Preparation method and application of biological enzyme immobilized carrier

Abstract

The invention discloses a preparation method and application of a biological enzyme immobilization carrier, and the preparation method comprises the following steps: S1, soaking dried calcium pectin powder in a pyrrole monomer aqueous solution, so that a pyrrole monomer uniformly permeates into calcium pectin gel particles along with water; s2, soaking the hydrogel obtained in the step S1 in a FeCl3 aqueous solution, and cross-linking the pyrrole monomer into polypyrrole; s3, the gel particles are put into a chitosan solution, the pH value of the chitosan solution is 4.0-4.5, stirring is conducted, chitosan is enriched on the surfaces of the calcium pectin gel particles to form a polyelectrolyte compound, and the chitosan is electrostatically adsorbed on the surfaces of the gel particles to obtain a biological enzyme fixed carrier with a gel core structure; according to the biological enzyme carrier with the gel solid structure, the characteristic that water in gel in the carrier does not flow is utilized, a substrate in reaction liquid is effectively enriched on the surface of the enzyme carrier, contact between enzyme and the substrate is improved, and the conversion efficiency of galactooligosaccharide is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological enzyme fixed carriers, and in particular to a preparation method and application of a biological enzyme fixed carrier. Background Art

[0002] In the case of immobilized enzymes, the enzyme itself is soluble in water. Through chemical or physical methods, the water-insoluble macromolecular carrier is combined with the free enzyme. After the enzyme is immobilized, its stability is significantly increased, which makes it easier to control and separate and can be used repeatedly. There are many forms of immobilized enzymes, including films, particles, lines, enzyme tubes, etc. according to different uses.

[0003] Compared with free enzymes, the immobilized enzyme process may cause partial inactivation of the free enzyme and a certain loss of enzyme activity.

[0004] However, since immobilized enzymes are fixed by carriers, the contact between enzymes and substrates requires mass transfer and diffusion compared to free enzymes, so the amount of enzyme or the concentration of substrates needs to be increased; however, too high a substrate concentration will reduce the effective concentration of water, reduce the diffusivity of water molecules, and reduce the rate of enzyme-catalyzed reactions. Excessive substrates will aggregate on the surface of enzyme molecules, and will not generate active intermediates or expose enzyme molecules, making it difficult to improve the conversion rate of the reaction. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a biological enzyme fixed carrier. The present invention proposes a biological enzyme carrier with a solid gel structure, which utilizes the property that water in the gel inside the carrier does not flow, effectively enriches the substrate in the reaction solution on the surface of the enzyme carrier, improves the contact between the enzyme and the substrate, and improves the conversion efficiency.

[0006] To solve this technical problem, the technical solution of the present invention is: a method for preparing a biological enzyme immobilization carrier, comprising the following steps: S1, soaking the dried calcium pectin powder in an aqueous solution of pyrrole monomer, so that the pyrrole monomer is uniformly infiltrated into the calcium pectin gel particles along with the water; S2, immersing the hydrogel obtained in S1 in an aqueous FeCl3 solution, and cross-linking the pyrrole monomers into polypyrrole; S3, putting the gel particles into a chitosan solution with a pH of 4.0 to 4.5, stirring, chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex, and chitosan is electrostatically adsorbed on the surface of the gel particles to obtain a bio-enzyme immobilization carrier with a gel core structure.

[0007] Preferably, the method for preparing the dried calcium pectin powder in step S1 comprises the following steps: S11, preparing a low-ester pectin and glucose mixture using deionized water, and adjusting the pH of the mixture to 3.0; S12, heating the mixed solution obtained in S11 in boiling water; S13, adding calcium chloride to the mixed solution heated in S12, and stirring rapidly and evenly; placing the obtained mixed solution in an environment of 3°C to 5°C to cool and place to obtain calcium pectin gel; S14. The calcium pectin gel is freeze-dried, crushed, and sieved through 250-300 meshes to obtain dry calcium pectin powder.

[0008] The present invention utilizes the porous property of the dry calcium pectin powder itself to cooperate with the diffusion and polymerization of the pyrrole monomer to effectively stabilize the structural stability of the obtained biological enzyme fixed carrier.

