High-stability immobilized mannanase, and preparation method and application thereof

Abstract

The application discloses a high-stability immobilized mannanase and a preparation method and application thereof, and belongs to the technical field of enzyme immobilization. The preparation method uses PAMAM modified SBA-15 / calcium alginate composite microspheres as a carrier to fix mannanase in an enzyme solution by adopting an embedding-covalent crosslinking method, so as to obtain the high-stability immobilized mannanase. The preparation method uses the PAMAM modified SBA-15 / calcium alginate composite microspheres as the carrier to fix the mannanase by adopting the embedding-covalent crosslinking synergistic method, so that the pH tolerance, the circulation stability and the hypoglycemic efficiency of the enzyme are improved, and the problems of the traditional immobilized enzyme, such as large enzyme activity loss, easy leakage and poor stability under high temperature, are solved.

Description

Technical Field

[0001] This invention belongs to the field of enzyme immobilization technology, specifically a highly stable immobilized mannanase, its preparation method, and its application. Background Technology

[0002] Mannanase is a key enzyme in the degradation of hemicellulose and has wide applications in feed, food, and energy. However, the free enzymes commonly used in industry suffer from poor stability, cannot be reused, and are difficult to separate, resulting in high costs and limiting large-scale application. Immobilized enzyme technology can improve enzyme stability and enable recycling, which is an effective way to reduce production costs.

[0003] Existing methods for immobilizing mannanase mainly include adsorption, encapsulation, and covalent cross-linking, using single materials such as alginate, chitosan, porous resin, and mesoporous silica as supports. For example, while calcium alginate has good biocompatibility, it has low mechanical strength, is easily broken, and prone to enzyme leakage; SBA-15 has a stable structure and large specific surface area, but few surface active groups, resulting in weak enzyme binding and easy detachment. Current technologies attempt to combine alginate with mesoporous silica to balance encapsulation effectiveness and mechanical strength. However, such composite materials still have significant shortcomings: first, the surface functional groups are limited, resulting in weak interactions with enzyme molecules, making it difficult to achieve a strong binding; second, the distribution of active sites on the support surface is uneven, and enzyme loading and immobilization efficiency still need improvement; third, immobilization strategies are mostly simple encapsulation or adsorption, failing to fundamentally solve the enzyme leakage problem. Summary of the Invention

[0004] To overcome the aforementioned technical problems, this invention provides a highly stable immobilized mannanase, its preparation method, and its application. This preparation method uses PAMAM-modified SBA-15 / calcium alginate composite microspheres as a carrier and employs an encapsulation-covalent cross-linking synergistic method to immobilize the mannanase, thereby improving the enzyme's pH tolerance, cycling stability, and glucose-lowering efficiency. This solves the problems of large enzyme activity loss, easy leakage, and poor stability at high temperatures associated with traditional immobilized enzymes.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention discloses a method for preparing highly stable immobilized mannanase. Using PAMAM-modified SBA-15 / calcium alginate composite microspheres as a carrier, mannanase in the enzyme solution is immobilized by an encapsulation-covalent cross-linking method to obtain highly stable immobilized mannanase.

[0007] According to some embodiments of the present invention, the composite microspheres are prepared by calcium ion crosslinking of dendritic PAMAM, mesoporous SBA-15, and sodium alginate.

[0008] According to some embodiments of the present invention, the mannanase is a mannanase derived from Trichoderma reesei.

[0009] According to some embodiments of the present invention, the dendritic PAMAM is a G3~G4 generation polyamide amine-amino group; wherein, if the generation is too low, there are insufficient sites; if it is too high, the steric hindrance is large and the diffusion is poor.

[0010] According to some embodiments of the present invention, mesoporous SBA-15 is a mesoporous molecular sieve, wherein the mesoporous SBA-15 has a grain size of 1~10 μm, a pore size of 6~11 nm, and a specific surface area ≥550 m². 2 / g.

