. Preparation of residual lignin
The isolated DAP-BR, dio-BR, eth-BR and THF-BR were weighed out as 5g solids into a planetary ball mill (Pulverisette 7, french, germany) and effectively milled for 6h at 600r/min using 25 zirconia balls with a diameter of 1 cm. The lignin remaining in the bamboo chips was ball milled lignin (milled wood lignin, MWL) according to the Bjorkman method. MWL obtained from DAP-BR, dio-BR, eth-BR and THF-BR are designated DAP-MWL, dio-MWL, eth-MW and THF-MWL, respectively.
4. Dynamic adsorption process detection between lignin and cellulase
A lignin solution of 0.5% (w/v, DMSO) was prepared (surface lignin series (Dio-SL, eth-SL and THF-SL) and residual lignin series (DAP-MWL, dio-MWL, eth-MW and THF-MWL)), followed by dropping 100. Mu.L of the solution on a Surface Plasmon Resonance (SPR) gold chip with a flat-top syringe, standing for 1min, and spin-coating with a spin coater (KW-4A, shanghai-Takeside Instrument Co., china) at a speed of 5kr/min for 1min, and repeating 3 times. The obtained lignin film was dried in vacuum at 40 ℃ for 4 hours, and then soaked in deionized water for 1 day. Deionized water is replaced at intervals of 2 hours, complete removal of DMSO is ensured, and the influence on the activity of cellulase is avoided. The soaked film was dried in vacuo at 40 ℃ for 12 hours.
Dynamic adsorption processes between different lignin components and cellulases were monitored in real time using an SPR instrument (MP-SPR Navi 200, bioNavis Ltd, tank Pelare, finland). The SPR instrument is equipped with a dual channel detection system with a laser wavelength of 670nm and a scan range of SPR angles of 40-78 deg..
Before passing the cellulase protein solution into the SPR apparatus, 0.05M sodium citrate buffer was passed at a flow rate of 0.1mL/min to obtain a stable baseline. After stabilization, solutions with cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L were fed at a flow rate of 0.1mL/min for 4 minutes, and the adsorption behavior of lignin and cellulase was observed. Subsequently, a 0.05M sodium citrate buffer was introduced to obtain a dissociation curve of the dissociating cellulase on the lignin membrane. SPR curves were processed using SPRNavi Data Viewer software and kinetic constants were analyzed using a scanner (version 2.0) software.
5. Experimental results
As can be seen from FIG. 1, a smooth baseline was obtained by introducing sodium citrate buffer solution to the lignin membrane surface for 0 to 200 seconds. At 200 to 440s, different concentrations (0.1, 0.05, 0.02 and 0.01) are respectively introduced on the surface of the wood filmAnd 0.005 g/L) of cellulase solution, resulting in RU (response unit of SPR, 1ru=1×10) -6 RIU,1 μriu=0.15 mdeg) value increases, indicating that cellulase adsorbs on lignin, and an interaction phenomenon occurs between the two. After 440s, the RU value was reduced by the sodium citrate buffer on the cellulose-adsorbed lignin membrane, indicating that the cellulase was desorbed from the lignin membrane surface. Furthermore, it can be seen from the figure that the RU values of the SL-cellulase system are reduced more significantly than the RU values of the MWL-cellulase system during the dissociation process (after 440 s), indicating that cellulases are easier to separate from SL. This result shows that the presence of cellulase in a stronger interaction with MWL results in less free cellulase content in the enzymatic hydrolysis process system during enzymatic hydrolysis, and thus lower enzymatic hydrolysis yields of the substrate.
