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CN-121975787-A - Enzyme immobilization carrier and application thereof

CN121975787ACN 121975787 ACN121975787 ACN 121975787ACN-121975787-A

Abstract

The invention relates to an enzyme immobilization carrier and application thereof, belonging to the technical field of enzyme immobilization, wherein the enzyme immobilization carrier comprises a magnetic Fe3O4-SiO2 nano core with the particle size of 20-50nm, a thermosensitive poly-N-isopropyl acrylamide hydrogel layer coated outside the magnetic core, amino mesoporous silica grafted on the surface of the hydrogel layer, with the pore size of 5-10nm and the specific surface area of more than or equal to 800m < 2 >/g, carbonyl reductase GJ3401 covalently immobilized on the mesoporous silica, coenzyme NADP crosslinked on the surface of the carrier through a PEG chain, sodium alginate added into the N-isopropyl acrylamide hydrogel layer, wherein the mass ratio of the sodium alginate is 1-3%, and the overall particle size of the carrier is 150-200 mu m.

Inventors

  • HE YI
  • CAO MENG
  • LU MENGYUN
  • WANG FUQUAN
  • Jiang nanshan

Assignees

  • 江苏阿尔法药业股份有限公司

Dates

Publication Date
20260505
Application Date
20260130

Claims (8)

  1. 1. An enzyme-immobilized carrier, comprising: Magnetic Fe3O4-SiO2 nano-cores with the particle size of 20-50nm; A temperature-sensitive poly-N-isopropyl acrylamide hydrogel layer coated outside the magnetic core; The amino mesoporous silica grafted on the surface of the hydrogel layer has a pore diameter of 5-10nm and a specific surface area of more than or equal to 800m < 2 >/g; Carbonyl reductase GJ3401 covalently immobilized on mesoporous silica; Coenzyme NADP cross-linked to the carrier surface via PEG chains.
  2. 2. The enzyme immobilization carrier according to claim 1, wherein sodium alginate is added into the N-isopropyl acrylamide hydrogel layer, the mass ratio of the sodium alginate is 1-3%, and the overall particle size of the carrier is 150-200 μm.
  3. 3. The enzyme-immobilized carrier according to claim 2, comprising the steps of: S1, polymerizing PNIPAM on the surface of a Fe3O4-SiO2 magnetic core; S2, adopting APTES to modify mesoporous silica and grafting amino; s3, covalently connecting the aminated mesoporous silica to the N-isopropyl acrylamide hydrogel layer; s4, adsorbing carbonyl reductase for 12 hours at the temperature of 7.0 and 4 ℃; s5, crosslinking the NADP to the PEG modification site by using EDC/NHS activation system.
  4. 4. The immobilized enzyme carrier of claim 3 wherein the reaction zone is controlled at 25-28 ℃, the separation zone is cooled to 25 ℃ or less to trigger swelling and sedimentation of N-isopropylacrylamide, and the carrier is recovered by coupling a magnetic field.
  5. 5. The immobilized enzyme carrier of claim 4, wherein 10-15% of NADP is added for each batch in the reaction process, and the diatomite filtration step is omitted.
  6. 6. The immobilized enzyme carrier of claim 5, wherein the mesoporous silica surface is modified with epoxy groups for enhancing the covalent immobilization efficiency of carbonyl reductase.
  7. 7. Use of an enzyme immobilization carrier in atorvastatin calcium, comprising the steps of: t1, adding a substrate A3 compound, isopropanol and a co-immobilization carrier into phosphate buffer with pH of 7.0; the reaction is stirred at the temperature of 28 ℃ until the residue of the A3 compound is less than 0.1%; t3, cooling to 25 ℃ to enable the carrier to settle, and recovering the carrier through magnetic separation; and T4, directly adding dichloromethane into the reaction solution for extraction, and carrying out reduced pressure distillation to obtain the A4 compound.
  8. 8. The use of an enzyme immobilization carrier in atorvastatin calcium according to claim 7, wherein the immobilization carrier is reused for more than or equal to 15 batches, and the total consumption of NADP is reduced to less than 30% of the free enzyme process.

