Bifunctional cellulose-based bioreactor as well as preparation method and application thereof

Abstract

The invention provides a bifunctional cellulose-based bioreactor as well as a preparation method and application thereof.According to the preparation method, cellulose is taken as a raw material, cellulose-based molecular imprinting hydrogel is prepared through a surface imprinting technology, and the imprinting material is green and degradable and has rich targeted recognition sites; meanwhile, the hydrotalcite-like compound is used as an immobilized enzyme carrier, and the hydrotalcite-like compound is of a nanosheet layer structure, has a large specific surface area and fixes more catalytic enzymes, so that the hydrotalcite-like compound immobilized enzyme system is prepared; cellulose-based molecularly imprinted hydrogel and a hydrotalcite-like immobilized enzyme system are subjected to a cross-linking reaction, a bifunctional cellulose-based bioreactor is obtained, and the bioreactor has the functions of targeted recognition and bioconversion and can adsorb a reaction substrate in a targeted mode, so that contact between the reaction substrate and the immobilized enzyme system is promoted, and the bioconversion efficiency is improved. Therefore, the catalytic efficiency is improved, the bifunctional cellulose-based bioreactor has the functions of targeted recognition and bioconversion at the same time, and a brand new research thought and technical means can be provided for structural modification of functional components of forest resources, especially flavonoid oxygen glycoside compounds.

Description

Technical Field

[0001] The present application relates to the technical field of bioreactors, and in particular to a dual-functional cellulose-based bioreactor, a preparation method and applications thereof. Background Art

[0002] A bioreactor is a system that uses biocatalysis (enzymes or organisms) to carry out biochemical reactions in vitro. It produces substances needed by humans by modifying organic substrates with the help of various biological systems. These systems include enzymes, microorganisms, animal cells, plant cells, etc. Bioreactors provide controllable conditions for the transformation of substances in biological systems. The catalytic and modification characteristics of bioreactors can increase the synthesis rate of natural products and greatly shorten the synthesis cycle. It has important theoretical and application value in the structural modification of high-value-added forest-derived natural products. Among them, immobilized enzymes that maintain enzyme activity have a significant impact on the conversion efficiency, cost and operability of bioreactors.

[0003] Substrate materials used to prepare bioreactors usually include organic materials such as alginate, carrageenan, chitosan and polyacrylamide, and inorganic materials such as silica, silicate, alumina, titania, other oxides, and metal-organic frameworks. However, different substrate materials have different effects on the activity of enzymes in bioreactors. Therefore, exploring new substrate materials with high reusability and good biocompatibility is crucial for the construction of bioreactors.

[0004] Cellulose has abundant active hydroxyl groups and can easily react with various functional groups to achieve structural modification. It is economical, environmentally friendly, renewable, and biocompatible, and is widely used in papermaking, textiles, food manufacturing, biomedicine, and other fields. Based on this, domestic and foreign scholars use functionalized cellulose as an enzyme immobilization matrix. However, ordinary cellulose immobilized enzyme materials have poor specific substrate recognition capabilities and do not have selective adsorption capabilities. In plant extracts where multiple functional components of forest resources coexist, when the immobilized enzyme comes into contact with these components, the limited conversion sites on the immobilized matrix will be competed for, thereby reducing its conversion efficiency for specific substrates. Summary of the invention

[0005] The present application provides a dual-functional cellulose-based bioreactor, a preparation method and application thereof, to solve the following technical problem: how to simultaneously improve the targeting recognition capability and conversion rate of the bioreactor.

[0006] In a first aspect, the present invention provides a method for preparing a bifunctional cellulose-based bioreactor, comprising the following steps:

[0007] preparing cellulose microspheres;

[0008] Using the cellulose microspheres as carriers to prepare cellulose-based molecularly imprinted hydrogels;

[0009] Using hydrotalcite-like substances as immobilized enzyme carriers to prepare hydrotalcite-like immobilized enzyme systems;

[0010] The cellulose-based molecular imprinting hydrogel and the hydrotalcite-like immobilized enzyme system are cross-linked to obtain a dual-functional cellulose-based bioreactor.

[0011] Optionally, the cellulose microspheres are prepared by reverse suspension polymerization, which specifically includes the following steps: fully mixing an oily medium and a surfactant and stirring and emulsifying to obtain an emulsion; mixing a cellulose solution, a sodium alginate solution and 3-(methacryloyloxy)propyltrimethoxysilane, mixing them with the emulsion by reverse suspension polymerization, adjusting the pH to neutral with concentrated hydrochloric acid and cross-linking with glutaraldehyde to obtain cellulose gel microspheres.

