A method for preparing immobilized lipase using complex modified amyloid fibrils

Abstract

This invention provides a method for preparing immobilized lipase using composite-modified protein amyloid protofibrils. The method first prepares β-lactoglobulin amyloid protofibrils by controlling pH and hydrothermal conditions, then polymerizes them into COF protofibrils via TFB and DB condensation. An immobilized carrier is obtained by composite modification with sulfopropyl betaine and pentamalloylmannose to adsorb and immobilize the lipase. In this invention, sulfopropyl betaine anchors to the carrier surface through electrostatic, hydrogen bonding, and hydrophobic interactions, forming a hydration film to prevent pore blockage and enhance the carrier's resilience. Pentamalloylmannose permeates the pores to form a stable interpenetrating structure, giving the carrier a high specific surface area and stability. Furthermore, it stabilizes the loaded lipase through multiple interactions, preventing conformational damage and detachment of the enzyme, while simultaneously creating a stable microenvironment for the lipase, inhibiting oxidation and denaturation inactivation. The immobilized lipase prepared by this invention exhibits high catalytic activity, excellent substrate mass transfer efficiency, and good reusability, and can efficiently catalyze oleic acid esterification reactions.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme immobilization technology, specifically relating to a method for preparing immobilized lipase using composite modified protein amyloid protoplasts. Background Technology

[0002] Lipases, as important biocatalysts, have shown broad application prospects in fields such as oil modification, biodiesel production, flavor ester synthesis, and the pharmaceutical industry. However, free lipases face bottlenecks in practical industrial applications, such as poor stability and difficulty in recycling and reusing, which seriously restricts their large-scale industrial application. Therefore, enzyme immobilization technology, by immobilizing enzyme molecules on a support, can effectively overcome the above-mentioned defects and has become a research hotspot in the field of biocatalysis.

[0003] Protein amyloid fibrils, as a new type of bionanomaterial, are highly ordered nanofiber structures formed by the self-assembly of proteins under certain conditions. They possess extremely high specific surface area, excellent mechanical strength, and good thermal stability. For example, Pilkington et al. first reported on the use of lysozyme amyloid fibrils as nanoscaffolds to immobilize enzymes, confirming that this material can effectively load enzyme molecules and maintain their catalytic activity. Existing research shows that various dietary proteins (such as whey protein, ovalbumin, and β-lactoglobulin) can form amyloid fibrils, and enzymes can be immobilized through physical adsorption or chemical cross-linking. Compared with traditional polysaccharide carriers such as cellulose nanocrystals and microcrystalline cellulose, protein fibrils have abundant active functional groups such as amino and carboxyl groups, facilitating chemical modification and functionalization. Their nanoscale fiber network structure provides sufficient enzyme binding sites and is also conducive to substrate diffusion and product release.

[0004] However, using single protein protofibrils as carriers still has problems such as strong hydrophilicity which is not conducive to the interfacial activation of lipases, insufficient mechanical strength which makes it difficult to meet the requirements of continuous reactions, and direct immobilization may lead to enzyme molecule leakage. Therefore, it is necessary to study how to improve the method of preparing immobilized lipases by composite modified protein amyloid protofibrils, so as to achieve high loading, high activity and high efficiency recovery of lipases, and expand the application of protein nanomaterials in the field of biocatalysis. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide a method for preparing immobilized lipases using composite-modified protein amyloid protofibrils. This method first prepares β-lactoglobulin amyloid protofibrils by controlling pH and hydrothermal conditions, then polymerizes them into COF protofibrils through TFB and DB condensation. The immobilized carrier is obtained by composite modification with sulfopropyl betaine and pentamalloylmannose to adsorb and immobilize the lipase. In this invention, sulfopropyl betaine is anchored to the carrier surface through electrostatic, hydrogen bonding, and hydrophobic interactions, forming a hydration film to prevent pore blockage and enhance the carrier's resilience. Pentamalloylmannose permeates the pores to form a stable interpenetrating structure, endowing the carrier with high specific surface area and stability. Furthermore, it can stabilize the loaded lipase through multiple interactions, preventing conformational damage and detachment of the enzyme, while simultaneously creating a stable microenvironment for the lipase, inhibiting oxidation and denaturation inactivation. The immobilized lipase prepared by this invention exhibits high catalytic activity, excellent substrate mass transfer efficiency, and good reusability, and can efficiently catalyze oleic acid esterification reactions.

