A method for preparing an acid-resistant immobilized enzyme and its application
By preparing co-immobilized enzymes in gluconic acid production, the problem of poor repetitive reactivity of immobilized enzymes in the prior art under low pH environment is solved, and continuous reaction within the pH 2-3 range and efficient reuse are achieved, reducing production costs.
Patent Information
- Application Number
- CN202411945096.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-04
AI Technical Summary
The existing immobilized enzyme preparation process cannot meet the low pH requirements in gluconic acid production process, and the repetitive reactivity is poor, which limits the development of immobilized enzyme one-step gluconic acid synthesis method.
The resin was washed with water and added catalase, cross-linking agent jingnipine and glucose solution for cross-linking, combined with glucose oxidase, jingnipine and glucose solution for cross-linking, to prepare a co-immobilized enzyme to improve the acidic reactivity and reusability of the enzyme.
The continuous reaction of immobilized enzymes within the pH range of 2-3 is achieved, eliminating pH adjustment and subsequent ion exchange steps, shortening the reaction period to within 10 hours, immobilized enzymes can be reused for more than 10 times, and the post-treatment steps are simplified, greatly reducing production costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biochemical engineering, and in particular to a preparation method of an acid-resistant immobilized enzyme and application thereof. Background Art
[0002] Gluconic acid, also known as dextrorotatory gluconic acid, is a sugar acid formed when the aldehyde group in the molecule of glucose is oxidized to a carboxyl group under the action of an oxidant or glucose oxidase. It is easily soluble in water, but not in most organic solvents such as ether, and has weak acidity. Gluconic acid can be widely used in the fields of food, medicine, etc., and can be used in the production of gluconolactone, an important food additive, and has great application value.
[0003] At present, there are three main methods for producing gluconic acid using glucose as raw material: chemical oxidation, microbial fermentation and enzyme method. Chemical oxidation often requires the participation of oxidants such as hydrogen peroxide, and the reaction needs to be carried out under special conditions such as high temperature and ultraviolet light. The production process has high energy consumption and cost, and there are few industrial applications. The microbial fermentation method generally uses Aspergillus niger as a production strain to continuously convert the gluconic acid produced by fermentation into derivatives such as sodium gluconate and calcium gluconate, and then generates gluconic acid through ion exchange. The conversion rate is high and the strain can be recycled. However, the microbial fermentation method has low production efficiency, low product purity, high operating requirements, and a large amount of industrial wastewater is generated during the ion exchange process, which has high treatment costs and causes environmental pollution. The enzyme method does not require aseptic operation, but if free enzymes are used, they cannot be recycled, the cost increases, and because they cannot tolerate acidity, they still need to generate gluconate first and then generate gluconic acid through ion exchange. Using immobilized enzymes to directly produce gluconic acid can effectively improve the acid resistance and stability of the enzyme, which is conducive to repeated use.
[0004] The preparation of immobilized enzymes mainly includes adsorption, embedding and cross-linking methods. The adsorption method uses an adsorbent to adsorb the enzyme on the surface to fix the enzyme. It is easy to operate, mild conditions, and has a high enzyme activity retention rate, but the prepared immobilized enzyme has poor reusability and is not suitable for use in an environment with intense ventilation and stirring. The embedding method encapsulates the enzyme in a gel or polymer film, which has a high immobilization efficiency, but the entry of the substrate and the release of the product are both difficult. The cross-linking method relies on bifunctional reagents to cross-link enzyme molecules to form a three-dimensional network structure. However, the reaction conditions of the cross-linking method are relatively intense, which has a greater impact on enzyme activity, and the mechanical properties of the immobilized enzyme obtained are poor, and it is generally used in combination with other immobilization methods. At present, the existing immobilized enzyme preparation process cannot meet the low pH requirements in the gluconic acid production process, and the repetitive reactivity is poor, which limits the development of the one-step synthesis of gluconic acid by immobilized enzymes. Summary of the invention
[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention provides a method for preparing an acid-resistant immobilized enzyme and its application, which can improve the enzyme activity retention rate of the immobilized enzyme, and improve its acid resistance and repeated reactivity.
