Preparation method and application of biological enzyme immobilization carrier

Abstract

The invention discloses a preparation method and application of a biological enzyme fixed carrier, comprising the following steps: S1, soaking dried calcium pectin powder in a pyrrole monomer aqueous solution, so that the pyrrole monomer is uniformly infiltrated into calcium pectin gel particles along with water; S2, soaking the hydrogel obtained in S1 in a FeCl3 aqueous solution, and cross-linking the pyrrole monomer into polypyrrole; S3, adding the gel particles into a chitosan solution with a pH value of 4.0 to 4.5, stirring, and enriching chitosan on the surface of the calcium pectin gel particles to form a polyelectrolyte complex, and electrostatically adsorbing chitosan on the surface of the gel particles to obtain a biological enzyme fixed carrier with a gel core structure. The invention provides a biological enzyme carrier with a solid gel structure, which utilizes the non-flowing property of water in the gel inside the carrier to effectively enrich 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 of oligogalactose.

Description

Technical Field

[0001] The present 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 immobilized enzymes, the enzyme itself is water-soluble. A water-insoluble macromolecular carrier is attached to the free enzyme through chemical or physical methods. This significantly increases the stability of the immobilized enzyme, making it easier to control, separate, and reuse. Immobilized enzymes come in a variety of forms, including films, granules, strips, and tubes, depending on their intended use.

[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, because immobilized enzymes are fixed to a carrier, contact between the enzyme and substrate requires mass transfer and diffusion, compared to free enzymes. Therefore, the enzyme dosage or substrate concentration must be increased. However, excessive substrate concentration reduces the effective water concentration, reduces the diffusivity of water molecules, and slows the enzymatic reaction rate. Excessive substrate will accumulate on the surface of the enzyme molecules, preventing the formation of active intermediates and the exposure of the enzyme molecules, making it difficult to improve the reaction conversion rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a biological enzyme immobilization carrier. The present invention proposes a biological enzyme carrier with a solid gel structure. By utilizing the property that water in the gel inside the carrier does not flow, the substrate in the reaction solution is effectively enriched on the surface of the enzyme carrier, thereby enhancing the contact between the enzyme and the substrate and improving 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:

[0007] S1. Soaking the dried calcium pectin powder in an aqueous solution of pyrrole monomer, so that the pyrrole monomer and the water evenly penetrate into the calcium pectin gel particles;

[0008] S2, soaking the hydrogel obtained in S1 in an aqueous FeCl3 solution, and cross-linking the pyrrole monomers into polypyrrole;

[0009] S3. Adding 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.

[0010] Preferably, the preparation method of the dried calcium pectin powder in step S1 comprises the following steps:

[0011] S11, using deionized water to prepare a low-ester pectin and glucose mixture, and adjusting the pH of the mixture to 3.0;

[0012] S12, heating the mixture obtained in S11 in boiling water;

[0013] S13, adding calcium chloride to the mixed solution heated in S12, and stirring rapidly and evenly; placing the resulting mixed solution in an environment of 3°C to 5°C to cool and obtain calcium pectin gel;

[0014] S14. The calcium pectin gel is freeze-dried, crushed, and sieved through 250-300 mesh to obtain dry calcium pectin powder.

[0015] The present invention utilizes the porous properties 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.

[0016] Preferably, the dried calcium pectin powder in step S1 is soaked for 6 to 8 hours at a soaking temperature of 4°C.

[0017] Preferably, the mass ratio of the dried calcium pectin powder to the pyrrole monomer in S1 is (8 to 15):1.

[0018] Preferably, the mass ratio of the gel particles in step S3, calculated as dry calcium pectin powder, to 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 the chitosan improves the stability of the calcium pectin gel particles.

[0019] Preferably, the concentration of the FeCl3 aqueous solution in step S2 is 0.089 mol / L. The present invention allows pyrrole monomers to penetrate into the interior of the gel particles by infiltration, and with the uniform distribution of water, ferric nitrate acts as an initiator to trigger the polymerization of pyrrole into polypyrrole, thereby stabilizing the structure of the calcium pectin gel particles through the formation of polypyrrole.

[0020] Another object of the present invention is to provide an application of a biological enzyme immobilization carrier. The present invention proposes a biological enzyme carrier with a solid gel structure. By utilizing the property that water in the gel inside the carrier does not flow, the substrate in the reaction solution is effectively enriched on the surface of the enzyme carrier, thereby improving the contact between the enzyme and the substrate and improving the conversion efficiency.

