An α-amylase immobilization carrier, its preparation method and application

Abstract

This invention discloses an α-amylase immobilization carrier, its preparation method, and its application, comprising the following steps: (1) preparation of mesoporous silica; (2) thiolization of mesoporous silica; (3) preparation of polyhydroxy mesoporous silica; and (4) preparation of the immobilization carrier. The α-amylase immobilization carrier provided by this invention uses chitosan-modified mesoporous silica as the carrier, which has a large surface area and numerous adsorption sites, allowing for stable binding with α-amylase, reducing α-amylase shedding, and improving the adsorption rate of α-amylase. The preparation method of this carrier is simple and easy to implement, the preparation process is green and environmentally friendly, pollution-free, and low in cost. This carrier can be reused multiple times while maintaining good enzyme activity, increasing the utilization rate of α-amylase and its carrier, and reducing costs.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme carrier preparation technology, specifically relating to an α-amylase immobilized carrier, its preparation method, and its application. Background Technology

[0002] Enzymes are biocatalysts with high catalytic efficiency, their catalytic rate being 10 times that of traditional catalysts. 7 -10 13 Enzymes exhibit high specificity to substrates and produce no byproducts during catalytic reactions, making them popular among producers. With the rapid development of materials science and the emergence of various novel enzyme immobilization technologies, the widespread application of enzymes in production has become possible.

[0003] Enzymes, with their high catalytic efficiency, possess unparalleled advantages over traditional catalysts. However, most enzymes are chemically proteins, exhibiting drawbacks such as easy inactivation, difficulty in storage and transportation, and poor recyclability, limiting their application in industrial production. Therefore, overcoming these shortcomings has become a hot research topic in the scientific community. Conventional enzyme catalytic reactions occur in aqueous solutions. Immobilized enzyme technology involves treating water-soluble enzymes using physical or chemical methods and immobilizing them on suitable supports (such as porous silica, activated carbon, porous glass, porous ceramics, diatomaceous earth, and alumina), rendering them insoluble in water while maintaining their enzyme activity. Immobilized enzymes not only retain high specificity and catalytic efficiency but also exhibit enhanced pH and thermal stability, increasing their lifespan. Furthermore, it simplifies production processes, facilitates enzyme recovery and purification of reaction products, and significantly improves product quality.

[0004] To address the problems associated with free enzymes, Chinese patent application No. 201510687008.4 discloses a carboxylated three-dimensional ordered mesoporous carbon-lysozyme composite material, its preparation method, and its applications. Lysozyme is covalently cross-linked onto the carboxylated three-dimensional ordered mesoporous carbon, resulting in immobilized enzymes with higher activity, better stability, and reusability. This patent uses carboxylated three-dimensional ordered mesoporous carbon as a carrier. The unique structure of this carbon—uniform pore size, orderly pore arrangement, and small interconnected windows between pores—facilitates the transfer of enzyme molecules within the pores, making it easier for them to reach the enzyme's active site. However, this patent still suffers from poor immobilization of free enzymes, hindering improvements in enzyme activity and operational stability. Whether it still provides good immobilization for α-amylase remains to be discussed.

[0005] α-Amylase can randomly act on the α-1,4-glycosidic bonds in starch to produce reducing sugars such as glucose. It has wide applications in the food, brewing, textile, and pharmaceutical industries. As a major industrial enzyme preparation, α-amylase is widely used in food processing, grain industry, alcohol industry, fermentation, and textile industry, making it one of the most widely used enzyme preparations in industrial production. However, pure α-amylase has poor stability, is extremely sensitive to temperature, pH, and organic solvents, is easily inactivated during processing, and cannot be reused. The product obtained from the reaction is difficult to purify and refine. Immobilizing α-amylase, however, allows for repeated recovery and reuse, improves enzyme stability, thereby improving product quality, and provides a simple and convenient way to directly obtain purified products. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an α-amylase immobilization carrier, its preparation method, and its application. This carrier has a large surface area and multiple adsorption sites, enabling it to stably bind with α-amylase, reducing α-amylase shedding, and improving the adsorption rate of α-amylase.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0009] (1) Preparation of mesoporous silica: Chlorohexadecylpyridine was added to deionized water and stirred until homogeneous. Then tetraethyl orthosilicate and ethanol solution were added to obtain a mixture. Next, polyethylene glycol octylphenyl ether, n-octyltrimethoxysilane and 1,3,5-trimethylbenzene were added. Ammonia was slowly added dropwise under stirring. After the addition was complete, the mixture was stirred and reacted. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere to obtain mesoporous silica.

