High-temperature-resistant and shear-resistant composite cellulase and preparation method thereof

Abstract

The present application relates to the technical fields of bioengineering and enzyme preparation, and particularly relates to a high-temperature-resistant and shear-resistant composite cellulase and a preparation method thereof. The composite cellulase is composed of a synergistically modified cellulase matrix, beta-glucosidase, xylanase, polyethylene glycol, magnesium chloride, trehalose and deionized water. The synergistically modified cellulase matrix is formed by the multiple synergistic effects of the induced dipole directional arrangement of enzyme molecules through an external electric field and the combination of interface hydrogen bond network under the action of graphene quantum dots, tris-hydroxymethyl aminomethane and metal salts, and by the endoglucanase and exoglucanase. The composite cellulase is synergistically regulated by multiple components, realizes the stabilization of enzyme molecule conformation, can still maintain high enzyme activity under high temperature and strong shear conditions, significantly improves the high-temperature resistance, shear stability and recycling performance, and is suitable for the fields of biomass degradation, textile treatment and organic waste resource utilization.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and enzyme preparation technology, specifically to a high-temperature resistant and shear-resistant composite cellulase and its preparation method. Background Technology

[0002] Cellulases are a class of complex enzyme systems that synergistically catalyze the degradation of cellulose into soluble oligosaccharides and glucose. They mainly include endoglucanases, exoglucanases, and β-glucosidases, and are widely used in the bioconversion of lignocellulose resources such as straw, enzymatic polishing of textiles, papermaking pulping, and the resource utilization of organic waste. Most existing cellulases are derived from microbial fermentation systems such as *Trichoderma reesei* and *Aspergillus niger*, and their optimal reaction temperature is typically 45–55℃.

[0003] In practical industrial applications, to improve reaction efficiency and production intensity, operation at temperatures of 60–80°C or even higher is often required, accompanied by strong mechanical stirring or pumping. Under these conditions, the spatial conformation of enzyme proteins is easily disrupted: on the one hand, high temperatures weaken internal hydrogen bonds and hydrophobic interactions, leading to irreversible thermal denaturation of proteins; on the other hand, high shear forces cause protein chain unwinding or local breakage, damaging the active site structure and resulting in a rapid decrease in enzyme activity. Furthermore, in complex substrate systems, a single enzyme component struggles to achieve efficient synergistic degradation of cellulose and hemicellulose structures, further reducing overall conversion efficiency.

[0004] In existing technologies, the addition of polyols, metal ions, or polymeric protective agents is commonly used to improve the stability of cellulase. However, these methods are mostly simple physical protections or single-mechanism interactions, making it difficult to effectively regulate the enzyme conformation at the molecular structural level. They also suffer from insufficient heat resistance, limited shear resistance, and poor long-term stability. Therefore, developing a composite cellulase system that achieves conformational stability through multi-scale structural regulation while also possessing heat resistance and shear resistance has become a pressing technical problem in this field. Summary of the Invention

[0005] To overcome the technical problems existing in the background art, such as the susceptibility of cellulase to thermal denaturation under high temperature conditions, the easy destruction of its structure under strong shear environments, and the difficulty of effectively controlling the enzyme conformation at the molecular level using existing stabilization methods, the present invention aims to provide a high-temperature and shear-resistant composite cellulase and its preparation method. The present invention introduces graphene quantum dots and tris(hydroxymethyl)aminomethane to construct an interface-controlled environment, and induces the dipole orientation of endoglucanase and exoglucanase molecules under an applied electric field. Simultaneously, hydrogen bonding networks are used to confine and fix the enzyme conformation, forming a synergistically modified cellulase matrix. Based on this, β-glucosidase, xylanase, polyethylene glycol, magnesium chloride, and trehalose are further compounded to construct a multi-component synergistically stable composite cellulase system. The present invention, through the construction of a directional confined structure and synergistic multi-component regulation, achieves improved stability of the cellulase while maintaining high enzyme activity under high temperature and strong shear conditions.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A high-temperature and shear-resistant composite cellulase, comprising the following raw materials in parts by weight: 30-60 parts of synergistically modified cellulase matrix; 10-30 parts of β-glucosidase; 10-30 parts of xylanase; 5-20 parts of polyethylene glycol; 1-8 parts of magnesium chloride; 1-8 parts of trehalose; and 50-200 parts of deionized water. The synergistically modified cellulase matrix is ​​a composite structure obtained by synergistic modification of enzyme molecular interface orientation and dipole rearrangement by applying an external electric field under the action of graphene quantum dots, tris(hydroxymethyl)aminomethane, and magnesium chloride, where endoglucanase and exoglucanase are involved. The external electric field mainly promotes the adjustment of enzyme molecular interface orientation and dipole rearrangement. The formation of enzyme conformational stability also depends on the synergistic effect of multiple synergistic actions such as graphene quantum dot interface adsorption, hydrogen bond network fixation, metal ion bridging, and nano-confining, combined with the interface hydrogen bond network freezing structure to form a directional confined structure.