Description

Enzyme immobilization carrier and application thereof Technical Field The invention relates to an enzyme immobilization carrier and application thereof, belonging to the field of enzyme immobilization. Background The catalytic reduction of carbonyl reductase to produce atorvastatin calcium intermediate is a key step of industrial production, and the traditional free enzyme process has the problems of easy enzyme deactivation, high coenzyme cost, difficult recovery and the like. The existing immobilized carrier can partially improve enzyme stability, but still has the challenges of low mass transfer efficiency, difficult coenzyme regeneration, complicated carrier separation step and the like. Therefore, there is a need to develop an immobilization carrier which has high enzyme activity stability, high-efficiency coenzyme recycling and easy separation and recovery. Disclosure of Invention The invention provides an enzyme immobilized carrier and application thereof, which realize efficient co-immobilization of enzyme and coenzyme and intelligent recovery of the carrier through a multistage structural design and a thermo-sensitive magnetic separation coupling strategy. The invention aims at realizing the following technical scheme, and an enzyme immobilization carrier comprises the following components: Magnetic Fe3O4-SiO2 nano-cores with the particle size of 20-50nm; A temperature-sensitive poly-N-isopropyl acrylamide hydrogel layer coated outside the magnetic core; The amino mesoporous silica grafted on the surface of the hydrogel layer has a pore diameter of 5-10nm and a specific surface area of more than or equal to 800m < 2 >/g; The mesoporous confinement effect provides a molecular sieve function and protects enzymes from being attacked by macromolecular inhibitors; Carbonyl reductase GJ3401 covalently immobilized on mesoporous silica; Coenzyme NADP cross-linked to the carrier surface via PEG chains. The PEG chain buffers mechanical stress and reduces coenzyme shedding. Further, sodium alginate is added into the N-isopropyl acrylamide hydrogel layer, the mass ratio of the sodium alginate is 1-3%, and the overall particle size of the carrier is 150-200 mu m. Furthermore, the sodium alginate directionally enhances and improves the shearing strength, optimizes the swelling rate, forms a calcium ion-carboxyl cross-linked network, solves the problem that the pure PNIPAM is easy to crack during phase transition, controls the size of a magnetic core to avoid magnetic aggregation, and ensures the response speed of a magnetic field. Further, the method comprises the following steps: S1, polymerizing PNIPAM on the surface of a Fe3O4-SiO2 magnetic core; S2, adopting APTES to modify mesoporous silica and grafting amino; s3, covalently connecting the aminated mesoporous silica to the N-isopropyl acrylamide hydrogel layer; s4, adsorbing carbonyl reductase for 12 hours at the temperature of 7.0 and 4 ℃; s5, crosslinking the NADP to the PEG modification site by using EDC/NHS activation system. Furthermore, the amino group and the epoxy group form a difunctional anchor, the stable connection of mesoporous silicon and hydrogel is realized through an amide bond, a covalent bond is formed between the mesoporous silicon and the hydrogel and an enzyme molecule lysine residue, the enzyme load is improved, and the half life at 50 ℃ is prolonged. Further, the temperature of the reaction zone is controlled at 25-28 ℃, and the separation zone triggers the N-isopropyl acrylamide to swell and settle by cooling to less than or equal to 25 ℃, and the carrier is recovered by coupling a magnetic field. Further, PNIPAM swells controllably at 25 ℃, the sedimentation velocity is increased by 4 times, the overswelling breakage rate is reduced from 3.8% to <1% compared with 20 ℃, and the recovery rate is more than 98.5%. Further, 10-15% of NADP is supplemented for each batch in the reaction process, and the diatomite filtering step is omitted. Furthermore, the batch supplementation strategy reduces the total consumption of NADP to 28% of free enzyme, breaks the bottleneck of coenzyme cost, eliminates diatomite filtration, improves the yield by directly extracting reaction liquid, and shortens the carrier recovery time from 45min to 8min. Furthermore, the mesoporous silica surface is modified with epoxy groups for enhancing the covalent immobilization efficiency of carbonyl reductase. An application of an enzyme immobilization carrier in atorvastatin calcium, comprising the following steps: t1, adding a substrate A3 compound, isopropanol and a co-immobilization carrier into phosphate buffer with pH of 7.0; the reaction is stirred at the temperature of 28 ℃ until the residue of the A3 compound is less than 0.1%; t3, cooling to 25 ℃ to enable the carrier to settle, and recovering the carrier through magnetic separation; and T4, directly adding dichloromethane into the reaction solution for extraction, and carrying out reduced pressure distillati