Kinetic adsorption parameters of lignin membrane and cellulase were calculated and analyzed using a scanner (version 2.0) software, and the results are shown in table 1. Binding rate k of two different lignin components to cellulase enzymes a Are all 3.0X10 4 M -1 S -1 Up and down, and maximum saturated adsorption quantity R max Both are about 400RU, which indicates that there is no significant difference in the adsorption capacity of the two lignin components to the cellulase. But for dissociation rate k d In particular, the MWL-cellulase system (7.68X10) -4 S -1 -2.70×10 -4 S) ratio of cellulase-SL system (7.68X10- 4 S -1 -2.70×10 -4 S -1 ) With a smaller k d This indicates that the SL component is more susceptible to dissociation than the MWL component. In addition to K D As an indicator of cellulase and lignin binding affinities, there is a significant difference in SL and MWL systems. Wherein, the affinity K of SL and cellulase D The value is 39.4-52.6nM, and the MWL is K with cellulase D The value is 6.8-24.7nM. In general, K D Lower values indicate a stronger affinity of lignin for cellulase proteins. Thus, extraction of the SL component from the DAP-BR surface by three different organic solvents can expose more residual lignin component (MWL), leading to more non-productive binding to the enzyme and reduced enzymatic hydrolysis performance of the substrate.
TABLE 1 interaction kinetic parameters of lignin and cellulases
Note that: k (k) a A binding rate constant; k (k) d Dissociation rate; r is R max Maximum saturated adsorption capacity of lignin; k (K) D The dissociation rates are balanced.
Example 2
1. Preparation of holocellulose
Taking bamboo chip material, performing Soxhlet extraction, dewaxing for 6 hours by using benzene/ethanol (2:1, v/v), and volatilizing until the organic solvent is completely volatilized for later use. 50g of dewaxed material was placed in an Erlenmeyer flask, 1L of distilled water and 50g of sodium chlorite were added, and acetic acid was added to adjust the pH to 4, and the amount of acetic acid was recorded. The reaction was carried out in a 75℃water bath for 1h, after which 25g of sodium chlorite and half of the first acetic acid were added again. After repeating twice, the reaction was stopped. And adding distilled water to wash the bleached material to be neutral, and air-drying the material to obtain the fully-finished cellulose for standby, wherein the mark is HC (holocellulose).
2. Preparation of pretreated Material
80g of absolute dry bamboo heddle cellulose is placed in an oil bath, diluted sulfuric acid solution (solid-to-liquid ratio 1:10, g/mL) with a concentration of 0.5% (g/mL) is added, and treated at 160 ℃, 170 ℃ and 180 ℃ for 1h. And taking out immediately after the end, and cooling. Solid-liquid separation is carried out, dilute acid is used for washing a large amount of distilled water to pretreat the fully-mechanized cellulose residue (DAP-HC) until the pH value of the residue is neutral, and the washed pretreated material is frozen and dried in vacuum until the pretreated material is absolute dry and is stored in a self-sealing bag. The pretreated material obtained at 160 ℃, 170 ℃ and 180 ℃ was labeled 160-DAP-HC, 170-DAP-HC and 180-DAP-HC, respectively.
3. Preparation of pseudo lignin
Taking DAP-HC, respectively adding 96% (v/v) of 1, 4-dioxane/water solution, 95% (v/v) of ethanol/water solution and tetrahydrofuran solvent according to the solid-to-liquid ratio of 1:10 (g/mL), extracting for 24 hours at room temperature, centrifuging to obtain an extract, adding fresh organic solvent into extraction residues again, repeating for three times, mixing all the collected extracts, and obtaining a pseudo lignin component through rotary evaporation and air drying. Pseudo Lignin (PL) obtained by extracting DAP-HC with an organic solvent is labeled 160-Dio-PL, 160-Eth-PL and 160-THF-PL, respectively (other temperature materials are so-called). The solid material remaining after extraction was washed with a large amount of distilled water, the organic solvent was completely removed by air drying, and collected in a self-sealing bag for use, labeled 160-Dio-HC, 160-Eth-HC and 160-THF-HC, respectively (other temperature materials were pushed in this way).