[0010] (2) Thioylation of mesoporous silica: The mesoporous silica obtained in step (1) is added to toluene, followed by the addition of γ-mercaptopropyltrimethoxysilane, and a constant temperature reaction is carried out to obtain thiolated mesoporous silica.

[0011] (3) Preparation of polyhydroxy mesoporous silica: The mercapto-modified mesoporous silica, 2-hydroxyethyl acrylate and azobisisobutyronitrile from step (2) are added to ethanol and the mercapto-olefin reaction is carried out under nitrogen atmosphere to obtain polyhydroxy mesoporous silica.

[0012] (4) Preparation of immobilized carrier: The polyhydroxy mesoporous silica, glutaraldehyde and chitosan acetic acid solution in step (3) are stirred and reacted. After the reaction is completed, sodium hydroxide solution is added and stirred for 10-30 min. Then, the mixture is filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0013] Preferably, in step (1), the mass ratio of hexadecyl chloride pyridine, tetraethyl orthosilicate, ethanol solution, polyethylene glycol octylphenyl ether, n-octyltrimethoxysilane, 1,3,5-trimethylbenzene, and ammonia is 1-3:30-50:100-150:1-3:30-50:10-25:60-100; the mass concentration of the ethanol solution is 30-60%, and the mass concentration of the ammonia is 20-25%.

[0014] Preferably, the stirring reaction in step (1) is carried out at a temperature of 50-80°C for 12-24 hours; the calcination temperature is 500-800°C and the calcination time is 4-8 hours.

[0015] Preferably, in step (2), the mass ratio of mesoporous silica to γ-mercaptopropyltrimethoxysilane is 50:30-50; the temperature of the isothermal reaction is 40-60℃ and the time is 1-2h.

[0016] Preferably, the mass ratio of thiolized mesoporous silica, 2-hydroxyethyl acrylate, and azobisisobutyronitrile in step (3) is 100:30-60:1-5.

[0017] Preferably, the temperature of the mercapto-alkene reaction in step (3) is 80-100℃ and the time is 15-25h.

[0018] Preferably, in step (4), the mass ratio of the polyhydroxy mesoporous silica, glutaraldehyde, chitosan acetic acid solution, and sodium hydroxide solution is 100:20-40:80-120:30-50; the concentration of the sodium hydroxide solution is 0.5-1 mol / L; and the stirring reaction temperature is 60-90℃ and the time is 0.5-2 h.

[0019] Preferably, the chitosan acetic acid solution in step (4) is prepared as follows: chitosan is added to an acetic acid solution with a concentration of 0.5-2 mol / L and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution; the mass ratio of chitosan to acetic acid solution is 5-10:100.

[0020] This invention also protects an α-amylase immobilization carrier prepared by the method described above.