[0011] According to some embodiments of the present invention, the preparation process of the composite microspheres is as follows: S01 Mesoporous SBA-15 is added to a dendritic PAMAM aqueous solution and stirred to graft the dendritic PAMAM onto the surface of SBA-15, thereby obtaining PAMAM-modified mesoporous SBA-15. S02. PAMAM-modified mesoporous SBA-15 was added to sodium alginate aqueous solution and stirred to obtain the carrier precursor solution; S03. The carrier precursor solution was added to the calcium chloride aqueous solution, stirred, and allowed to stand for cross-linking to form PAMAM-modified SBA-15 / calcium alginate composite microspheres.

[0012] In S01, the mass ratio of SBA-15 to the aqueous solution of the dendritic PAMAM is 1:2 to 1:4; In S01, the mass concentration of the dendritic PAMAM aqueous solution is 5-10%; In S01, the stirring is carried out at 25~30℃ and 80~100r / min for 1.5~2.0h. In S01, after the grafting is completed, the solid product is centrifuged at 8000~10000r / min for 10~15min, washed with deionized water 2~3 times, and then vacuum dried at 60~70℃ for 3~4h to obtain PAMAM modified mesoporous SBA-15. In S02, the mass ratio of PAMAM modified mesoporous SBA-15 to sodium alginate aqueous solution is 1:3 to 1:5. In SO2, the mass concentration of the sodium alginate aqueous solution is 1.8~2.5%; In S02, the stirring is carried out at 25~30℃ and 60~80r / min for 30~40min; In S03, the volume of the calcium chloride aqueous solution is 8 to 10 times that of the carrier precursor solution; In SO3, the mass concentration of the calcium chloride aqueous solution is 1.5%~2.2%; In S03, the process of adding the precursor liquid is to add it to the calcium chloride aqueous solution by dripping it with a peristaltic pump at a dripping rate of 15-20 drops / min, and stirring at a low speed of 30-50 r / min during the dripping process; In S03, the settling time is 2-3 hours; In step S03, after standing, the surface is washed 3-4 times with deionized water to remove residual calcium ions, resulting in a dendritic PAMAM-modified mesoporous SBA-15 / calcium alginate composite microsphere carrier. After draining the water, it is ready for use.

[0013] According to some embodiments of the present invention, the steps of the encapsulation-covalent crosslinking method are as follows: the composite microsphere carrier is added to the enzyme solution at a solid-liquid ratio of 1 / 8 to 12 g / mL, and the mixture is stirred and encapsulated in the dark at 20 to 28°C and 60 to 100 r / min for 1.0 to 1.5 h to obtain the enzyme-carrier complex; the encapsulated enzyme-carrier complex is added to the crosslinking agent solution and crosslinked in the dark for 2.0 to 3.0 h to obtain the crosslinked product.

[0014] According to some embodiments of the present invention, the liquid-to-solid ratio of the enzyme solution to the composite microsphere carrier is 8-12 mL / g.

[0015] According to some embodiments of the present invention, the initial enzyme activity of the enzyme solution is 800~1200 U / mL.

[0016] According to some embodiments of the present invention, the pH of the enzyme solution is 5.0 to 6.0, and the pH is adjusted using an acetate-sodium acetate buffer solution.

[0017] According to some embodiments of the present invention, the pH of the system is kept stable at 5.0 to 6.0 during the encapsulation process.

[0018] According to some embodiments of the present invention, the solid-liquid ratio of the enzyme-carrier complex and the cross-linking agent solution is 1 / 6 to 8 g / mL.

[0019] According to some embodiments of the present invention, the crosslinking agent solution contains 0.004%~0.01% glutaraldehyde, 0.016%~0.04% polyethylene glycol diglycidyl ether and the balance water.

[0020] According to some embodiments of the present invention, the crosslinking is performed under light-protected and sealed conditions at 4~8°C and 40~60 r / min.

[0021] According to some embodiments of the present invention, after cross-linking, the cross-linking product is washed 3 to 5 times with an acetate-sodium acetate buffer solution at pH 5.0 to 6.0, and then dried to obtain a highly stable immobilized mannanase.

[0022] This invention discloses a highly stable immobilized mannanase prepared by the aforementioned method.