[0006] Technical solution: A method for preparing immobilized lipase using composite modified protein amyloid protofibrils, comprising the following steps: S1. Weigh β-lactoglobulin and prepare a protein solution with a mass concentration of 1-5%. Then, perform a hydrothermal reaction at pH 2-4 to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. TFB and acetic acid were added to the β-lactoglobulin amyloid fibrils dispersion for pre-activation, DB was added for polycondensation reaction, and the mixture was centrifuged, washed and dried to obtain β-lactoglobulin amyloid fibrils COF; S3. β-lactoglobulin amyloid fibrils COF were modified by using sulfopropyl betaine and pentagalloylmannose to obtain a composite immobilized carrier; S4. The composite immobilization carrier is pre-activated with glutaraldehyde, then immersed in CALB lipase solution for adsorption and immobilization, and washed and dried to obtain immobilized CALB lipase.

[0007] Furthermore, the conditions for the hydrothermal reaction in S1 are a stirring speed of 200-800 rpm, a reaction temperature of 80-95℃, and a heating time of 4-12 h.

[0008] Furthermore, the amount of TFB added in S2 is 1-5%, based on the volume of the β-lactoglobulin amyloid fibrillary dispersion; the pre-activation conditions are a reaction temperature of 25-35℃, a stirring speed of 400-500rpm, and a reaction time of 15-30min; the molar ratio of acetic acid to TFB is (0.5-1):1; and the molar ratio of TFB to DB is 1:(0.5-1.5).

[0009] Furthermore, the conditions for the polycondensation reaction in S2 are a reaction temperature of 40-55℃, a stirring speed of 700-900rpm, and a reaction time of 1-4h.

[0010] Furthermore, the amount of sulfopropyl betaine added in S3 is 8-15%, and the amount of pentagalloylmannose added is 5-12%, based on the mass of β-lactoglobulin amyloid fibrils COF.

[0011] Furthermore, the conditions for composite modification in S3 are pH value 6.0-8.0, treatment temperature 25-60℃, and treatment time 3-6h.

[0012] Furthermore, the concentration of glutaraldehyde in S4 is 0.5-1.0%, and the pre-activation time is 1-2.5 h.

[0013] Furthermore, the concentration of CALB lipase solution in S4 is 5-10 mg / mL, and the ratio of composite immobilization carrier to enzyme solution is 1 g: (5-10) mL; the adsorption and immobilization conditions are a reaction temperature of 35-50℃ and an immobilization time of 4-8 h.

[0014] Immobilized CALB lipase prepared by any of the methods described above.

[0015] Furthermore, the immobilized CALB lipase is used in the synthesis of fatty acid glycerides. Beneficial effects

[0016] 1. This invention employs a composite modification of β-lactoglobulin amyloid fibrils (COFs) with sulfopropyl betaine and galloylmannose. On one hand, the positively charged quaternary ammonium groups and negatively charged sulfonic acid groups of sulfopropyl betaine can electrostatically interact with the amino and carboxyl groups on the surface of β-lactoglobulin amyloid fibrils (COFs) to achieve rapid physical adsorption and anchoring. Furthermore, the hydroxyl groups in both molecules can form high-density intermolecular hydrogen bonds, and the hydrophobic water regions can hydrophobically associate with the hydrophobic regions of the fibrils. On the other hand, sulfopropyl betaine forms a dense hydration film on the surface of β-lactoglobulin amyloid fibrils (COFs), which not only prevents pore blockage caused by β-lactoglobulin amyloid fibril aggregation but also enhances the stress resistance of the β-lactoglobulin amyloid fibril backbone while preserving the β-lactoglobulin amyloid fibrils. The original three-dimensional porous ordered framework structure of the protofibrils provides a suitable spatial structure for subsequent composite modification with galloylmannose. On the other hand, galloylmannose fully penetrates into the pores of the β-lactoglobulin amyloid protofibrils COF, and its hydroxyl groups can form hydrogen bonds with the active groups on the surface of the β-lactoglobulin amyloid protofibrils COF to bind in situ. At the same time, galloylmannose molecules initially aggregate through hydrogen bonds and π-π stacking to form nanoscale small molecule aggregates, which further entangle and cross-link with the network of β-lactoglobulin amyloid protofibrils COF to form a highly interpenetrating and stable composite structure with high specific surface area, high structural stability, and high bioadhesion, providing a good carrier basis for the immobilization of lipases.