[0012] Optionally, the mass ratio of the oily medium to the surfactant is 120g-130g:1g-3g; the mass ratio of the cellulose solution, the sodium alginate solution and the 3-(methacryloyloxy)propyltrimethoxysilane is 80g-84g:10g-15g:1-2g.

[0013] Optionally, the cellulose microspheres are used as carriers to prepare cellulose-based molecularly imprinted hydrogels, specifically comprising:

[0014] The template molecule and the functional monomer are subjected to a prepolymerization reaction, and then the cellulose microspheres, N,N'-methylenebisacrylamide, 3-sulfopropyl methacrylate and ammonium persulfate are added to carry out a polymerization reaction to obtain a polymer hydrogel;

[0015] removing the template molecules in the polymer hydrogel to obtain a cellulose-based molecularly imprinted hydrogel;

[0016] Wherein, the molar ratio of the template molecule to the functional monomer is 1:3 to 1:9;

[0017] The molar ratio of the template molecule to N,N'-methylenebisacrylamide is 1:18 to 1:54;

[0018] The molar ratio of the template molecule to ammonium persulfate is 1:1 to 1:3;

[0019] The molar ratio of the template molecule to 3-sulfopropyl methacrylate is 1:5 to 1:10;

[0020] The mass ratio of template molecules to cellulose gel microspheres is 1:2 to 1:15.

[0021] Optionally, the template molecule and the functional monomer are subjected to a prepolymerization reaction, specifically comprising: conducting a prepolymerization reaction of the template molecule and the functional monomer in a methanol solution, wherein the prepolymerization reaction temperature is room temperature and the reaction time is 4 hours to 8 hours.

[0022] Optionally, the functional monomer includes one of 4-vinylpyridine, 2-vinylpyridine, acrylamide and α-methylacrylic acid.

[0023] Optionally, the polymerization reaction is carried out under the protection of an inert gas, the reaction temperature is 60° C. to 80° C., and the reaction time is 2 h to 4 h.

[0024] Optionally, the removing the template molecules in the polymer hydrogel specifically includes: ultrasonically eluting the template molecules in the polymer hydrogel using a methanol / acetic acid solution, and detecting the template molecules in the eluent until the template molecules are completely removed.

[0025] Optionally, the hydrotalcite-like material is magnesium / aluminum hydrotalcite-like material.

[0026] Optionally, the preparation method of the magnesium / aluminum hydrotalcite includes the following steps: heating MgCl2·6H2O and AlCl3·6H2O in a molar ratio of 1:1 to 3:1 in an alkaline solution at 60 to 80°C for 8 to 12 hours, filtering with a filter membrane, redispersing the mixture in a 0.5M Na2CO3 solution, stirring for 1 to 3 hours, filtering, washing, and drying to obtain the magnesium / aluminum hydrotalcite.

[0027] Optionally, a hydrotalcite-like immobilized enzyme system is prepared by using a hydrotalcite-like as an immobilized enzyme carrier, specifically comprising: activating the hydrotalcite-like with glutaraldehyde, washing with deionized water, and then using the activated hydrotalcite-like to immobilize the catalytic enzyme. During the immobilization process, the catalytic enzyme dosage is 5 to 50 U / mL, the immobilization time is 2 to 10 hours, and the immobilization temperature is 15 to 50°C.

[0028] Optionally, the mass ratio of the cellulose-based molecular imprinting hydrogel to the hydrotalcite-like immobilized enzyme system is 1:2 to 1:3.

[0029] Optionally, the cross-linking reaction time of the cellulose-based molecular imprinting hydrogel and the hydrotalcite-like immobilized enzyme system is 6 hours to 36 hours.

[0030] In a second aspect, the present invention further provides a bifunctional cellulose-based bioreactor, which is prepared by the preparation method of the bifunctional cellulose-based bioreactor described in the first aspect.

[0031] In a third aspect, the present invention further provides a process in which the bifunctional cellulose-based bioreactor described in the second aspect is used for targeted recognition and biotransformation of flavonoid glycosides.

[0032] Optionally, the bifunctional cellulose-based bioreactor is used to target and recognize phlorizin and convert the phlorizin into phlorretin.

[0033] Optionally, the bifunctional cellulose-based bioreactor is used to target and identify phlorizin and convert the phlorizin into phlorretin, specifically comprising: extracting the leaves of Ligustrum lucidum with hot water, and using the cellulose bioreactor to convert the phlorizin in the water extract into phlorretin.

[0034] Optionally, the solid-liquid ratio of the leaves of Ligusticum chuanxiong during the extraction process is 2g / L~20g / L, the ratio of the cellulose bioreactor to the water extract of Ligusticum chuanxiong during the conversion process is 20mg:0.2~0.5mL, the conversion temperature is 30℃~50℃, and the conversion time is 30min~120min.