[0008] Optionally, the synergistically modified cellulase matrix comprises the following raw materials in parts by weight: 30-60 parts of endoglucanase; 20-50 parts of exoglucanase; 1-10 parts of graphene quantum dots; 5-25 parts of tris(hydroxymethyl)aminomethane; 1-8 parts of magnesium chloride; and 50-150 parts of deionized water.

[0009] Optionally, the method for preparing the synergistically modified cellulase matrix includes the following steps:

[0010] (1) Dissolve the endoglucanase and exoglucanase in deionized water to obtain an enzyme solution;

[0011] (2) Add graphene quantum dots with carboxyl, hydroxyl or amino groups on the surface, tris(hydroxymethyl)aminomethane buffer and magnesium chloride to the enzyme solution, and incubate at pH 6.0 to 8.5 to load endoglucanase and exoglucanase onto the surface of graphene quantum dots through electrostatic interaction, hydrogen bonding and / or magnesium ion bridging, so that enzyme molecules form an interfacial orientation structure on the surface of graphene quantum dots characterized by the exposure of catalytic domains on the outside;

[0012] (3) Post-process the directionally arranged enzyme intermediate to obtain the synergistically modified cellulase matrix.

[0013] Optionally, the reaction conditions in step (1) are: pH 5.8 to 6.2, temperature 22 to 28°C, stirring speed 150 to 300 rpm, and dissolution time 20 to 60 min.

[0014] Optionally, the reaction conditions in step (2) are: an applied electric field strength of 0.8 to 1.5 V / cm, a temperature of 28 to 35°C, a stirring speed of 300 to 500 rpm, and a processing time of 40 to 90 min.

[0015] Optionally, the reaction conditions in step (3) are: a reaction time of 60 to 120 min, pre-freezing at -40 to -50 °C, freeze-drying at a vacuum of 10 to 50 Pa for 24 to 48 h, or spray drying at an inlet air temperature of 140 to 160 °C.

[0016] Optionally, a method for preparing a high-temperature and shear-resistant composite cellulase includes the following steps:

[0017] S1, the crude cellulase solution obtained from fermentation is centrifuged to remove cell impurities, and the supernatant is obtained. The supernatant is then concentrated by ultrafiltration to obtain concentrated enzyme solution.

[0018] S2, β-glucosidase, xylanase, polyethylene glycol, magnesium chloride, and trehalose are added to the concentrated enzyme solution for compound mixing to obtain a compound enzyme solution;

[0019] S3 involves mixing the compound enzyme solution with the synergistically modified cellulase matrix, followed by drying or liquid stabilization treatment to obtain a high-temperature and shear-resistant composite cellulase.

[0020] Optionally, the reaction conditions for step S1 are: centrifugation speed of 4000-8000 rpm, centrifugation time of 10-30 min, ultrafiltration molecular weight cutoff of 10-30 kDa, and concentration factor of 2-5 times.

[0021] Optionally, the reaction conditions in step S2 are a temperature of 25–40°C, a stirring speed of 200–500 rpm, and a mixing time of 30–120 min.

[0022] Optionally, the reaction conditions in step S3 are: mixing time of 30 to 90 min, followed by pre-freezing at -40 to -50 °C, and freeze-drying at a vacuum of 10 to 50 Pa for 24 to 48 h, or spray drying at an inlet air temperature of 140 to 160 °C.