[0017] 2. This invention utilizes composite modified β-lactoglobulin amyloid fibrils (COFs) for immobilizing lipase, achieving efficient and stable loading. The amide bonds and hydroxyl groups in the composite modified β-lactoglobulin amyloid fibrils (COFs) can form high-density intermolecular hydrogen bonds with the polar groups on the lipase surface, enabling rapid and tight adhesion between the enzyme molecule and the carrier and preventing enzyme molecule detachment. Furthermore, the positively charged quaternary ammonium group and negatively charged sulfonic acid group of sulfopropyl betaine can form electrostatic adsorption and charge matching with charged amino acid residues on the enzyme molecule surface to avoid enzyme activity impairment caused by conformational distortion due to single electrostatic interaction. In addition, the hydrophobic fragments and aromatic hydrophobic regions of the galloyl group in the composite modified β-lactoglobulin fibrils (COFs) can specifically hydrophobically associate with the hydrophobic regions of the lipase, further stabilizing the immobilization of lipase molecules by the composite modified β-lactoglobulin fibrils (COFs).

[0018] 3. This invention employs composite-modified β-lactoglobulin amyloid fibrils (COFs) for immobilizing lipase. The hydration film formed by sulfopropyl betaine on the carrier surface provides a stable ion-buffered microenvironment for the lipase, reducing the impact of sudden changes in the external acid-base environment on lipase activity and preventing protein denaturation and inactivation caused by direct contact of enzyme molecules with organic solvents. Simultaneously, the hydration film reduces the mutual influence between lipase molecules. Furthermore, the rigid framework of the β-lactoglobulin amyloid fibrils (COFs) provides structural isolation for lipase molecules, dispersing immobilized lipase molecules within the carrier channels and gel network, preventing aggregation and self-degradation of lipase molecules to ensure good catalytic activity. The polyphenolic structure of galloylmannose can scavenge free radicals in the reaction system, inhibiting oxidative inactivation of the lipase, thereby maintaining high stability and high catalytic efficiency of the immobilized lipase.

[0019] 4. The immobilized lipase prepared in this invention can efficiently catalyze oleic acid esterification and has excellent reusability. On the one hand, the immobilization support is composed of a rigid amyloid protofibril COF three-dimensional porous framework and a flexible galloylmannose gel network. The high specific surface area and interconnected pores provide sufficient channels for the diffusion of oleic acid and alcohol substrates, improving the mass transfer rate of the reaction system. At the same time, the interfacial hydration membrane regulated by sulfopropyl betaine can adsorb and enrich oleic acid, and can also allow alcohol substrates to quickly penetrate to the lipase active site. Meanwhile, the reaction products can be promptly removed from the active site to prevent product aggregation. Inhibition leads to a decrease in the catalytic rate of immobilized enzymes; on the other hand, the β-lactoglobulin amyloid fibrils COF backbone has high mechanical strength, is resistant to acid and alkali, and is resistant to solvent erosion. Combined with galloylmannose supramolecular gel, it has a stable structure in the reaction system and will not swell, break, or dissolve to maintain an interpenetrating porous structure. Each cycle can provide a stable microenvironment and mass transfer channel for lipase catalysis. The experimental results show that after repeated use, the catalytic efficiency of immobilized lipase for oleic acid esterification reaction is basically stable, thus achieving efficient conversion of oleic acid and excellent reusability. Attached Figure Description

[0020] Figure 1 The relative activities of the immobilized CALB lipase prepared in Example 8 and Comparative Examples 1-6 at different temperatures; Figure 2 The relative activities of the immobilized CALB lipases prepared in Example 8 and Comparative Examples 1-6 at different pH values; Figure 3 The relative activities of the immobilized CALB lipase prepared in Example 8 and Comparative Examples 1-6 at different cycles are shown. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: Example 1