[0023] The optimal reaction temperature for the highly stable immobilized mannanase is ≤80℃, and preferably 20~70℃.

[0024] The optimal reaction pH for the highly stable immobilized mannanase is 4-7.

[0025] The present invention also discloses the application of the aforementioned highly stable immobilized mannanase in polysaccharide degradation.

[0026] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0027] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes dendritic PAMAM modified mesoporous SBA 15 / Calcium alginate composite microspheres serve as carriers, combined with encapsulation The two-step covalent cross-linking immobilization process significantly improves the immobilization efficiency and stability of mannanase.

[0028] 2. This invention utilizes mesoporous SBA 15. Dendritic PAMAM is compounded with calcium alginate, SBA 15. It provides a high specific surface area and regular pores to ensure sufficient contact between the substrate and the enzyme; the surface of the PAMAM dendritic macromolecule is rich in amino groups, providing a large number of active sites for the covalent binding of enzyme molecules; the calcium alginate gel network achieves gentle encapsulation and structural support for the enzyme.

[0029] 3. The preparation method of the present invention has high enzyme immobilization efficiency, which is due to the introduction of PAMAM which improves the surface reactivity of the carrier. At the same time, the mild embedding conditions effectively protect the spatial conformation of the enzyme and avoid the enzyme from denaturation and inactivation during the immobilization process.

[0030] 4. The stability of the highly stable immobilized mannanase of this invention is significantly better than that of free enzymes and traditional immobilized enzymes, and it is expected to be applied in the fields of feed, food, and biomass degradation. Detailed Implementation

[0031] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0032] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0033] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be combined arbitrarily; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for specific parameters, it is understood that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this invention, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed herein; "0-5" is merely a shortened representation of these numerical combinations. Furthermore, when a parameter is described as an integer greater than or equal to 2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0035] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0036] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0037] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0038] Unless otherwise specified, the term "or" is inclusive in this invention. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0039] The raw material information used in the following examples is as follows: Dendritic PAMAM originates from Xi'an Qiyue Biotechnology, specifically G3-NH2 dendritic polyamide amine-amino grouped; Mesoporous SBA-15 (mesoporous molecular sieve) originates from Zhuoran Environmental Protection Technology Co., Ltd., and has a pore size of 1~10μm, a pore size of 6~11nm, and a specific surface area ≥550m². 2 / g; Mannanase enzyme solution: derived from Trichoderma reesei, with an initial enzyme activity of 1000 U / mL, prepared with acetate-sodium acetate buffer at pH 5.5, for later use; This includes, but is not limited to, the models from the above manufacturers.

[0040] Example 1 The preparation method of the highly stable immobilized mannanase in this embodiment is as follows: Preparation of S1. PAMAM-modified SBA-15 / calcium alginate composite microsphere carrier S01. Weigh 1.0g of dried mesoporous SBA-15 and slowly add it to 3.0g of 7.5% dendritic PAMAM aqueous solution (the mass ratio of SBA-15 to dendritic PAMAM aqueous solution is 1:3); place the mixture in a constant temperature magnetic stirrer at 28℃ and 90r / min and stir for 1.8h to allow the dendritic PAMAM to be fully grafted onto the surface of SBA-15.

[0041] After the reaction was completed, the mixture was centrifuged at 9000 r / min for 12 min and the solid product was collected. The solid product was washed three times with deionized water, and centrifuged (8000 r / min, 10 min) after each wash to remove ungrafted free PAMAM. The washed solid product was placed in a vacuum drying oven at 65℃ and vacuum dried for 3.5 h to obtain PAMAM-modified mesoporous SBA-15. After grinding, it was passed through an 80-mesh sieve for later use.

[0042] S02. Weigh 1.0g of the PAMAM-modified mesoporous SBA-15 prepared above and slowly add it to 4.0g of sodium alginate aqueous solution with a mass concentration of 2.0% (the mass ratio of PAMAM-modified mesoporous SBA-15 to sodium alginate aqueous solution is 1:4); place the mixture in a constant temperature magnetic stirrer at 28℃ and 70r / min and stir and disperse for 35min to obtain the carrier precursor solution.