[0009] Preferably, the dried calcium pectin powder in step S1 is soaked for 6 to 8 hours at a soaking temperature of 4°C.

[0010] Preferably, the mass ratio of the dried calcium pectin powder to the pyrrole monomer in S1 is (8 to 15):1.

[0011] Preferably, the mass ratio of the gel particles in step S3, calculated as dry calcium pectin powder and chitosan, is (1.1 to 1.3): 1. The present invention utilizes the three-dimensional spatial structure of the calcium pectin gel particles to effectively load chitosan to form a polyelectrolyte complex, and improves the stability of the calcium pectin gel particles through chitosan.

[0012] Preferably, the concentration of the FeCl3 aqueous solution in step S2 is 0.089 mol / L. In the present invention, the pyrrole monomer is infiltrated into the interior of the gel particles by infiltration, and the water is evenly dispersed. The ferric nitrate is used as an initiator to initiate the polymerization of pyrrole into polypyrrole, thereby stabilizing the structure of the calcium pectin gel particles through the formation of polypyrrole.

[0013] Another object of the present invention is to provide an application of a biological enzyme fixed carrier. The present invention proposes a biological enzyme carrier with a solid gel structure, which utilizes the property that water in the gel inside the carrier does not flow, effectively enriches the substrate in the reaction solution on the surface of the enzyme carrier, thereby improving the contact between the enzyme and the substrate and improving the conversion efficiency.

[0014] To solve this technical problem, the technical solution of the present invention is: the application of the bio-enzyme immobilization carrier prepared by the present invention comprises the following steps: A1, adding β-galactosidase to the dispersed solution of the biological enzyme immobilization carrier, and adsorbing the β-galactosidase through chitosan; A2, placing the calcium pectin gel-chitosan adsorbed β-galactosidase obtained in A1 in a glutaraldehyde aqueous solution for cross-linking to obtain calcium pectin gel-chitosan solidified β-galactosidase; A3. The pectin calcium gel-chitosan solidified β-galactosidase obtained in S2 is added to a lactose solution, and the lactose solution is circulated. The circulating lactose solution passes through the three-dimensional pores formed by the accumulation of the biological enzyme immobilization carrier and contacts with the β-galactosidase on the surface of the biological enzyme immobilization carrier to generate oligogalactose.

[0015] Preferably, the mass ratio of β-galactosidase to chitosan in the enzyme immobilization carrier in step A1 is: (1.0 mg to 1.5 mg): 1 g.

[0016] The process conditions for the lactose solution circulation in A3 are preferably as follows: The cycle temperature is 45°C to 52°C and the cycle time is 10 hours to 14 hours.

[0017] By adopting the above technical solution, the beneficial effects of the present invention are: The present invention uses calcium pectin gel particles cross-linked with polypyrrole as a core structure for loading chitosan, and subsequently cooperates with glutaraldehyde to cross-link chitosan and beta-galactosidase, wherein the glutaraldehyde cross-linking includes chitosan distributed on the surface of the calcium pectin gel particles, and chitosan as a shell structure of the carrier stabilizes the structure of the calcium pectin gel particles; in the production process of oligogalactose, due to the immobility of water in the pectin high gel particles, the lactose solution flows through the surface of the calcium pectin gel-chitosan, thereby ensuring the contact between the lactose solution and the beta-galactosidase, and the present invention utilizes the flow of lactose, flows through the carrier stack to form a spatial flow channel with a three-dimensional structure, that is, flows through the carrier surface, thereby improving the contact between lactose and the enzyme, and the carrier obtained by the present invention can be used repeatedly and maintains a relatively stable conversion efficiency; The present invention controls the lactose solution to circulate through the surface of β-galactosidase in a flow mode to improve the contact between the substrate and the enzyme, that is, the effective concentration of water will not be reduced due to excessively high substrate concentration, the diffusivity of water molecules will not be reduced, and the enzyme-catalyzed reaction rate will not be reduced. It also avoids excessive substrate from aggregating on the surface of the enzyme molecules, ensures that the enzyme molecules are exposed, and improves the conversion rate of the reaction. DETAILED DESCRIPTION

[0018] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0019] Example 1 This embodiment discloses a method for preparing a biological enzyme immobilization carrier, comprising the following steps: S1, soaking the dried calcium pectin powder in an aqueous solution of pyrrole monomer, so that the pyrrole monomer is uniformly infiltrated into the calcium pectin gel particles along with the water; The dried calcium pectin powder was soaked for 6 hours at a soaking temperature of 4°C.