[0021] This invention also protects the use of the aforementioned α-amylase immobilization carrier in immobilizing α-amylase.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The method for preparing the α-amylase immobilization carrier provided by the present invention involves two steps of adding different silicon sources and preparing mesoporous silica particles under hydrothermal reaction conditions. First, tetraethyl orthosilicate is emulsified and dispersed into a microemulsion under the action of hexadecylpyridine chloride. Then, n-octyltrimethoxysilane containing long-chain branches is added to the mixture under the dispersion of polyethylene glycol octylphenyl ether. At the same time, pore-expanding agent 1,3,5-trimethylbenzene is introduced, which can adjust the particle size and pore size of mesoporous silica in the subsequent synthesis process, providing a large specific surface area and surface roughness. Then, ammonia water is added for hydrothermal reaction, and finally calcination is carried out to obtain mesoporous silica. The present invention adds two silicon sources in batches, so that the prepared silica has a small particle size and a large surface pore size, which is beneficial to increasing the loading capacity of α-amylase. Then, the mesoporous silica is reacted with γ-mercaptopropyltrimethoxysilane. The process involves reacting silanes to introduce thiol groups onto the surface, which improves the efficiency of subsequent modification reactions. Then, the thiolized mesoporous silica undergoes a thiol-alkene click reaction with 2-hydroxyethyl acrylate, allowing the 2-hydroxyethyl acrylate to attach to the silica surface, resulting in polyhydroxy mesoporous silica. This increases the number of hydroxyl groups on the silica surface and improves its hydrophilic wettability, facilitating subsequent cross-linking reactions. Finally, the polyhydroxy mesoporous silica is cross-linked with a chitosan-acetic acid solution under glutaraldehyde, grafting a large number of chitosan molecules onto the silica surface, increasing its branching degree. This allows for the attachment of more α-amylase, increasing the loading capacity. Furthermore, the covalent bond between chitosan and α-amylase prevents enzyme detachment during use, improving the reusability of the immobilized enzyme.

[0024] (2) The α-amylase immobilization carrier provided by the present invention uses chitosan-modified mesoporous silica as a carrier, which has a large surface area and many adsorption sites, can stably bind with α-amylase, reduce the shedding of α-amylase, and improve the adsorption rate of α-amylase; the preparation method of the carrier is simple and easy, the preparation process is green and environmentally friendly, pollution-free, and low in cost; the carrier can be reused many times and still maintains good enzyme activity, thus improving the utilization rate of α-amylase and its carrier and reducing costs. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The chitosan was purchased from Beijing Solarbio Technology Co., Ltd.

[0027] Example 1

[0028] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0029] (1) Preparation of mesoporous silica: 2g of hexadecyl chloride pyridine was added to 300mL of deionized water and stirred until homogeneous. Then, 40g of tetraethyl orthosilicate and 130g of 50% ethanol solution were added to obtain a mixture. Next, 2g of polyethylene glycol octylphenyl ether, 40g of n-octyltrimethoxysilane and 20g of 1,3,5-trimethylbenzene were added. Under stirring, 80g of 25% ammonia solution was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted at 70℃ for 18h. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere at 700℃ for 6h to obtain mesoporous silica.

[0030] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by the addition of 40g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 50℃ for 1.5h. After the reaction was completed, the silica was filtered and dried to obtain thiolated mesoporous silica.

[0031] (3) Preparation of polyhydroxy mesoporous silica: 100g of mercapto-modified mesoporous silica, 50g of 2-hydroxyethyl acrylate and 3g of azobisisobutyronitrile from step (2) were added to 700mL of ethanol and the mercapto-olefin reaction was carried out under a nitrogen atmosphere at a temperature of 90℃ for 20h. After the reaction was completed, the silica was filtered, washed and dried to obtain polyhydroxy mesoporous silica.

[0032] (4) Preparation of immobilized carrier: 100g of polyhydroxy mesoporous silica, 30g of glutaraldehyde and 100g of chitosan acetic acid solution from step (3) were stirred and reacted at 80℃ for 1h. After the reaction was completed, 40g of sodium hydroxide solution with a concentration of 1mol / L was added and stirred for 20min. Then, the mixture was filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0033] The chitosan acetic acid solution in step (4) is prepared as follows: 8g of chitosan is added to 100g of acetic acid solution with a concentration of 1mol / L, and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution.

