Screening and identification method of alcohol dehydrogenase based on NAD (Nicotinamide Adenine Diuncleotide) (P) (Phosphate) H (Hydrogen) fluorescence

A technology of alcohol dehydrogenase and identification method, which is applied in the field of screening and identification of alcohol dehydrogenase based on NAD(P)H fluorescence, can solve the problems of high reagent toxicity, cumbersome process, and many reagents, and achieve the interference factors of the reaction Less, simple operation process, high accuracy

Inactive Publication Date: 2012-06-27
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the traditional method, the NBT / PMS detection method has been improved. This method requires more reagents, the reagents used are more toxic and the process is more cumbersome.

Method used

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  • Screening and identification method of alcohol dehydrogenase based on NAD (Nicotinamide Adenine Diuncleotide) (P) (Phosphate) H (Hydrogen) fluorescence
  • Screening and identification method of alcohol dehydrogenase based on NAD (Nicotinamide Adenine Diuncleotide) (P) (Phosphate) H (Hydrogen) fluorescence
  • Screening and identification method of alcohol dehydrogenase based on NAD (Nicotinamide Adenine Diuncleotide) (P) (Phosphate) H (Hydrogen) fluorescence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Excitation and emission wavelengths of coenzymes NADH and NADPH

[0033] The reduced coenzyme NADH and NADPH, the oxidized coenzyme NAD + and NADP + Prepare aqueous solutions with different concentration gradients from 0.01 to 1mM respectively, take 300μl and put them into a 96-well microplate, measure the fluorescence intensity at the excitation wavelength of 360nm and emission wavelength of 460nm, the results are shown in figure 1 . It can be seen from the figure that at the excitation wavelength of 360nm and the emission wavelength of 460nm, the reduced coenzyme has fluorescence, while the oxidized coenzyme does not emit fluorescence.

[0034] Prepare 0.5mM NADH and NADPH aqueous solution, set the excitation wavelength to 360nm, measure the fluorescence value in the emission spectrum range of 400-500nm, and obtain the fluorescence spectrum diagram, see figure 2 . Depend on figure 2 It can be seen that the optimum emission wavelength of NADH and NADP...

Embodiment 2

[0035] Example 2: Relationship between temperature, pH, organic solvent, substrate, etc. and NAD(P)H fluorescence

[0036] (1) The relationship between temperature and NAD(P)H fluorescence

[0037] Prepare 0.5mM NAD(P)H aqueous solution, take 300μl and fill it into a 96-well microwell plate, place it overnight in a temperature gradient of 4-70°C for 16h, and seal it with plastic wrap during the period to prevent volatilization from affecting the fluorescence value. The fluorescence values ​​were measured at the optimum wavelength, and the results are shown in Figure 4 . Depend on Figure 4 It can be seen that the temperature has basically no effect on the fluorescence value of NADH within this range; the autofluorescence of NADPH is weakened at high temperature, but according to Example 1 figure 1 As a result of the analysis, its fluorescence was still significant.

[0038] (2) The relationship between pH and NAD(P)H fluorescence

[0039] Prepare 0.5mM NAD(P)H solution wit...

Embodiment 3

[0045] Example 3: Using 1-phenylethanol as a substrate for rapid screening of alcohol dehydrogenase-producing bacteria

[0046] Take 150 μl of 1-phenylethanol and spread it on the LB plate, and absorb it for a while, which is the enrichment plate. Weigh 0.5 grams of soil samples (from Hubei, Inner Mongolia, Shandong, etc.), place them in a 2ml centrifuge tube (containing 1ml of sterile water), dilute 1000 times with sterile water, and take 100μl to spread evenly on the enrichment plate. Incubate at 30°C for 48h. Then, colonies with different shapes on the plate were selected and inoculated on the LB slant, and cultured at 30°C for 24h. Take 2 inoculum loops of bacteria from the obtained slant and put them in 60 μl of non-sterile water, and shake to make a bacterial suspension for primary screening. The total volume of the reaction system is 300 μl, including glycine-NaOH buffer solution (total concentration 50 mM) at pH 10.0, 0.5% (V / V) swirling 1-phenylethanol, 1.0 mM NAD ...

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Abstract

The invention provides a fast screening and identification method of alcohol dehydrogenase based on NAD (Nicotinamide Adenine Diuncleotide) (P) (Phosphate) H (Hydrogen) fluorescence, which comprises the following steps of: based on the characteristic of the alcohol dehydrogenase taking NAD or NADP as coenzyme, taking a 96-pore micro-pore plate as a reaction vessel, adding a sample to be detected in a reaction system to oxidize alcohol, converting oxidized coenzyme incapable of transmitting the fluorescence into reduced coenzyme NADH or NADPH capable of transmitting the fluorescence under the excitation of a certain exciting light while the alcohol is oxidized and screening alcohol dehydrogenase producing strains through determining the change of fluorescence intensity of reaction liquid. The invention has the beneficial effects that compared with the existing alcohol dehydrogenase producing strain screening method, the fast screening and identification method has the characteristics of short time consumed, high accuracy, fewer reagent required and high throughput screening; and moreover, the reaction system is simple, the operation process is simple and convenient, and the interference factors in the reaction are less.

Description

(1) Technical field [0001] The invention relates to a method for screening and identifying alcohol dehydrogenases based on NAD(P)H fluorescence, which is applicable to all alcohol dehydrogenases with NAD(H) or NADP(H) as coenzymes. (2) Background technology [0002] Alcohol dehydrogenases (ADHs, E.C.1.1.1.1) are enzymes that use NAD(H) or NADP(H) as a coenzyme to transfer electrons and catalyze the oxidation of alcohols or the reduction of ketones and aldehydes. Alcohol dehydrogenase is widely found in microorganisms such as bacteria, yeast and mold, and has important physiological significance in intracellular metabolism. Alcohol dehydrogenase is also an important class of biocatalysts, which can prepare pharmaceutical intermediates-chiral alcohols through selective oxidation or asymmetric reduction, and are being used more and more in the fields of biochemical and industrial catalysis. [0003] At present, the methods for screening alcohol dehydrogenase mainly include the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C12Q1/04G01N21/64
Inventor 应向贤汪钊杨池熊斌
Owner ZHEJIANG UNIV OF TECH
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