[0020] The mass ratio of the dry calcium pectin powder to the pyrrole monomer is 10:1.

[0021] S2, immersing the hydrogel obtained in S1 in an aqueous FeCl3 solution, and cross-linking the pyrrole monomers into polypyrrole; The concentration of the FeCl3 aqueous solution in step S2 is 0.089 mol / L.

[0022] S3, putting the gel particles into a chitosan solution with a pH of 4.0, stirring, chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex, and chitosan is electrostatically adsorbed on the surface of the gel particles to obtain a bio-enzyme immobilization carrier with a gel core structure.

[0023] In the present embodiment, the mass ratio of the gel particles in step S3, calculated as dry calcium pectin powder, to chitosan is 1.2:1.

[0024] The preparation method of the dried calcium pectin powder in step S1 in this embodiment comprises the following steps: S11. Prepare a low-ester pectin and glucose mixture using deionized water, wherein the mass fraction of low-ester pectin (esterification degree 35%, total galacturonic acid 88%) is 1%, and the mass fraction of glucose is 10%; adjust the pH of the mixture to 3.0; S12, placing the mixed solution obtained in S11 in boiling water and heating for 2 minutes; S13, adding calcium chloride to the mixed solution heated in S12, with the mass ratio of calcium ion to low-ester pectin being 30 mg:1 g, and stirring rapidly and evenly; placing the obtained mixed solution in an environment of 3° C. to 5° C. and cooling for 24 hours to obtain calcium pectin gel; S14. The calcium pectin gel is freeze-dried, crushed, and sieved through 300 mesh to obtain dry calcium pectin powder.

[0025] The present invention utilizes the porous property of the dry calcium pectin powder itself to cooperate with the diffusion and polymerization of the pyrrole monomer to effectively stabilize the structure of the obtained biological enzyme fixed carrier.

[0026] This embodiment also discloses the application of the bio-enzyme immobilization carrier, comprising the following steps: A1, adding β-galactosidase to the dispersed solution of the biological enzyme immobilization carrier, and adsorbing the β-galactosidase through chitosan; The mass ratio of β-galactosidase to chitosan in the enzyme immobilization carrier in step A1 is: 1.0 mg: 1 g.

[0027] The process conditions for adsorbing β-galactosidase by chitosan in step A1 of this embodiment are as follows: The adsorption was carried out on a shaker at 15°C and 280 r / min for 5 hours.

[0028] A2, placing the calcium pectin gel-chitosan adsorbed β-galactosidase obtained in A1 in a 2% glutaraldehyde aqueous solution for cross-linking for 3 hours to obtain calcium pectin gel-chitosan solidified β-galactosidase; A3. The pectin calcium gel-chitosan solidified β-galactosidase obtained in S2 is added to a lactose solution, and the lactose solution is circulated. The circulating lactose solution passes through the three-dimensional pores formed by the accumulation of the biological enzyme immobilization carrier and contacts with the β-galactosidase on the surface of the biological enzyme immobilization carrier to generate oligogalactose.

[0029] The process conditions for the lactose solution circulation in A3 are as follows: The circulating temperature was 48° C. to 52° C., the circulating time was 10 hours, and the liquid circulating pump circulated the reaction liquid at a flow rate of 50 mL / min.

[0030] In this example, 500 g / L lactose solution was pumped into a 20 L reaction tank as a raw material. The three compartments in the reaction tank were separated layer by layer by four layers of screens along the axial direction of the tank body and loaded with the β-galactosidase immobilized by calcium pectin gel-chitosan as a biological enzyme immobilization carrier prepared in this example.

[0031] When the cycle time is reached, the reaction liquid is pumped out and separated using continuous simulated moving bed technology to obtain oligosaccharides. The separated lactose is then recovered and the reaction continues.

