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A high-throughput screening method for trans-anethole oxygenase mutants, mutant strains and mutants
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A brain oxygenase and mutant technology, applied in the field of bioengineering, can solve the problem of low catalytic efficiency, achieve good application value and improve catalytic efficiency
Active Publication Date: 2021-08-24
JIANGNAN UNIV
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[0003] Trans-anethole oxygenase (TAO), first discovered by Han et al. in Pseudomonas putidaJYR-1, the enzyme can oxidize trans-anethole to synthesize p-anisaldehyde, and can also oxidize isoeugenol , isosafrole and p-methoxyisoeugenol, but the catalytic efficiency is not high, E.coli BL21(DE3)(pET-TAO) contained 0.5mmol·L -1 Glucose and 1mmol·L -1 0.63mmol·L -1 p-anisaldehyde and vanillin, 0.38mmol·L -1 jasminal and veratraldehyde
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Embodiment 1
[0029] Establishment of high-throughput screening methods:
[0030] (1) Effect of different alkali solutions on color development
[0031] Measure 1mol·L respectively -1 The influence of NaOH-50% ethanol solution and KOH-50% ethanol solution on color development, record OD 470 and stability (OD within 1h 470 The ratio of the absolute value of the maximum difference to the current use value), as shown in Table 1.
[0032] Table 1 The influence of different alkali solutions on color development
[0033] lye NaOH KOH OD 470
2.86 1.814 stability 0.016 0.055
[0034] The results show that the OD using NaOH-50% ethanol solution 470 Higher response and better stability.
[0035] (2) Effect of different alkali concentrations on color development
[0036] Measure 0.5~3.0mol·L respectively -1 The effect of NaOH on color development, record OD 470 and stability, as shown in Table 2.
[0037] Table 2 Effects of different alkali concentrations ...
Embodiment 2
[0052] Taking the synthetic product jasmonal as an example, take 50 μL of jasmonal standard solution (concentration is 0-1000 mg L -1 ), add 50~300μL of 2,4-dinitrophenylhydrazine developer, react at 10~40℃ for 5~40min, add 0.5~3mol·L -1 NaOH-(0~80%) ethanol solution, reacted at 10~40°C for 5~60min, took it out, and measured its absorbance value OD by microplate reader 470 , with the jasminal concentration as the abscissa, and the OD470 value as the ordinate, draw a standard curve, and obtain the calculation formula as y=0.00496x+0.09445, R 2 is 0.9995, and the detection concentration range of jasmonal is 0~700mg·L -1 . This method can be used as a high-throughput screening method.
Embodiment 3
[0054] The establishment of the tao mutation library:
[0055] (1) Error-prone PCR to establish a tao mutation library
[0056] According to the trans-anethole oxygenase gene in Pseudomonas putida JYR-1, tao was synthesized and connected to PGEX-6P-1 to obtain the recombinant plasmid PGEX-6P-1-tao, which was used as a template to design error-prone PCR primers taoF, taoD, set Mg 2+ Concentration, Mn 2+ Concentrations are 7~10mmol·L -1 And 30~250μmol·L -1 , error-prone PCR using common rTaq polymerase. Primers are as follows:
[0061] Use nucleic acid gel electrophoresis to detect PCR products, purify and recover the PCR products, and then use a one-step cloning kit to connect to the linear vector PGEX-6P-1, and transform into E.coli BL21 (DE3) super competent, spread culture , picked a single ...
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Abstract
The invention discloses a high-throughput screening method for mutants of trans-anethole oxygenase, mutant strains and mutants, including performing error-prone PCR with trans-anethole oxygenase gene as a template to obtain trans-anethole Brain oxygenase mutant library; the trans-anethole oxygenase reaction substrate is added into the solution containing the trans-anethole oxygenase mutant for catalytic reaction, and the reaction solution is mixed with 2,4-dinitrate Base phenylhydrazine for color reaction, and add alkali solution and organic solvent to react, and measure the absorbance. In the present invention, a mutation library is constructed by error-prone PCR, and 2,4-dinitrophenylhydrazine is used as a color reagent, and the OD is measured by a microplate reader. 470 Based on the high-throughput screening method, the mutant strains for the synthesis of jasmonal recombinant strains are screened. This method has the advantages of high efficiency, precision and speed. The catalytic efficiency is significantly improved and has good application value.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-throughput screening method for trans-anethole oxygenase mutants, mutant strains and mutants. Background technique [0002] Acrylbenzene type essential oil is a kind of acrylbenzene compound extracted from plant essential oil by physical method. The common feature of this type of compound is that it has a propenyl side chain and a methoxy substituent on the benzene ring. Group, often used as a starting material for the synthesis of valuable spices and drugs with a benzene ring structure (such as vanillin, jasminal, anisaldehyde, etc.). Due to the existence of chemically synthesized fragrances in the production of fragrances: the residual chemical substances in the fragrance compounds are harmful to human health, chemical synthesis will bring environmental pollution, and the stereoselectivity of chemical synthesis reactions is poor, resulting in the produ...
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