Method for improving biological enzyme stability by using Eudragit as carrier
By covalently crosslinking biological enzymes with Eudragit vector, the problems of high cost and insufficient stability in the pulp and paper industry application are solved, and the high stability and recycling of biological enzymes are achieved, and the production costs are reduced.
Patent Information
- Application Number
- CN202510288963.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-05-02
AI Technical Summary
The application of biological enzymes in the pulp and paper industry is limited by high cost and insufficient stability, which limits its large-scale industrial application.
Eudragit is used as a carrier to change the solubleness of Eudragit by adjusting pH and activate it through EDC, covalent cross-linking of biological enzymes is carried out to improve the stability and recovery of biological enzymes.
It significantly improves the stability and reusability of biological enzymes, reduces the loss and production costs of enzymes, and broadens the application prospects of biological enzyme pretreatment in the pulp and paper industry.
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Figure CN119913768A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of light industry pulping and papermaking, in particular to a method for improving the stability of biological enzymes by using Eudragit as a carrier. Background Art
[0002] The pulp and paper industry is one of the largest chemical industries in the world. Bio-enzyme technology and bio-enzyme pretreatment utilize the specificity, high efficiency and environmental friendliness of enzymes to degrade cellulose, hemicellulose and lignin in wood fibers, improve fiber morphology and improve pulp performance, and have gradually become a research hotspot in the pulp and paper industry. However, bio-enzyme technology still faces many challenges in practical applications, such as high cost and poor stability.
[0003] In order to improve the application of bio-enzymes in the papermaking industry, the pH-sensitive carrier Eudragit was selected as the immobilization carrier. Eudragit is a pH-responsive polymer with intelligent slow-release control function. Its mechanism of action in bio-enzyme immobilization includes: pH sensitivity-under different pH conditions, Eudragit exhibits different solubility and stability, protects the activity of enzymes in acidic environments, and releases enzymes in neutral or alkaline environments; physical adsorption and chemical cross-linking-Eudragit fixes enzymes through physical adsorption or chemical cross-linking, enhances enzyme stability and reduces loss. Eudragit's mechanical strength and chemical stability ensure that it maintains high enzyme activity after repeated use.
[0004] Bio-enzyme pretreatment technology has broad application prospects in the pulp and paper industry, but its high cost and lack of stability limit its large-scale industrial application. Summary of the invention
[0005] In view of the deficiencies in the prior art, the present invention proposes a method for improving the stability of biological enzymes by using Eudragit as a carrier, wherein the solubility of Eudragit is changed by adjusting the pH and activated by EDC, and biological enzymes are added for covalent cross-linking, and finally better stability and recyclability are obtained.
[0006] In order to achieve the above technical objectives, the present invention proposes A method for improving the stability of a biological enzyme by using Eudragit as a carrier, comprising the following steps: step 1, carrier pretreatment: adding a pH-sensitive Eudragit carrier to deionized water, adjusting the pH to 6-8 with 0.01 mol / L-0.03 mol / L NaOH, to form a fully dissolved carrier solution; step 2, carrier activation: adding EDC with a concentration of 0.1%-0.4% (w / v) to the carrier solution of step (1), and activating for 0.5-2h; step 3, enzyme immobilization: adding a biological enzyme to the activated carrier solution, wherein the amount of the biological enzyme added is 0.5%-4% (w / v), and the immobilization treatment reaction is performed for 4-12 hours; step 4, post-treatment: adjusting the pH of the mixed solution obtained in step 3 to 3.0-5.0, collecting the precipitate after centrifugation, and dissolving the precipitate in a phosphate buffer solution with a pH of 4.5-5.5 to obtain an immobilized enzyme preparation.
[0007] Furthermore, the Eudragit carrier is Eudragit L-100 or Eudragit S-100.
[0008] Furthermore, the biological enzyme in step 3 is pectinase.
[0009] Furthermore, in step 3, the immobilization treatment equipment adopts a constant temperature shaker.
[0010] Furthermore, in step 3, the reaction temperature of the constant temperature shaker is 20-60° C., and the shaking speed is 60-120 rpm.
