High-throughput screening method of nitrile invertase

A technology for nitrile converting enzymes and screening methods, which is applied in the field of high-throughput screening of nitrile converting enzymes, can solve the problems of poor versatility, high equipment requirements, time-consuming and laborious, etc., and achieve the effects of simple operation, rapid detection, and convenient rapid screening

Inactive Publication Date: 2010-09-22
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In order to overcome the shortcomings of the traditional nitrile hydratase screening model, such as time-consuming and labor-intensive screening, high equipment requirements, and poor versatility, the present invention provides a high-throughput screening method for nitrile converting enzymes based on complexation color reaction. Not only for high-throughput screening of nitrile hydratases, but also for rapid screening of amidases and nitrilases

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  • High-throughput screening method of nitrile invertase
  • High-throughput screening method of nitrile invertase
  • High-throughput screening method of nitrile invertase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Screening of nitrile hydratase-producing bacteria containing 2-amino-2,3-dimethylbutyronitrile hydration activity

[0038] After culturing the microorganisms to be screened, they were centrifuged, washed with normal saline, evenly suspended in distilled water, and prepared as OD 600 =10 bacterial suspension. Take 5ml of the bacterial suspension and place it in a stoppered Erlenmeyer flask (50ml), then put it in a 30°C water bath to preheat for 5min, and add 20μl of 2-amino-2,3-dimethylbutyronitrile to start the reaction. Samples were taken after 15 minutes, and centrifuged at 10,000 rpm for 5 minutes. Take 100 μl of the supernatant in a 96-well plate, and add FeSO with a concentration of 100 mM successively 4 and FeCl 3 100 μl of each aqueous solution, and observe the color change. Joined FeSO successively 4 and FeCl 3 After aqueous solution, the experimental group whose supernatant first changed from colorless to light green and then to yellow containe...

Embodiment 2

[0040] Example 2: Screening of nitrile hydratase-producing bacteria containing β-aminopropionitrile hydration activity

[0041] After culturing the microorganisms to be screened, make a bacterial suspension according to the method of Example 1, take 10ml of the bacterial suspension and place it in a stoppered Erlenmeyer flask (50ml), then put it in a water bath at 20°C for preheating for 10min, add 30μl of β -Aminopropionitrile starts to react. Samples were taken after 10 minutes, and centrifuged at 12,000 rpm for 4 minutes. Take 200 μl of the supernatant in a 96-well plate, and add FeCl with a concentration of 1.0M successively 2 and Fe(NO 3 ) 3 20 μl of each aqueous solution, and observe the color change. Add FeCl successively 2 and Fe(NO 3 ) 3 After aqueous solution, the experimental group whose supernatant first changed from colorless to light green and then to yellow contained nitrile hydratase.

[0042] Controlled by colorimetry, the strains Rhodococcus sp.ZJB-09...

Embodiment 3

[0043] Example 3: Screening of amidase-producing bacteria containing 2-aminobutyramide hydrolysis activity

[0044] After culturing the microorganisms to be screened, make a bacterial suspension according to the method of Example 1, take 7ml of the bacterial suspension and place it in a stoppered Erlenmeyer flask (50ml), then put it into a water bath at 40°C for preheating for 4min, add 20 μl of 2 -Aminobutanamide starts to react. Samples were taken after 40 min, and centrifuged at 10,000 rpm for 6 min. Take 180 μl of the supernatant in a 96-well plate, and add FeCl at a concentration of 200 mM 2 and Fe(NO 3 ) 3 60 μl of each aqueous solution, and observe the color change. Add FeCl successively 2 and Fe(NO 3 ) 3 After aqueous solution, the supernatant does not undergo a significant color change and eventually shows Fe 3+ The yellow test group of the ionic solution contains amidase.

[0045] The strain Delftia tsuruhatensis ZJB-05174 (CCTCC No: M 205114) can catalyze t...

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Abstract

The invention provides a high-throughput screening method of nitrile invertase, which comprises the following steps of: dissolving a sample to be tested in distilled water; adding a substrate namely an amino-nitrile compound, a hydroxy-nitrile compound or an amide compound into the mixture until the concentration of the substrate is 1 to 100 mM; performing a conversion reaction in a water bath at the temperature of between 10 and 50 DEG C for 10 to 120 minutes; taking the conversion liquid, adding Fe2<+> ion aqueous solution and Fe3<+> ion aqueous solution into the conversion liquid successively to perform a color reaction, wherein the quantity ratio of the added substrate to the Fe2<+> to the F3<+> is 1:1-3:1-3, the time interval for adding the two ions is 0 to 10 minutes; and judging the type of the contained nitrile invertase according to color changes of the solution. The complex color reaction-based high-throughput screening method of the nitrile invertase can better overcome the defects of the conventional screening method, can quickly identify whether the tested sample has the nitrile hydratase, amidase or nitrilase through simple color contrasts, has the advantages of simple and convenient operation, quick detection, economical efficiency, practicability and the like, and greatly facilitates the quick screening of the nitrile hydratase, the amidase or the nitrilase.

Description

(1) Technical field [0001] The invention relates to a high-throughput screening method for nitrile converting enzymes. (2) Background technology [0002] Nitrile compounds are important pharmaceutical, pesticide and fine chemical intermediates. There are three ways of microbial degradation of nitriles: oxidation, reduction and hydrolysis. The latter can be divided into two classes according to the different enzyme systems involved in the hydrolysis reaction: the one is nitrilase (nitrilase, E.C.3.5.5.1), which directly hydrolyzes nitrile into carboxylic acid and ammonia; Under the action of nitrile hydratase, E.C.4.2.1.84), nitrile is hydrated to form amide. If there is amidase (amidase, E.C.3.5.1.4) in the microorganism, carboxylic acid and ammonia will be further generated under its action, and the reaction needs to be completed in two steps (BiotechLett.1994, 16:47-50), such as figure 1 shown. [0003] At present, the industrial application of nitrile hydratase (NHase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/527C12Q1/34G01N21/78
Inventor 郑裕国林志坚郑仁朝雷利华戴昌龙沈寅初
Owner ZHEJIANG UNIV OF TECH
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