[0022] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 1.0 g of β-lactoglobulin and add it to 99.0 mL of water to prepare a protein solution with a mass concentration of 1%. Adjust the pH to 2.0 with 1 M hydrochloric acid and heat under reflux at 80℃ and 200 rpm for 12 h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion under ultrasonic power of 200 W and ultrasonic frequency of 20 kHz for 30 min to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 1.0 g TFB and 0.36 mL acetic acid to 100 mL β-lactoglobulin amyloid fibrils dispersion, pre-activate at 25 °C and 400 rpm for 30 min, then add 0.47 g DB and heat to 40 °C, and perform polycondensation reaction at 700 rpm for 4 h; after the reaction is completed, centrifuge the product at 8000 rpm for 20 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 2g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, 0.16g of sulfopropyl betaine and 0.1g of pengaloylmannose were added, and the mixture was subjected to composite modification treatment at 25℃ and 200rpm for 6h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. 2g of the composite immobilization carrier was pre-activated in 20mL of 0.5% glutaraldehyde solution for 2.5h, then centrifuged and washed. The carrier was transferred to 20mL of CALB lipase solution with a concentration of 5mg / mL and immobilized at 35℃ and 100rpm for 8h. After immobilization, the carrier was washed three times with 50mL of phosphate buffer and freeze-dried to obtain immobilized CALB lipase. Example 2

[0023] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 2.0g of β-lactoglobulin and add it to 98.0mL of water to prepare a protein solution with a mass concentration of 2%. Adjust the pH to 2.5 with 1M hydrochloric acid and heat under reflux at 82℃ and 300rpm for 10h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion under ultrasonic power of 250W and ultrasonic frequency of 25kHz for 25min to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 1.5 g TFB and 0.4 mL acetic acid to 100 mL of β-lactoglobulin amyloid fibrils dispersion, pre-activate at 27 °C and 420 rpm for 25 min, then add 0.66 g DB and heat to 42 °C, and perform polycondensation reaction at 750 rpm for 3.5 h; after the reaction, centrifuge the product at 9000 rpm for 15 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 3g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, and 0.27g of sulfopropyl betaine and 0.18g of pengaloylmannose were added. The mixture was subjected to composite modification treatment at 30℃ and 300rpm for 5.5h, and then centrifuged and washed to obtain the composite immobilized carrier. S4. Immerse 3g of the composite immobilization carrier in 30mL of 0.6% glutaraldehyde solution for 2h for pre-activation, then centrifuge and wash. Transfer the carrier to 25mL of CALB lipase solution with a concentration of 6mg / mL and immobilize at 37℃ and 150rpm for 8h. After immobilization, wash three times with 50mL of phosphate buffer and freeze-dry to obtain immobilized CALB lipase. Example 3

[0024] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 3.0g of β-lactoglobulin and add it to 97.0mL of water to prepare a protein solution with a mass concentration of 3%. Adjust the pH to 3.0 with 1M hydrochloric acid and heat under reflux at 85℃ and 400rpm for 8h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion under ultrasonic power of 300W and ultrasonic frequency of 30kHz for 22min to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 2.0 g TFB and 0.5 mL acetic acid to 100 mL of β-lactoglobulin amyloid fibrils dispersion, pre-activate at 28 °C and 450 rpm for 22 min, then add 0.85 g DB and heat to 45 °C, and perform polycondensation reaction at 800 rpm for 3 h; after the reaction, centrifuge the product at 8000 rpm for 15 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 4g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, 0.4g of sulfopropyl betaine and 0.28g of pengaloylmannose were added, and the mixture was subjected to composite modification treatment at 35℃ and 400rpm for 5h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. 3.75 g of the composite immobilization carrier was pre-activated in 30 mL of 0.7% glutaraldehyde solution for 2.5 h, then centrifuged and washed. The carrier was transferred to 30 mL of CALB lipase solution with a concentration of 7 mg / mL and immobilized at 40 °C and 200 rpm for 6 h. After immobilization, the carrier was washed three times with 50 mL of phosphate buffer and freeze-dried to obtain immobilized CALB lipase. Example 4