[0035] The above technical solution provided by the present invention has the following advantages compared with the prior art:

[0036] The invention provides a dual-functional cellulose-based bioreactor, a preparation method and an application thereof. The preparation method uses cellulose as a raw material and prepares a cellulose-based molecular imprinted hydrogel by surface imprinting technology. The imprinted material is green and degradable and has abundant target recognition sites. At the same time, a hydrotalcite-like substance is used as an immobilized enzyme carrier. The hydrotalcite-like substance is a nanosheet structure and has a large specific surface area, and can immobilize more catalytic enzymes on its surface, thereby preparing a hydrotalcite-like immobilized enzyme system. The cellulose-based molecular imprinted hydrogel and the hydrotalcite-like immobilized enzyme system are cross-linked to obtain a dual-functional cellulose-based bioreactor. The bioreactor has the functions of targeted recognition and biotransformation at the same time, can target and adsorb reaction substrates, and then promote the contact between the reaction substrates and the immobilized enzyme system, thereby improving the catalytic efficiency and providing a new research idea and technical means for the structural modification of functional components of forest resources, especially flavonoid oxygen glycoside compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0038] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0039] Figure 1 This is a schematic diagram of the preparation process of cellulose-based molecularly imprinted hydrogel in the examples of this application;

[0040] Figure 2 The scanning electron micrographs of the cellulose-based microspheres, cellulose-based molecularly imprinted hydrogels and cellulose-based non-molecularly imprinted hydrogels provided in Example 1 of the present application;

[0041] Figure 3 This is a schematic diagram of the preparation process of magnesium / aluminum hydrotalcite in the embodiment of the present application;

[0042] Figure 4 Atomic force microscope images of hydrotalcite-like, β-glucosidase and hydrotalcite-like immobilized β-glucosidase system in the examples of the present application;

[0043] Figure 5 A diagram showing the mechanism of constructing a dual-functional cellulose-based bioreactor in an embodiment of the present application;

[0044] Figure 6 This is a scanning electron microscope image of the hydrotalcite-like immobilized enzyme system and the bifunctional cellulose-based bioreactor in the examples of the present application;

[0045] Figure 7 This is a diagram of the mechanism of targeted recognition and conversion of phlorizin by a dual-functional cellulose-based bioreactor in an example of this application. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0047] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0048] In this application, in the absence of any contrary description, the terms including "including" and the like mean "including but not limited to". In this article, relational terms such as "first" and "second" and the like are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist, for example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plurality" refers to two or more. "At least one", "the following at least one (individual)" or similar expressions thereof refer to any combination of these items, including any combination of singular (individual) or plural (individual). For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or plural, respectively.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0050] In a first aspect, the present invention provides a method for preparing a bifunctional cellulose-based bioreactor, comprising the following steps:

[0051] preparing cellulose microspheres;

[0052] Using the cellulose microspheres as carriers to prepare cellulose-based molecularly imprinted hydrogels;

[0053] Using hydrotalcite-like substances as immobilized enzyme carriers to prepare hydrotalcite-like immobilized enzyme systems;

[0054] The cellulose-based molecular imprinting hydrogel and the hydrotalcite-like immobilized enzyme system are cross-linked to obtain a dual-functional cellulose-based bioreactor.

[0055] In the above embodiment, cellulose is used as raw material to prepare cellulose-based molecular imprinted hydrogel by surface imprinting technology. The imprinted material is green and degradable and has abundant target recognition sites. At the same time, hydrotalcite-like is used as an immobilized enzyme carrier. The hydrotalcite-like is a nanosheet structure with a large specific surface area and can immobilize more catalytic enzymes on its surface, thereby preparing a hydrotalcite-like immobilized enzyme system. The cellulose-based molecular imprinted hydrogel and the hydrotalcite-like immobilized enzyme system are cross-linked to obtain a bifunctional cellulose-based bioreactor.

[0056] As an optional embodiment, the cellulose microspheres are prepared by reverse suspension polymerization, which specifically includes the following steps: fully mixing the oily medium and the surfactant and stirring and emulsifying to obtain an emulsion; mixing the cellulose solution, the sodium alginate solution and 3-(methacryloyloxy)propyltrimethoxysilane, mixing them with the emulsion by reverse suspension polymerization, adjusting the pH to neutral with concentrated hydrochloric acid and cross-linking with glutaraldehyde to obtain cellulose microspheres.

[0057] In the above embodiment, the prepared cellulose microspheres have a porous structure, which can greatly increase the specific surface area. At the same time, the mechanical strength of the cellulose material is relatively high, and can provide good support.