[0006] The preparation method of the immobilized enzyme provided by the present invention is achieved by the following technical scheme:
[0007] (1) washing the resin twice with water, adding catalase, cross-linking agent genipin and glucose solution for cross-linking;
[0008] (2) Glucose oxidase, genipin and glucose solution were added for cross-linking, and the mixture was allowed to stand at 4°C overnight and then washed thoroughly with water to obtain a co-immobilized enzyme.
[0009] In the step (1), the concentration of catalase is 500-1000 U / mL, the concentration of genipin is 0.25%-0.5%, and the concentration of glucose solution is 50-100 g / L.
[0010] The cross-linking conditions in step (1) are: stirring temperature of 20-30° C., stirring speed of 100-300 rpm, and reaction time of 2-4 h.
[0011] In step (2), the concentration of glucose oxidase is 50-100 U / mL, the concentration of genipin is 0.25%-0.5%, and the concentration of glucose solution is 50-100 g / L
[0012] The cross-linking conditions in step (2) are: stirring temperature of 20-30° C., stirring speed of 100-300 rpm, and reaction time of 2-4 h.
[0013] The present invention also provides an application of the co-immobilized enzyme prepared above in the production of gluconic acid, and the specific technical scheme is as follows:
[0014] (1) The co-immobilized enzyme prepared above is added to a glucose solution, and air is introduced to start the reaction to prepare gluconic acid, wherein the initial glucose concentration is 200-300 g / L, the ventilation volume is 4 vvm, the tank pressure is 0.05-0.1 MPa, the reaction temperature is 30-40° C., the stirring speed is 400 rpm, and the initial addition amount of the immobilized enzyme is 2%-5% of the solution mass.
[0015] (2) When the concentration of glucose solution is lower than 10 g / L, the reaction is considered to be complete. The gluconic acid product is obtained by filtration, and the immobilized enzyme is recovered and washed for the next reaction.
[0016] The method of the present invention is used to prepare the glucose oxidase-catalase co-immobilized enzyme, which can effectively improve the acidic reactivity of the enzyme, so that the co-immobilized enzyme can continue to react within the range of pH 2-3, eliminating the pH adjustment and subsequent ion exchange steps, and the reaction cycle is shortened to less than 10 hours. The prepared immobilized enzyme can be reused more than 10 times, the post-processing steps are simplified, and the production cost is greatly reduced. The gluconic acid product produced by the immobilized enzyme only needs to be filtered and enzyme-removed in one step, and the product purity is high. DETAILED DESCRIPTION
[0017] The present invention is further described in conjunction with specific implementation cases, and those skilled in the art can better understand and grasp the present invention by this, rather than limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.
[0018] 1. Glucose oxidase activity determination method
[0019] The glucose oxidase activity of the co-immobilized enzyme was determined by acid-base titration. 9.5 mL of 20 g / L glucose solution and 0.5 g of the immobilized enzyme to be tested were added to a conical flask, and 0.5 mL of deionized water was added as a blank control. The reaction was shaken at 30 ° C and 180 r / min for 1 hour. After taking it out, 10 mL of 0.1 mol / L sodium hydroxide solution and two drops of phenolphthalein were immediately added, and titrated with 0.1 mol / L hydrochloric acid standard titration solution. The glucose oxidase activity was calculated according to the following formula, where V0 is the volume of the standard hydrochloric acid solution consumed by the blank sample, V is the volume of the standard hydrochloric acid solution consumed by the sample to be tested, c is the concentration of the hydrochloric acid standard solution, and n is the dilution multiple.
[0020] Glucose oxidase activity = (V0-V)×c×1000÷60×n.
[0021] 2. Catalase activity assay
[0022] The activity of immobilized catalase was determined by sodium thiosulfate titration. 30% hydrogen peroxide solution was diluted 400 times with 0.02mol / L phosphate buffer as substrate solution and preheated to 30°C. 0.5g of immobilized enzyme was added to a conical flask, placed in a 30°C water bath shaker, 5mL of substrate solution was added and reacted for 5min. After taking it out, 2mL of sulfuric acid solution, 1mL of potassium iodide solution, 1 drop of ammonium molybdate and 2-3 drops of starch indicator were added in sequence, and titrated with sodium thiosulfate until the blue color faded. The catalase activity was calculated according to the following formula, where V0 is the volume of the standard sodium thiosulfate solution consumed by the blank sample, V is the volume of the standard sodium thiosulfate solution consumed by the sample to be tested, f is the concentration of the sodium thiosulfate solution, and N is the dilution multiple.