[0023] The beneficial effects of this invention are:

[0024] The external electric field applied in this invention is mainly used to promote the regulation of enzyme molecule interface orientation and dipole rearrangement. The formation of enzyme conformational stability also depends on the synergistic effects of multiple processes, such as graphene quantum dot interface adsorption, hydrogen bond network fixation, metal ion bridging, and nano-confinement, which transform the disordered conformation of endoglucanase and exoglucanase into an ordered oriented structure, reducing the probability of conformational collapse under high temperature conditions at the molecular scale. At the same time, the high specific surface area interface constructed by graphene quantum dots enhances the stability of the local microenvironment, and the dense hydrogen bond network formed by tris(hydroxymethyl)aminomethane freezes the conformation of the oriented enzyme molecules, achieving structural confinement and fixation. The above multiple effects synergistically construct an "orientation-confinement-stability" composite structure, which enables enzyme molecules to effectively disperse stress and maintain the integrity of the active center when subjected to mechanical shear force, thereby maintaining a high enzyme activity retention rate under 80℃ and high shear conditions, significantly improving the high temperature resistance and shear stability of the composite cellulase. Attached Figure Description

[0025] The invention will now be further described with reference to the accompanying drawings.

[0026] Figure 1 This is a comparison chart of the performance test results of samples with different ratios. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. However, the present invention is not limited to the following embodiments. Equivalent adjustments made without departing from the spirit and essence of the present invention should also be considered to fall within the protection scope of the present invention.

[0028] Example 1: The purpose of this example is to verify that the composite cellulase of the present invention still has basic stability under low component dosage and mild conditions.

[0029] S1, 30 parts of endoglucanase and 20 parts of exoglucanase were added to 50 parts of deionized water, the pH was adjusted to 5.8, and the mixture was stirred at 150 rpm for 20 min at 22 °C to obtain an enzyme solution; then 1 part of graphene quantum dots, 5 parts of tris(hydroxymethyl)aminomethane and 1 part of magnesium chloride were added, and the mixture was treated at 28 °C and 300 rpm for 40 min under an external electric field of 0.8 V / cm to obtain a directionally aligned enzyme intermediate; the directionally aligned enzyme intermediate was reacted for another 60 min, pre-frozen at −40 °C, and freeze-dried under a vacuum of 50 Pa for 24 h to obtain 30 parts of synergistically modified cellulase matrix;

[0030] S2. The crude cellulase solution obtained from fermentation is centrifuged at 4000 rpm for 10 min to obtain the supernatant. The supernatant is then concentrated twice using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain the concentrated enzyme solution. 10 parts of β-glucosidase, 10 parts of xylanase, 5 parts of polyethylene glycol, 1 part of magnesium chloride, and 1 part of trehalose are added to the concentrated enzyme solution. The mixture is stirred at 200 rpm for 30 min at 25°C to obtain the compound enzyme solution.

[0031] S3. After mixing the compound enzyme solution with the synergistic modified cellulase matrix for 30 min, it was pre-frozen at −40℃ and freeze-dried at a vacuum of 50 Pa for 24 h to obtain a high-temperature resistant and shear-resistant compound cellulase.

[0032] Example 2: The purpose of this example is to obtain a composite cellulase system with optimal overall performance.

[0033] S1, 45 parts of endoglucanase and 35 parts of exoglucanase were added to 100 parts of deionized water, the pH was adjusted to 6.0, and the mixture was stirred at 200 rpm for 40 min at 25 °C to obtain an enzyme solution; then 5 parts of graphene quantum dots, 15 parts of tris(hydroxymethyl)aminomethane and 4 parts of magnesium chloride were added, and the mixture was treated at 30 °C and 400 rpm for 60 min under an external electric field of 1.0 V / cm to obtain a directionally aligned enzyme intermediate; the directionally aligned enzyme intermediate was reacted for another 90 min, pre-frozen at −45 °C, and freeze-dried under a vacuum of 30 Pa for 36 h to obtain 45 parts of synergistically modified cellulase matrix;

[0034] S2, the crude cellulase solution obtained from fermentation was centrifuged at 6000 rpm for 20 min to obtain the supernatant, which was then concentrated 3 times using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain the concentrated enzyme solution; 20 parts of β-glucosidase, 20 parts of xylanase, 12 parts of polyethylene glycol, 4 parts of magnesium chloride, and 4 parts of trehalose were added to the concentrated enzyme solution, and the mixture was stirred at 350 rpm for 60 min at 30℃ to obtain the compound enzyme solution;

[0035] S3. After mixing the compound enzyme solution with the synergistic modified cellulase matrix for 60 min, it was pre-frozen at −45℃ and freeze-dried at a vacuum of 30 Pa for 36 h to obtain a high-temperature resistant and shear-resistant compound cellulase.