[0025] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 4.0g of β-lactoglobulin and add it to 96.0mL of water to prepare a protein solution with a mass concentration of 4%. Adjust the pH to 3.5 with 1M hydrochloric acid and heat under reflux at 82℃ and 700rpm for 5h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion for 15min under ultrasonic power of 450W and ultrasonic frequency of 40kHz to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 4.5 g TFB and 0.9 mL acetic acid to 100 mL of β-lactoglobulin amyloid fibrils dispersion, pre-activate at 30 °C and 450 rpm for 15 min, then add 2 g DB and heat to 50 °C, and perform polycondensation reaction at 750 rpm for 1.5 h; after the reaction, centrifuge the product at 8000 rpm for 10 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 8g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, 1.1g of sulfopropyl betaine and 0.9g of pengaloylmannose were added, and the mixture was subjected to composite modification treatment at 55℃ and 500rpm for 3.5h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. Immerse 8g of the composite immobilization carrier in 50mL of 0.9% glutaraldehyde solution for 1h for pre-activation, then centrifuge and wash. Transfer the carrier to 50mL of CALB lipase solution with a concentration of 9mg / mL and fix it at 37℃ and 150rpm for 4.5h. After fixation, wash three times with 50mL of phosphate buffer and freeze-dry to obtain immobilized CALB lipase. Example 5

[0026] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 4.0g of β-lactoglobulin and add it to 96.0mL of water to prepare a protein solution with a mass concentration of 4%. Adjust the pH to 3.5 with 1M hydrochloric acid and heat under reflux at 90℃ and 600rpm for 6h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion for 15min under ultrasonic power of 400W and ultrasonic frequency of 35kHz to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 4.0 g TFB and 0.8 mL acetic acid to 100 mL β-lactoglobulin amyloid fibrils dispersion, pre-activate at 32 °C and 480 rpm for 20 min, then add 1.7 g DB and heat to 50 °C, and perform polycondensation reaction at 750 rpm for 2 h; after the reaction, centrifuge the product at 9000 rpm for 15 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 6g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 7.5, and 0.85g of sulfopropyl betaine and 0.65g of pengaloylmannose were added. The mixture was subjected to composite modification treatment at 50℃ and 500rpm for 4h, and then centrifuged and washed to obtain the composite immobilized carrier. S4. 6g of the composite immobilization carrier was pre-activated in 50mL of 0.6% glutaraldehyde solution for 1.5h, then centrifuged and washed. The carrier was transferred to 50mL of CALB lipase solution with a concentration of 8mg / mL and immobilized at 45℃ and 250rpm for 5h. After immobilization, the carrier was washed three times with 50mL of phosphate buffer and freeze-dried to obtain immobilized CALB lipase. Example 6

[0027] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 3.0g of β-lactoglobulin and add it to 97.0mL of water to prepare a protein solution with a mass concentration of 3%. Adjust the pH to 3.0 with 1M hydrochloric acid and heat under reflux at 87℃ and 500rpm for 8h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion under ultrasonic power of 350W and ultrasonic frequency of 35kHz for 20min to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 3.0 g TFB and 0.65 mL acetic acid to 100 mL of β-lactoglobulin amyloid fibrils dispersion, pre-activate at 27 °C and 420 rpm for 20 min, then add 1.2 g DB and heat to 45 °C, and perform polycondensation reaction at 800 rpm for 2.5 h; after the reaction, centrifuge the product at 9000 rpm for 10 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 5g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 7.0, 0.6g of sulfopropyl betaine and 0.4g of pengaloylmannose were added, and the mixture was subjected to composite modification treatment at 40℃ and 450rpm for 4.5h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. Immerse 5g of the composite immobilization carrier in 45mL of 0.75% glutaraldehyde solution for 1.5h for pre-activation, then centrifuge and wash. Transfer the carrier to 35mL of 7.5mg / mL CALB lipase solution and immobilize at 40℃ and 250rpm for 6h. After immobilization, wash three times with 50mL phosphate buffer and freeze-dry to obtain immobilized CALB lipase. Example 7