[0023] Catalase activity = (V0-V)×f / 2×1000×N / 5.
[0024] Example 1 Preparation of immobilized enzyme by direct mixing method
[0025] The resin was washed three times with 2 times the volume of water. Genipin was used as a cross-linking agent and the following cross-linking solution was added to the resin: glucose oxidase concentration of 100 U / mL, catalase concentration of 1000 U / mL, genipin concentration of 0.25%, glucose concentration of 50 g / L. The reaction was carried out at a stirring speed of 300 rpm and a stirring temperature of 30°C for 6 hours to obtain a co-immobilized enzyme.
[0026] The glucose oxidase activity of the immobilized enzyme was measured to be 462.77 U / g resin, and the catalase activity was 814.79 U / g resin. The direct mixing method directly mixes the carrier, enzyme solution and cross-linking agent, and the preparation process is simple and suitable for large-scale production.
[0027] Example 2 Effect of cross-linking agent concentration on co-immobilized enzyme
[0028] The resin was washed three times with 2 times the volume of water, and the following cross-linking solution was added: glucose oxidase concentration of 100U / mL, catalase concentration of 1000U / mL, genipin concentration of 0.5%, glucose concentration of 50g / L. The reaction was carried out at a stirring speed of 300rpm and a stirring temperature of 30°C for 6h to obtain a co-immobilized enzyme.
[0029] The glucose oxidase activity of the immobilized enzyme was measured to be 395.28U / g resin, and the catalase activity was 982.61U / g resin. Glucose oxidase is an aerobic dehydrogenase with a homodimeric structure, and catalase has a tetrameric structure. In theory, more cross-linking agents are required to completely immobilize catalase in the pores inside the carrier. As the concentration of genipin increased, the glucose oxidase activity of the immobilized enzyme decreased, but the catalase activity increased significantly, proving that high concentrations of genipin are beneficial to the immobilization of catalase.
[0030] Example 3 Effect of enzyme addition order on co-immobilized enzymes
[0031] The resin was washed three times with 2 times the volume of water, and the following cross-linking solution was added: catalase concentration of 1000U / mL, genipin concentration of 0.25%, glucose concentration of 50g / L, and reacted at a stirring speed of 300rpm and a stirring temperature of 30°C for 3h to obtain a single immobilized catalase. After filtering and fully washing the single immobilized enzyme, the following cross-linking solution was added: glucose oxidase concentration of 100U / mL, genipin concentration of 0.25%, glucose concentration of 50g / L, and reacted at a stirring speed of 300rpm and a stirring temperature of 30°C for 3h to obtain a co-immobilized enzyme.
[0032] The glucose oxidase activity of the immobilized enzymes was measured to be 408.37 U / g resin, and the catalase activity was 932.61 U / g resin. Glucose oxidase and catalase may have a certain competitive relationship during immobilization. Immobilizing the tetrameric catalase with a larger molecular weight first can significantly improve the catalase activity, and has no significant effect on the glucose oxidase activity.
[0033] Example 4 Effect of gradient concentration cross-linking agent immobilization on co-immobilized enzyme
[0034] The resin was washed three times with 2 times the volume of water, and the following cross-linking solution was added: catalase concentration 1000U / mL, genipin concentration 0.5%, glucose concentration 50g / L, and reacted at a stirring speed of 300rpm and a stirring temperature of 30°C for 3h to obtain a single immobilized catalase. After filtering and fully washing the single immobilized enzyme, the following cross-linking solution was added: glucose oxidase concentration 100U / mL, genipin concentration 0.25%, glucose concentration 50g / L, and reacted at a stirring speed of 300rpm and a stirring temperature of 30°C for 3h to obtain a co-immobilized enzyme.