[0036] Example 3: The purpose of this example is to verify the limiting stability of the system under high component content and reinforcement conditions.

[0037] S1, 60 parts of endoglucanase and 50 parts of exoglucanase were added to 150 parts of deionized water, the pH was adjusted to 6.2, and the mixture was stirred at 300 rpm for 60 min at 28 °C to obtain an enzyme solution; then 10 parts of graphene quantum dots, 25 parts of tris(hydroxymethyl)aminomethane and 8 parts of magnesium chloride were added, and the mixture was treated at 35 °C and 500 rpm for 90 min under an external electric field of 1.5 V / cm to obtain a directionally aligned enzyme intermediate; the directionally aligned enzyme intermediate was reacted for another 120 min, pre-frozen at −50 °C, and freeze-dried under a vacuum of 10 Pa for 48 h to obtain 60 parts of synergistically modified cellulase matrix;

[0038] S2. The crude cellulase solution obtained from fermentation was centrifuged at 8000 rpm for 30 min to obtain the supernatant. The supernatant was then concentrated 5 times using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain the concentrated enzyme solution. 30 parts of β-glucosidase, 30 parts of xylanase, 20 parts of polyethylene glycol, 8 parts of magnesium chloride, and 8 parts of trehalose were added to the concentrated enzyme solution. The mixture was stirred at 500 rpm for 120 min at 40℃ to obtain the compound enzyme solution.

[0039] S3. After mixing the compound enzyme solution with the synergistic modified cellulase matrix for 90 min, it was pre-frozen at −50℃ and freeze-dried at a vacuum of 10 Pa for 48 h to obtain a high-temperature resistant and shear-resistant compound cellulase.

[0040] Comparative Example 1: The purpose of this comparative example is to verify the effect of the lack of electric field-induced orientation on the synergistic modified structure.

[0041] S1, 45 parts of endoglucanase and 35 parts of exoglucanase were added to 100 parts of deionized water, the pH was adjusted to 6.0, and the mixture was stirred at 200 rpm for 40 min at 25 °C to obtain an enzyme solution; then 5 parts of graphene quantum dots, 15 parts of tris(hydroxymethyl)aminomethane and 4 parts of magnesium chloride were added, and the mixture was treated at 30 °C and 400 rpm for 60 min without an external electric field to obtain a non-directional enzyme intermediate; the non-directional enzyme intermediate was reacted for another 90 min, pre-frozen at −45 °C, and freeze-dried at 30 Pa vacuum for 36 h to obtain 45 parts of single-interface modified cellulase matrix;

[0042] S2, the crude cellulase solution obtained from fermentation was centrifuged at 6000 rpm for 20 min to obtain the supernatant, which was then concentrated 3 times using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain the concentrated enzyme solution; 20 parts of β-glucosidase, 20 parts of xylanase, 12 parts of polyethylene glycol, 4 parts of magnesium chloride, and 4 parts of trehalose were added to the concentrated enzyme solution, and the mixture was stirred at 350 rpm for 60 min at 30℃ to obtain the compound enzyme solution;

[0043] S3. The compound enzyme solution was mixed with a single interface-modified cellulase matrix for 60 min, pre-frozen at −45℃, and then freeze-dried at a vacuum of 30 Pa for 36 h to obtain the compound cellulase.

[0044] Comparative Example 2: The purpose of this comparative example is to verify the effect of the lack of interface confinement materials on the synergistic modification structure.