[0043] S03. Add the carrier precursor solution to the peristaltic pump and slowly add it dropwise at a rate of 18 drops / min to a 2.0% calcium chloride aqueous solution. During the dropwise addition, use a low-speed magnetic stirring at 40 r / min to ensure that the added precursor solution is evenly dispersed and initially formed.

[0044] After the addition is complete, stop stirring and place the beaker in a constant temperature environment of 25℃ for 2.5 hours to allow sodium alginate and calcium ions to fully crosslink and form stable composite microspheres.

[0045] S04. After settling, wash the composite microspheres four times with deionized water. After each wash, gently blot the surface moisture with filter paper to remove residual calcium ions on the surface of the microspheres. After draining the water, the PAMAM-modified SBA-15 / calcium alginate composite microsphere carrier is obtained for later use.

[0046] S2. Immobilization of mannanase by encapsulation-covalent cross-linking method Step 1: Embedding Process Weigh 1.0 g of the prepared composite microsphere carrier and add it to 10.0 mL of the prepared mannanase solution (the liquid-to-solid ratio of enzyme solution to composite microsphere carrier is 10 mL / g); adjust the pH of the system to 5.5 and keep the pH of the system stable; place the mixed system in a constant temperature magnetic stirrer at 25℃ and 80 r / min, and stir and embed for 1.0 h in the dark to allow the mannanase to be fully embedded in the gel network of the composite microspheres, thus obtaining the enzyme-carrier complex.

[0047] Step 2: Covalent cross-linking process The crosslinking agent solution contains 0.007% glutaraldehyde, 0.028% polyethylene glycol diglycidyl ether, and the balance water by mass percentage. The enzyme-carrier complex was added to the crosslinking agent solution (the solid-liquid ratio of the enzyme-carrier complex to the crosslinking agent solution was 1:7 g / mL); the mixture was placed in a constant temperature magnetic stirrer at 6℃ and 50 r / min, and crosslinked in the dark for 2.5 h to obtain the crosslinked product.

[0048] Step 3: Post-processing The cross-linking product was washed four times with an acetate-sodium acetate buffer solution at pH 5.5. After each wash, the product was centrifuged to remove unbound free enzyme and residual cross-linking agent. The washed product was then placed in a vacuum drying oven at 50°C and dried to constant weight, which yielded highly stable immobilized mannanase.

[0049] Example 2 The difference between this embodiment and Embodiment 1 is as follows: The concentration of PAMAM aqueous solution in S01 is 10wt%, SBA The mass ratio of 15:PAMAM aqueous solution is 1:2; The other raw materials, steps and parameters are the same as in Example 1.

[0050] Example 3 The difference between this embodiment and Embodiment 1 is as follows: The concentration of PAMAM aqueous solution in S01 is 3wt%, SBA The mass ratio of 15:PAMAM aqueous solution is 1:1; The other raw materials, steps and parameters are the same as in Example 1.

[0051] Example 4 The difference between this embodiment and Embodiment 1 is as follows: The mass concentration of sodium alginate aqueous solution in SO2 is 3.0%; The other raw materials, steps and parameters are the same as in Example 1.

[0052] Example 5 The difference between this embodiment and Embodiment 1 is as follows: Step S1 does not include step S03. Instead, the carrier precursor liquid prepared in S02 is allowed to stand and then subjected to S04 to obtain PAMAM modified SBA-15 / calcium alginate composite microsphere carrier. The other raw materials, steps and parameters are the same as in Example 1.

[0053] Example 6 The difference between this embodiment and Embodiment 1 is as follows: The crosslinking agent solution in S2 contains 0.01% glutaraldehyde and the balance is water; The other raw materials, steps and parameters are the same as in Example 1.

[0054] Comparative Example 1 The difference between this comparative example and Example 1 is as follows: S1 does not include the grafting step of mesoporous SBA-15 with SO1. Instead, the mesoporous SBA-15 is directly subjected to the subsequent SO2 and SO3 steps to finally obtain SBA-15 / calcium alginate composite microspheres. The other raw materials, steps and parameters are the same as in Example 1.

