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High throughput screening method of alcohol dehydrogenase

A technology of alcohol dehydrogenase and screening method, applied in the field of enzyme screening, can solve the problems of unsuitable whole cell screening, large background interference, high coenzyme cost, etc., achieve high-throughput culture and screening, low background interference, and rapid detection accurate effect

Inactive Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the disadvantages of high coenzyme cost, large background interference and unsuitability for whole cell screening in the existing alcohol dehydrogenase screening process

Method used

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  • High throughput screening method of alcohol dehydrogenase
  • High throughput screening method of alcohol dehydrogenase
  • High throughput screening method of alcohol dehydrogenase

Examples

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Embodiment 1

[0041] The present invention is further described below in conjunction with specific examples, but the scope of protection of the present invention is not limited thereto: Embodiment 1: Taking the microorganism that reduces 4-chlorophenyl-pyridin-2-yl-methanone dehydrogenase as an example, to Full-wavelength scanning of red to reddish-brown phenylhydrazone compounds generated by each component in the enzymatic reaction system and 2,4-dinitrophenylhydrazine

[0042]In order to accurately measure the content of the red to reddish-brown phenylhydrazone compound generated by ketone and 2,4-dinitrophenylhydrazine, the present invention measures the characteristic absorption of various compounds and 2,4-dinitrophenylhydrazine in the reaction system respectively. wavelength. The specific operation method is to mix 4-chlorophenyl-pyridin-2-yl-methanone (CPMK), glucose, glucono-γ-lactone, 4-chlorophenyl-pyridin-2-yl-methanol (CPMA), Glucose dehydrogenase and NADPH were prepared into a...

Embodiment 3

[0043] Prepare 0.5-5mM CPMK, benzophenone, phenylacetophenone, 4-chlorobenzophenone, 2,4'-dichlorodiphenylketone, 3,4'-dichlorodiphenylmethanone Ketone, 4,4'-dichlorodiphenyl ketone, acetophenone, 4-chloroacetophenone, 4-aminoacetophenone, 2-naphthophenone, 2-butanone, 2,3-butanedione Ketone, 2,3-pentanedione, 2,3-hexanedione, and 2,5-hexanedione solutions, pipette 100 μL of the solution into a 2 mL EP tube in turn, add 100 μL of 2,4-dinitrophenylhydrazine solution (20mM, containing 0.3% sulfuric acid), react in a shaker at 30°C, 120rpm for 30min, add 1mL KOH solution (1.0M) and 0.8mL deionized water, and measure the absorbance value of sixteen kinds of ketone samples at 500nm , plotting the linear relationship between different concentrations of ketone and the absorbance value (attached Figure 3-17 ). It can be seen that several aliphatic or aryl substituted ketones have a good linear relationship within a certain concentration range and the 500nm wavelength absorbance val...

Embodiment 4

[0044] Embodiment 4: Comparison of the consistency of chromogenic method and liquid phase detection method

[0045] The strain Kluyveromyces sp.CCTCC M2011385 preserved in the laboratory (preserved in China Center for Type Culture Collection (CCTCC), address: China, Wuhan, Wuhan University, 430072, preservation number NO.: M2011385, preservation date: November 11, 2011 ) is a strain that has been confirmed to contain alcohol dehydrogenase. With a final concentration of 2mM CPMK as the substrate and 2.5mM glucose as the auxiliary substrate, dissolve in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (10mL, 100mM) at pH 7.0, add Kluyveromycessp.CCTCC M2011385 wet cells (0.2g / 10mL ), sampled 100 μL every 20 min, added 100 μL 2,4-dinitrophenylhydrazine solution (20 mM, containing 0.3% sulfuric acid), reacted in a shaker at 30 ° C, 120 rpm for 30 min, and then added 1 mL KOH solution (1.0 M) and 0.8 mL of deionized water, and measure the absorbance of these sa...

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Abstract

The invention relates to a high throughput screening method of alcohol dehydrogenase, and provides a high throughput screening method for aliphatic series or aryl-substituted ketone alcohol dehydrogenase in 96-well plates. According to the method, aliphatic series or aryl substituted ketone is used for performing a chromogenic reaction with 2,4-dinitrophenylhydrazine under alkaline conditions. A deep-hole plate is used for performing micromation cell culture, no cell wall breaking processing needs to be performed, a traditional screening method of detecting additionally-added expensive coenzyme NAD(P)H absorbance changes is broken through, and the method has the advantages of being low in cost, easy and convenient to operate, sensitive in reaction, accurate in detection, low in equipment requirement and high in universality, and the activity and reaction conversion of alcohol dehydrogenase in a whole-cell sample can be rapidly detected.

Description

technical field [0001] The invention relates to an enzyme screening method, in particular to a high-throughput screening method for alcohol dehydrogenase, and belongs to the field of biotechnology. Background technique [0002] Chiral alcohols are recognized as very important and widely used intermediates in organic synthesis. Many drugs use chiral alcohols as the main building blocks, such as the antihistamine bepotastine besilate and the lipid-lowering drug Lipitor and the antiplatelet agglutinin clopidogrel. Asymmetric reduction of prochiral carbonyl compounds to synthesize chiral alcohols is the strategy with the highest atom economy and the most potential for application and development. [0003] Alcohol dehydrogenase (ADH) is an important oxidoreductase in organisms, which can reversibly catalyze the reduction of carbonyl groups and the oxidation of hydroxyl groups, and is widely used in the fields of biocatalysis and biomedicine. Alcohol dehydrogenase can catalyze c...

Claims

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Application Information

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IPC IPC(8): C12Q1/32C12Q1/02
CPCC12Q1/32C12Q1/02C12Q2304/00C12Q2326/00
Inventor 倪晔许国超周婕妤韩瑞枝董晋军
Owner JIANGNAN UNIV
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