[0011] Furthermore, the buffer solution in step 4 is a phosphate buffer solution with a concentration of 0.01 to 0.1 mol / L and a usage amount of 5% to 10% (w / v).
[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention can effectively immobilize the biological enzyme on the surface and pores of the carrier, and because Eudragit is reversibly soluble, it can also provide great convenience for the recycling of the biological enzyme through simple separation means (filtration, pH adjustment), reduce enzyme loss and reduce production costs. In addition, the reversible solubility of the carrier also allows the enzyme to be released when needed, further improving the utilization efficiency of the enzyme and the flexibility of the reaction. Immobilization of the biological enzyme by the carrier can improve the thermal stability and pH stability of the biological enzyme, and still maintain a high activity after multiple recycling.
[0013] In summary, by immobilizing the bio-enzyme on the Eudragit carrier, not only the stability of the enzyme in a complex reaction environment is significantly improved, but also its reusability is enhanced, thereby greatly reducing the cost of enzyme use, providing a broader development prospect for bio-enzyme pretreatment in the pulp and paper industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 To compare the relative enzyme activity of pectinase covalently immobilized on Eudragit L-100 under different temperature and pH conditions; Figure 2 To compare the relative enzyme activity of pectinase covalently immobilized on Eudragit S-100 under different temperature and pH conditions; Figure 3 Relative enzyme activity of non-immobilized pectinase under different temperature and pH conditions. DETAILED DESCRIPTION
[0015] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form, i.e., not intended to limit the scope of protection of the present invention.
[0016] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.
[0017] Example 1 This embodiment discloses a method for improving the stability of a biological enzyme using Eudragit as a carrier, which specifically comprises the following steps: Step 1, immobilization of pectinase: Step 11, carrier pretreatment: 1% (w / v) Eudragit L-100 was added to deionized water and the pH was adjusted to 6 by 0.01 mol / L NaOH to form a fully dissolved carrier solution; Step 12, carrier activation: add 0.2% (w / v) EDC to the fully dissolved Eudragit series carrier solution, and activate the carrier for 0.5 h; Step 13, adding 0.5% (v / v) pectinase to the activated carrier solution, and immobilizing it on a constant temperature shaker for 8 hours, wherein the shaker is set at a temperature of 30°C and a rotation speed of 60 rpm; Step 14, enzyme immobilization: adjusting the pH of the immobilized pectinase carrier mixed solution to 3, collecting the precipitate by centrifugation, and dissolving the precipitate immobilized pectinase in a 0.01 mol / L phosphate buffer solution with a pH of 4.5 at a dissolution concentration of 5% (w / v) to obtain an immobilized enzyme preparation; Step 2: pH and thermal stability test of immobilized enzyme: The enzyme activity was determined by the DNS method. In this process, the substrate of the immobilized enzyme was pectin dissolved in a buffer solution with a pH of 5 to 9. The reaction temperature was controlled at 30 to 70°C. The relative enzyme activity of immobilized enzyme under different temperature and pH conditions is as follows Figure 1 shown.
[0018] Example 2 Step 1, immobilization of pectinase: Step 11, carrier pretreatment: 1% (w / v) Eudragit S-100 was added to deionized water and the pH was adjusted to 8 by 0.03 mol / L NaOH to form a fully dissolved carrier solution; Step 12, carrier activation: add 0.2% (w / v) EDC to the fully dissolved Eudragit series carrier solution, and activate the carrier for 2 h; Step 13, enzyme immobilization: 4% (v / v) pectinase was added to the activated carrier solution and immobilized on a constant temperature shaker for 8 h, wherein the shaker was set at 60°C and 120 rpm; Step 14, post-treatment: adjust the pH of the immobilized pectinase carrier mixed solution to 5, collect the precipitate by centrifugation, dissolve the precipitated immobilized pectinase in a 0.1 mol / L phosphate buffer solution with a pH of 5.5 at a dissolution concentration of 10% (w / v) to obtain an immobilized enzyme preparation.
[0019] Step 2: pH and thermal stability test of immobilized enzyme: The enzyme activity was determined by the DNS method. In this process, the substrate of the immobilized enzyme was pectin dissolved in a buffer solution with a pH of 5 to 9; the action temperature was controlled at 30 to 70°C.