[0032] Example 2 The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0033] Example 3 The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0034] Example 4 The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0035] Table 1 Process parameters for preparing enzyme immobilization carriers in Examples 1 to 4

[0036] Table 2 Process parameters for preparing galacto-oligosaccharides using enzyme-immobilized carriers in Examples 1 to 4

[0037] Comparative Example 1 This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps: S1. Pump 500 g / L lactose solution as raw material into a 20 L reaction tank, and add β-galactosidase into the reaction tank; the mass ratio of the added β-galactosidase to the volume of the lactose solution is 25 mg / 1 L; S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours; S3. Separate the reaction solution using continuous simulated moving bed technology to obtain galacto-oligosaccharides, and separate lactose to recover and continue the reaction.

[0038] Comparative Example 2 This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps: S1, 500 g / L lactose solution is pumped into a 20 L reaction tank as a raw material, and the three compartments in the reaction tank are separated layer by layer by four layers of screens along the axial direction of the tank body and loaded with β-galactosidase immobilized by chitosan; S2, the cycle temperature is 48°C-52°C, and the cycle time is 10 hours; When the lactose solution flows through the surface of chitosan, it comes into contact with β-galactosidase and reacts; In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0039] S3. The reaction liquid is pumped out, and galacto-oligosaccharides are separated using continuous simulated moving bed technology, and the separated lactose is recovered to continue the reaction.

[0040] The volume ratio of the input fixed β-galactosidase to the lactose solution is 25 mg / 1L; The preparation method of chitosan-immobilized β-galactosidase in this comparative example comprises the following steps: S11, dripping a chitosan solution with a pH of 4.0 dropwise into a large amount of 1 mol / L NaOH aqueous solution, allowing to react, then washing the chitosan microspheres with distilled water until neutral, and collecting the microspheres by filtration.

[0041] S12, placing the chitosan microspheres of S11 in a glutaraldehyde aqueous solution, allowing the reaction to stand, adding β-galactosidase, the mass ratio of β-galactosidase to chitosan being 1.0 mg:1 g, the chitosan adsorbing β-galactosidase, cross-linking in a 2% glutaraldehyde aqueous solution for 3 hours, to obtain chitosan-solidified β-galactosidase.

[0042] Table 3 Conversion of lactose into galacto-oligosaccharides

[0043] The compressive strength of the particle structure of the immobilized enzyme was further tested after different immersion times. When sampling, 30 particles were placed on the sample table of the compressive strength meter with tweezers. The average value of the test results was taken as the compressive strength. The specific data are shown in Table 4.

[0044] Table 4 Structural stability of enzyme carriers immobilized by soaking in Examples 1 to 4 and Comparative Example 2

[0045] As shown in Table 4, the carrier for immobilizing enzymes of the present invention has a stable structure after being subjected to compression and has good biocompatibility, and is suitable for the preparation of galacto-oligosaccharides.

[0046] The immobilized enzymes obtained in Examples 1 to 4 and Comparative Example 2 were used to prepare galacto-oligosaccharides and were reused 5 times. Compared with the first use, the galacto-oligosaccharide content and the conversion retention rate were shown in Table 5.

[0047] Table 5 Conversion rate of the fifth preparation of galacto-oligosaccharides by immobilized enzyme in Examples 1 to 4 and Comparative Example 2