[0028] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 5.0g of β-lactoglobulin and add it to 95.0mL of water to prepare a protein solution with a mass concentration of 5%. Adjust the pH to 4.0 with 1M hydrochloric acid and heat under reflux at 95℃ and 500rpm for 4h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion for 10min under ultrasonic power of 500W and ultrasonic frequency of 40kHz to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 5.0 g TFB and 1.1 mL acetic acid to 100 mL β-lactoglobulin amyloid fibrils dispersion, pre-activate at 35 °C and 500 rpm for 15 min, then add 2.5 g DB and heat to 42 °C, and perform polycondensation reaction at 800 rpm for 1 h; after the reaction, centrifuge the product at 9000 rpm for 15 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 5g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, 0.75g of sulfopropyl betaine and 0.6g of pengaloylmannose were added, and the mixture was subjected to composite modification treatment at 60℃ and 400rpm for 3h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. Immerse 6g of the composite immobilization carrier in 50mL of 1.0% glutaraldehyde solution for 1h for pre-activation, then centrifuge and wash. Transfer the carrier to 50mL of 10mg / mL CALB lipase solution and fix it at 50℃ and 350rpm for 4h. After fixation, wash three times with 50mL phosphate buffer and freeze-dry to obtain immobilized CALB lipase. Example 8

[0029] A method for preparing immobilized lipase using composite modified protein amyloid protofibrils includes the following steps: S1. Weigh 3.0g of β-lactoglobulin and add it to 97.0mL of water to prepare a protein solution with a mass concentration of 3%. Adjust the pH to 3.0 with 1M hydrochloric acid and heat under reflux at 85℃ and 500rpm for 8h to obtain a β-lactoglobulin dispersion. Sonicate the β-lactoglobulin dispersion under ultrasonic power of 350W and ultrasonic frequency of 35kHz for 20min to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. Add 3.0 g TFB and 0.7 mL acetic acid to 100 mL of β-lactoglobulin amyloid fibrils dispersion, pre-activate at 30 °C and 450 rpm for 20 min, then add 1.2 g DB and heat to 45 °C, and perform polycondensation reaction at 750 rpm for 2.5 h; after the reaction, centrifuge the product at 9000 rpm for 15 min, wash three times each with 50 mL DMF, 50 mL methanol and 50 mL water, and freeze-dry to obtain β-lactoglobulin amyloid fibrils COF; S3. 5g of β-lactoglobulin amyloid fibrils COF were dispersed in 100mL of phosphate buffer at pH 6.0, 0.7g of sulfopropyl betaine and 0.45g of pentamalloylmannose were added, and the mixture was subjected to composite modification treatment at 45℃ and 350rpm for 4h. After centrifugation and washing, the composite immobilized carrier was obtained. S4. Immerse 5g of the composite immobilization carrier in 40mL of 0.8% glutaraldehyde solution for 1.5h for pre-activation, then centrifuge and wash. Transfer the carrier to 40mL of CALB lipase solution with a concentration of 8mg / mL and immobilize at 40℃ and 250rpm for 6h. After immobilization, wash three times with 50mL of phosphate buffer and freeze-dry to obtain immobilized CALB lipase. Comparative Example 1

[0030] The difference between this comparative example and Example 8 is that sulfopropyl betaine is replaced with sulfonyl betaine; the remaining operations are the same as in Example 8. Comparative Example 2

[0031] The difference between this comparative example and Example 8 is that pentagalloylmannose is replaced with mannose, and the remaining operations are the same as in Example 8. Comparative Example 3

[0032] The difference between this comparative example and Example 8 is that only pentagalloylmannose was used for modification; the remaining operations are the same as in Example 8. Comparative Example 4

[0033] The difference between this comparative example and Example 8 is that only sulfopropyl betaine is used for modification, while the remaining operations are the same as in Example 8. Comparative Example 5

[0034] The difference between this comparative example and Example 8 is that TFB and DB are not added; the remaining operations are the same as in Example 8. Comparative Example 6

[0035] The difference between this comparative example and Example 8 is that the composite modification of sulfopropyl betaine and pentagalloylmannose is not used; the remaining operations are the same as in Example 8.

[0036] Performance testing (1) Enzyme immobilization efficiency Accurately measure 50 mL of CALB lipase solution and determine the initial protein concentration using the BCA method, denoted as C0. Immerse the composite immobilization carrier in the above enzyme solution for adsorption and immobilization. After immobilization, collect the residual enzyme solution and centrifuge at 4000 rpm for 10 min to remove suspended particles. Determine the residual protein concentration using the same method, denoted as C1. Simultaneously, collect the immobilized enzyme and wash it three times with 50 mL of phosphate buffer (pH 7.0, 0.1 mol / L). Combine the washing buffers and determine the protein concentration, denoted as C2. Enzyme immobilization efficiency (%) = (C0V0 - C1V1 - C2V2) / C0V0 × 100%, where V0 is the initial enzyme solution volume, V1 is the residual enzyme solution volume, and V2 is the washing buffer volume.

