A high -throughput filtering method of a tyrosine phenol lobic enzyme high -vitality strain

A technology of tyrosine phenol and screening method, which is applied in the direction of biochemical equipment and method, measuring device, microorganism determination/inspection, etc., can solve the problem of large number of mutants in the mutation library, and achieve rapid detection, simple operation, and easy The effect of mechanized automation

Active Publication Date: 2021-10-15
ZHEJIANG UNIV OF TECH
View PDF10 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the wide variety of tyrosine phenol lyases in nature, the number of mutant library mutants obtained by enzyme molecular transformation is huge (usually containing 10 4 ~10 6 mutants), it is difficult to efficiently screen for tyrosine phenol lyases with improved catalytic performance by traditional detection methods such as chromatography

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A high -throughput filtering method of a tyrosine phenol lobic enzyme high -vitality strain
  • A high -throughput filtering method of a tyrosine phenol lobic enzyme high -vitality strain
  • A high -throughput filtering method of a tyrosine phenol lobic enzyme high -vitality strain

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a high-throughput screening method for strains with high activity of tyrosine phenol lyase. The method is as follows: adding the wet thallus obtained by fermentation and cultivation of the strain to be tested into the substrate reaction liquid as a sample of the strain to be tested , the reaction solution was centrifuged on a shaking table to obtain a supernatant; the supernatant was added to the color reaction solution, left to stand at room temperature for color development, and the absorbance was measured at 465nm. According to the standard curve of sodium pyruvate, the pyruvate in the supernatant was obtained. content of sodium, and then obtain the activity of tyrosine phenol lyase in the strain sample to be tested, thereby screen out the strain with high activity of tyrosine phenol lyase; With the improved tyrosine phenol lyase and its mutants, the time required for analyzing one sample is shortened from 20 minutes to only 1 minute for analyzing nearly 60 samples, and the error of the measured enzyme activity is controlled within 3%.

Description

(1) Technical field [0001] The invention relates to a high-throughput screening method for tyrosine phenol lyase and mutants thereof with improved levodopa synthesis ability. (2) Background technology [0002] Tyrosine phenol-lyase (TPL), also known as β-tyrosinase, is a kind of pyridoxal-5'-phosphate (PLP)-dependent lyase. TPL can catalyze the α,β-elimination reaction of L-tyrosine to generate pyruvate, phenol and ammonia. This reaction is a reversible reaction. If catechol is used instead of phenol, the enzyme can catalyze and generate levodopa. The catalytic mechanism can be expressed by the following formula: [0003] [0004] Levodopa is an important active substance in biological organisms and is the main drug for the treatment of Parkinson's disease. The principle of its treatment of Parkinson's disease is: dopamine, a derivative of levodopa, is an important neurotransmitter for the treatment of Parkinson's disease, but it cannot pass through the blood-brain barr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityPatents(China)
IPC IPC(8): C12Q1/527C12Q1/04C12N15/70
CPCC12N15/70C12Q1/527G01N2333/988
Inventor汤晓玲郑仁朝郑裕国索慧刘潇
OwnerZHEJIANG UNIV OF TECH