[0058] As an optional embodiment, the mass ratio of the oily medium and the surfactant is 120g~130g:1g~3g; the mass ratio of the cellulose solution, the sodium alginate solution and the 3-(methacryloyloxy)propyltrimethoxysilane is 80g~84g:10g~15g:1g~2g.

[0059] As an optional embodiment, the cellulose microspheres are used as carriers to prepare cellulose-based molecularly imprinted hydrogels, specifically comprising:

[0060] The template molecule and the functional monomer are subjected to a prepolymerization reaction, and then the cellulose microspheres, a crosslinking agent (N,N'-methylenebisacrylamide), a hydrophilic monomer (3-sulfopropyl methacrylate) and an initiator (ammonium persulfate) are added to carry out a polymerization reaction to obtain a polymer hydrogel;

[0061] removing the template molecules in the polymer hydrogel to obtain a cellulose-based molecularly imprinted hydrogel;

[0062] In the above embodiment, the target molecule is selected as the template molecule, and a stable complex is formed with the functional monomer through a prepolymerization reaction, and then cellulose microspheres, a cross-linking agent, a hydrophilic monomer and an initiator are added thereto to carry out a polymerization reaction, wherein the cellulose microspheres are used as a carrier to provide a larger specific surface area and mechanical strength, N,N'-methylenebisacrylamide is used as a cross-linking agent to enhance the stability of the polymer network, the hydrophilic monomer 3-sulfopropyl methacrylate is added to further increase the hydrophilicity of the polymer, and ammonium persulfate is used as an initiator to initiate the entire polymerization reaction, and finally the polymer hydrogel is obtained by centrifugation, filtration and other means.

[0063] Wherein, the molar ratio of the template molecule to the functional monomer is 1:3 to 1:9;

[0064] The molar ratio of the template molecule to N,N'-methylenebisacrylamide is 1:18 to 1:54;

[0065] The molar ratio of the template molecule to ammonium persulfate is 1:1 to 1:3;

[0066] The molar ratio of the template molecule to 3-sulfopropyl methacrylate is 1:5 to 1:10;

[0067] The mass ratio of template molecules to cellulose gel microspheres is 1:2 to 1:15.

[0068] In the above embodiment, the ratio of the amount of each reactant is controlled, and the reason is to ensure that the template molecule can effectively interact with the functional monomer and other components to form a polymer hydrogel with specific recognition sites. Among them, the ratio between the template molecule and the functional monomer determines the stability of the complex and the specificity of the polymer. A higher ratio of functional monomers can provide more binding sites, which helps to form a more stable complex, so the number of functional monomers needs to be greater than the template molecule, but a high ratio of functional monomers may cause too many functional monomers to be unused, so the molar ratio of the template molecule to the functional monomer is controlled to be 1:3 to 1:9. Exemplarily, the molar ratio of the template molecule to the functional monomer can be 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9.

[0069] The reason for controlling the proportion of the cross-linking agent is that the amount of the cross-linking agent determines the pore size and structural stability of the polymer network. If the amount of the cross-linking agent is too much, the polymer network will be too tight and the pores will be too small, which is not conducive to the recognition of the target molecule; if the amount is too low, the polymer structure will be unstable. Therefore, the molar ratio of the template molecule to the cross-linking agent is controlled within the range of 1:18 to 1:54. Under the premise of ensuring the stability of the polymer, the network is more open and the pores are moderate, which is conducive to the recognition of the target molecule. Exemplarily, the molar ratio of the template molecule to the cross-linking agent can be 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 or 1:54.

[0070] The purpose of controlling the amount of hydrophilic monomer is to make the final polymer have a certain hydrophilicity and ensure the stability of the polymer hydrogel in aqueous solution. If the hydrophilic monomer is less than 1 / 10 of the molar mass of the template molecule, the hydrophilicity of the final product cannot be ensured. If the amount of hydrophilic monomer exceeds 1 / 5 of the molar mass of the template molecule, the polymer will be overly hydrophilic, affecting the formation of recognition sites in the functional monomer. Therefore, the molar ratio of the template molecule to the hydrophilic monomer is controlled to be 1:5 to 1:10. For example, the molar ratio of the template molecule to the hydrophilic monomer can be set to 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0071] Cellulose gel microspheres as carrier materials can provide a larger surface area and better mechanical strength. If the amount of cellulose gel microspheres used is small, less than 1 / 15 of the mass of the template molecule, it cannot provide a sufficiently large surface area and mechanical strength. However, if the mass of the cellulose gel microspheres is greater than 1 / 2 of the mass of the template molecule, it may affect the stability of the polymer network. Therefore, the mass ratio of the template molecule to the cellulose gel microspheres is controlled to be 1:2 to 1:15. Exemplarily, the mass ratio of the template molecule to the cellulose gel microspheres can be 1:2, 1:3, 1:4, 1:5, 16, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15.