[0035] The glucose oxidase activity of the immobilized enzyme was measured to be 438.37 U / g resin, and the catalase activity was 1075.86 U / g resin. The co-immobilized enzyme was prepared by successively immobilizing catalase and glucose oxidase with a gradient concentration of cross-linking agent, which can improve the catalase activity without affecting the glucose oxidase activity.
[0036] Example 5 Rapid evaluation of the acid resistance of co-immobilized enzymes
[0037] An initial reaction solution with a glucose concentration of 200 g / L and a gluconic acid concentration of 100 g / L was prepared, and the co-immobilized enzyme was added at a 2% enzyme addition amount, the reaction temperature was controlled at 40°C, the stirring speed was 400 rpm, and a 3% hydrogen peroxide solution was added. After the reaction was completed, glucose was added to make the glucose concentration in the solution 200 g / L. When the glucose concentration in the solution could not continue to decrease, the reaction was considered to be completed, and the reaction time of the co-immobilized enzyme was recorded. The results are shown in Table 1.
[0038] Table 1 Rapid evaluation of acid resistance of co-immobilized enzymes
[0039]
[0040]
[0041] As can be seen from Table 1, at the end of the reaction, the solution pH has dropped from the initial 1.8 to 1.3. Adding protective agent glucose, using gradient concentration cross-linking agent genipin cross-linking, and fixing double enzymes in sequence are all conducive to the continuous reaction of the immobilized enzyme under acidic conditions, and can increase the reaction time under acidic conditions by about 5 hours. The immobilized enzyme prepared by the present invention can react continuously for 15 hours at an extremely low pH below 2, and the acid resistance evaluation results of the immobilized enzyme are consistent with the enzyme activity determination results.
[0042] Example 6 Evaluation of the application of co-immobilized enzymes
[0043] Add 2.5 L of glucose solution with a concentration of 200 g / L to a 3.5 L bioreactor, control the reaction temperature to 40°C, the stirring speed to 400 rpm, the ventilation volume to 600 L / h (4 vvm), and the tank pressure to 0.05 MPa, and calibrate the dissolved oxygen electrode. Add 2% of the solution mass of the co-immobilized enzyme prepared in Example 1-4 to start the reaction. Filter out the immobilized enzyme after the reaction, wash it thoroughly, filter it, and then put it into the bioreactor for repeated reaction. When the residual sugar concentration cannot be reduced to less than 10 g / L when the reaction time is extended, it is considered that the repeated reaction cannot be continued.
[0044] At the same time, a control group was set up. The specific steps are as follows:
[0045] Control group 1: Effect of cross-linking agent on immobilized enzyme
[0046] The resin was washed three times with 2 times the volume of water. Glutaraldehyde was used as the cross-linking agent. The following cross-linking solution was added to the resin: glucose oxidase concentration of 100 U / mL, catalase concentration of 1000 U / mL, glutaraldehyde concentration of 2.0%, glucose concentration of 50 g / L. The reaction was carried out at a stirring speed of 300 rpm and a stirring temperature of 30°C for 6 hours to obtain a co-immobilized enzyme.
[0047] The glucose oxidase activity of the immobilized enzyme was measured to be 284.43 U / g resin, and the catalase activity was 788.13 U / g resin. Compared with Example 1, it was shown that the cross-linking agent glutaraldehyde had some damage to the activity of the co-immobilized enzyme.
[0048] Control group 2: Effect of protective agent glucose on co-immobilized enzyme
[0049] The resin was washed three times with 2-fold volume of water, and the following cross-linking solution was added: glucose oxidase concentration of 100 U / mL, catalase concentration of 1000 U / mL, and genipin concentration of 0.25%. The reaction was carried out at a stirring speed of 300 rpm and a stirring temperature of 30°C for 6 hours to obtain a co-immobilized enzyme.
[0050] The glucose oxidase activity of the immobilized enzyme was measured to be 325.61U / g resin, and the catalase activity was 803.59U / g resin. Theoretical analysis shows that the substrate can occupy the active pocket of the enzyme molecule during the cross-linking process, thereby protecting the enzyme molecule from damage by the cross-linking agent. The dual enzyme activity and application effect of Example 1 are significantly better than those of Control Group 2, indicating that the substrate glucose can protect the dual enzyme activity, especially the activity of glucose oxidase, during the cross-linking process.
