Fluorescent detecting method for nitrile hydrolitic enzyme activity
A nitrilase and fluorescence detection technology, which is applied in the field of fluorescence detection of nitrilase activity, can solve the problems of lack of fluorescent probe detection theory and method, limited large-scale application, low sensitivity, etc., and achieves stable properties, simple equipment, high purity effect
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Embodiment 1
[0023] Take 3.90 g of salicylaldehyde and 1.86 g of hydroxylamine hydrochloride and add 0.066 mol of sodium hydroxide solution. Then the reaction mixture was stirred and reacted in a water bath at 50°C for 2 hours to obtain a uniform color (yellow) solution. The pH was adjusted with 36% acetic acid until it was neutral. When placed in an ice bath, there would be The crystals were separated out, filtered, washed with water, and air-dried to obtain 2.50 g (salicylic aldoxime) of the product. The yield of the product was 67.8%. Then, 1.00 g of salicylidene oxime and 0.50 g of acetic anhydride were added to the reactor, heated and stirred with an oil bath (160° C.) to reflux and reacted for 10 hours. After heating and refluxing, the reaction liquid is rotary distilled to distill off the unreacted acetic anhydride, and 10% NaOH solution is added to the remaining liquid after distillation until the liquid is a clear liquid, and then an appropriate amount of absolute ethanol is added. Th...
Embodiment 2
[0030] Add 2.00 g of 5-chlorosalicylic aldehyde and 1.00 g of hydroxylamine hydrochloride to 30 ml of NaOH solution (2mol / ml). (At this time, the resulting solution is not uniform and contains a small amount of solid particles. Therefore, in the reaction Add a small amount of absolute ethanol to the solution to dissolve a small amount of solid particles in the reaction solution) and stir the reaction mixture in a water bath at 50°C for 2 hours, then add 36% acetic acid to adjust the pH to neutral, white The powdery solid precipitates, and then the reactor is placed in an ice water bath to stand for a while, and then filtered. The white powdery solid obtained is 5-chlorosalicylic aldoxime, and the yield of this product is more than 90%. Then, 1.00 g of 5-chlorosalicylic aldoxime and 0.50 g of acetic anhydride were added to the reactor, heated and stirred with an oil bath (160° C.) to reflux, and reacted for 7 hours. After heating and refluxing, the reaction liquid is rotary distill...
Embodiment 3
[0036] Take 1.00g 5-fluorosalicylic aldehyde and 0.50g hydroxylamine hydrochloride and add them to 30ml NaOH (2mol / ml) solution, and stir the reaction mixture in a water bath at 50℃ for 2 hours, then add 36% acetic acid to adjust The pH is neutral, and a white powdery solid precipitates out. The reactor is then placed in an ice water bath for a while, and then filtered. The white powdery solid obtained is 5-fluorosalicylic aldoxime. The yield of this product is 95% . Then 1.00 g of 5-fluorosalicylic aldoxime and 0.50 g of acetic anhydride were added to the reactor, heated and stirred with an oil bath (160° C.) to reflux, and reacted for 8 hours. After heating and refluxing, the reaction liquid is rotary distilled to distill off the unreacted acetic anhydride, and 10% NaOH solution is added to the remaining liquid after distillation until the liquid is a clear liquid, and then an appropriate amount of absolute ethanol is added. The liquid was uniformly colored (yellow in color), an...
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