[0045] S1, 45 parts of endoglucanase and 35 parts of exoglucanase were added to 100 parts of deionized water, the pH was adjusted to 6.0, and the mixture was stirred at 200 rpm for 40 min at 25 °C to obtain an enzyme solution; then 15 parts of tris(hydroxymethyl)aminomethane and 4 parts of magnesium chloride were added, and the mixture was treated at 30 °C and 400 rpm for 60 min under an external electric field of 1.0 V / cm to obtain a directionally aligned enzyme intermediate; the directionally aligned enzyme intermediate was further reacted for 90 min, pre-frozen at −45 °C, and freeze-dried under a vacuum of 30 Pa for 36 h to obtain 45 parts of a single directionally modified cellulase matrix;

[0046] S2, the crude cellulase solution obtained from fermentation was centrifuged at 6000 rpm for 20 min to obtain the supernatant, which was then concentrated 3 times using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain the concentrated enzyme solution; 20 parts of β-glucosidase, 20 parts of xylanase, 12 parts of polyethylene glycol, 4 parts of magnesium chloride, and 4 parts of trehalose were added to the concentrated enzyme solution, and the mixture was stirred at 350 rpm for 60 min at 30℃ to obtain the compound enzyme solution;

[0047] S3. The compound enzyme solution was mixed with a single directional modified cellulase matrix and treated for 60 min, then pre-frozen at −45℃ and freeze-dried at a vacuum of 30 Pa for 36 h to obtain the compound cellulase.

[0048] Comparative Example 3: The purpose of this comparative example is to verify the change in the overall system stability when the synergistic modified structure is missing.

[0049] S1. The crude cellulase solution obtained from fermentation is centrifuged at 6000 rpm for 20 min to obtain the supernatant, which is then concentrated three times using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain the concentrated enzyme solution.

[0050] S2, add 20 parts of β-glucosidase, 20 parts of xylanase, 12 parts of polyethylene glycol, 4 parts of magnesium chloride, and 4 parts of trehalose to the concentrated enzyme solution, and stir at 350 rpm for 60 min at 30℃ to obtain the compound enzyme solution.

[0051] S3. After directly treating the compound enzyme solution for 60 min, it was pre-frozen at −45℃ and freeze-dried at a vacuum of 30 Pa for 36 h to obtain the compound cellulase.

[0052] Performance testing:

[0053] 1. High Temperature Resistance Test Method

[0054] The composite cellulases obtained in the examples and comparative examples were prepared into enzyme solutions with an activity of 100 U / mL using an acetate-sodium acetate buffer solution at pH 5.0. 10 mL of each solution was placed in a sealed test tube and treated in an 80°C water bath for 2 hours. After treatment, the solutions were rapidly cooled to room temperature in an ice-water bath. Then, 1 mL of the enzyme solution was added to a reaction system containing 50 mg of filter paper strips and 4 mL of buffer solution. The reaction was carried out at 50°C for 60 minutes. After the reaction, DNS reagent was added, and the mixture was incubated in a 100°C water bath for 5 minutes. After cooling, the absorbance was measured at 540 nm. The reducing sugar content was calculated based on the glucose standard curve, and the enzyme activity retention rate was calculated with the enzyme activity of the untreated enzyme solution as 100%.

[0055] 2. Shear resistance test method

[0056] The composite cellulases obtained in the examples and comparative examples were prepared into enzyme solutions with an enzyme activity of 100 U / mL. 50 mL of each solution was placed in a reactor equipped with a mechanical stirrer and continuously stirred at 1000 rpm for 120 min at 30°C. After stirring, 1 mL of the solution was taken and reacted at 50°C for 60 min according to the filter paper enzyme activity assay method. The amount of reducing sugar generated was determined by the DNS method, and the enzyme activity loss rate was calculated using the unsheared enzyme solution as a control.

[0057] 3. Test methods for cellulose degradation performance

[0058] Using microcrystalline cellulose as a substrate, 0.5 g was weighed and added to 50 mL of acetate-sodium acetate buffer solution with a pH of 5.0. The composite cellulase obtained in the examples and comparative examples was added to make the final enzyme activity concentration 50 U / g substrate. The reaction was carried out at 50 °C with stirring at 200 rpm for 8 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 min, and the supernatant was collected. The reducing sugar content was determined by the DNS method. The cellulose conversion rate was calculated based on the initial amount of substrate to evaluate the degradation efficiency of the enzyme.

