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Trans-anethole oxygenase mutant, and mutant strain

A technology of trans-anethole and brain oxygenase, applied in the field of bioengineering, can solve the problems of low catalytic efficiency and achieve the effect of improving production efficiency and reducing costs

Active Publication Date: 2018-11-06
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[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 In different substrate solutions, reacted for 6h, can synthesize 0.63mmol·L-1 p-anisaldehyde and vanillin, 0.38mmol·L -1 jasminal and veratraldehyde

Method used

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  • Trans-anethole oxygenase mutant, and mutant strain
  • Trans-anethole oxygenase mutant, and mutant strain
  • Trans-anethole oxygenase mutant, and mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Establishment of high-throughput screening methods:

[0026] (1) Effect of different alkali solutions on color development

[0027] 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.

[0028] Table 1 The influence of different alkali solutions on color development

[0029] lye

NaOH

KOH

OD 470

2.86

1.814

stability

0.016

0.055

[0030] The results show that the OD using NaOH-50% ethanol solution 470 Higher response and better stability.

[0031] (2) Effect of different alkali concentrations on color development

[0032] 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.

[0033] Table 2 Effects of different alkali c...

Embodiment 2

[0048] 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

[0050] The establishment of the tao mutation library:

[0051] (1) Error-prone PCR to establish a tao mutation library

[0052] 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:

[0053] taoF: ttccaggggcccctgggatccGGATCCATGGAGGACATCATGC

[0054] taoD: gtcacgatgcggccgctcgagCTCGAGTCAGTTAGTCCTCAAGTCG

[0055] The PCR program was set as follows:

[0056]

[0057] 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 trans-anethole oxygenase mutant, and a mutant strain. When the amino acid sequence of the trans-anethole oxygenase mutant is compared with the amino acid sequence of wild-type trans-anethole oxygenase, it is found that the 319th site amino acid proline is replaced by serine, and the amino acid sequence of the trans-anethole oxygenase mutant is represented by SEQ ID NO 1.According to a construction method, error-prone PCR is adopted, random mutation of trans-anethole oxygenase is realized, introduction into E.Coil BL21(DE3) is carried out, and the mutant strain 4C7 isobtained through screening. The conversion rates of the trans-anethole oxygenase mutant on anisaldehyde and veratraldehyde are 2.05 and 2.38 times of those of an original bacterial strain; the trans-anethole oxygenase mutant is more suitable for industrialized production; production cost is reduced; and production efficiency is increased obviously.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a trans-anethole oxygenase mutant and a mutant strain thereof. 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 product being a racemate, etc. defect. At...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/70C12N1/21C12R1/19
CPCC12N9/0083
Inventor 王玉琴郑璞刘思琴陈鹏程闻鹏
Owner JIANGNAN UNIV
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