[0034] Example 2

[0035] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0036] (1) Preparation of mesoporous silica: 1g of hexadecyl chloride pyridine was added to 300mL of deionized water and stirred until homogeneous. Then, 30g of tetraethyl orthosilicate and 100g of 30% ethanol solution were added to obtain a mixture. Then, 1g of polyethylene glycol octylphenyl ether, 30g of n-octyltrimethoxysilane and 10g of 1,3,5-trimethylbenzene were added. Under stirring, 60g of 20% ammonia solution was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted at 50℃ for 24h. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere at 500℃ for 8h to obtain mesoporous silica.

[0037] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by the addition of 30g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 40℃ for 2h. After the reaction was completed, the mixture was filtered and dried to obtain thiolated mesoporous silica.

[0038] (3) Preparation of polyhydroxy mesoporous silica: 100g of mercapto-modified mesoporous silica, 30g of 2-hydroxyethyl acrylate and 1g of azobisisobutyronitrile from step (2) were added to 700mL of ethanol and the mercapto-olefin reaction was carried out under a nitrogen atmosphere at a temperature of 80℃ for 25h. After the reaction was completed, the silica was filtered, washed and dried to obtain polyhydroxy mesoporous silica.

[0039] (4) Preparation of immobilized carrier: 100g of polyhydroxy mesoporous silica, 20g of glutaraldehyde and 80g of chitosan acetic acid solution from step (3) were stirred and reacted at 60℃ for 2h. After the reaction was completed, 30g of sodium hydroxide solution with a concentration of 0.5mol / L was added and stirred for 30min. Then, the mixture was filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0040] The chitosan acetic acid solution in step (4) is prepared as follows: 5g of chitosan is added to 100g of acetic acid solution with a concentration of 0.5mol / L, and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution.

[0041] Example 3

[0042] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0043] (1) Preparation of mesoporous silica: 3g of hexadecyl chloride pyridine was added to 400mL of deionized water and stirred until homogeneous. Then, 50g of tetraethyl orthosilicate and 150g of 60% ethanol solution were added to obtain a mixture. Next, 3g of polyethylene glycol octylphenyl ether, 50g of n-octyltrimethoxysilane and 25g of 1,3,5-trimethylbenzene were added. Under stirring, 100g of 25% ammonia solution was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted at 80℃ for 12h. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere at 800℃ for 4h to obtain mesoporous silica.

[0044] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by 50g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 60℃ for 1h. After the reaction was completed, the mixture was filtered and dried to obtain thiolated mesoporous silica.

[0045] (3) Preparation of polyhydroxy mesoporous silica: 100g of mercapto-modified mesoporous silica, 60g of 2-hydroxyethyl acrylate and 5g of azobisisobutyronitrile from step (2) were added to 700mL of ethanol and the mercapto-olefin reaction was carried out under a nitrogen atmosphere at a temperature of 100℃ for 15h. After the reaction was completed, the silica was filtered, washed and dried to obtain polyhydroxy mesoporous silica.

[0046] (4) Preparation of immobilized carrier: 100g of polyhydroxy mesoporous silica, 40g of glutaraldehyde and 120g of chitosan acetic acid solution from step (3) were stirred and reacted at a temperature of 90℃ for 0.5h. After the reaction was completed, 50g of sodium hydroxide solution with a concentration of 1mol / L was added and stirred for 10min. Then, the mixture was filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0047] The chitosan acetic acid solution in step (4) is prepared as follows: 10g of chitosan is added to 100g of acetic acid solution with a concentration of 2mol / L, and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution.

[0048] Comparative Example 1

[0049] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0050] (1) Preparation of mesoporous silica: 2g of hexadecyl chloride pyridine was added to 300mL of deionized water and stirred until homogeneous. Then, 40g of tetraethyl orthosilicate was added to obtain a mixture. Next, 80g of ammonia water with a mass concentration of 25% was slowly added dropwise under stirring. After the addition was completed, the mixture was stirred and reacted at a temperature of 70℃ for 18h. After the reaction was completed, the mixture was filtered, washed, dried, and calcined under a nitrogen atmosphere at a temperature of 700℃ for 6h to obtain mesoporous silica.