[0072] As an optional implementation, the template molecule and the functional monomer are subjected to a prepolymerization reaction, specifically comprising: conducting a prepolymerization reaction of the template molecule and the functional monomer in a methanol solution, wherein the prepolymerization reaction temperature is 20°C to 30°C, i.e. room temperature; and the reaction time is 4h to 8h.

[0073] As an optional embodiment, the functional monomer includes one of 4-vinylpyridine, 2-vinylpyridine, acrylamide and α-methacrylic acid.

[0074] As an optional embodiment, the template molecule is phlorizin.

[0075] As an optional implementation manner, the polymerization reaction is carried out under the protection of an inert gas, the reaction temperature is 60° C. to 80° C., and the reaction time is 2 h to 4 h.

[0076] As an optional implementation, the removing the template molecules in the polymer hydrogel specifically includes: ultrasonically eluting the template molecules in the polymer hydrogel using a methanol / acetic acid solution, and detecting the template molecules in the eluent until the template molecules are completely removed.

[0077] As an optional implementation, the hydrotalcite-like substance is a magnesium / aluminum hydrotalcite-like substance.

[0078] Hydrotalcite has anion exchange properties, and high-valent anions can replace low-valent anions between layers. Since 3-sulfopropyl methacrylate is added when preparing cellulose-based molecular imprinting hydrogel, the sulfonic acid group in 3-sulfopropyl methacrylate is inserted into the interlayer region of hydrotalcite by anion exchange, so that the hydrotalcite-immobilized enzyme is successfully embedded in the cellulose-based molecular imprinting hydrogel, so that the two successfully undergo cross-linking reaction. As an optional embodiment, the preparation method of the magnesium / aluminum hydrotalcite includes the following steps: MgCl2·6H2O and AlCl3·6H2O in a molar ratio of 1:1 to 3:1 are heated in an alkaline solution at 60 to 80°C for 8 to 12 hours, filtered with a filter membrane, and then redispersed in a 0.5M Na2CO3 solution and stirred for 1 to 3 hours, filtered, washed, and dried to obtain magnesium / aluminum hydrotalcite.

[0079] As an optional embodiment, hydrotalcite is used as an immobilized enzyme carrier to prepare a hydrotalcite immobilized enzyme system, specifically comprising: activating the hydrotalcite with glutaraldehyde, washing with deionized water, and then using the activated hydrotalcite to immobilize the catalytic enzyme. During the immobilization process, the catalytic enzyme dosage is 5U / mL to 50U / mL, the immobilization time is 2h to 10h, and the immobilization temperature is 15°C to 50°C.

[0080] As an optional embodiment, the type of the catalytic enzyme is selected according to the actual type of catalytic reaction, such as β-glucosidase.

[0081] As an optional embodiment, the mass ratio of the cellulose-based molecularly imprinted hydrogel to the hydrotalcite-like immobilized enzyme system is 1:2 to 1:3.

[0082] In the above embodiment, the mass ratio of the cellulose-based molecular imprinted hydrogel and the hydrotalcite-like immobilized enzyme system is controlled, and its purpose is to provide a certain target molecule capture ability and corresponding catalytic effect while ensuring that the reactor has a certain mechanical strength and stability. If the amount of cellulose-based molecular imprinted hydrogel is large, it may lead to stronger target molecule recognition ability and capture ability, and can absorb more target molecules, but due to the small proportion of the hydrotalcite-like immobilized enzyme system, the amount of catalytic enzyme is small, the catalytic ability is weak, and the reaction efficiency is reduced; on the contrary, if the amount of cellulose-based molecular imprinted hydrogel is small, the target molecule recognition ability and capture ability may be weaker, and insufficient target molecules can be absorbed, but due to the high proportion of the hydrotalcite-like immobilized enzyme system, the amount of catalytic enzyme is surplus, and the yield is also reduced. Therefore, the mass ratio of the cellulose-based molecular imprinted hydrogel and the hydrotalcite-like immobilized enzyme system is controlled to be 1:2 to 1:3.

[0083] In a second aspect, the present invention further provides a bifunctional cellulose-based bioreactor, which is prepared by the preparation method of the bifunctional cellulose-based bioreactor described in the first aspect.

[0084] In a third aspect, the present invention further provides a process in which the bifunctional cellulose-based bioreactor described in the second aspect is used for targeted recognition and biotransformation of flavonoid glycosides.

[0085] As an optional embodiment, the bifunctional cellulose-based bioreactor is used to target and recognize phlorizin and convert the phlorizin into phlorretin.

