Application of monoamine oxidase in preparation of tropinone

A technology of monoamine oxidase and tropinone, applied in the field of enzyme catalysis and synthetic biology, can solve the problems of insufficient stability and low expression, and achieve the effects of improving the cyclization reaction, solving the rate-limiting step, and having high application value.

Pending Publication Date: 2022-04-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The P450 of plants is a membrane protein, which has defects such as low expression, insufficient stability, and difficulty in enzyme engineering. Therefore, it is necessary to develop a more efficient 4-(1-methyl-2-pyrrolidinyl)-3-oxo The biocatalytic reaction of converting butyric acid into tropinone will be a breakthrough technology for improving the biosynthetic yield of tropinone and realizing the microbial production of tropin drugs

Method used

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  • Application of monoamine oxidase in preparation of tropinone
  • Application of monoamine oxidase in preparation of tropinone
  • Application of monoamine oxidase in preparation of tropinone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of Monoamine Oxidase Heterologous Expression Strain

[0028] Take Escherichia coli competent cells E.coli BL21 out of the -80°C refrigerator and place them on ice, pipette 1 microliter of the constructed monoamine oxidase expression plasmid pET28a-MAO into the competent cells, mix gently, Ice-bathed for 30 minutes, then placed in a 42°C water bath for heat shock for 90 seconds, immediately placed on ice for 2 minutes, and added 1 mL of LB to the transformation system. After cultivating on a shaker at 37°C for 40-60 minutes, take out the low-speed centrifugation for 3 minutes, discard most of the supernatant, gently blow the resuspended bacteria with a pipette gun, and spread it on the LA plate containing 50 μg / mL kanamycin antibiotic Above, cultivate in a 37°C incubator for 8-12 hours, and the obtained transformant with kanamycin resistance is the monoamine oxidase heterologous expression strain.

Embodiment 2

[0029] Example 2: Heterologous expression of monoamine oxidase

[0030] Pick the monoclonal transformants obtained in Example 1 and inoculate them into 4mL LB (containing 50 μg / mL kanamycin antibiotic), and cultivate them at 37°C until OD 600 =0.6, transferred to 400mL LB (containing 50μg / mL kanamycin antibiotic), cultured on a shaker at 37°C until OD 600 =0.6, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce expression at 18° C. for 18-20 h.

Embodiment 3

[0031] Example 3: Biotransformation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid to produce tropinone

[0032] Take the bacterial strain that has completed heterologous expression in Example 2, collect the bacteria by centrifugation, resuspend with 4 mL of M9 medium, and add the substrate 4-(1-methyl-2-pyrrolidinyl)-3- Oxobutyric acid, transformed at 4°C for 120 hours, added 8 mL of ethyl acetate, extracted, centrifuged to take the supernatant ethyl acetate layer, evaporated to dryness and resuspended with 1 mL of acetonitrile, centrifuged to take the supernatant to detect the formation of tropinone by LC-MS (Mass spectrometry results such as Figure 4 ), the results showed that the conversion rate of this reaction was as high as 90%.

[0033] LC-MS detection conditions:

[0034] Instrument: SHIMADZU LCMS-2020UPLC-MS;

[0035] Analytical column: R227-32015-03Shim-pack Velox Biphenyl (2.7μm, 2.1x100 mm, Shimadzu Corporation), the flow rate is 0.4mL min -1 , the detecte...

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Abstract

The invention discloses application of monoamine oxidase in preparation of tropinone, and belongs to the technical field of enzyme catalysis and synthetic biology. The amino acid sequence of the monoamine oxidase provided by the invention is as shown in SEQ ID NO.1. The invention provides application of the monoamine oxidase in preparation of tropinone, and 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid is catalyzed to be converted into tropinone. The method for preparing the tropinone is green, mild and environment-friendly, the catalytic efficiency of the tropinone is remarkably improved compared with that of P450, and the tropinone has application prospects.

Description

technical field [0001] The invention belongs to the technical field of enzyme catalysis and synthetic biology, and more specifically relates to the application of a monoamine oxidase in the preparation of tropinone. Background technique [0002] Monoamine oxidase (monoamine oxidase, MAO) is a flavin-dependent amine oxidase, which was first discovered in animals and widely exists in organisms. This enzyme can catalyze the metabolism of monoamines, oxidative deamination, and generate the metabolite hydrogen peroxide, which plays an important role in the process of amine metabolism in the body, so it has attracted much attention (Iacovino, L.etal.2018, The structure of monoamine oxidases: past, present, and future. Journal Of Neural Transmission, 125, 1567-1579.). The monoamine oxidase MAO-N discovered from the microorganism Aspergillus niger in 1995 is the first case of monoamine oxidase derived from invertebrates discovered so far. It can oxidize simple terminal linear fatty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12N9/06C12N15/53C12N15/70C12N15/81C12N1/19C12N1/21C12R1/19
Inventor 瞿旭东林芝
Owner WUHAN UNIV
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