[0059] 4. Cyclic stability test method

[0060] The composite cellulase obtained in the examples and comparative examples was added to the above cellulose degradation system and reacted at 50°C for 8 hours. After the reaction was completed, the mixture was centrifuged at 10,000–12,000 rpm for 10 minutes to allow the synergistically modified composite enzyme particles to settle. The supernatant was discarded, and the settled composite enzyme particles were added back to an equal amount of fresh substrate system to continue the reaction. The recovered enzyme was then added back to an equal amount of fresh substrate system to continue the reaction. The above steps were repeated for 5 cycles. After each reaction, the enzyme activity was measured, and the enzyme activity retention rate after each cycle was calculated with the enzyme activity of the first reaction as 100% to evaluate the reusability stability of the composite cellulase.

[0061] Table 1. Performance test results of the composite cellulase

[0062] Example 1 72 75 78 70 Example 2 89 93 91 88 Example 3 84 90 87 82 Comparative Example 1 60 65 70 58 Comparative Example 2 55 68 68 55 Comparative Example 3 42 50 60 40

[0063] According to Table 1 and Figure 1 As shown, different embodiments and comparative examples exhibit significant differences in high-temperature resistance, shear resistance, cellulose degradation efficiency, and cycle stability. Overall, the embodiments of the present invention demonstrate superior performance compared to the comparative examples, indicating that the constructed synergistic modified structure has a significant effect on improving the overall stability of the composite cellulase.

[0064] Regarding high-temperature resistance, the enzyme activity retention rates of Examples 1-3 were 72%, 89%, and 84%, respectively, significantly higher than those of Comparative Examples 1-3 (60%, 55%, and 42%). Example 2 showed the best performance, indicating that the synergistic modified structure constructed through electric field-induced directional alignment and interfacial hydrogen bond network can effectively inhibit conformational collapse of enzyme molecules under high-temperature conditions and improve the thermal stability of the enzyme protein. In contrast, the comparative examples, lacking key modification factors, were more prone to thermal denaturation, leading to a rapid decline in enzyme activity.

[0065] Regarding shear resistance, the enzyme activity retention rates of all examples were above 75%, with Example 2 reaching 93%, significantly better than the 50%–68% of the comparative examples. This result indicates that the directional confinement structure in the synergistically modified cellulase matrix can disperse external stress under mechanical shearing, reducing enzyme molecule structural damage and thus maintaining high catalytic activity. In the comparative examples, the lack of synergistic modification structures or key confinement factors led to more susceptible structural damage to the enzyme molecules under shearing.

[0066] Regarding cellulose degradation performance, the conversion rates of all examples reached over 78%, with Example 2 reaching 91%, significantly higher than the 60%–70% of the comparative examples. This indicates that after synergistic modification, the enzyme system not only maintained high stability but also promoted synergistic effects among multiple enzymes, improving substrate degradation efficiency. In particular, the well-matched component ratios and modification conditions in Example 2 ensured that the enzyme active site maintained a good structure, thereby achieving a higher conversion rate.

[0067] Regarding cycle stability, the enzyme activity retention rate of the examples remained above 70% after 5 cycles, with Example 2 reaching 88%, which was much higher than the 40% to 58% of the comparative examples. This indicates that the synergistic modified structure constructed in this invention can maintain the integrity of the enzyme molecular structure during multiple uses, significantly improving the reusability of the composite cellulase. In contrast, the comparative examples showed significant enzyme activity decay after multiple cycles due to insufficient structural stability.

[0068] In summary, this invention achieves targeted regulation and confined stability of enzyme molecular structure by constructing a synergistically modified cellulase matrix, resulting in composite cellulase that significantly outperforms existing technologies in terms of high temperature resistance, shear resistance, and cyclic stability. Furthermore, Example 2 demonstrates the best performance across all performance indicators, fully showcasing the superiority of the technical solution of this invention.

Claims

1. A high-temperature resistant and shear-resistant composite cellulase, characterized in that, The cellulase comprises the following raw materials in parts by weight: 30-60 parts of synergistically modified cellulase matrix; 10-30 parts of β-glucosidase; 10-30 parts of xylanase; 5-20 parts of polyethylene glycol; 1-8 parts of magnesium chloride; 1-8 parts of trehalose; and 50-200 parts of deionized water. The synergistically modified cellulase matrix is ​​a composite structure obtained by synergistic modification of enzyme molecular interface orientation and dipole rearrangement under the action of graphene quantum dots, tris(hydroxymethyl)aminomethane, and magnesium chloride, with an external electric field mainly used to promote the adjustment of enzyme molecular interface orientation and dipole rearrangement. The formation of enzyme conformational stability also depends on the synergistic effect of multiple synergistic actions such as graphene quantum dot interface adsorption, hydrogen bond network fixation, metal ion bridging, and nano-confining, combined with the interface hydrogen bond network frozen structure to form a directional confined structure.