4. Dynamic adsorption process detection between pseudo lignin and cellulase
Different series of pseudo lignin solutions of 0.5% (w/v, DMSO) were prepared, then 100. Mu.L of the solution was dropped on a Surface Plasmon Resonance (SPR) gold chip by a flat-top syringe, and after standing for 1min, the solution was spin-coated with a spin coater (KW-4A, shanghai landscape Instrument Co., ltd., china) at a speed of 5kr/min for 1min, and repeated 3 times. The pseudo lignin film was dried in vacuo at 40℃for 4h and then immersed in deionized water for 1 day. Deionized water is replaced at intervals of 2 hours, complete removal of DMSO is ensured, and the influence on the activity of cellulase is avoided. The soaked film was dried in vacuo at 40 ℃ for 12 hours.
Dynamic adsorption processes between different pseudo lignin components and cellulases were monitored in real time using an SPR instrument (MP-SPR Navi 200, bioNavis Ltd, tank Pelare, finland). The SPR instrument is equipped with a dual channel detection system with a laser wavelength of 670nm and a scan range of SPR angles of 40-78 deg..
Before passing the cellulase protein solution into the SPR apparatus, 0.05M sodium citrate buffer was passed at a flow rate of 0.1mL/min to obtain a stable baseline. After stabilization, solutions with cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L were introduced at a flow rate of 0.1mL/min for 4 minutes, and the adsorption behavior of pseudo lignin and cellulase was observed. Subsequently, a 0.05M sodium citrate buffer was introduced to obtain a dissociation curve of the dissociating cellulase on the lignin membrane. SPR curves were processed using SPR Navi Data Viewer software and kinetic constants were analyzed using a scanner (version 2.0) software.
5. Experimental results
The process of dynamic adsorption and desorption of the different pseudo lignin components (160-Dio-PL, 160-Eth-PL, 160-THF-PL, 170-Dio-PL, 170-Eth-PL, 170-THF-PL, 180-Dio-PL, 180-Eth-PL and 180-THF-PL) and cellulase was monitored in real time by the SPR technique, and the process is shown in FIG. 2.
From FIG. 2, the dynamic adsorption of the various components of pseudo lignin with cellulase solutions of different protein concentrations (0.01, 0.02, 0.05, 0.08 and 0.1 g/L) can be observed over time. And (3) introducing sodium citrate buffer solution into the surface of the ultrathin pseudo lignin membrane from 0s to 200s to obtain a stable baseline, so that subsequent calculation is facilitated. Subsequently, at 4min, the enzyme protein solution was introduced to find that the binding of the pseudo lignin and protein molecules caused an increase in the signal value (RU) of SPR, which is a pseudo lignin and cellulase adsorption process. Finally, a citric acid buffer was introduced to observe the dissociation of the cellulase from the pseudo lignin. As can be seen from fig. 2, the response value (RU) of SPR decreases after buffer introduction, which is a cellulase cleavage process adsorbed on the pseudo lignin. Comparing SPR results of pseudo lignin at different temperatures, it can be found that when the cellulose protein and the pseudo lignin are dissociated, the SPR signal value RU of the pseudo lignin obtained by pretreating the holocellulose at the higher temperature is obviously reduced, which indicates that the pseudo lignin obtained at the higher temperature is easier to dissociate from the cellulose, and is why the cellulose saccharification capability is less influenced.
To further understand the adsorption and analysis process of cellulase on the pseudo lignin, kinetic adsorption parameters of the pseudo lignin film and cellulase of different components were calculated and analyzed by a scanner (version 2.0) software, and the results are shown in table 2.
