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Immobilized enzyme as well as preparation method and application thereof

A technology of immobilized enzymes and reductases, which is applied in the direction of immobilized enzymes, biochemical equipment and methods, fixed on/in organic carriers, etc., can solve the problems of difficult recycling of enzymes, achieve stable immobilization effects, and improve The effect of recycling efficiency

Active Publication Date: 2020-04-07
JILIN ASYMCHEM LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The main purpose of the present invention is to provide a kind of immobilized enzyme, its preparation method and its application, to solve the problem that this type of enzyme is difficult to recycle in the prior art

Method used

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  • Immobilized enzyme as well as preparation method and application thereof
  • Immobilized enzyme as well as preparation method and application thereof
  • Immobilized enzyme as well as preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0050] In the second typical embodiment of the present application, a method for preparing immobilized enzymes is provided, the preparation method comprising: pre-treating the cross-linking agent with a polymer to obtain a processed cross-linking agent; Amino resin carrier is modified by reagent to obtain modified carrier; enzyme is fixed on the modified carrier to obtain immobilized enzyme; enzyme is selected from transaminase, ketoreductase, monooxygenase, ammonia lyase, ene reductase, imine reduction Any one of enzymes, amino acid dehydrogenases, and nitrilases.

[0051] The preparation method of the present application pre-treats the cross-linking agent with a polymer, so that the cross-linking agent has more network structure, and then uses the cross-linking agent with more network structure to modify the amino resin carrier , so that the carrier also has more network structures, so when the enzyme is immobilized on the modified carrier, the immobilization effect on the e...

Embodiment 1

[0084] Example 1: Immobilized transaminase

[0085] Take 1 g of amino resin, wash it with 20 mM phosphate buffer solution (referred to as PB, pH7.0) and set aside.

[0086]Add 160 μL of glutaraldehyde (50% aqueous solution) to 4 mL of PB buffer (20 mM, pH 7.0), and add an appropriate ratio of PEG or PEI. After incubating at room temperature for 30-60 min, add the above-mentioned amino resin to the solution, followed by 20 Incubate at -25°C for 1-2h. The modified amino resin was obtained after filtration, and then 4 mL of enzyme solution (20-25 mg / mL protein, including the main enzyme and its corresponding coenzyme) was added to the modified amino resin, and then incubated at 20° C. for 16-24 h. Finally, wash 3 times with 20 mM PB (pH 8.0 containing 0.5M NaCl).

[0087] Immobilized transaminase activity and reusability test:

[0088]

[0089] In a 10mL reaction bottle, add 0.3mL MeOH, dissolve 0.1g of main material 1 or main material 2, add 4eq isopropylamine hydrochlorid...

Embodiment 2

[0094] Embodiment 2: Conversion rate and reutilization test of immobilized ketoreductases (ketoreductases)

[0095] The immobilization method is the same as in Example 1

[0096] Immobilized enzyme activity and reusability detection:

[0097]

[0098] In a 10mL reaction flask, add 0.5mL isopropanol (IPA), dissolve 0.1g of the main material 3 or 4, and add 0.5mL0.1M PB 7.0 and 1-10mg NAD+, then add 0.05g enzyme powder or by 0.1- 0.3g of immobilized enzyme prepared by enzyme powder, stirred at 30°C for 16-20h. The conversion rate of the system was detected by GC. After each round of reaction, the immobilized enzyme was separated and reused in the next round of reaction, and the number of reuse was investigated. The response data is as follows:

[0099] Table 8:

[0100]

[0101]

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PUM

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Abstract

The invention provides an immobilized enzyme as well as a preparation method and an application thereof. The immobilized enzyme comprises an enzyme and an amino resin carrier for immobilizing the enzyme, the enzyme is selected from any one of transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase and nitrilase, the amino resin carrieris the cross-linking agent modified amino resin carrier, and a cross-linking agent is a cross-linking agent treated by a high polymer. The amino resin carrier is modified by adopting the cross-linking agent treated by the high polymer, so that enzymes immobilized on the amino resin carrier can form netted cross-linking, the immobilization effect of the enzymes is more stable, and the cyclic utilization efficiency of the enzymes is improved.

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] The immobilization method of a single enzyme has been reported in the prior art, but the immobilization methods suitable for dif...

Claims

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Application Information

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IPC IPC(8): C12N11/091
CPCC12N11/08C12N9/1096C12N9/0006C12N9/0073C12N9/88C12N9/001C12N9/0018C12N9/78C12N9/0044C12Y206/01C12Y101/01C12Y114/13022C12Y403/01C12Y103/01C12Y305/05001C12Y104/0102
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郝明敏高妍妍
Owner JILIN ASYMCHEM LAB CO LTD
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