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Isoeugenol monooxygenase mutant with high thermal stability and high activity and application of isoeugenol monooxygenase mutant

A high thermal stability, isoeugenol technology, applied in the field of bioengineering, can solve the problems of poor thermal stability of IEM, unable to meet industrial production and other problems

Active Publication Date: 2021-07-23
CHINA FORTUNE WAY CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] After combining and mutating the above three amino acids, a three-site mutant with significantly improved thermal stability and specific activity was obtained, which solved the existing IEM has poor thermal stability and cannot meet the problems of industrial production

Method used

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  • Isoeugenol monooxygenase mutant with high thermal stability and high activity and application of isoeugenol monooxygenase mutant
  • Isoeugenol monooxygenase mutant with high thermal stability and high activity and application of isoeugenol monooxygenase mutant
  • Isoeugenol monooxygenase mutant with high thermal stability and high activity and application of isoeugenol monooxygenase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of Isoeugenol Monooxygenase Gene and Construction of Recombinant Engineering Bacteria

[0044] According to the protein NCBI library, a section of isoeugenol monooxygenase gene (the amino acid sequence is shown in NCBI accession number ACP17973.1 and the nucleotide sequence is shown in NCBI accession number FJ851547) was obtained from Pseudomonas nitroreductors Jin1 ), and submitted to Shanghai Jierui Bioengineering Co., Ltd. for synthesis, the amino acid sequence of this isoeugenol monooxygenase IEM is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.7 . By upstream primer 5'-CGC CATATG ATGGCGAGACTCAACCGCAAC-3' (underlined base is the recognition site of restriction endonuclease Nde I) and downstream primer 5'-CCG CTCGAG TTAAGGTCTGGGTACCCAGCA-3' (the underlined base is the restriction endonuclease Xho I recognition site) amplifies the target gene, and PCR amplification uses the PrimeSTAR Max high-fidelity polymerase of Biotech (Beijin...

Embodiment 2

[0049] Construction of isoeugenol monooxygenase mutants based on whole plasmid PCR

[0050] Using the recombinant plasmid pET21a-IEM as a template, the plasmid containing the mutant gene was amplified in vitro by PCR.

[0051] The primers used for site-directed mutagenesis are (the underline is the mutation site):

[0052] K83R primer-F:5'-CATCAGTCGCTGGGTT AGG ACCGCTCGATTCACG-3'

[0053] K83R primer-R:5'-CGTGAATCGAGCGGT CCT AACCCAGCGACTGATG-3'

[0054] K95R primer-F:5'-GAACGACTAGCGCGA AGA TCGCTATTTGGCATG-3'

[0055] K95R primer-R:5'-CATGCCAAATAGCGA TCT TCGCGCTAGTCGTTC-3'

[0056] L273F primer-F:5'-GTCAGATTCGCTGG TTC AAGGCACCGGCGCTC-3'

[0057] L273F primer-R:5'-GAGCGCCGGTGCCTT GAA CCAGCGAATCTGAC-3'

[0058] PCR reaction system is as follows: (primer concentration is 10 μ mol / L)

[0059]

[0060] The PCR amplification program was: pre-denaturation at 95°C for 10 min; denaturation at 98°C for 10 s, annealing at 56°C for 15 s, extension at 72°C for 75 s, 24 c...

Embodiment 3

[0065] Inducible expression of parental and mutant strains

[0066] Plate activation: use an inoculation loop to dip the glycerol tube bacterial solution, line the LB-resistant solid plate (containing 100 μg / mL Amp) in four areas, and incubate it upside down at 37°C for 12 hours in a constant temperature incubator.

[0067] Seed culture: Pick a single colony from a solid plate and inoculate it into LB liquid medium (containing 100 μg / mL Amp), with a liquid volume of 50 mL / 250 mL, and culture on a shaker at 37°C and 200 rpm for 12 hours.

[0068]Shake Flask Fermentation: Put the seed solution cultivated for 12 hours into the fermentation medium (containing 100 μg / mL Amp) at an inoculum size of 1%, and the liquid volume is 50 mL / 250 mL. First, cultivate it at 37 ° C and 200 rpm to the cell concentration OD 600 =0.6-0.8, after the liquid is cooled to room temperature, then add IPTG with a final concentration of 0.1 mM for induction, and induce expression at 25°C for 12 hours. Af...

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Abstract

The invention discloses an isoeugenol monooxygenase mutant and application thereof, and belongs to the field of bioengineering. The isoeugenol monooxygenase gene derived from Pseudomonas nitroreducens Jin1 is subjected to site-specific mutagenesis, so that the 83rd site of the coded amino acid sequence is mutated from lysine K to arginine R, the 95th site of the coded amino acid sequence is mutated from lysine K to arginine R, and the 273rd site of the coded amino acid sequence is mutated from leucine to phenylalanine F. The specific activity of the isoeugenol monooxygenase mutant K83R / K95R / L273F is 2.1 times that before mutation, and the half-life periods of the isoeugenol monooxygenase mutant K83R / K95R / L273F at 25 DEG C, 30 DEG C and 35 DEG C are respectively improved by 3.0 times, 12.0 times and 24.7 times. The isoeugenol monooxygenase mutant disclosed by the invention can catalyze propenyl on a benzene ring of a substrate isoeugenol to oxidize in a relatively wide temperature range under the action of oxygen so as to generate vanillin, can be widely applied to industries such as food, chemical engineering and pharmacy, and has a wide prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a mutant of isoeugenol monooxygenase with high thermal stability and high activity and application thereof. Background technique [0002] Isoeugenol Monooxygenase (Isoeugenol Monooxygenase) is referred to as IEM, which uses isoeugenol as a substrate to generate vanillin by oxidatively cleaving the propenyl group connected to the benzene ring without the presence of any coenzymes and cofactors. And vanillin (4-hydroxy-3-methoxybenzaldehyde), also known as vanillin, is widely used in the fields of food, chemical industry and medicine due to its unique aroma and properties. In food, vanillin can be added to cakes and ice cream as a flavoring agent to enhance its aroma and flavor; it is often used in the chemical industry to test the presence of resorcinol and tannic acid; it is also used in the production of many drugs in the pharmaceutical industry Raw materials...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P7/24C12R1/19
CPCC12N9/0004C12N15/70C12P7/24Y02P20/54
Inventor 徐毅陆馨怡马宝娣王倩吴小梅张磊刘胜利
Owner CHINA FORTUNE WAY CO
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