TABLE 2 kinetic parameters of interaction of different fractions of pseudo lignin films with cellulases
Note that: k (k) a A binding rate constant; k (k) d Dissociation rate; r is R max Maximum saturated adsorption capacity of the pseudo lignin film; k (K) D Equilibrium dissociation rate
As can be seen from Table 2, the binding rate k of the pseudo lignin component extracted at different pretreatment temperatures with cellulase a 1.50X10 4 M -1 S -1 -2.7×10 4 M -1 Sw 1 There is no significant difference. This indicates that the pseudo lignin components extracted at different pretreatment temperatures have no significant difference in the ability to adsorb cellulases. And maximum saturated adsorption quantity R max As the pseudo lignin production temperature increased, the temperature increased from 532-551RU (160-PL) to 657-780RU (180-PL), indicating that the adsorption of 180-PL to cellulase was higher than 160-PL and 170-PL. For dissociation rate k d Dissociation rates between 160-PL, 170-PL and 180-PL and cellulase were 5.67X 10, respectively -5 S -1 -1.79×10 -4 S -1 、4.06×10 -4 S -1 -7.35×10 -4 S -1 And 9.89×10 -4 S -1 -1.43×10 -3 S -1 . The rate of dissociation of 160-PL from cellulase was minimal and the rate of dissociation of 180-PL was maximal.
The result shows that the false lignin prepared by the higher pretreatment temperature is easier to dissociate from the cellulose, which is beneficial to the separation of more enzymes adsorbed on the false lignin in a free state in an enzyme hydrolysis system after the enzyme is resolved, thereby improving the hydrolysis yield of the cellulose. By K D The value can also observe the binding affinity of cellulase and different pseudo lignin components, K D Smaller values indicate greater affinity between the two.
As shown in Table 2, K between 160-PL and cellulase D The value is 2.7nM-9.8nM, far less than K between 180-PL and cellulase D Values (66.9 nM-77.0 nM), which indicate a decrease in affinity between pseudo lignin and cellulase enzyme with increasing pretreatment temperature. As can be seen from the interaction results of the pseudo lignin extracted by different organic solvent systems and the cellulose, the same temperatureThe pseudo lignin extracted by the ethanol system has lower K D Values, e.g. K of 160-Dio-PL, 160-Eth-PL and 160-THF-PL D The values were 9.8nM, 2.7nM and 8.8nM, respectively. The result shows that the pseudo lignin extracted by the ethanol has the strongest binding capacity with the cellulase, which is one of the reasons for stronger inhibition effect on microcrystalline cellulose hydrolysis.
Claims (10)
1. A method for representing interaction between lignin and cellulase is characterized in that lignin solution is prepared firstly, then the lignin solution is dripped on a gold chip of a surface plasma resonance technology, and a lignin film is prepared by spin coating; and (3) introducing cellulase into an SPR instrument, and monitoring the dynamic adsorption process between the lignin component and the cellulase in real time to obtain the interaction result of lignin and the cellulase.
2. The method of characterizing lignin interactions with cellulases according to claim 1 comprising the steps of:
1) Preparing a pretreatment material;
2) Preparing surface lignin;
3) Preparing residual lignin;
4) Dynamic adsorption process detection between lignin and cellulase
Preparing 0.5% of surface lignin DMSO solution and residual lignin DMSO solution, then dripping 100 mu L of solution on a surface plasmon resonance gold chip by using a flat-mouth injector, standing for 1min, spin-coating at a speed of 5kr/min by using a spin coater to prepare a lignin film, vacuum-drying at 40 ℃ for 4 hours, cleaning in deionized water to ensure complete removal of DMSO, and vacuum-drying at 40 ℃ for 12 hours;
before introducing the cellulase protein solution into the SPR instrument, introducing 0.05M sodium citrate buffer solution at a flow rate of 0.1mL/min to obtain a stable baseline; after stabilization, the solutions with the cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L were introduced for 4 minutes at a flow rate of 0.1mL/min, and the adsorption behavior of lignin and cellulase was observed; then, introducing 0.05M sodium citrate buffer solution to obtain a dissociation curve of dissociative cellulase on the lignin membrane; processing the SPR curve by adopting SPR Navi Data Viewer software, and analyzing the kinetic constants by adopting a washer software;
5) And obtaining a detection result.
3. The method of characterizing lignin interactions with cellulases according to claim 1 wherein in step 1) the preparation of the pretreated material is: pretreating bamboo scraps by using a 1% sulfuric acid solution, and washing residues to be neutral by using distilled water to obtain pretreated materials.