[0055] Comparative Example 2 The difference between this comparative example and Example 1 is as follows: Step S2 does not include step two; instead, the enzyme-carrier complex obtained in step one is directly subjected to the post-processing in step three. The lack of cross-linking leads to weak enzyme binding and poor stability. The other raw materials, steps and parameters are the same as in Example 1.

[0056] Test Example 1—pH Stability Test The highly stable immobilized mannanase and free mannanase (with enzyme activity equal to that of the highly stable immobilized mannanase) prepared in Example 1 were placed in buffer solutions of pH 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, and incubated at 25°C for 2 hours. The residual enzyme activity was then measured using the DNS method (substrate: 0.5% glucomannan) under optimal conditions, with the highest enzyme activity being 100%. The test results are shown in Table 1.

[0057]

[0058] Test Example 2—Thermal Stability Test The highly stable immobilized mannanase and free mannanase (with enzyme activity equal to that of the highly stable immobilized mannanase) prepared in Example 1 were placed in water baths at 40℃, 50℃, 55℃, and 60℃ for 1 hour, respectively, and then rapidly cooled to determine the residual enzyme activity; the test results are shown in Table 2.

[0059]

[0060] Application examples The highly stable immobilized mannanase prepared in the above examples and comparative examples was applied in the following ways, and the application results are shown in Table 3. (1) Polysaccharide degradation rate 100 mL of 0.5% glucomannan substrate solution was placed in a constant temperature water bath reactor and preheated at 50 °C for 10 min. Highly stable immobilized mannanase was added at an enzyme addition rate of 200 U / g substrate, and the reaction was carried out at 50 °C and 150 rpm for 2 h for catalytic degradation. During the reaction, 0.5 mL samples were taken every 20 min, inactivated in a boiling water bath for 10 min, and the supernatant was collected after centrifugation to determine the reducing sugar content. After the reaction, the immobilized enzyme was filtered and recovered, washed with buffer, and used for the next batch of degradation.

[0061] The control group used free mannanase (with the same enzyme activity as the immobilized enzyme), and the enzyme addition was also based on 200 U / g substrate.

[0062] Finally, the content of reducing sugars produced by degradation was determined using the DNS method, and the polysaccharide degradation rate was calculated.

[0063] (2) Stability of repeated use After recovering the immobilized mannanase from the experimental group and the free mannanase from the control group, they were repeatedly used for glucomannan degradation. The reaction conditions were the same each time, and the relative enzyme activity was measured after 10 cycles.

[0064]

[0065] Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this invention. It should be understood that the above descriptions are merely specific embodiments of this invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for preparing a highly stable immobilized mannanase, characterized in that, Mannanase in enzyme solution was immobilized using PAMAM-modified SBA-15 / calcium alginate composite microspheres as carriers via an encapsulation-covalent cross-linking method to obtain highly stable immobilized mannanase. The composite microspheres are prepared by cross-linking dendritic PAMAM, mesoporous SBA-15, and sodium alginate.

2. The method for preparing highly stable immobilized mannanase as described in claim 1, characterized in that, The mannanase is a mannanase derived from Trichoderma reesei; And / or, the dendritic PAMAM is a G3~G4 generation polyamide amine-amino group; And / or, the mesoporous SBA-15 has a grain size of 1~10μm, a pore size of 6~11nm, and a specific surface area ≥550m². 2 / g.

3. The method for preparing highly stable immobilized mannanase as described in claim 1, characterized in that, The preparation process of the composite microspheres is as follows: S01 Mesoporous SBA-15 is added to a dendritic PAMAM aqueous solution and stirred to graft the dendritic PAMAM onto the surface of SBA-15, thereby obtaining PAMAM-modified mesoporous SBA-15. S02. PAMAM-modified mesoporous SBA-15 was added to sodium alginate aqueous solution and stirred to obtain the carrier precursor solution; S03. The carrier precursor solution was added to the calcium chloride aqueous solution, stirred, and allowed to stand for crosslinking to form PAMAM-modified SBA-15 / calcium alginate composite microspheres.