2. The high-temperature resistant and shear-resistant composite cellulase according to claim 1, characterized in that, The synergistically modified cellulase matrix comprises the following raw materials in parts by weight: 30-60 parts of endoglucanase; 20-50 parts of exoglucanase; 1-10 parts of graphene quantum dots; 5-25 parts of tris(hydroxymethyl)aminomethane; 1-8 parts of magnesium chloride; and 50-150 parts of deionized water.

3. A high-temperature resistant and shear-resistant composite cellulase according to claim 1 or 2, characterized in that, The method for preparing the synergistic modified cellulase matrix includes the following steps: (1) Dissolve the endoglucanase and exoglucanase in deionized water to obtain an enzyme solution; (2) Add graphene quantum dots with carboxyl, hydroxy or amino groups on the surface, tris(hydroxymethyl)aminomethane buffer and magnesium chloride to the enzyme solution, and incubate at pH 6.0 to 8.5 to load endoglucanase and exoglucanase onto the surface of graphene quantum dots through electrostatic interaction, hydrogen bonding and / or magnesium ion bridging, so that enzyme molecules form an interfacial orientation structure on the surface of graphene quantum dots characterized by the exposure of catalytic domains on the outside; (3) Post-process the directionally arranged enzyme intermediate to obtain the synergistically modified cellulase matrix.

4. The high-temperature resistant and shear-resistant composite cellulase according to claim 3, characterized in that, The reaction conditions for step (1) are: pH 5.8–6.2, temperature 22–28°C, stirring speed 150–300 rpm, and dissolution time 20–60 min.

5. The high-temperature resistant and shear-resistant composite cellulase according to claim 3, characterized in that, The reaction conditions for step (2) are: an applied electric field strength of 0.8 to 1.5 V / cm, a temperature of 28 to 35°C, a stirring speed of 300 to 500 rpm, and a processing time of 40 to 90 min.

6. The high-temperature resistant and shear-resistant composite cellulase according to claim 3, characterized in that, The reaction conditions for step (3) are: a reaction time of 60 to 120 min, pre-freezing at -40 to -50 °C, freeze-drying at a vacuum of 10 to 50 Pa for 24 to 48 h, or spray drying at an inlet air temperature of 140 to 160 °C.

7. A method for preparing a high-temperature resistant and shear-resistant composite cellulase, characterized in that, The preparation method includes the following steps: S1, the crude cellulase solution obtained from fermentation is centrifuged to remove cell impurities, and the supernatant is obtained. The supernatant is then concentrated by ultrafiltration to obtain concentrated enzyme solution. S2, β-glucosidase, xylanase, polyethylene glycol, magnesium chloride, and trehalose are added to the concentrated enzyme solution for compound mixing to obtain a compound enzyme solution; S3 involves mixing the compound enzyme solution with the synergistically modified cellulase matrix, followed by drying or liquid stabilization treatment to obtain a high-temperature and shear-resistant composite cellulase.

8. The method for preparing a high-temperature resistant and shear-resistant composite cellulase according to claim 7, characterized in that, The reaction conditions for step S1 are: centrifugation speed of 4000-8000 rpm, centrifugation time of 10-30 min, ultrafiltration molecular weight cutoff of 10-30 kDa, and concentration factor of 2-5 times.

9. The method for preparing a high-temperature resistant and shear-resistant composite cellulase according to claim 7, characterized in that, The reaction conditions for step S2 are a temperature of 25–40°C, a stirring speed of 200–500 rpm, and a mixing time of 30–120 min.

10. The method for preparing a high-temperature resistant and shear-resistant composite cellulase according to claim 7, characterized in that, The reaction conditions for step S3 are: mixing time of 30 to 90 minutes, followed by pre-freezing at -40 to -50°C, freeze-drying at a vacuum of 10 to 50 Pa for 24 to 48 hours, or spray drying at an inlet air temperature of 140 to 160°C.

Images