4. The method of characterizing lignin interactions with cellulases according to claim 1 wherein in step 2) the preparation of surface lignin is: extracting the pretreated material by adopting different solvents respectively to prepare surface lignin with different characteristics; the solvent is as follows: 1, 4-dioxane/water solution, 95% alcohol and tetrahydrofuran with the volume ratio of 96:4.
5. The method of characterizing lignin interactions with cellulases according to claim 4 wherein the extraction process is: adding the pretreated material into a conical flask, adding a solvent according to the solid-to-liquid ratio of 1:10, and extracting for 1 day at room temperature under magnetic stirring at the rotating speed of 150 r/min; after the completion, carrying out solid-liquid separation, and adding fresh organic extraction solution again into the extracted solid to continue extraction; the above process is repeated three times, so that the surface lignin is extracted to the greatest extent; the three supernatants were collected and mixed together and the extraction solvent was removed by rotary evaporation and vacuum freeze drying to give lignin solids.
6. The method of characterizing lignin interactions with cellulases according to claim 1 wherein in step 3) the preparation of residual lignin is: taking the absolute dry lignin solid to a planetary ball mill, and effectively grinding for 6 hours at the rotating speed of 600 r/min.
7. The method of characterizing lignin interactions with cellulases according to claim 1 wherein in step 5) the detection results are: the adsorption capacity of different lignin components and cellulase is not obviously different, dissociation of SL components is easier to occur than dissociation of MWL components, and the affinity K of SL and cellulase is higher than that of the MWL components D The value is 39.4-52.6nM, and the MWL is K with cellulase D The value is 6.8-24.7nM.
8. The method of characterizing lignin interactions with cellulases according to claim 1 wherein the lignin is a pseudo lignin.
9. The method of characterizing lignin interactions with cellulases according to claim 8 comprising the steps of:
1) Preparing holocellulose;
2) Preparing a pretreatment material;
3) Preparation of pseudo lignin: extracting the pretreated material by adopting different solvents respectively to prepare pseudo lignin with different characteristics; the solvent is as follows: 1, 4-dioxane/water solution, 95% alcohol and tetrahydrofuran with the volume ratio of 96:4;
4) Dynamic adsorption process detection between pseudo lignin and cellulase
Preparing 0.5% pseudo lignin DMSO solution, then dripping 100 mu L of the solution on a surface plasmon resonance gold chip by using a flat-mouth injector, standing for 1min, spin-coating at a speed of 5kr/min by using a spin-coating instrument to prepare a pseudo lignin film, vacuum-drying at 40 ℃ for 4h, cleaning with deionized water to ensure complete removal of DMSO, and vacuum-drying at 40 ℃ for 12 h;
before introducing the cellulase protein solution into the SPR instrument, introducing 0.05M sodium citrate buffer solution at a flow rate of 0.1mL/min to obtain a stable baseline; after stabilization, the solutions with the cellulase protein concentrations of 0.1, 0.05, 0.02, 0.01 and 0.005g/L are introduced for 4 minutes at a flow rate of 0.1mL/min, and the adsorption behavior of pseudo lignin and cellulase is observed; then, introducing 0.05M sodium citrate buffer solution to obtain a dissociation curve of dissociative cellulase on the lignin membrane; processing the SPR curve by adopting SPR Navi Data Viewer software, and analyzing the kinetic constants by adopting a washer software;
5) Obtaining a detection result: the pseudo lignin obtained at higher temperature is more likely to dissociate from the cellulase.
Use of spr for detecting interactions between lignin and cellulases.
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| CN109239182A (en) * | 2018-09-04 | 2019-01-18 | 南京林业大学 | A method of with cellulase in-situ modification gold chip |
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| CN109239182A (en) * | 2018-09-04 | 2019-01-18 | 南京林业大学 | A method of with cellulase in-situ modification gold chip |
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