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Monooxygenase MpdA, coding gene mpdA thereof and application of monooxygenase MpdA and coding gene mpdA thereof in synthesis of vitamin E precursor

A monooxygenase, encoding gene technology, applied in the high field of biology, can solve problems such as incompetent specific synthesis

Active Publication Date: 2021-11-05
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological synthesis requires specific microorganisms or enzymes, but so far there is no microorganism or enzyme that can specifically synthesize 2,3,5-trimethylhydroquinone

Method used

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  • Monooxygenase MpdA, coding gene mpdA thereof and application of monooxygenase MpdA and coding gene mpdA thereof in synthesis of vitamin E precursor
  • Monooxygenase MpdA, coding gene mpdA thereof and application of monooxygenase MpdA and coding gene mpdA thereof in synthesis of vitamin E precursor
  • Monooxygenase MpdA, coding gene mpdA thereof and application of monooxygenase MpdA and coding gene mpdA thereof in synthesis of vitamin E precursor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The cloning of embodiment 1 gene mpdA

[0032] (1) Extraction and sequencing of bacterial genome total DNA

[0033]The strain Mycobacterium neoaurum B5-4 and its mutant strain Mycobacterium neoaurum B5-4M were respectively cultured in large quantities in liquid LB medium (30°C, 200rpm, 48h), and then the total genomic DNA of the two strains with high purity and large fragments was extracted by CTAB method. Dissolve them in deionized water respectively and store them at -20°C. For specific methods, refer to the "Refined Molecular Biology Experiment Guide" edited by F. Osper et al. The genomic DNA of the obtained two bacterial strains was entrusted to a sequencing company for sequencing.

[0034] (2) Functional identification of gene mpdA

[0035] By comparing and analyzing the genomes of the strain Mycobacterium neoaurum B5-4 and its mutant strains, it was found that a DNA fragment of about 23kb was missing in the mutant strain. This fragment was analyzed in combinatio...

Embodiment 2

[0037] Example 2 The whole cell catalytic method synthesis of 2,3,5-trimethylhydroquinone

[0038] (1) Construction of engineering strains

[0039] Using the total DNA of strain B5-4 as a template, use forward primer (SEQ ID NO.3): 5'-ACCGAGCTCAGATCTACTAGTATGCAATTTTCCAAAGTTGG-3' and reverse primer (SEQ ID NO.4): 5'-ACACTGGCGGCCGTTACTAGTTCACAGCCACGGGGTATTCGG-3' to amplify out of the gene mpdA. PCR amplification program: denaturation at 95°C for 3 min; denaturation at 95°C for 1.5 min, annealing at 53°C for 0.5 min, extension at 72°C for 1.5 min, and 25 cycles; extension at 72°C for 10 min, and cooling to room temperature. The obtained gene mpdA fragment was introduced into the SpeI restriction site of the plasmid pRESQ to construct the recombinant plasmid pMPDA; then, the recombinant plasmid was transformed into the strain Rhodococcus qingshengii YL-1 (CCTCC AB 2017132) (Li C, Zhang J, WuZG, Cao L, Yan X, Li SP.2012.Biodegr adation of buprofezin by Rhodococcussp.strain YL-1is...

Embodiment 3

[0042] Example 3 The enzyme-catalyzed synthesis of 2,3,5-trimethylhydroquinone

[0043] (1) Preparation of crude enzyme solution

[0044] Strain YL-mpdA was cultured to the logarithmic phase in LB medium, collected by centrifugation at 5000rpm for 10min, washed twice with PBS buffer, resuspended in 10ml of PBS buffer, and ultrasonically broken in an ice-water bath for 10-15 minutes (AutoScience, UH-650B ultrasonic processor, 30% intensity), centrifuged at 12000rpm for 30min, and collected the supernatant, which was the prepared crude enzyme solution.

[0045] (2) Functional determination of 2,3,6-trimethylphenol catalyzed by crude enzyme solution

[0046] The reaction system of 2,3,6-trimethylphenol catalyzed by crude enzyme solution is: 0.5mM 2,3,6-trimethylphenol, 0.2mM NADH, 0.02mM FAD, 10ml of crude enzyme solution is added. After reacting at 30°C for 2 hours, an equal volume of dichloromethane was added to terminate the reaction and the product was extracted by vigorous...

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Abstract

The invention discloses monooxygenase MpdA, a coding gene mpdA thereof and application of the monooxygenase MpdA and the coding gene mpdA thereof in synthesis of a vitamin E precursor. An amino acid sequence of the MpdA is SEQ ID NO. 2, the whole length of the amino acid sequence comprises 413 amino acids, a nucleotide sequence of the coding gene of the MpdA is SEQ ID NO.1, and the whole length of the nucleotide sequence is 1242bp. The MpdA is a monooxygenase which is found for the first time and can catalyze the conversion of 2,3,6-trimethylphenol into the vitamin E precursor 2,3,5-trimethylhydroquinone. The 2,3,6-trimethylphenol can be efficiently converted into the 2,3,5-trimethylhydroquinone by using a MpdA-containing engineering bacterium cell or MpdA-containing enzyme liquid. The 2,3,5-trimethylhydroquinone is one of two precursors for synthesizing the vitamin E, and the 2,3,6-trimethylphenol monooxygenase MpdA and the coding gene mpdA thereof have huge application potential in synthesis of the vitamin E precursor 2,3,5-trimethylhydroquinone by a biological method.

Description

technical field [0001] The invention belongs to the field of biological high technology, and relates to monooxygenase MpdA and its encoding gene mpdA and the application of both in the synthesis of vitamin E precursor. Background technique [0002] Vitamin E has various physiological functions such as anti-oxidation, promoting the secretion of sex hormones, and reducing cardiovascular diseases, and is widely used in food, health care products, cosmetics, clinical, pharmaceutical and other industries. As the demand for vitamin E continues to rise, it is far from being able to meet people's needs only by extracting it from natural plant leaves or seeds. More than 80% of vitamin E on the market comes from industrial synthesis. [0003] At present, the main route of industrial synthesis of vitamin E is the condensation of 2,3,5-trimethylhydroquinone and isophytic alcohol. The biofermentative synthesis of isophytic alcohol has been successfully commercialized in recent years, bu...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/74C12N1/21C12P7/66C12R1/01
CPCC12N9/0073C12N15/74C12P7/66Y02A50/30
Inventor 闫新纪俊宾
Owner NANJING AGRICULTURAL UNIVERSITY
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