[0051] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by the addition of 40g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 50℃ for 1.5h. After the reaction was completed, the silica was filtered and dried to obtain thiolated mesoporous silica.

[0052] (3) Preparation of polyhydroxy mesoporous silica: 100g of mercapto-modified mesoporous silica, 50g of 2-hydroxyethyl acrylate and 3g of azobisisobutyronitrile from step (2) were added to 700mL of ethanol and the mercapto-olefin reaction was carried out under a nitrogen atmosphere at a temperature of 90℃ for 20h. After the reaction was completed, the silica was filtered, washed and dried to obtain polyhydroxy mesoporous silica.

[0053] (4) Preparation of immobilized carrier: 100g of polyhydroxy mesoporous silica, 30g of glutaraldehyde and 100g of chitosan acetic acid solution from step (3) were stirred and reacted at 80℃ for 1h. After the reaction was completed, 40g of sodium hydroxide solution with a concentration of 1mol / L was added and stirred for 20min. Then, the mixture was filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0054] The chitosan acetic acid solution in step (4) is prepared as follows: 8g of chitosan is added to 100g of acetic acid solution with a concentration of 1mol / L, and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution.

[0055] Comparative Example 2

[0056] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0057] (1) Preparation of mesoporous silica: 2g of hexadecyl chloride pyridine was added to 300mL of deionized water and stirred until homogeneous. Then, 40g of tetraethyl orthosilicate and 130g of 50% ethanol solution were added to obtain a mixture. Next, 2g of polyethylene glycol octylphenyl ether, 40g of n-octyltrimethoxysilane and 20g of 1,3,5-trimethylbenzene were added. Under stirring, 80g of 25% ammonia solution was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted at 70℃ for 18h. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere at 700℃ for 6h to obtain mesoporous silica.

[0058] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by the addition of 40g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 50℃ for 1.5h. After the reaction was completed, the silica was filtered and dried to obtain thiolated mesoporous silica.

[0059] (3) Preparation of immobilized carrier: 100g of polyhydroxy mesoporous silica, 30g of glutaraldehyde and 100g of chitosan acetic acid solution from step (2) were stirred and reacted at 80℃ for 1h. After the reaction was completed, 40g of sodium hydroxide solution with a concentration of 1mol / L was added and stirred for 20min. Then, the mixture was filtered, washed and dried to obtain the α-amylase immobilized carrier.

[0060] The chitosan acetic acid solution in step (3) is prepared as follows: 8g of chitosan is added to 100g of acetic acid solution with a concentration of 1mol / L, and stirred until the chitosan is completely dissolved to obtain the chitosan acetic acid solution.

[0061] Comparative Example 3

[0062] A method for preparing an α-amylase immobilization carrier includes the following steps:

[0063] (1) Preparation of mesoporous silica: 2g of hexadecyl chloride pyridine was added to 300mL of deionized water and stirred until homogeneous. Then, 40g of tetraethyl orthosilicate and 130g of 50% ethanol solution were added to obtain a mixture. Next, 2g of polyethylene glycol octylphenyl ether, 40g of n-octyltrimethoxysilane and 20g of 1,3,5-trimethylbenzene were added. Under stirring, 80g of 25% ammonia solution was slowly added dropwise. After the addition was completed, the mixture was stirred and reacted at 70℃ for 18h. After the reaction was completed, the mixture was filtered, washed and dried, and then calcined under a nitrogen atmosphere at 700℃ for 6h to obtain mesoporous silica.