[0021] To solve this technical problem, the technical solution of the present invention is: the application of the biological enzyme immobilization carrier prepared by the present invention comprises the following steps:

[0022] A1. Add β-galactosidase to the dispersed solution of the enzyme immobilization carrier, and adsorb the β-galactosidase via chitosan;

[0023] A2, placing the pectin calcium gel-chitosan adsorbed β-galactosidase obtained in A1 in a glutaraldehyde aqueous solution for cross-linking to obtain pectin calcium gel-chitosan solidified β-galactosidase;

[0024] 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 oligomeric galactose.

[0025] Preferably, the mass ratio of β-galactosidase in step A1 to chitosan in the enzyme immobilization carrier is: (1.0 mg to 1.5 mg): 1 g.

[0026] The process conditions for the lactose solution circulation in A3 are preferably as follows:

[0027] The cycle temperature is from 45°C to 52°C and the cycle time is from 10 hours to 14 hours.

[0028] By adopting the above technical solution, the beneficial effects of the present invention are:

[0029] The present invention uses calcium pectin gel particles cross-linked with polypyrrole as a core structure for loading chitosan, and subsequently uses glutaraldehyde to cross-link chitosan and β-galactosidase, wherein the glutaraldehyde crosslinking includes chitosan distributed on the surface of the calcium pectin gel particles, and the chitosan serves as a shell structure of the carrier to stabilize the structure of the calcium pectin gel particles. During 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 contact between the lactose solution and the β-galactosidase. The present invention utilizes the flow of lactose to flow through the stacked carriers to form a spatial flow channel with a three-dimensional structure, that is, it flows through the carrier surface, thereby improving the contact between lactose and the enzyme. The carrier obtained by the present invention can be repeatedly used and maintains a relatively stable conversion efficiency.

[0030] The present invention controls the lactose solution to circulate across the surface of β-galactosidase in a flow manner, thereby improving the contact between the substrate and the enzyme. This prevents excessively high substrate concentration from reducing the effective concentration of water, the diffusivity of water molecules, and the speed of the enzymatic reaction. It also prevents excess substrate from aggregating on the surface of the enzyme molecules, thereby ensuring exposure of the enzyme molecules and improving the conversion rate of the reaction. DETAILED DESCRIPTION

[0031] In order to further explain the technical solution of the present invention, the present invention is described in detail below through specific embodiments.

[0032] Example 1

[0033] This embodiment discloses a method for preparing a biological enzyme immobilization carrier, comprising the following steps:

[0034] S1. Soaking the dried calcium pectin powder in an aqueous solution of pyrrole monomer, so that the pyrrole monomer and the water evenly penetrate into the calcium pectin gel particles;

[0035] The dried calcium pectin powder was soaked for 6 hours at a soaking temperature of 4°C.

[0036] The mass ratio of dry calcium pectin powder to pyrrole monomer is 10:1.

[0037] S2, soaking the hydrogel obtained in S1 in an aqueous FeCl3 solution, and cross-linking the pyrrole monomers into polypyrrole;

[0038] The concentration of the FeCl3 aqueous solution in step S2 is 0.089 mol / L.

[0039] S3. Adding the gel particles into a chitosan solution with a pH of 4.0 and stirring, chitosan is enriched on the surface of the calcium pectin gel particles to form a polyelectrolyte complex. Chitosan is electrostatically adsorbed on the surface of the gel particles to obtain a bio-enzyme immobilization carrier with a gel core structure.

[0040] In this embodiment, the mass ratio of the gel particles in step S3, calculated as dry calcium pectin powder, to chitosan is 1.2:1.

[0041] The preparation method of the dried calcium pectin powder in step S1 in this embodiment comprises the following steps:

[0042] S11. Prepare a mixture of low-ester pectin and glucose using deionized water, wherein the mass fraction of low-ester pectin (35% esterification, 88% total galacturonic acid) is 1% and the mass fraction of glucose is 10%. Adjust the pH of the mixture to 3.0.

[0043] S12, placing the mixture obtained in S11 in boiling water and heating for 2 minutes;

[0044] 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; cooling the resulting mixed solution in an environment of 3° C. to 5° C. for 24 hours to obtain calcium pectin gel;

[0045] S14. The calcium pectin gel is freeze-dried, crushed, and sieved through 300 mesh to obtain dry calcium pectin powder.

