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In-vitro enzyme activity application of citrus-oxygen-methyltransferase CitOMT2

A technology of citrus oxymethylation and transferase, applied in the fields of genetic engineering technology and enzymology

Inactive Publication Date: 2020-01-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that there is a FOMT that can effectively catalyze the 3 / 5 / 7 position in citrus, but the FOMT that can simultaneously catalyze 3' / 5' / 7 has not been reported in plants.

Method used

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  • In-vitro enzyme activity application of citrus-oxygen-methyltransferase CitOMT2
  • In-vitro enzyme activity application of citrus-oxygen-methyltransferase CitOMT2
  • In-vitro enzyme activity application of citrus-oxygen-methyltransferase CitOMT2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Cloning of CitOMT2 full length

[0022] (1) Experimental method

[0023] 1. Extraction of RNA from the peel of 'Oukan' by CTAB method

[0024] Cut the fresh 'Oukan' peel into cubes, add liquid nitrogen to quick-freeze and grind; take 0.3g sample powder and add it to 4ml CTAB / β-mercaptoethanol extract preheated at 65°C in advance, vortex and mix well Continue heating at 65°C for 5 minutes; add 4ml of chloroform:isoamyl alcohol (24:1 / V:V) extract to the mixture, vortex and mix well, then centrifuge at 15°C and 10,000rpm for 10min; absorb the supernatant Liquid to a new centrifuge tube, add 4ml of the above extract, repeat the extraction, after centrifugation, draw the supernatant to a new centrifuge tube; add 1 / 4 volume of 10M lithium chloride solution to the obtained supernatant, Stand overnight at 4°C; the next day, centrifuge at 4°C, 10,000rpm for 30min, discard the supernatant; add 400μL of SSTE preheated at 65°C to the precipitate, tap to dissolve the pr...

Embodiment 2

[0033] Example 2: Expression and purification of CitOMT2 recombinant protein

[0034] (1) Experimental method

[0035] 1. Construction of target gene recombinant cells

[0036] According to the CitOMT2 full-length sequence, the primer pair SEQ:No.5 and SEQ:No.6 with the pET32a restriction site are designed, and the CitOMT2-T plasmid obtained in Example 1 is used as a template, according to the amplification shown in Example 1. The amplification system and program cloned the full-length sequence of CitOMT2 (without terminator), and constructed the CitOMT2-PET32a recombinant plasmid. The recombinant plasmid with correct sequencing was transformed into the E. coli expression strain BL21(DE3)pLysS(Promega), the positive recombinant cells were screened by ampicillin (Amp) resistance medium and colony PCR, and the corresponding bacterial solution was added to an equal volume of 50 % glycerol stored at -80°C.

[0037] 2. Prokaryotic expression of target gene and purification of re...

Embodiment 3

[0041] Embodiment 3: CitOMT2 enzyme activity assay in vitro

[0042] (1) Experimental method

[0043] 1. CitOMT2 regioselectivity assay for different substrates

[0044] The regioselectivity of CitOMT2 recombinant protein was determined by seven substrates including luteolin, quercetin, 3-methylquercetin, eriodictyol, quercetin, myricetin and baicalein. The enzyme activity reaction system was 200 μL, including 25 μL of recombinant protein, 1 mM of S-adenosylmethionine, 200 μM of substrate, and the reaction buffer was Tris-HCl (pH 8.0). After the reaction solution was mixed, it was incubated in a water bath at 37°C for 2 hours, and an equal volume of methanol was added to mix well to terminate the reaction. The supernatant was centrifuged and passed through a 0.22 μm organic filter membrane for subsequent high-performance liquid chromatography (HPLC) and mass spectrometry analysis.

[0045] HPLC analysis condition: Agilent 1260HPLC system; Sunfire C18ODS chromatographic colum...

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Abstract

The invention provides an in-vitro enzyme activity application of citrus-oxygen-methyltransferase CitOMT2. The CitOMT2 recombinant protein obtained through prokaryotic expression can efficiently catalyze the methylation of various flavonoids at specific sites in vitro. That is to say, luteolin, quercetin, 3-methylquercetin,eriodictyol, quercetin and myricetin are catalyzed to be methylated at thehydroxyl group in the meta-position of a B ring to generate corresponding 3'or 3' / 5' methylated derivatives. Baicalein is catalyzed to be methylated at the 7 site of hydroxy to generate 7-methylbaicalein. According to the invention, an oxygen-methyltransferase CitOMT2 capable of catalyzing the 3' / 5' / 7 sites of flavonoids is identified for the first time. The multi-site catalytic property of CitOMT2 helps to quickly and accurately expand the diversity of O-methylated flavonoids, and the oxygen-methyltransferase CitOMT2has broad prospects and application value.

Description

technical field [0001] The invention relates to the fields of genetic engineering technology and enzymology, in particular to an in vitro enzyme activity application of citrus oxygen methyltransferase CitOMT2. Background technique [0002] Flavonoids are important small molecular compounds that widely exist in plants. So far, there are more than 10,000 types of flavonoids. The structural diversity of flavonoids lays the foundation for their multiple biological activities in the human body. This structural diversity is mainly formed by a series of modification reactions, among which O-methylation is one of the most important modifications. It has been reported that the structure of flavonoids modified by O-methylation increases lipophilicity and bioavailability, which is more conducive to exerting their biological activities, such as hypoglycemic, anti-tumor, anti-oxidation, anti-inflammation, and anti-depression. [0003] O-methylated flavonoids currently used in bioactiv...

Claims

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Application Information

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IPC IPC(8): C12P17/06C12N9/10C12N15/70
CPCC12N9/1007C12N15/70C12P17/06C12Y201/01068
Inventor 孙崇德刘晓娟王岳曹锦萍陈昆松
Owner ZHEJIANG UNIV
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