[0064] (2) Thiolization of mesoporous silica: The mesoporous silica (50g) obtained in step (1) was added to 500mL of toluene, followed by the addition of 40g of γ-mercaptopropyltrimethoxysilane. The reaction was carried out at 50℃ for 1.5h. After the reaction was completed, the silica was filtered and dried to obtain thiolated mesoporous silica.

[0065] (3) Preparation of polyhydroxy mesoporous silica: 100g of thiolized mesoporous silica, 50g of 2-hydroxyethyl acrylate and 3g of azobisisobutyronitrile from step (2) were added to 700mL of ethanol and the thiol-olefin reaction was carried out under a nitrogen atmosphere at a temperature of 90℃ for 20h. After the reaction was completed, the silica was filtered, washed and dried to obtain polyhydroxy mesoporous silica, which is the α-amylase immobilization carrier.

[0066] The carriers prepared in Examples 1-3 and Comparative Examples 1-3 were used to immobilize α-amylase. The specific methods are as follows: 0.5 g of α-amylase (Shanghai Lizhu Pharmaceutical Co., Ltd., isoelectric point 5.4, molecular weight 96000) was dissolved in 100 mL of 0.01 M PBS aqueous solution with pH 7.2. The solution was magnetically stirred at 1000 rpm for 1 h. After stirring, the solution was centrifuged at 9500 rpm for 10 min to remove a small amount of undissolved impurities. The supernatant was filtered with chemically pure separation filter paper to obtain a clear α-amylase solution. 0.2 g of the α-amylase immobilization carrier was added to 10 mL of the α-amylase solution. The solution was shaken at 250 rpm for 30 min at 25 °C and stored in a refrigerator at 4 °C for 24 h. After storage, the solution was centrifuged at 3000 rpm for 5 min. The enzyme content of the supernatant was determined by UV-Vis spectrophotometer. The amount of α-amylase adsorbed on the carrier was calculated based on the concentration of the α-amylase solution before and after adsorption.

[0067] Enzyme activity assay: One unit of α-amylase activity is defined as the amount of enzyme required to liquefy 1g of soluble starch (Tianjin Beilian Fine Chemicals Development Co., Ltd.) in 1 hour at 60℃ and pH=6.0. 2mg of enzyme powder was weighed and dissolved in 5mL of phosphate buffer (pH=6.0). Using 20g / L soluble starch as the substrate and 0.088g / L iodine solution as the indicator, the α-amylase activity was determined colorimetrically. The initial α-amylase activity was found to be 8.0U / mg. The initial concentration of α-amylase was 0.5mg / mL.

[0068] Reusability test: The activity of immobilized α-amylase was measured at a certain temperature (37℃) and a certain pH (7.0). After each measurement, the immobilized α-amylase was separated by filtration and the carrier was washed with PBS before the next round of reaction was carried out. The residual activity of α-amylase after each cycle was recorded, and the highest data of α-amylase activity was defined as 100% (i.e., the data of 0 cycles).

[0069] Table 1. Performance of α-amylase immobilization carriers in Examples 1-3 and Comparative Examples 1-3

[0070] Adsorption capacity (mg / g) enzyme activity % Reuse rate (15 times) Example 1 132.6 5.3 74.6％ Example 2 127.2 5.0 70.3％ Example 3 131.5 5.1 71.9％ Comparative Example 1 85.4 3.8 53.8％ Comparative Example 2 76.8 4.2 49.6％ Comparative Example 3 73.9 3.9 42.7％

[0071] As can be seen from the data in Table 1 above, the α-amylase immobilization carrier prepared by this invention has a high adsorption capacity, and after immobilizing α-amylase with it, α-amylase can still maintain high activity. Furthermore, it can stably bind with α-amylase, reducing the shedding of α-amylase and increasing the number of times it can be reused, thus showing good application prospects.

