Enzyme immobilization method and application

A technology for immobilizing enzymes and enzyme liquids, applied to biochemical equipment and methods, and enzymes immobilized on/in organic carriers, etc., can solve problems such as difficulty in ensuring repeatability, limited application range, and complicated processes, and achieve Improve the space-time yield, good repeatability, easy to obtain the effect

Inactive Publication Date: 2016-09-28
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These carriers currently used for enzyme immobilization are all prepared by the laboratory itself, the process

Method used

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  • Enzyme immobilization method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 Preparation of carbonyl reductase ChKRED20 immobilized enzyme

[0028] The construction method, heterologous expression, enzyme purification, etc. of the recombinant bacteria of carbonyl reductase ChKRED20 (NCBI accession number: KC342020) are conventional methods in the art. Restriction sites EcoR I and Sal I were added to the two ends of the enzyme gene, and the pET 28a (+) carrier fragment after the same double digestion was connected to obtain the recombinant plasmid pET28a-ChKRED20, which was then transformed into Escherichia coli BL21 ( DE3) to construct recombinant bacteria. The enzyme after heterologous expression of the gene in Escherichia coli has 6 histidines at the N-terminal, which constitutes a histidine tag and can be combined with metal affinity resin.

[0029] Induced expression of carbonyl reductase ChKRED20: Pick a single clone of the recombinant bacteria into LB (containing 50 μg / ml kanamycin) medium, culture at 37°C and 200 rpm for 16 ...

Embodiment 2

[0039] Embodiment 2 Carbonyl reductase ChKRED20 immobilized enzyme and the comparison of pure enzyme biocatalytic efficiency

[0040] (1) Reaction system of pure enzyme: Potassium phosphate buffer (100mM, pH 7.0) contains the following components, 10% (v / v) isopropanol, 0.2g / l NAD + , 2g / l ChKRED20 pure enzyme and substrate 2-chloro-1-(3,4-difluorophenyl)ethanone, concentration 100g / l. 40°C, 200rpm.

[0041] (2) Reaction system of immobilized enzyme: the preparation method of immobilized enzyme is the same as in Example 1. The immobilized enzyme is 2g / l (the concentration refers to the amount of target enzyme contained in the immobilized enzyme), and the rest of the conditions are the same as the pure enzyme reaction system.

[0042]There is no significant difference between the two in the catalytic efficiency at the initial stage of the reaction (the reaction time is 1 hour, and the conversion rate is about 45%). As time goes on, the catalytic efficiency of the immobilized ...

Embodiment 3

[0044] Example 3 Carbonyl reductase ChKRED20 immobilized enzyme catalyzes different concentrations of substrates

[0045] When preparing the immobilized enzyme, the crude enzyme solution was used instead of the pure enzyme solution, and the rest were the same as in Example 1.

[0046] Reaction system: Potassium phosphate buffer (100mM, pH 7.0), 10% (v / v) isopropanol, 0.2g / lNAD + , 5 g / l ChKRED20 immobilized enzyme (the amount of the target enzyme contained), and the substrate 2-chloro-1-(3,4-difluorophenyl)ethanone. The reaction conditions were 40 °C, 200 rpm.

[0047] When the substrate concentration is 50g / l, the time required for complete conversion (conversion rate>99%) is 6h; when the concentration is 100g / l, the time required for complete conversion is 10h; when the substrate concentration is 150g / l, the time required for complete conversion is 24h. Space time yield (Space time yield) is the highest when the substrate concentration is 100g / l, which is 10g / (l.h). When...

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Abstract

The invention discloses a simple and convenient method for recombinase immobilization. According to the method, the characteristic that a histidine label of recombinant protein and metal ion affinity chromatography resin are combined specifically is utilized, and therefore recombinase is immobilized on the resin, and the immobilized recombinase is prepared. Immobilized carbonyl reductase is used for continuous production of chiral alcohol, and the space time yield is greatly increased.

Description

technical field [0001] The invention belongs to the technical field of enzyme immobilization, specifically relates to a simple method for immobilizing recombinant enzymes with histidine tags, and provides a method for applying carbonyl reductase immobilized enzymes to biocatalyze the production of chiral alcohols. Background technique [0002] Biocatalytic processes have typical advantages, such as high reaction efficiency, high stereoselectivity, mild reaction conditions, less use of organic solvents, and environmental friendliness. Used in chemical production. However, natural enzymes often have some disadvantages, such as poor stability and the formation of a mixed system that is difficult to separate from the product after the biocatalytic reaction. These shortcomings are not conducive to the application of enzymes in industry. In addition, the high cost of enzymes is also one of the key factors restricting their application. Using enzyme immobilization technology can ...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12P7/22C12P7/62
CPCC12N9/0006C12N11/04C12N11/10C12P7/22C12P7/62C12Y101/01184Y02P20/50
Inventor 吴中柳刘艳
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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