4. The method for preparing highly stable immobilized mannanase as described in claim 3, characterized in that, At least one of the following conditions (1) to (12) must be met: (1) In S01, the mass ratio of SBA-15 to the aqueous solution of the dendritic PAMAM is 1:2 to 1:4; (2) In S01, the mass concentration of the dendritic PAMAM aqueous solution is 5~10%; (3) In S01, the stirring is carried out at 25~30℃ and 80~100r / min for 1.5~2.0h; (4) In S01, after the grafting is completed, the solid product is centrifuged at 8000~10000r / min for 10~15min, washed with deionized water 2~3 times, and then vacuum dried at 60~70℃ for 3~4h to obtain PAMAM modified mesoporous SBA-15. (5) In S02, the mass ratio of PAMAM modified mesoporous SBA-15 to sodium alginate aqueous solution is 1:3 to 1:5; (6) In SO2, the mass concentration of the sodium alginate aqueous solution is 1.8~2.5%; (7) In S02, the stirring is carried out at 25~30℃ and 60~80r / min for 30~40min; (8) In SO3, the volume of the calcium chloride aqueous solution is 8 to 10 times that of the carrier precursor solution; (9) In SO3, the mass concentration of the calcium chloride aqueous solution is 1.5%~2.2%; (10) In S03, the process of adding the precursor liquid is to add the feedstock to the calcium chloride aqueous solution by dripping with a peristaltic pump at a dripping rate of 15~20 drops / min, and stirring at a low speed of 30~50 r / min during the dripping process; (11) In S03, the settling time is 2~3h; (12) In S03, after standing, the surface is washed with deionized water 3 to 4 times to remove residual calcium ions, and the dendritic PAMAM modified mesoporous SBA-15 / calcium alginate composite microsphere carrier is obtained. After draining the water, it is ready for use.

5. The method for preparing highly stable immobilized mannanase as described in claim 1, characterized in that, The steps of the encapsulation-covalent crosslinking method are as follows: the composite microsphere carrier is added to the enzyme solution at a solid-liquid ratio of 1 / 8 to 12 g / mL, and the mixture is stirred in the dark at 20 to 28°C and 60 to 100 r / min for 1.0 to 1.5 h to obtain the enzyme-carrier complex; the encapsulated enzyme-carrier complex is added to the crosslinking agent solution and crosslinked in the dark for 2.0 to 3.0 h to obtain the crosslinked product.

6. The method for preparing highly stable immobilized mannanase as described in claim 5, characterized in that, The liquid-to-solid ratio of the enzyme solution to the composite microsphere carrier is 8-12 mL / g; And / or, the initial enzyme activity of the enzyme solution is 800~1200 U / mL; And / or, the pH of the enzyme solution is 5.0 to 6.

0.

7. The method for preparing highly stable immobilized mannanase as described in claim 5, characterized in that, The pH of the system was kept stable at 5.0-6.0 during the encapsulation process; And / or, the solid-liquid ratio of the enzyme-carrier complex and the cross-linking agent solution is 1 / 6 to 8 g / mL; And / or, the crosslinking agent solution contains 0.004%~0.01% glutaraldehyde, 0.016%~0.04% polyethylene glycol diglycidyl ether and the balance water; And / or, the crosslinking is performed under light-protected, sealed conditions at 4~8°C and 40~60 r / min; And / or, after cross-linking, the cross-linking product is washed 3 to 5 times with an acetate-sodium acetate buffer solution at pH 5.0 to 6.0, and then dried to obtain a highly stable immobilized mannanase.

8. A highly stable immobilized mannanase, characterized in that, It was prepared by the aforementioned method.

9. The highly stable immobilized mannanase as described in claim 8, characterized in that, The optimal reaction temperature for the highly stable immobilized mannanase is ≤80℃, and more preferably 20~70℃. And / or, the optimal reaction pH for the highly stable immobilized mannanase is 4-7.

10. The application of the highly stable immobilized mannanase as described in any one of claims 8 to 9 in polysaccharide degradation.

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