[0046] 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.

[0047] This embodiment also discloses the application of a biological enzyme immobilization carrier, comprising the following steps:

[0048] A1. Add β-galactosidase to the dispersed solution of the enzyme immobilization carrier, and adsorb the β-galactosidase via chitosan;

[0049] The mass ratio of β-galactosidase to chitosan in the enzyme immobilization carrier in step A1 is: 1.0 mg:1 g.

[0050] The process conditions for adsorbing β-galactosidase by chitosan in step A1 of this embodiment are as follows:

[0051] The adsorption was carried out on a shaker at 15°C and 280 r / min for 5 hours.

[0052] A2, placing the pectin calcium gel-chitosan adsorbed β-galactosidase obtained in A1 in a 2% glutaraldehyde aqueous solution for cross-linking for 3 hours to obtain pectin calcium gel-chitosan solidified β-galactosidase;

[0053] 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 oligomeric galactose.

[0054] The process conditions for the lactose solution circulation in A3 are as follows:

[0055] The circulating temperature was 48° C. to 52° C., the circulating time was 10 hours, and the liquid circulating pump circulated the reaction solution at a flow rate of 50 mL / min.

[0056] 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 screen along the axial direction of the tank body and loaded with β-galactosidase immobilized in this example using pectin calcium gel-chitosan as a biological enzyme immobilization carrier.

[0057] 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 recovered and the reaction is continued.

[0058] Example 2

[0059] The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0060] Example 3

[0061] The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0062] Example 4

[0063] The main differences between this embodiment and embodiment 1 are shown in Table 1 and Table 2.

[0064] Table 1 Process parameters for preparing enzyme immobilization carriers in Examples 1 to 4

[0065]

[0066] Table 2 Process parameters for preparing galacto-oligosaccharides using enzyme-immobilized carriers in Examples 1 to 4

[0067]

[0068] Comparative Example 1

[0069] This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps:

[0070] 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;

[0071] S2, the cycle temperature is 45°C to 48°C, and the cycle time is 10 hours;

[0072] S3. Separate the reaction solution using continuous simulated moving bed technology to obtain galacto-oligosaccharides, and recover the separated lactose to continue the reaction.

[0073] Comparative Example 2

[0074] This comparative example discloses a method for preparing galacto-oligosaccharide, comprising the following steps:

[0075] S1. A 500 g / L lactose solution was pumped into a 20 L reaction tank as a raw material. The reaction tank was then loaded with chitosan-immobilized β-galactosidase in three compartments separated by four layers of mesh along the axial direction of the tank.

[0076] S2, the cycle temperature is 48 ° C -52 ° C, and the cycle time is 10 hours;

[0077] When the lactose solution flows through the surface of chitosan, it comes into contact with β-galactosidase and reacts;

[0078] In step S2, the liquid circulation pump circulates the reaction solution at a flow rate of 50 mL / min.

[0079] S3. The reaction liquid is pumped out and separated using continuous simulated moving bed technology to obtain oligosaccharides, and the separated lactose is recovered to continue the reaction.

[0080] The ratio of the mass of the immobilized β-galactosidase to the volume of the lactose solution is 25 mg / 1 L;

[0081] The preparation method of chitosan-immobilized β-galactosidase in this comparative example comprises the following steps:

[0082] S11. Add chitosan solution with a pH of 4.0 dropwise into a large amount of 1 mol / L NaOH aqueous solution, let it stand for reaction, then wash the chitosan microspheres with distilled water until neutral, and collect the microspheres by filtration.

[0083] S12. The chitosan microspheres prepared in S11 were placed in a glutaraldehyde aqueous solution, allowed to react, and β-galactosidase was added thereto at a mass ratio of β-galactosidase to chitosan of 1.0 mg:1 g. The chitosan adsorbed the β-galactosidase and cross-linked in a 2% glutaraldehyde aqueous solution for 3 hours to obtain chitosan-solidified β-galactosidase.

[0084] Table 3 Conversion of lactose into galacto-oligosaccharides

[0085]

[0086] 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.

[0087] Table 4 Structural stability of enzyme carriers immobilized by soaking in Examples 1 to 4 and Comparative Example 2

[0088]

[0089] As shown in Table 4, the carrier for immobilizing enzymes of the present invention has a stable structure after compression and good biocompatibility, and is suitable for the preparation of galacto-oligosaccharides.

