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Immobilized enzyme, preparation method therefor and application of immobilized enzyme

A technology for immobilizing enzymes and reductases, applied in the directions of immobilized enzymes, biochemical equipment and methods, oxidoreductases, etc., can solve the problem of difficulty in realizing the recycling of sensitive enzyme activities, and achieve the effect of improving mechanical stability.

Active Publication Date: 2020-06-05
ASYMCHEM LIFE SCI TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The main purpose of the present invention is to provide an immobilized enzyme, its preparation method and its application, so as to solve the problem that it is difficult to realize the activity and recycling of sensitive enzymes in the prior art

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  • Immobilized enzyme, preparation method therefor and application of immobilized enzyme
  • Immobilized enzyme, preparation method therefor and application of immobilized enzyme
  • Immobilized enzyme, preparation method therefor and application of immobilized enzyme

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preparation example Construction

[0039] In the second typical embodiment of the present application, a method for preparing an immobilized enzyme is provided. The preparation method includes: using an activator to activate the cationic polymer to obtain an activated cationic polymer; combining the enzyme with the activated The cationic polymer is immobilized to obtain an immobilized enzyme; wherein, the activator is a polyol-unmodified cross-linking agent, a polyol-modified cross-linking agent or a cofactor, and the enzyme is selected from any one of the following: transaminase, ketoreductase, mono Oxygenase, ammonia lyase, ene reductase, imine reductase, amino acid dehydrogenase and nitrilase.

[0040] In the preparation method of the above-mentioned immobilized enzyme, the immobilized enzyme can be obtained by first activating PEI with glutaraldehyde or a cofactor, and then combining with the enzyme. The method is simple and is not easy to affect the activity of the enzyme during the immobilization process,...

Embodiment 1

[0085] Example 1: Preparation of glutaraldehyde-activated PEI covalently immobilized enzyme aggregates (GA: Glutaraldehyde, PEI: Polyethylene imine, GP: GA activated PEI, glutaraldehyde-activated PEI)

[0086] PEG-modified glutaraldehyde: dilute glutaraldehyde with water to a final concentration of 20 g / 100mL, add 2-10g / 100mL PEG 2000 or PEG 6000, mix at room temperature for 1-3 h and set aside.

[0087] Preparation of activated PEI solution 1: Add 2 g of glutaraldehyde and 4 g of PEI (3-70 KDa) to 100 mL of aqueous solution, pH 8.0, and stir for 3 h.

[0088] Preparation of activated PEI solution 2: add 4 g PEI (3 KDa) to 100 mL aqueous solution, pH 8.0, and PEG-modified glutaraldehyde (the final concentration of glutaraldehyde in the solution is 2 g / 100mL) and stir for 3 h .

[0089] Immobilization: 10 mL of enzyme (100-200 mg / mL enzyme protein content, correspondingly containing 3-10 mg / mL cofactor) was placed in a round bottom shaker flask, cooled to 5-10 °C, and mixed fo...

Embodiment 2

[0090] Example 2: Preparation of covalently immobilized enzyme aggregates in PEI activated by cofactors

[0091] Cofactor-activated PEI solution preparation: PEI (Mw=3-70 KDa) was diluted with water to a final PEI concentration of 1g / 100mL~2 g / 100mL and a pH value of 7.0~11.0. Depending on enzyme needs, add 3-10 mg / mL of each enzyme cofactor to the PEI solution and mix for 30-60 min at room temperature.

[0092] Immobilization: At 4-10 °C, add 7.5 mL of cofactor-activated PEI solution dropwise to 5 mL of enzyme solution (enzyme protein content is 30-80 mg / mL, correspondingly contains cofactor), mix for 30- After 60 min, add the cross-linking agent glutaraldehyde or PEG-modified glutaraldehyde, and make the final concentration of glutaraldehyde 0.2-1 g / 100mL. After mixing for 30-60 min, centrifuge to collect the insoluble precipitate, and pass through a 30-mesh sieve. After natural drying, the precipitate was soaked in 0.1 M PB (pH 7.0-8.0, containing 0.5-0.9M NaCl) for 3 hou...

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Abstract

The invention provides an immobilized enzyme, a preparation method therefor and application of the immobilized enzyme. The immobilized enzyme comprises activated polyethylene imine (PEI) and an enzymecovalently bound onto the activated PEI; the enzyme is selected from any one of the following enzymes: transaminase, ketoreductase, cyclohexanone monooxygenase, phenylalanine ammonia lyase, ene reductase, imine reductase, amino acid dehydrogenase and nitrilase. The enzyme and the PEI are immobilized, and the enzyme is immobilized between a poly-net structure formed by the PEI, so that the mechanical stability is improved, immobilization of the enzyme is realized, and the situation that the activity of the enzyme is reduced due to direct covalent bonding of the enzyme and cross-linking agentsincluding glutaraldehyde and the like during immobilization is avoided.

Description

technical field [0001] The invention relates to the field of immobilized enzymes, in particular to an immobilized enzyme, its preparation method and application. Background technique [0002] The use of microbial cells or isolated or engineered enzymes has enabled major advances in biocatalysis and transformed manufacturing. Many classes of enzymes such as acyltransferases, amidases, transaminases, ketoreductases, oxidases, monooxygenases, and hydrolases are used in the production of reactions involving antibiotics, herbicides, pharmaceutical intermediates, and new-age therapeutics . [0003] When free enzymes are used as biocatalysts, there is a great waste of enzymes because it is very difficult to recover water-soluble enzymes. The water-insoluble immobilized enzymes can be easily recovered by very simple filtration after each cycle. [0004] It is well known to use carriers or supports for the immobilization of free enzymes. However, solid phase supports have the dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/089C12N11/098C12N11/18
CPCC12N11/18C12N9/1096C12N9/0006C12N9/0073C12N9/001C12N9/78C12N9/0028C12N9/88C12N9/0014C12Y206/01C12Y101/01002C12Y114/13022C12Y103/01C12Y305/05001C12Y403/01C12N11/089C12N11/098C12N9/0016C12Y103/01031C12Y104/01009C12Y104/0102C12Y403/01024C12N11/08C12R2001/01C12R2001/645C12R2001/075Y02P20/50C12N11/06
Inventor 洪浩詹姆斯·盖吉肖毅张娜罗杰斯卡·维亚撒·威廉姆斯崔瑜霞赵佳东高妍妍傅涵
Owner ASYMCHEM LIFE SCI TIANJIN
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