[0072] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an α-amylase immobilization carrier, characterized by, The method comprises the following steps: (1) Preparation of mesoporous silica: chlorohexadecylpyridine is added into deionized water and stirred uniformly, then tetraethyl orthosilicate and ethanol solution are added to obtain a mixed solution; polyethylene glycol octylphenyl ether, n-octyltrimethoxysilane and 1, 3, 5-trimethylbenzene are added, ammonia water is slowly added dropwise under stirring, and stirring reaction is performed after the dropwise addition is completed; after the reaction is completed, filtration, washing, drying and calcination under a nitrogen atmosphere are performed to obtain mesoporous silica; (2) Thiolation of mesoporous silica: the mesoporous silica obtained in step (1) is added into toluene, then γ-mercaptopropyltrimethoxysilane is added, and constant temperature reaction is performed to obtain thiolated mesoporous silica; (3) Preparation of polyhydroxyl mesoporous silica: the thiolated mesoporous silica, 2-hydroxyethyl acrylate and azobisisobutyronitrile in step (2) are added into ethanol, and thiol-alkene reaction is performed under a nitrogen atmosphere to obtain polyhydroxyl mesoporous silica; (4) Preparation of immobilized carrier: the polyhydroxyl mesoporous silica, glutaraldehyde and chitosan acetic acid solution in step (3) are subjected to stirring reaction, sodium hydroxide solution is added after the reaction is completed, stirring is performed for 10-30 min, then suction filtration, washing and drying are performed to obtain the immobilized carrier of α-amylase.

2. The method for preparing the α-amylase immobilization carrier according to claim 1, wherein, In step (1), the mass ratio of chlorohexadecylpyridine, tetraethyl orthosilicate, ethanol solution, polyethylene glycol octylphenyl ether, n-octyltrimethoxysilane, 1, 3, 5-trimethylbenzene and ammonia water is 1-3:30-50:100-150:1-3:30-50:10-25:60-100; the mass concentration of the ethanol solution is 30-60%, and the mass concentration of the ammonia water is 20-25%.

3. The method for preparing the α-amylase immobilization carrier according to claim 1, wherein the α-amylase is immobilized on the carrier by the method of cross-linking. In step (1), the temperature of the stirring reaction is 50-80°C, and the time is 12-24 h; the temperature of the calcination is 500-800°C, and the calcination time is 4-8 h.

4. The method for preparing the α-amylase immobilization carrier according to claim 1, wherein, In step (2), the mass ratio of the mesoporous silica and γ-mercaptopropyltrimethoxysilane is 50:30-50; the temperature of the constant temperature reaction is 40-60°C, and the time is 1-2 h.

5. The method for preparing the α-amylase immobilization carrier according to claim 1, characterized in that, In step (3), the mass ratio of the thiolated mesoporous silica, 2-hydroxyethyl acrylate and azobisisobutyronitrile is 100:30-60:1-5.

6. The method for preparing the α-amylase immobilization carrier according to claim 1, characterized in that, In step (3), the temperature of the thiol-alkene reaction is 80-100°C, and the time is 15-25 h.

7. The method for preparing the α-amylase immobilization carrier according to claim 1, characterized in that, In step (4), the mass ratio of the polyhydroxyl mesoporous silica, glutaraldehyde, chitosan acetic acid solution and sodium hydroxide solution is 100:20-40:80-120:30-50; the concentration of the sodium hydroxide solution is 0.5-1 mol / L; the temperature of the stirring reaction is 60-90°C, and the time is 0.5-2 h.

8. The method for preparing the α-amylase immobilization carrier according to claim 1, characterized in that, In step (4), the chitosan acetic acid solution is prepared as follows: chitosan is added into an acetic acid solution with a concentration of 0.5-2 mol / L, and stirring is performed until the chitosan is completely dissolved to obtain the chitosan acetic acid solution; the mass ratio of the chitosan and the acetic acid solution is 5-10:

100.

9. An α-amylase immobilization carrier obtainable by the method according to any one of claims 1 to 8.

10. Use of an α-amylase immobilization carrier according to claim 9 for immobilizing α-amylase.