A screening method for β-hydroxycarboxylate hydroxyl oxidase
A technology of hydroxycarboxylate hydroxyoxidase and screening method, applied in the biological field, can solve the problems of complicated operation, expensive equipment and the like, and achieve the effects of convenient operation and accurate flux
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Embodiment 1
[0013] Example 1: FeCl 3 Chromogenic solution analysis
[0014] Weigh Rhodococcus Rhodococcus sp. CCZU10-1 resting cells (wet weight) were suspended in 2 mL of phosphate buffer solution system (pH 7.0, 100 mmol / L) to form a 10% (mass concentration) cell suspension, and the final concentration of 100 mmol / L was added The substrate 4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester was mixed evenly and then oxidative transformation reaction was carried out at 30°C and 160 r / min for 6 h. After the reaction was completed, an equal volume of ethyl acetate solution was added for extraction, and further color development was carried out in a 24-well plate. Extraction solution: color developer A (FeCl 3 Solution; 0.04 mol / L): Chromogenic agent B (HCl; 0.1 mol / L)=20:800:800 (μL: μL: μL), shaking reaction at 20°C for 30 min, using 9 times After the volume of absolute ethanol was mixed, measure its UV absorption value at 510 nm wavelength (standard curve Y 底物(mmol / L) =49.25×OD 510 ;...
Embodiment 2
[0015] Example 2: Fe 2 (SO 4 ) 3 Chromogenic solution analysis
[0016] Weigh Rhodococcus Rhodococcus sp. CCZU10-1 resting cells (wet weight) were suspended in 2 mL of phosphate buffer solution system (pH 7.0, 100 mmol / L) to form a 10% (mass concentration) cell suspension, and the final concentration of 100 mmol / L was added The substrate 4,4,4-trifluoro-3-hydroxybutyric acid ethyl ester was mixed evenly and then oxidative conversion reaction was carried out at 30°C and 160 r / min for 6 h. After the reaction was completed, an equal volume of ethyl acetate solution was added for extraction, and further color development was carried out in a 24-well plate. Extraction solution: color developer A (Fe 2 (SO 4 ) 3 solution; 0.08 mol / L): Chromogen B (H 2 SO 4 ; 0.3 mol / L)=20:800:800 (μL: μL:μL), shake the reaction at 35 ℃ for 15 min, mix the color reaction solution with 9 times the volume of absolute ethanol, and measure its wavelength at 510nm Under the UV absorbance value (...
Embodiment 3
[0017] Example 3: Fe(ClO 4 ) 3 Chromogenic solution analysis
[0018] Weigh 0.1 g Rhodococcus Rhodococcus Sp. CCZU10-1 (CGMCC No. 4911) resting cells (wet weight) were suspended in 2 mL phosphate buffer solution system (pH 7.0, 100 mmol / L), and substrate 4,4 was added to a final concentration of 100 mmol / L , Ethyl 4-trifluoro-3-hydroxybutyrate, after mixing evenly, carry out the oxidation conversion reaction at 30°C and 160 r / min for 6 hours. Add an equal volume of ethyl acetate solution after the reaction to extract, and further develop color in a 24-well plate, extracting solution: chromogenic agent A (Fe(ClO 4 ) 3 solution; 0.07 mol / L): Chromogen B (HClO 4 ; 0.1 mol / L)=20:800:800 (μL: μL: μL), shake the reaction at 30°C for 20 min, mix the color reaction solution with 9 times the volume of absolute ethanol, and measure its wavelength at 510 nm UV absorption value at wavelength (Y 底物（mmol / L） =666.67×OD 510 ; 2 =0.9923). The conversion rate of the product 4,4,4-tri...
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