[0037] (2) Catalytic activity 200 μL of p-NPP ethanol solution (5 mg / mL) was added to a mixture of immobilized CALB (30 mg) and phosphate buffer (1.8 mL 0.1 mol / L pH 7.0). The mixture was incubated at 37 °C and 160 rpm for 5 min in a constant temperature water bath. After filtration through a 0.45 μm filter, the absorbance of the supernatant containing p-nitrophenol was measured spectrophotometrically at 410 nm. The hydrolytic activity unit (U) was defined as the amount of lipase required to release 1 μmol of p-nitrophenol per minute after p-NPP-catalyzed hydrolysis under the above conditions.

[0038] Immobilized lipase powder (10 mg) was added to a mixture of lauric acid (0.5 mmol), octanol (2 mmol), and cyclohexane (5 mL). The resulting mixture was reacted in a constant temperature water bath shaker at 40 °C and 160 rpm for 2 h. The supernatant was collected, and the conversion rate of lauric acid was determined by the hot ethanol method. Esterification activity unit (U) is defined as the amount of lipase required to consume 1 μmol of lauric acid per hour under the above determination conditions.

[0039] Table 1 Enzyme immobilization efficiency and catalytic activity of Examples 1-8 and Comparative Examples 1-6

[0040] As shown in Table 1, the enzyme immobilization efficiency, hydrolysis activity, and esterification activity of Examples 1-8 are all superior to those of Comparative Examples 1-6. This indicates that the directional growth of COF frameworks on the surface of β-lactoglobulin amyloid fibrils forms a structural carrier with a high specific surface area. Furthermore, the composite modification with sulfopropyl betaine and pentamalloylmannose can further enhance the binding force between CALB lipase and the carrier and maintain the conformation of the active center to preserve catalytic activity by forming hydrogen bonds and hydrophobic interactions with the enzyme protein through polyphenolic hydroxyl groups, and providing electrostatic stability and a hydrophilic microenvironment.

[0041] (3) Stability of immobilized enzymes Immobilized CALB lipase was incubated in constant temperature water baths at 30℃, 40℃, 50℃, 60℃, and 70℃ for 2 hours. After cooling, the residual enzyme activity was measured according to the hydrolysis activity determination method in (2) above. The enzyme activity of the unincubated sample was taken as 100%, and the relative enzyme activity was calculated. Immobilized CALB lipase was dispersed in buffer solutions at pH 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 and placed at room temperature for 2 hours. After adjusting to the optimal pH, the residual enzyme activity was measured. The enzyme activity of the sample treated at pH 7.0 was taken as 100%, and the relative enzyme activity was calculated.

[0042] Depend on Figure 1 and Figure 2 It can be seen that as temperature and pH increase, the relative activity of immobilized CALB lipase shows a trend of first increasing and then decreasing. Moreover, the relative activity of the immobilized CALB lipase prepared in Example 8 is higher than that of Comparative Examples 1-6. This indicates that the composite modified β-lactoglobulin amyloid fibrils (COF) used in this invention can better protect the catalytic activity of CALB lipase under unsuitable temperature and pH. Example 9

[0043] Validation experiment of immobilized CALB lipase-catalyzed fatty acid glyceride synthesis Glycerol and oleic acid were accurately weighed into a three-necked flask according to a molar ratio of 4:1. Initial water (2.5% of the total substrate mass) was added, and the mixture was stirred at room temperature for 10 min to homogenize the system. The oil bath temperature was then set to 50℃ and equilibrated for 15-20 min. After the reaction system temperature stabilized, immobilized CALB lipase (5% of the total substrate mass) was added to initiate the reaction. The reaction was continued at 300 rpm and 50℃ for 12 h. After the reaction, the sample was centrifuged at 3500 × g for 5 min. The upper oil phase was collected for oleic acid conversion analysis. The oleic acid conversion rate was based on the amount of oleic acid consumed per milligram of immobilized lipase. The change in acid value was used to reflect the oleic acid conversion rate. The acid value was determined according to GB 5009.229-2016 National Food Safety Standard for Determination of Acid Value in Food. Oleic acid conversion rate (%) = (A0 - A i ) / A0×100%, where A i Let A0 be the acid value of the reaction system at a certain moment, and A0 be the acid value of the system at the beginning of the reaction.