[0051] Table 2 Comparison of reaction conditions for preparing gluconic acid by one-step method using immobilized enzyme
[0052]
[0053] The use of glutaraldehyde as a cross-linking agent has a greater impact on the immobilized enzyme. Hydrogen peroxide accumulation has occurred after 14 hours of reaction, and it is impossible to completely catalyze the reaction of 200g / L glucose. When glucose is not added as a protective agent and genipin is used as a cross-linking agent, the immobilized enzyme reaction cannot reduce the glucose concentration to below 10g / L after 14 hours, but the immobilized enzyme still has catalytic activity at this time, but the reaction rate is too slow. Adding the protective agent glucose can achieve a single reuse of the immobilized enzyme, but the second reaction rate has dropped significantly. After increasing the concentration of genipin, the fixation effect of catalase is better, the number of repeated reactions has been improved to a certain extent, and it can be reused 3 times. The rate drops after the fourth reaction. The number of repeated reactions is increased to 4 times by fixing catalase and glucose oxidase in batches. By using the method of adding a protective agent, fixing a gradient concentration cross-linking agent, and fixing the two enzymes in turn to prepare the immobilized enzyme, the reaction rate and the number of repeated reactions of the immobilized enzyme have been significantly improved. After 10 repeated reactions, it can still smoothly catalyze the complete decomposition of glucose. The immobilized enzyme prepared by the present invention realizes the one-step production of gluconic acid and reduces the production cost of gluconic acid.
[0054] In summary, the present invention can effectively improve the acidic reactivity of the enzyme by preparing the glucose oxidase-catalase co-immobilized enzyme, so that the co-immobilized enzyme can continue to react within the pH range of 2-3, eliminating the pH adjustment and subsequent ion exchange steps, achieving one-step production under acidic conditions, and shortening the reaction cycle to less than 10 hours. The prepared immobilized enzyme can be reused more than 10 times, the post-processing steps are simplified, and the production cost is greatly reduced.
Claims
1. A method for preparing an acid-resistant immobilized enzyme, characterized in that: The following steps are involved: (1) The resin was washed twice with water, and catalase, genipin and glucose solution were added to carry out cross-linking reaction; (2) Glucose oxidase, genipin and glucose solution were added to carry out cross-linking reaction. The mixture was allowed to stand at 4°C overnight and then washed thoroughly with water to obtain co-immobilized enzyme.
2. The preparation method according to claim 1, characterized in that In the step (1), the concentration of catalase is 500-1000 U / mL, the concentration of genipin is 0.25%-0.5%, and the concentration of glucose solution is 50-100 g / L.
3. The preparation method according to claim 1 or 2, characterized in that: In the step (2), the concentration of glucose oxidase is 50-100 U / mL, the concentration of genipin is 0.25%-0.5%, and the concentration of glucose solution is 50-100 g / L.
4. The preparation method according to claim 3, characterized in that: The parameters of the cross-linking reaction in steps (1) and (2) are stirring temperature 20-30° C., stirring speed 100-300 rpm, and reaction time 2-4 h.
5. Use of the co-immobilized enzyme prepared by the preparation method according to claim 1 in producing gluconic acid products.
6. The use according to claim 5, characterized in that The co-immobilized enzyme is put into the glucose solution, air is introduced to start the reaction, and the solution is filtered to obtain the finished gluconic acid product.
7. The use according to claim 6, characterized in that The amount of immobilized enzyme added in the reaction is 2%-5% of the solution mass, the concentration of glucose solution is 200-300 g / L, the ventilation volume is 4 vvm, the tank pressure is 0.05-0.1 MPa, the reaction temperature is 30-40°C, and the stirring speed is 200-600 rpm.
8. The use according to claim 6 or 7, characterized in that When the concentration of the glucose solution was lower than 10 g / L, the reaction was stopped, and the co-immobilized enzyme was recovered by filtration and washed.