[0090] 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 conversion retention rate were shown in Table 5.

[0091] Table 5 Conversion rate of the fifth preparation of galacto-oligosaccharides by immobilized enzyme in Examples 1 to 4 and Comparative Example 2

[0092]

[0093] In conjunction with Table 5, it can be seen that the present invention uses gel as the inner core of enzyme carrier to cooperate with carrier surface chitosan in the solid state in neutral aqueous solution, and the immobility of water in pectin high gel particles, realizes the lactose solution for controlling flow to gather and flow from the distribution position of stacking immobilized enzyme, lactose solution fully flows through from the enzyme surface, ensures that lactose solution is fully contacted with beta-galactosidase, promotes the contact of lactose with enzyme;The present invention is used for the carrier of immobilized enzyme to have both stable structure, good compression resistance, while ensuring that enzyme is contacted with lactose solution, the carrier obtained by the present invention can be used repeatedly, and maintains relatively stable conversion efficiency.Compared to pure chitosan as enzyme carrier in comparative example 2, the chitosan balls with porous structure are difficult to ensure that lactose solution is fully enriched relative to enzyme in the flow process, and compared to Examples 1 to 4 of the present invention, the dispersibility of enzyme load is also limited, therefore, the production method of oligomeric galactose proposed by the present invention has 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 manner, 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 excess substrate will not be aggregated on the surface of the enzyme molecules, ensuring that the enzyme molecules are exposed and improving the conversion rate of the reaction. Examples 1 to 4 are achieved by infiltrating pyrrole monomers into the interior of the gel particles by infiltration, accompanied by uniform distribution of water dispersion, and ferric nitrate as an initiator triggering pyrrole to self-polymerize into polypyrrole, thereby stabilizing the structure of the calcium pectin gel particles through polypyrrole formation; the gel particles are then put into a chitosan solution, and chitosan is electrostatically adsorbed on the surface of the gel particles. Glutaraldehyde is then used to achieve crosslinking between chitosan and chitosan, as well as between chitosan and enzyme, thereby obtaining a structurally stable carrier for enzyme fixation. As the number of cycles increases, Examples 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 and the water evenly penetrate into the calcium pectin gel particles; The mass ratio of dried calcium pectin powder to pyrrole monomer in S1 is (8 to 15):1; S2, soaking the calcium pectin gel particles infiltrated with pyrrole monomers obtained in S1 in an FeCl3 aqueous solution, and cross-linking the pyrrole monomers to form polypyrrole; S3, adding the pectin calcium gel particles cross-linked with polypyrrole into a chitosan solution, wherein the pH of the chitosan solution is 4.0 to 4.5, and stirring, so that chitosan is enriched on the surface of the pectin calcium gel particles cross-linked with polypyrrole to form a polyelectrolyte complex, and chitosan is electrostatically adsorbed on the surface of the pectin calcium gel particles cross-linked with polypyrrole to obtain a biological enzyme immobilization carrier with a gel core structure; In step S3, the mass ratio of the calcium pectin gel particles cross-linked with polypyrrole to chitosan is (1.1 to 1.3):1 based on the dry calcium pectin powder.

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

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

5. Use of the enzyme immobilization carrier prepared by the preparation method according to any one of claims 1 to 4, characterized in that: The following steps are involved: A1. Add β-galactosidase to the dispersed solution of the enzyme immobilization carrier, and adsorb the β-galactosidase via chitosan; A2, placing the pectin calcium gel-chitosan adsorbed β-galactosidase obtained in A1 in a glutaraldehyde aqueous solution for cross-linking to obtain pectin calcium gel-chitosan solidified β-galactosidase; A3. Adding the pectin calcium gel-chitosan solidified β-galactosidase obtained in A2 into a lactose solution, and circulating the lactose solution. The circulating lactose solution passes through the three-dimensional pores formed by the accumulation of the biological enzyme immobilization carrier and contacts the β-galactosidase on the surface of the biological enzyme immobilization carrier to generate oligomeric galactose.

6. The use according to claim 5, characterized in that: The mass ratio of β-galactosidase in step A1 to chitosan in the enzyme immobilization carrier is: (1.0 mg to 1.5 mg): 1 g.

7. The use according to claim 5, characterized in that: The process conditions for the lactose solution circulation in A3 are as follows: The cycle temperature is from 45°C to 52°C and the cycle time is from 10 hours to 14 hours.

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