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Immobilized enzyme carrier, immobilized enzyme and preparation method

An immobilized enzyme carrier, immobilized enzyme technology, applied to biochemical equipment and methods, fixed on or in inorganic carriers, fixed on/in organic carriers, etc., can solve the problem of unsatisfactory reusability, Difficulty in recovery and reuse, obstacles to large-scale application, etc., to achieve good operational stability, good activity recovery rate, and good enzyme loading effect

Pending Publication Date: 2021-11-16
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, from an industrial point of view, free enzymes also have many disadvantages, such as unsatisfactory reusability, short catalytic life, low operational stability against temperature, pH and some environmental factors, and difficulties in recycling and reuse, which seriously hinder their development. Large-scale application at the industrial level

Method used

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  • Immobilized enzyme carrier, immobilized enzyme and preparation method
  • Immobilized enzyme carrier, immobilized enzyme and preparation method
  • Immobilized enzyme carrier, immobilized enzyme and preparation method

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Experimental program
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Effect test

preparation example Construction

[0048] 1. Preparation of immobilized enzyme carrier

[0049] 1.1 Effect of ESMP on the performance of immobilized enzyme carrier

[0050] 0.5g Fe 3 o 4 (Preparation conditions refer to Fe in MCESM1 3 o 4 Preparation method) and 20 mL filtered chitosan solution (3.5% mass concentration) were stirred at room temperature for 1 h, and 10% sodium hydroxide solution (25% ethanol: 75% water, v / v) was added dropwise thereto to obtain Large particles were washed sequentially with deionized water, ethanol solution (50% ethanol: 50% water, v / v) and deionized water until neutral. The resulting large particles were incubated at 50°C in 20mmol / L genipin solution, rinsed with deionized water after 5h, and the BG and Fe 3 o 4 / CS was added to a 10mL reactor at a mass ratio of 1:8, β-glucosidase was added to 1mL PBS buffer (pH7.0) and incubated at 4°C for 3h, and Fe was collected under the action of a magnetic field. 3 o 4 / CS / ESMP@BG. The measured enzyme activity was 52.4%.

[0051]...

Embodiment 1

[0066] Embodiment 1: magnetic chitosan eggshell membrane (Fe 3 o 4 / CS / ESMP) comprises the following steps:

[0067] Step 1: dissolving chitosan powder in 2% (w / v) acetic acid aqueous solution, stirring at 50° C. for 4 h to obtain a 0.035 g / mL chitosan solution, and then filtering it with gauze. In 20mL containing FeCl 2 4H 2 O 0.89g, FeCl 3 ·6H 2 In O 2.42g aqueous solution, the 2% (w / v) acetic acid aqueous solution of chitosan (deacetylation degree ≥ 85%, viscosity is 50mPa.s) that the mass percent concentration after adding 20mL filter wherein is 0.035g / mL , stirred at room temperature for 1 h, added 1.4 g ESMP to it, mixed and stirred for 2 h, then raised the temperature to 80 ° C, added ammonia water as a precipitant, stirred for 1 h, and then added dropwise 10% sodium hydroxide solution (25% ethanol : 75% water, v / v), the obtained large particles were washed with deionized water, ethanol (50% ethanol: 50% water, v / v) and deionized water successively until neutral. ...

Embodiment 2

[0069] Embodiment 2: Preparation of immobilized β-glucosidase

[0070] The BG prepared by embodiment 1 and Fe 3 o 4 / CS / ESMP was added to the 10mL reactor according to the mass ratio of 1:8, and β-glucosidase was added to 1mL PBS buffer (pH 7.0) and incubated at 4°C for 3h, and the β-glucosidase was immobilized on the cross-linked substrate. After the particles were loaded, all samples were washed with sodium phosphate buffer solution at pH 7.0 until no protein was detected, and Fe was collected under the action of a magnetic field. 3 o 4 / CS / ESMP@BG.

[0071] In order to further confirm the prepared Fe 3 o 4 In the presence of the target β-glucosidase on / CS / ESMP, our detection of Fe 3 o 4 / CS / ESMP and Fe 3 o 4 / CS / ESMP@BG performed confocal laser scanning microscopy (CLSM) analysis with fluorescein isothiocyanate-labeled Fe 3 o 4 Confocal microscopy analysis of / CS / ESMP@BG see Figure 6 . From Figure 6 In the sample morphology images shown in panels A and C, i...

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Abstract

The invention discloses an immobilized enzyme carrier, an immobilized enzyme and a preparation method. According to the method, macromolecular particles of chitosan are attached to the surface of an egg shell membrane, magnetic Fe3O4 is uniformly precipitated and distributed on the egg shell membrane in a co-precipitation mode, the prepared magnetic Fe3O4 / CS / ESMP serves as an enzyme carrier of immobilized beta-glucosidase, the rigidity and tolerance of an enzyme structure are improved, and good operation stability is achieved; in addition, the prepared Fe3O4 / CS / ESM@BG can be repeatedly used, and the operation is simple and convenient. The Fe3O4 / CS / ESMP@BG is easy to separate and collect under the action of a magnetic field so as to be recycled, the use efficiency of enzyme is improved, and the prepared Fe3O4 / CS / ESMP@BG can be applied to biosynthesis of genipin.

Description

technical field [0001] The invention belongs to the technical field of immobilized enzymes, and in particular relates to an immobilized enzyme carrier, an immobilized enzyme and a preparation method. Background technique [0002] Genipin is an aglycone of geniposide, which is found in Rubiaceae gardenia, geranium, Eucommia and other plants. It is reported that genipin has antioxidant, anti-inflammatory, anti-tumor, anti-depressant, anti-fungal, anti-angiogenic, anti-diabetic, immunosuppressant effects, used for the treatment of jaundice, edema and hypertension, and can inhibit some cancers. The proliferation of cells, including leukemia, breast cancer, prostate cancer and hepatocellular carcinoma, makes genipin widely used in biomedicine. Some studies have also reported the great ability of genipin to generate cross-links between proteins and polymers such as collagen, gelatin, chitosan, etc. In addition, genipin is safer for food processing due to its lower cytotoxicity. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/14C12N11/10C12N9/42
CPCC12N11/14C12N11/10C12N9/2445C12Y302/01021
Inventor 陈峻青汤玉琪
Owner SOUTHEAST UNIV
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