Monoamine oxidase and gene and application thereof

A technology of monoamine oxidase and gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve rare and other problems

Inactive Publication Date: 2018-08-03
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, except for MAO-N and CHAO, which have been widely studied, the di

Method used

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  • Monoamine oxidase and gene and application thereof
  • Monoamine oxidase and gene and application thereof
  • Monoamine oxidase and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1. Acquisition of Monoamine Oxidase Encoding Gene

[0095] By analyzing the genome sequence of Pseudomonas strain, a 1248bp monoamine oxidase sequence was determined, a pair of specific primers were designed to amplify by PCR technology, and finally the full-length coding frame sequence of the gene was obtained. The specific method is as follows: using the extracted genomic DNA as a template, using primers 1: 5'-CCATGGGCCGTATAGCAATCATCG-3' and 2: 5'-CTCGAGTCACAGGTGCTCTCCGAAATG-3', and introducing restriction sites for PCR amplification. The PCR reaction system is as follows: 12.5 μL of 2×TaqPCR Master Mix, 1 μL of genomic DNA, 0.5 μL of upstream and downstream primers, ddH 2 O 10.5 μL. PCR reaction conditions: pre-denaturation at 95°C for 10 min; denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 72°C for 90 s as a cycle; extension at 72°C for 10 min, 30 cycles. The PCR product was electrophoresed on a 0.9% agarose gel to verify whether a ...

Embodiment 2

[0096] Example 2. Construction of monoamine oxidase-encoding gene recombinant expression vector

[0097] Using the plasmid pET28b(+) as the expression vector of the enzyme protein, a prokaryotic recombinant expression vector of the monoamine oxidase gene MAO5 was constructed. The specific method is as follows: extract the recombinant plasmid pGEM-T-MAO5, use restriction endonuclease NCOI / wxya Treat the recombinant plasmid, and use T4 DNA ligase (NEB) to connect the gene fragment with the expression vector pET28b(+) treated with the same restriction endonuclease to construct the expression vector pET28b(+)-MAO5. The constructed intracellular expression vector pET28b(+)-MAO5 was transformed into Escherichia coli BL21(DE3) by the heat shock method, spread on a plate containing kanamycin resistance, cultured at 37°C overnight, and clones were randomly selected for Colony PCR identification.

Embodiment 3

[0098] Example 3. Recombinant expression and detection of monoamine oxidase protein

[0099] The recombinant Escherichia coli BL21(DE3) / pET28b-MAO5 containing the intracellular expression recombinant plasmid pET28b-MAO5 after identification in Example 2 was respectively treated with LB liquid medium containing 50 μg / ml kanamycin resistance at 37°C, Cultivate at 250rpm for 12h, then inoculate with 1% inoculum (v / v) into fresh LB liquid medium containing 50μg / ml kanamycin resistance, and cultivate at 37˚C and 250rpm to the cell concentration OD 600About 0.6, then add IPTG with a final concentration of 0.01mM to the LB liquid medium, induce culture at 20°C, 250rpm for 20h, centrifuge at 4°C, 8000rpm for 5min, and collect the bacterial cells containing recombinant monoamine oxidase.

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Abstract

The invention discloses a monoamine oxidase from Pseudomonas sp., a coding gene, a recombinant expression vector, a recombination expression transformant, and the synthesis application of the monoamine oxidase in tetrahydroquinoline chiral amine compounds. The coding gene of the monoamine oxidase MAO5-ZMU is from Pseudomonas sp. ZMU-T01. The amino acid sequence of monoamine oxidase protein is shown in SEQID No.1, and the corresponding coding gen nucleotide sequence is shown in SEQIDNo.2; the monoamine oxidase gene can be connected with an expression carrier to construct to obtain a recombination expression plasmid; the recombination expression plasmid is converted to escherichia coli to obtain genetically engineered bacteria with monoamine oxidase activity. The genetically engineered bacteria with monoamine oxidase activity can be used for effectively catalyzing 2-methyl-1,2,3,4-tetrahydroquinoline compounds for oxidation splitting, has the characteristics of high selectivity, moderatereaction condition, environment protection and the like and can be used for the biosynthesis of the chiral tetrahydroquinoline compounds.

Description

technical field [0001] The invention relates to a monoamine oxidase, a coding gene and a recombinant expression carrier, a new Escherichia coli strain producing the monoamine oxidase, and the application of the enzyme in the synthesis of chiral amine compounds. Background technique [0002] Monoamine oxidase belongs to the oxidoreductase family, which can oxidize amine compounds to generate corresponding imines, and at the same time reduce oxygen to hydrogen peroxide, which is one of the ways for organisms to degrade amine compounds. As an important class of compounds, chiral amines widely exist in clinical medicine, agricultural chemicals, and chiral resolving agents. Chemical synthesis of chiral amines mainly includes metal-catalyzed asymmetric reduction and small-molecule catalyst-catalyzed asymmetric synthesis. The chemical synthesis of chiral amines can often obtain chiral compounds with high enantiopurity, but environmentally harmful heavy metals and organic solvents ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P41/00C12P17/12
Inventor 陈永正万南微邓国忠秦磊
Owner ZUNYI MEDICAL UNIVERSITY
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