[0044] Table 2 shows the oleic acid conversion rates of the immobilized CALB lipases prepared in Example 8 and Comparative Examples 1-6.

[0045] As shown in Table 2, the oleic acid conversion rate of the immobilized CALB lipase prepared in Example 8 when catalyzing the synthesis of fatty acid glycerides is significantly better than that of Comparative Examples 1-6. This is because the immobilized CALB lipase prepared in this invention has higher catalytic activity. Example 10

[0046] Reusability of immobilized CALB lipase After the reaction in Example 9 was completed (as the first round of reaction), the immobilized lipase was washed three times with tert-amyl alcohol, and then the washed sample was placed in a fume hood to air dry. The air-dried immobilized lipase was used in the next reaction under the same test method as in Example 9. The activity of the immobilized lipase was expressed as relative activity, and the relative activity (%) of the lipase was defined as the ratio of the oleic acid conversion obtained from each cycle to the fatty acid conversion obtained from the first cycle.

[0047] Depend on Figure 3 It can be seen that the immobilized CALB lipase prepared in Example 8 can be repeated about 9 times, while the comparative examples 1-6 can be repeated about 6 times. After 9 cycles, the catalytic activity of comparative examples 1-6 has been greatly lost, while the catalytic activity of Example 8 is still close to 50%. Therefore, the immobilized CALB lipase prepared in this invention has good reusability.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing immobilized lipase using composite modified protein amyloid protofibrils, characterized in that, Includes the following steps: S1. Weigh β-lactoglobulin and prepare a protein solution with a mass concentration of 1-5%. Then, perform a hydrothermal reaction at pH 2-4 to obtain a β-lactoglobulin amyloid fibrils dispersion. S2. TFB and acetic acid were added to the β-lactoglobulin amyloid fibrils dispersion for pre-activation, DB was added for polycondensation reaction, and the mixture was centrifuged, washed and dried to obtain β-lactoglobulin amyloid fibrils COF; S3. β-lactoglobulin amyloid fibrils COF were modified by using sulfopropyl betaine and pentagalloylmannose to obtain a composite immobilized carrier; S4. The composite immobilization carrier is pre-activated with glutaraldehyde, then immersed in CALB lipase solution for adsorption and immobilization, and washed and dried to obtain immobilized CALB lipase.

2. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The conditions for the hydrothermal reaction in S1 are: stirring speed 200-800 rpm, reaction temperature 80-95℃, and heating time 4-12 h.

3. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The amount of TFB added in S2 is 1-5%, based on the volume of the β-lactoglobulin amyloid fibrillary dispersion; the pre-activation conditions are a reaction temperature of 25-35℃, a stirring speed of 400-500rpm, and a reaction time of 15-30min; the molar ratio of acetic acid to TFB is (0.5-1):1; and the molar ratio of TFB to DB is 1:(0.5-1.5).

4. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The conditions for the polycondensation reaction in S2 are: reaction temperature 40-55℃, stirring speed 700-900rpm, and reaction time 1-4h.

5. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The amount of sulfopropyl betaine added in S3 is 8-15%, and the amount of pentagalloylmannose added is 5-12%, based on the mass of β-lactoglobulin amyloid fibrils COF.

6. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The conditions for composite modification in S3 are: pH value 6.0-8.0, treatment temperature 25-60℃, and treatment time 3-6h.

7. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The concentration of glutaraldehyde in S4 is 0.5-1.0%, and the pre-activation time is 1-2.5 h.

8. The method for preparing immobilized lipase using composite modified protein amyloid protofibrils according to claim 1, characterized in that, The concentration of CALB lipase solution in S4 is 5-10 mg / mL, and the ratio of composite immobilization carrier to enzyme solution is 1 g: (5-10) mL; the adsorption and immobilization conditions are a reaction temperature of 35-50℃ and an immobilization time of 4-8 h.

9. Immobilized CALB lipase prepared by the method according to any one of claims 1-8.

10. The application of the immobilized CALB lipase according to claim 9 in the synthesis of fatty acid glycerides.

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