[0086] As an optional embodiment, the bifunctional cellulose-based bioreactor is used to target and identify phlorizin and convert the phlorizin into phlorretin, specifically comprising: extracting the leaves of Ligustrum lucidum with hot water, and using the cellulose bioreactor to convert the phlorizin in the water extract into phlorretin.

[0087] As an optional implementation method, the solid-liquid ratio of the leaves of the Ligusticum chuanxiong during the extraction process is 2g / L~20g / L, the ratio of the cellulose bioreactor to the Ligusticum chuanxiong water extract during the conversion process is 20mg:0.2~0.5mL, the conversion temperature is 30℃~50℃, and the conversion time is 30min~120min.

[0088] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the conditions recommended by the manufacturer are followed.

[0089] Example 1

[0090] This embodiment provides a dual-function cellulose-based bioreactor, comprising the following steps:

[0091] S1, preparation of cellulose microspheres;

[0092] S2, using the cellulose microspheres as carriers to prepare cellulose-based molecularly imprinted hydrogels;

[0093] S3, using the hydrotalcite-like substance as an enzyme carrier to prepare a hydrotalcite-like enzyme immobilization system;

[0094] S4, cross-linking the cellulose-based molecular imprinting hydrogel and the hydrotalcite-like immobilized enzyme system to obtain a bifunctional cellulose-based bioreactor.

[0095] Among them, step S1 specifically includes: fully mixing the oily medium liquid paraffin and the surfactant Tween 80 in a mass ratio of 125g:2.5g, and stirring and emulsifying at 60°C for 30 minutes to obtain an emulsion; adding a cellulose solution with a mass concentration of 2%, a sodium alginate solution with a mass concentration of 2% and 3-(methacryloyloxy)propyltrimethoxysilane in a ratio of 84g:15g:1g to the emulsion, and continuing to stir the emulsion for 3 hours using a reverse suspension polymerization method, adjusting the pH to neutral with concentrated hydrochloric acid, adding 10mL of a 20% glutaraldehyde solution for cross-linking for 30 minutes, and then passing the obtained solid through a 60-mesh sieve and soaking in hot water for 2 hours to obtain cellulose gel microspheres.

[0096] Step S2 specifically includes: prepolymerizing the template molecule phlorizin and the functional monomer 4-vinylpyridine in a molar ratio of 1:7 in a methanol solution for 8 hours, adding 129.5 mg of cellulose hydrogel microspheres, 1 mmol 3-sulfopropyl methacrylate, 647.5 mg of N,N'-methylenebisacrylamide and 45.3 mg of ammonium persulfate, fully mixing, and polymerizing at 80°C for 4 hours under nitrogen protection to obtain a polymer hydrogel; ultrasonically eluting the phlorizin in the polymer hydrogel using a methanol / acetic acid solution (methanol: acetic acid mass ratio of 9:1), detecting the phlorizin content using high performance liquid chromatography, and if the phlorizin is not completely removed, repeating the phlorizin removal operation until the phlorizin is completely removed to obtain a cellulose-based molecularly imprinted hydrogel.

[0097] Step S3 specifically includes:

[0098] The principle of preparing magnesium / aluminum hydrotalcite is as follows Figure 3 As shown, 100 mL of an aqueous solution of MgCl2·6H2O (150 mM) and AlCl3·6H2O (50 mM) was mixed with 100 mL of a Tris-HCL buffer solution (pH = 10) and heated at 80°C for 12 h to obtain a magnesium / aluminum hydrotalcite. The magnesium / aluminum hydrotalcite was filtered using a 0.22 μm filter membrane and then redispersed in a 0.5 M Na2CO3 solution, stirred at room temperature for 1 h, filtered, washed three times with distilled water, and dried in an oven at 80°C to obtain a magnesium / aluminum hydrotalcite.

[0099] 10 mg of magnesium / aluminum hydrotalcite was activated with 5% glutaraldehyde for 30 min and then washed three times with deionized water. The activated hydrotalcite was incubated with 0.5 mL of β-glucosidase (20 U / mL) at 25° C. for 4 h under shaking to obtain a hydrotalcite-immobilized β-glucosidase system. Figure 4 The atomic force microscope images of hydrotalcite-like, β-glucosidase and hydrotalcite-like immobilized β-glucosidase system. Figure 4It can be seen that the prepared hydrotalcite-like material has a sheet-like structure with a thickness of about 1.5 nm, and β-glucosidase is immobilized on the surface of the hydrotalcite-like material.

