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Monoamine oxidase and application thereof

A monoamine oxidase, oxidation reaction technology, applied in applications, enzymes, biochemical equipment and methods, etc., can solve the problems of low substrate concentration, low activity of MAON401, and difficulty in realizing industrial-scale application.

Pending Publication Date: 2022-07-12
SUZHOU BAIFUAN ENZYME TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In 2010, U.S. Patents US 8178333 and US 8859784 disclosed a monoamine oxidase derived from Aspergillus oryzae, which was more active than wild-type monoamine oxidase derived from Aspergillus niger, but less stable
However, the activity of MAON401 is still very low, and there is severe substrate and product inhibition, resulting in a final substrate loading of only 65g / L (J.Am.Chem.Soc.2012,134,6467-6472)
[0008] In 2014, Chinese patent CN201410441595.4 disclosed a novel monoamine oxidase derived from the environment, which can asymmetrically oxidize 6,6-dimethyl-3-azabicyclo[3.1.0]hexane or (3aR,6aS) -Octahydrocyclopenta[c]pyrrole, generating (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-ene and (3S,7R)-3 -Azabicyclo[3,3,0]oct-2-ene, which can be used as intermediates for the further synthesis of boceprevir and telaprevir respectively, but the substrate concentration is still very low, and the highest concentration only reaches 100mM, Difficult to achieve industrial scale application
[0009] In summary, (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-ene (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-ene ( Naimatevir, boceprevir precursor), and (3S,7R)-3-azabicyclo[3,3,0]oct-2-ene (telaprevir precursor) have high stereoselectivity and environmental friendliness, etc., but there are still many shortcomings. Currently known monoamine oxidases have problems such as low catalytic activity, low substrate tolerance concentration and low production efficiency.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Gene cloning of monoamine oxidase

[0086] The monoamine oxidase with obvious activity to 6,6-dimethyl-3-azabicyclo[3.1.0]hexane was predicted by bioinformatics analysis method, and then cloned and expressed to verify its function. Using this method, three novel monoamine oxidase genes were cloned from Penicillium sp., Zymoseptoria sp. and Alternaria sp., respectively.

[0087] As an example, using the genomic DNA of Penicillium as a template, a complete nucleic acid molecule encoding the monoamine oxidase SEQ ID No. 1 is obtained by using conventional technical methods in the art (such as polymerase chain reaction PCR). The synthetic primers involved are:

[0088] Upstream primer: CCG GAATTC atgacctctcgcgacggt,

[0089] Downstream primer: CCG CTCGAG tagccgaagccgtgg.

[0090]The underlined sequence of the upstream primer is the restriction site of EcoR I, and the underlined sequence of the downstream primer is the restriction site of Xho I.

[0091] U...

Embodiment 2

[0094] Example 2: Preparation of monoamine oxidase recombinant expression vector and recombinant expression transformants

[0095] The monoamine oxidase gene obtained by PCR amplification in Example 1 and the empty vector plasmid pET28a were double digested with restriction enzymes EcoR I and Xho I at 37°C for 12h respectively. The double-enzyme digested product was verified by agarose gel electrophoresis, and then purified and recovered. The obtained linearized pET28a plasmid was ligated with the purified target gene fragment with T4 DNA ligase at 16 °C overnight, and the ligated product was transformed into the large intestine. Bacillus E.coli BL21 (DE3) competent cells (Novagen), and evenly spread on LB agar plates containing 50 μg / mL kanamycin, placed in a 37 ° C incubator for about 12 hours, and the long The colonies that came out were verified by colony PCR, and clones with positive colony PCR were picked for sequencing verification. After the sequence was verified to b...

Embodiment 3

[0099] Example 3: Construction of monoamine oxidase mutants and high-throughput plate screening

[0100] Monoamine oxidase PsMAO was used as the parent, and error-prone PCR technology was used to construct a random mutant library of monoamine oxidase: using pET28a-PsMAO as a template and the upstream primer and downstream primer in Example 1 as primer pairs, error-prone PCR was performed with rTaq DNA polymerase , to build a random mutation library. PCR system (50 μL): 0.5 μL of rTaq DNA polymerase, 10×PCR buffer (Mg 2+ Plus) 5.0 μL, dNTP Mixture (2.0 mM each) 4.0 μL, MnCl with a final concentration of 100 μmol / L 2 , pET28a-PsMAO plasmid 0.5ng, upstream and downstream primers (10μM) each 2μL, add sterilized distilled water to make up to 50μL. PCR reaction procedure: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 94°C for 30s; (3) annealing at 55°C for 30s; (4) extension at 72°C for 2 min; steps (2) to (4) were carried out for a total of 30 cycles ; The final ex...

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Abstract

The invention provides an application of monoamine oxidase SEQ ID Nos.1-3 or a mutant thereof in catalyzing desymmetric oxidation reaction of prochiral nitrogen heterocyclic compounds, which comprises the following steps: by taking a nitrogen heterocyclic compound as a substrate, carrying out catalytic oxidation reaction by using the monoamine oxidase SEQ ID Nos.1-3 or the mutant thereof in an oxidation environment to generate chiral imine.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, in particular to the application of a monoamine oxidase in catalyzing the desymmetric oxidation reaction of latent chiral nitrogen heterocyclic compounds to synthesize chiral imines. Background technique [0002] Chiral amines widely exist in natural biologically active molecules, are important structural units of chiral drugs, and are also important synthetic precursors widely used in the fields of pharmacy and fine chemical industry. It is estimated that about 40% of chiral drugs currently on the market contain building blocks of chiral amines. For example, Paxlovid, an oral drug developed by Pfizer in the United States, can reduce the risk of hospitalization or death in patients with mild or moderate COVID-19 by about 89% (Science, 2021, 374 (6575). ): 1586-1593). On December 22, 2021, Palovide received emergency use authorization from the U.S. Food and Drug Administration (FDA) for the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P17/10
CPCC12N9/0022C12Y104/03004C12P17/10
Inventor 欧阳鹏飞钱小龙戴忆思张雄寅张傲南
Owner SUZHOU BAIFUAN ENZYME TECH
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