High-flux screening method for high-activity bacteria strain of tyrosine phenol-lyase
A technology of tyrosine phenol and screening method, which is applied in biochemical equipment and methods, measuring devices, microbial determination/inspection, etc., can solve the problem of large number of mutants in the mutation library, and achieve rapid detection, easy mechanized and automatic operation , the effect of easy operation
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Embodiment 1
[0031] Correspondence between embodiment 1 sodium pyruvate concentration and absorbance value
[0032] Prepare 0-100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM) sodium pyruvate solutions with ultrapure water. Reaction system 10mL, successively add 1mL 250g / L NaOH aqueous solution, 200μL sodium pyruvate solution of different concentrations, 6.7mL ultrapure water, 100μL salicylaldehyde, 2mL 250g / L NaOH aqueous solution, among them shake well after adding salicylaldehyde Make it fully react with sodium pyruvate to develop color. Leave it at room temperature for 2 h, and measure the absorbance at 465 nm with a microplate reader. Take the absorbance as the ordinate, and the concentration of sodium pyruvate as the abscissa, draw the absorbance curve, the result is as follows figure 2 . The higher the concentration of sodium pyruvate, the greater the absorbance value, and there is a linear relationship between the two: Y=0.0434X+0.3046, R 2 = 0.9998.
Embodiment 2
[0033] The determination of embodiment 2 color reaction time
[0034] Prepare 0-100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM) sodium pyruvate solutions with ultrapure water. To a reaction system of 10 mL, add 1 mL of 250 g / L NaOH aqueous solution, 200 μL of sodium pyruvate solutions of different concentrations, 6.7 mL of ultrapure water, 100 μL of salicylaldehyde, and 2 mL of 250 g / L NaOH aqueous solution, and mix well. Place it at room temperature, take samples every 10 minutes and measure the absorbance at 465 nm with a microplate reader, and the reaction time is 2 hours. Take the absorbance as the ordinate, and the color reaction time as the abscissa, draw the absorbance curve, the result is as follows image 3 . It shows that the absorbance value tends to be stable after more than 1 hour of color reaction.
Embodiment 3
[0035] Under the embodiment 3 without thalline reaction system, the corresponding relationship between the concentration of sodium pyruvate and the absorbance value
[0036] The final concentration of the substrate reaction solution (pH 7.0-8.0) consists of: catechol 5g / L, sodium pyruvate (0, 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40g / L ), ammonium acetate 50g / L, sodium sulfite 1g / L, EDTA-2Na2g / L, pyridoxal phosphate (PLP) 1mM, and the solvent is ultrapure water. Different concentrations of sodium pyruvate were reacted sequentially at 30°C and 150 rpm on a shaker for 30 minutes, and 400 μL of 1M HCl was added to terminate the reaction to obtain reaction solutions of different concentrations of sodium pyruvate.
[0037] The color reaction steps are as follows: 10 mL of color reaction system, 1 mL of 250 g / L NaOH aqueous solution, 200 μL of reaction solutions with different concentrations of sodium pyruvate, 6.7 mL of ultrapure water, 100 μL of salicylaldehyde, 2 mL of 250 g / L NaOH aqu...
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