[0048] It can be seen from Table 5 that the present invention uses gel as the inner core of the enzyme carrier to cooperate with the carrier surface chitosan in a solid state in a neutral aqueous solution, and the immobility of water in the pectin high gel particles, and the lactose solution that controls the flow gathers and flows from the distribution position of the stacked immobilized enzyme, and the lactose solution fully flows from the enzyme surface, ensuring that the lactose solution is fully in contact with β-galactosidase, and promoting the contact of lactose with the enzyme; the carrier for the immobilized enzyme of the present invention has a stable structure and good compression resistance, and at the same time ensures that the enzyme is in contact with the lactose solution, and the carrier obtained by the present invention can be used repeatedly and repeatedly, and a relatively stable conversion efficiency is maintained. Compared with pure chitosan as an enzyme carrier in Comparative Example 2, the chitosan sphere with a porous structure is difficult to ensure that the lactose solution is fully enriched relative to the enzyme during the flow process, and compared with Examples 1 to 4 of the present invention, the dispersibility of the enzyme load is also limited, therefore, the production method of oligomeric galactose proposed by the present invention has a high conversion rate and can be repeatedly used. The present invention controls the lactose solution to circulate through the surface of beta-galactosidase in a flow mode, thereby improving the contact between the substrate and the enzyme, that is, the effective concentration of water will not be reduced due to excessive substrate concentration, the diffusivity of water molecules will be reduced, the enzymatic reaction speed will be reduced, and the excessive substrate will not be aggregated on the surface of the enzyme molecules, so as to ensure that the enzyme molecules are exposed and improve the conversion rate of the reaction. Embodiments 1 to 4 are carried out by infiltrating the pyrrole monomer into the interior of the gel particles by means of infiltration, accompanied by the uniform distribution of water dispersion, and ferric nitrate is used as an initiator to trigger that pyrrole can be self-polymerized into polypyrrole, thereby stabilizing the structure of the calcium pectin gel particles through the formation of polypyrrole; the gel particles are then put into a chitosan solution, and the chitosan is electrostatically adsorbed on the surface of the gel particles, and glutaraldehyde is used to realize the crosslinking between chitosan and chitosan and chitosan and the enzyme, so as to obtain a structurally stable carrier for enzyme fixation, and as the number of cycles increases, Embodiments 1 to 4 have a more stable conversion efficiency.

Claims

1. A method for preparing a biological enzyme immobilization carrier, characterized in that: The following steps are involved: S1, soaking the dried calcium pectin powder in an aqueous solution of pyrrole monomer, so that the pyrrole monomer is uniformly infiltrated into the calcium pectin gel particles along with the water; S2, immersing the hydrogel obtained in S1 in an aqueous FeCl3 solution, and cross-linking the pyrrole monomers into polypyrrole; S3, putting the gel particles into a chitosan solution with a pH of 4.0 to 4.5, stirring, chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex, and chitosan is electrostatically adsorbed on the surface of the gel particles to obtain a bio-enzyme immobilization carrier with a gel core structure.

2. The preparation method according to claim 1, characterized in that: The method for preparing the dried calcium pectin powder in step S1 comprises the following steps: S11, preparing a low-ester pectin and glucose mixture using deionized water, and adjusting the pH of the mixture to 3.0; S12, heating the mixed solution obtained in S11 in boiling water; S13, adding calcium chloride to the mixed solution heated in S12, and stirring rapidly and evenly; placing the obtained mixed solution in an environment of 3°C to 5°C to cool and place to obtain calcium pectin gel; S14. The calcium pectin gel is freeze-dried, crushed, and sieved through 250-300 meshes to obtain dry calcium pectin powder.

3. The preparation method according to claim 1, characterized in that: The dried calcium pectin powder in step S1 is soaked for 6 to 8 hours at a soaking temperature of 4°C.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the dried calcium pectin powder to the pyrrole monomer in S1 is (8 to 15):

1.

5. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of the gel particles based on dry calcium pectin powder to chitosan is (1.1 to 1.3):

1.

6. The preparation method according to claim 1, characterized in that: The concentration of the FeCl3 aqueous solution in step S2 is 0.089 mol / L.

7. Use of the enzyme immobilization carrier obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The following steps are involved: A1, adding β-galactosidase to the dispersed solution of the biological enzyme immobilization carrier, and adsorbing the β-galactosidase through chitosan; A2, placing the calcium pectin gel-chitosan adsorbed β-galactosidase obtained in A1 in a glutaraldehyde aqueous solution for cross-linking to obtain calcium pectin gel-chitosan solidified β-galactosidase; A3. The pectin calcium gel-chitosan solidified β-galactosidase obtained in S2 is added to a lactose solution, and the lactose solution is circulated. The circulating lactose solution passes through the three-dimensional pores formed by the accumulation of the biological enzyme immobilization carrier and contacts with the β-galactosidase on the surface of the biological enzyme immobilization carrier to generate oligogalactose.

8. The use according to claim 7, characterized in that: The mass ratio of β-galactosidase to chitosan in the enzyme immobilization carrier in step A1 is: (1.0 mg to 1.5 mg): 1 g.

9. The use according to claim 7, characterized in that: The process conditions for the lactose solution circulation in A3 are as follows: The cycle temperature is 45°C to 52°C and the cycle time is 10 hours to 14 hours.

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