[0100] Step S4 specifically includes:

[0101] 10 mg of cellulose-based imprinted hydrogel and 10 mg of hydrotalcite-like immobilized enzyme system were cross-linked at 4°C for 6 h to obtain a bifunctional cellulose-based bioreactor. The construction mechanism of the bifunctional cellulose-based bioreactor is as follows: Figure 5 As shown. Figure 5 It can be seen that 3-sulfopropyl methacrylate was added in the preparation of cellulose-based molecularly imprinted hydrogel. Since hydrotalcite-like particles have anion exchange properties, that is, high-valent anions can replace low-valent anions between layers, the sulfonic acid groups in 3-sulfopropyl methacrylate were inserted into the interlayer region of hydrotalcite-like particles through anion exchange, thereby successfully embedding the hydrotalcite-like immobilized enzyme into the cellulose-based molecularly imprinted hydrogel.

[0102] Figure 6 (Left) is the scanning electron microscopy image of the hydrotalcite-like immobilized β-glucosidase system. Figure 6 (Right) is a scanning electron microscope image of a bifunctional cellulose-based bioreactor. From the comparison of the two images, it can be seen that the hydrotalcite-like immobilized β-glucosidase system has been successfully embedded in the cellulose-based molecularly imprinted hydrogel.

[0103] Example 2

[0104] This example uses the bifunctional cellulose-based bioreactor prepared in Example 1 to carry out targeted recognition and conversion of phlorizin, and its mechanism diagram is as follows: Figure 7 As shown, the following steps are included:

[0105] First, 20 mg of the bifunctional cellulose-based bioreactor was dispersed in 180 μL of a buffer solution, wherein the buffer solution was an acetic acid-sodium acetate buffer solution; then, 20 μL of a 1 mg / mL phlorizin solution was added to the buffer solution, and the solution was converted in a water bath at 50°C for 2 hours. Finally, the phlorizin content in the solution after the reaction was determined by RP-HPLC.

[0106] Through detection and calculation, it was found that the conversion rate of phlorizin in the dual-functional cellulose-based bioreactor in this embodiment was 80.7%.

[0107] Example 3

[0108] This example uses the bifunctional cellulose-based bioreactor prepared in Example 1 to identify and transform phlorizin in the water extract of Ligusticum chuanxiong.

[0109] Preparation of the extract of leaves of Zingiber officinale: Add 1 g of Zingiber officinale leaves into 50 mL of distilled water, perform ultrasonic extraction at 50°C for 30 min, and obtain the Zingiber officinale water extract after filtration. After testing, the phloretin content in the Zingiber officinale water extract is 0.48 μg / g.

[0110] Targeted recognition and transformation: The bifunctional cellulose-based bioreactor (20 mg) obtained in Example 1 was placed in 0.2 mL of the water extract of Ligusticum chuanxiong, and transformed at 50°C for 2 h. The phloretin content in the Ligusticum chuanxiong extract after transformation was tested to be 12.55 μg / g. It can be seen that the phloretin content in the Ligusticum chuanxiong extract was increased by 26 times through the targeted recognition and transformation process of the bifunctional cellulose-based bioreactor.

[0111] Recycling of the bifunctional cellulose-based bioreactor: The water extract of the reacted Ligusticum chuanxiong leaves is filtered to obtain a bifunctional cellulose-based bioreactor, which is then washed and reused. After testing, the conversion efficiency of the phloridzin in the bioreactor remains above 70% after 5 consecutive cycles.

[0112] Comparative Example 1

[0113] This comparative example uses a hydrotalcite-like immobilized β-glucosidase system to convert phlorizin, comprising the following steps:

[0114] First, 20 mg of the hydrotalcite-immobilized β-glucosidase system was dispersed in 180 μL of a buffer solution, which was an acetic acid-sodium acetate buffer solution; then, 20 μL of a 1 mg / mL phlorizin solution was added to the buffer solution, and the mixture was converted in a water bath at 50°C for 2 hours. Finally, the phlorizin content in the solution after the reaction was determined by RP-HPLC.

[0115] The test showed that the conversion rate of phlorizin by the hydrotalcite-like immobilized β-glucosidase system was 60.5%.

[0116] Comparative Example 2

[0117] In this comparative example, a hydrotalcite-like β-glucosidase system was used to identify and transform phlorizin in the water extract of Zingiber officinale leaves and osmanthus seeds.

[0118] Preparation of the extract of leaves of Zingiber officinale: Add 1 g of Zingiber officinale leaves into 50 mL of distilled water, perform ultrasonic extraction at 50°C for 30 min, and obtain the Zingiber officinale water extract after filtration. After testing, the phloretin content in the Zingiber officinale water extract is 0.48 μg / g.