[0020] The relative enzyme activity of immobilized enzyme under different temperature and pH conditions is as follows Figure 2 shown.
[0021] Comparative Example pH and thermal stability test of unimmobilized pectinase: The enzyme activity was determined by the DNS method. In this process, the substrate of pectinase was pectin dissolved in a buffer solution with a pH of 5 to 9, and the action temperature was controlled at 30 to 70°C. The relative enzyme activity of immobilized enzyme under different temperature and pH conditions is as follows Figure 3 shown.
[0022] Depend on Figure 1It can be seen that at pH 5-8, the relative enzyme activity of pectinase fixed by Eudragit L-100 gradually increased with the increase of pH, and the relative enzyme activity was the highest at pH 8; and the relative enzyme activity of the fixed pectinase gradually increased with the increase of temperature, and the relative enzyme activity was the highest at 60 ℃.
[0023] Depend on Figure 2 It can be seen that at pH 5-8, the relative enzyme activity of pectinase fixed by Eudragit S-100 gradually increased with the increase of pH, and the relative enzyme activity was the highest at pH 8; and the relative enzyme activity of the fixed pectinase gradually increased with the increase of temperature, and the relative enzyme activity was the highest at 60 ℃.
[0024] Depend on Figure 3 It can be seen that at pH 5-8, the relative enzyme activity of the unimmobilized pectinase gradually increased with the increase of pH, and the relative enzyme activity was the highest at pH 6; and the relative enzyme activity of the immobilized pectinase gradually increased with the increase of temperature, and the relative enzyme activity was the highest at 50 ℃.
[0025] The comparison showed that the pectinase immobilized on the two carriers ( Figure 1 , 2 ) is lower than that of the unimmobilized enzyme ( Figure 3 ), and there were significant improvements in pH adaptability (especially alkaline environment) and temperature stability (high temperature tolerance), indicating that immobilization technology can improve the application performance of pectinase and broaden its applicable conditions.
[0026] It should be noted that the above contents are further detailed descriptions of the present invention in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions; the dimensional data of this embodiment does not limit the technical solution, but only shows one of the specific working conditions. For ordinary technicians in the technical field to which the present invention belongs, several simple improvements and modifications can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.
Claims
1. A method for improving the stability of biological enzymes using Eudragit as a carrier, characterized in that: The following steps are involved: Step 1, carrier pretreatment: add a pH-sensitive Eudragit carrier to deionized water and adjust the pH to 6-8 with 0.01mol / L-0.03mol / L NaOH to form a fully dissolved carrier solution; Step 2, carrier activation: add EDC with a concentration of 0.1%-0.4% (w / v) to the carrier solution of step (1) and activate it for 0.5-2h; Step 3, enzyme immobilization: add biological enzyme to the activated carrier solution, the addition amount of the biological enzyme is 0.5%-4% (w / v), and the immobilization treatment reaction is carried out for 4-12 hours; Step 4, post-treatment: adjust the pH of the mixed solution obtained in step 3 to 3.0-5.0, collect the precipitate after centrifugation, and dissolve the precipitate in a buffer solution with a pH of 4.5-5.5 to obtain an immobilized enzyme preparation.
2. The method for improving the stability of biological enzymes using Eudragit as a carrier according to claim 1, characterized in that: The Eudragit carrier is Eudragit L-100 or Eudragit S-100.
3. The method for improving the stability of biological enzymes using Eudragit as a carrier according to claim 1, characterized in that: The biological enzyme in step 3 is pectinase.
4. The method for improving the stability of biological enzymes using Eudragit as a carrier according to claim 1, characterized in that: In step 3, the immobilization treatment equipment adopts a constant temperature shaker.
5. The method for improving the stability of biological enzymes using Eudragit as a carrier according to claim 1, characterized in that: In step 3, the reaction temperature of the constant temperature shaker is 20-60° C., and the shaking speed is 60-120 rpm.
6. The method for improving the stability of biological enzymes using Eudragit as a carrier according to claim 1, characterized in that: The buffer solution in step 4 is a phosphate buffer solution with a concentration of 0.01 to 0.1 mol / L and a usage amount of 5% to 10% (w / v).