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Monoamine oxidase from Aspergillus flavus and application thereof in chiral amine intermediate preparation

A monoamine oxidase, biocatalyst technology, applied in biochemical equipment and methods, enzymes, applications, etc., can solve problems such as high price and limited application

Inactive Publication Date: 2019-06-21
CHENGDU SOURCEBIO LIMITED LIABILITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The asymmetric hydrogenation reduction of imines catalyzed by transition metals is another important way to obtain chiral amines. However, this method also has many shortcomings: such as the need for activated imine substrates; the need to use transition metals, such as Rh, Ru, Ir and Ti, but the high price limits their application in industrialization

Method used

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  • Monoamine oxidase from Aspergillus flavus and application thereof in chiral amine intermediate preparation
  • Monoamine oxidase from Aspergillus flavus and application thereof in chiral amine intermediate preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Genomic DNA Extraction

[0044] Using the CTAB method to extract genomic DNA, the operation method is as follows:

[0045] Strain culture: a small amount of Aspergillus flavus ( Aspergillus flavus 12) The slant seeds were inoculated in Aspergillus medium, cultivated at 30°C and 230 rpm for 24-36 hours to prepare seed liquid, and transferred to Aspergillus medium at an inoculum amount of 0.5%-2% for expanded cultivation, culture conditions: 30°C, 230rpm, 48~72h. Aspergillus medium composition: 0.3% KH 2 PO 4 ;0.15% MgSO 4 •7H 2 O; 0.1% yeast extract; 0.06% peptone; 2% glucose; 20% potato.

[0046] Take 0.5~1.0g of mycelia into a mortar, add quartz sand and liquid nitrogen to grind and pulverize. Transfer the powder to a 2.0 ml EP tube, add 600 ul of DNA extraction solution (500 μl per 50 mg of bacteria), shake, mix, and incubate at 55°C for 60 minutes. Use 4 mol / L NaCl to adjust the NaCl concentration of the solution to 1.4 mol / L, then add 1 / 10 volume ...

Embodiment 2

[0047] Example 2: Gene Hunting Clonal Amplification Af MAO6-3 gene

[0048] By performing sequence comparison and homology analysis on the reported monoamine oxidase gene, design amalgamative primers according to the N-terminal and C-terminal conserved sequences, and use the genomic DNA obtained in Example 1 as a template to perform PCR to clone and amplify the monoamine oxidase gene. .

[0049] The PCR reaction system is 50ul, respectively by 5ul ×10 pfu DNA polymerase buffer (with Mg 2+ ), 1 ul primer F, 1 ul primer R, 4 ul dNTPs, 1 ul genomic DNA, 0.5 ul Pfu DNA polymerase and 37.5 ul ddH 2 O composition. Wherein, the primers used for PCR amplification are:

[0050] primer F: 5ˊ- GGA TCC ATG GCA CCA CCT TCA AAC GAA -3ˊ (restriction site B am H I), primer R: 5ˊ- GAA TTC TCA AAG CCT CGA TCG CAA CGT TTC-3ˊ (restriction site E coR I);

[0051] The PCR conditions were pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing a...

Embodiment 3

[0065] Embodiment 3: will Af MAO6-3 Gene Construction in Genetically Engineered Strains

[0066] For plasmid pMD19T- Af MAO6-3 was double digested. Enzyme digestion system 40 ul, from 33 ul pMD19T- Af MAO6-3, 1.0ul BSA, 1.0ul B amH I, composed of 4 ul NEB Buffer 3, add 1.0 ul after digestion at 37°C for 3 hours E coRI, continue to digest at 37°C for 3 hours.

[0067] The vector pET28a was double digested. Enzyme digestion system 40 ul, composed of 33 ul pET28a, 1.0 ul BSA, 1.0 ul B amH I, composed of 4 ul NEB Buffer 3, add 1.0 ul after digestion at 37°C for 3 hours E For coR I, continue to digest at 37°C for 3 hours.

[0068] The digested product was recovered and purified using a gel extraction kit.

[0069] Ligation: 10 ul system, 6 ul Af MAO6-3 ( B amH I / E coR I), 2 ul pET28a ( B amH I / E coR I), 1 ul T4 DNA ligase Buffer, 1 ul T4 DNA ligase. Ligation was performed overnight at 16°C.

[0070] Transformation and culture: the connected 10 ul vector pET2...

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Abstract

The invention belongs to the technical field of biology, relates to genetic engineering, biotransformation, bioconversion and biological catalysis, and organic synthesis technology, and discloses a monoamine oxidase gene from Aspergillus flavus and application thereof in chiral amine intermediate preparation using the monoamine oxidase gene to catalyze asymmetric oxidation reaction. The monoamineoxidase AaMAO7-6 gene is 1419 bp and encodes monoamine oxidase formed by 470 amino acids. After the recombinant monoamine oxidase built by the monoamine oxidase gene is expressed, the whole-cell or enzyme is used for transforming cis-7-aza-bicyclo[3,3,0]octane to generate the key chiral intermediate of Telaprevir. The preparation method is mild in reaction conditions, high in stereoselectivity, easy to achieve industrialization and the like.

Description

technical field [0001] The invention belongs to the field of applied microorganisms and enzyme engineering, and specifically relates to a strain of Aspergillus flavus and a novel monoamine oxidase encoded by its genome, and uses the monoamine oxidase derived from the strain as a biocatalyst to prepare several chiral amine intermediates. Background technique [0002] Chiral amine is an important and key drug structural unit. At present, about 40% of drugs contain chiral amine structure. Efficient manufacturing technology of chiral amine is a key technology necessary for the progress and development of chiral drug manufacturing industry. At present, the preparation of chiral amines is mainly based on chemical asymmetric resolution and synthesis, while the biocatalytic production of chiral amines mainly uses lipase and transaminase as catalysts, and the research and use of monoamine oxidase (MAO) is less. [0003] Optical activity Δ 1 -Pyrrolidine compounds are key chiral inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P21/02
Inventor 王海波
Owner CHENGDU SOURCEBIO LIMITED LIABILITY
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