[0119] Targeted identification and conversion: The hydrotalcite-like β-glucosidase system (20 mg) was placed in 0.2 mL of the water extract of the leaves of the ginger and converted at 50°C for 2 hours. The phloretin content in the extract of the leaves of the ginger after conversion was tested to be 9.91 μg / g.

[0120] Comparative Example 3

[0121] This comparative example uses the hydrotalcite-like β-glucosidase system and the bifunctional cellulose-based bioreactor provided in Example 1 to transform polydatin, including the following steps:

[0122] First, the hydrotalcite-like β-glucosidase system and the bifunctional cellulose-based bioreactor were dispersed in 180 μL of buffer solution, and then 20 μL of 1 mg / mL polydatin solution was added and converted in a 50°C water bath for 2 hours. Then, the resveratrol content obtained was determined by RP-HPLC.

[0123] After testing, it was found that the conversion rate of polydatin by the hydrotalcite-like β-glucosidase system was 46.3%, while the relative conversion rate of genistin by the bifunctional cellulose-based bioreactor was only 34.5%. The reason is that the bifunctional cellulose-based bioreactor provided in Example 1 has no targeted recognition effect on polydatin, and its imprinting layer hinders the contact between polydatin and β-glucosidase, so the bifunctional cellulose-based bioreactor does not improve the conversion efficiency of polydatin.

[0124] Comparative Example 4

[0125] In this comparative example, the hydrotalcite-like β-glucosidase system and the bifunctional cellulose-based bioreactor are dispersed in 180 μL buffer solution, and then 20 μL of 1 mg / mL genistin solution is added, and the genistin content obtained is determined by RP-HPLC after conversion in a 50°C water bath for 2 hours. The conversion rate of the hydrotalcite-like β-glucosidase system to genistin is 41.8%, while the relative conversion rate of the bifunctional cellulose-based bioreactor to genistin is 34.3%. The reason is that the bifunctional cellulose-based bioreactor has no targeted recognition effect on genistin, and its imprinting layer hinders the contact of genistin with β-glucosidase, and does not improve the conversion efficiency.

[0126] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for preparing a dual-functional cellulose-based bioreactor, characterized in that: The following steps are involved: preparing cellulose microspheres; Using the cellulose microspheres as carriers to prepare cellulose-based molecularly imprinted hydrogels; Using hydrotalcite-like substances as immobilized enzyme carriers to prepare hydrotalcite-like immobilized enzyme systems; The cellulose-based molecular imprinting hydrogel and the hydrotalcite-like immobilized enzyme system are cross-linked to obtain a dual-functional cellulose-based bioreactor.

2. The preparation method according to claim 1, characterized in that: The method of using the cellulose microspheres as a carrier to prepare a cellulose-based molecularly imprinted hydrogel specifically comprises: The template molecule and the functional monomer are subjected to a prepolymerization reaction, and then the cellulose microspheres, the crosslinking agent, the hydrophilic monomer and the initiator are added to carry out a polymerization reaction to obtain a polymer hydrogel; removing the template molecules in the polymer hydrogel to obtain a cellulose-based molecularly imprinted hydrogel; Wherein, the molar ratio of the template molecule to the functional monomer is 1:3 to 1:9; The molar ratio of template molecule to cross-linking agent is 1:18 to 1:54; The molar ratio of template molecule to initiator is 1:1 to 1:3; The molar ratio of template molecule to hydrophilic monomer is 1:5 to 1:10; The mass ratio of template molecules to cellulose gel microspheres is 1:2 to 1:

15.

3. The preparation method according to claim 2, characterized in that: The template molecule and the functional monomer are subjected to a prepolymerization reaction, specifically comprising: the template molecule and the functional monomer are subjected to a prepolymerization reaction in a solvent, wherein the prepolymerization reaction time is 4 hours to 8 hours.

4. The preparation method according to claim 2, characterized in that: The functional monomer includes one of 4-vinylpyridine, 2-vinylpyridine, acrylamide and α-methylacrylic acid.

5. The preparation method according to claim 2, characterized in that: The polymerization reaction is carried out under the protection of inert gas, the reaction temperature is 60° C. to 80° C., and the reaction time is 2 h to 4 h.

6. The preparation method according to claim 1, characterized in that: The hydrotalcite-like substance is selected from magnesium / aluminum-like hydrotalcite.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the cellulose-based molecular imprinting hydrogel to the hydrotalcite-like immobilized enzyme system is 1:2 to 1:

3.

8. A dual-function cellulose-based bioreactor, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 7.

9. The bifunctional cellulose-based bioreactor according to claim 8 is used for the targeted recognition and biotransformation process of flavonoid glycosides.

10. The dual-functional cellulose-based bioreactor of claim 8 is used for targeted recognition of phlorizin and conversion of the phlorizin into phlorretin.

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