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Phenylacetone monooxygenase and application thereof in preparation of prazole drugs

A technology of phenylacetone monooxygenase and acetone monooxygenase, which is applied in the application field of phenylacetone monooxygenase and in the preparation of prazoles, can solve the problems such as the overall efficiency difference of esomeprazole, Achieve the effects of improving substrate binding efficiency, improving activity, and reducing engineering costs

Pending Publication Date: 2021-09-24
FUZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the asymmetric synthesis of optically pure esomeprazole or other prazole compounds catalyzed by phenylacetone monooxygenase (PAMO), which has oxidation activity to phenylacetone.
At the same time, the above-mentioned patents all use free enzyme reaction, and need to add formate dehydrogenase or carbonyl reductase to realize the regeneration of coenzyme NADPH. There is a patent (CN 108239618 A) that reports monooxygenase and carbonyl reductase (or isopropanol dehydrogenase) ) co-expression for the synthesis of esomeprazole, but there is no report on the co-expression of monooxygenase and formate dehydrogenase or glucose dehydrogenase, the expression levels of different dehydrogenases in E. coli are different, and different monooxygenase The co-expression combination and co-expression mode of oxygenase and dehydrogenase directly affect the soluble expression of monooxygenase and the regeneration of coenzyme, which leads to the difference in the overall efficiency of biocatalytic synthesis of esomeprazole

Method used

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  • Phenylacetone monooxygenase and application thereof in preparation of prazole drugs
  • Phenylacetone monooxygenase and application thereof in preparation of prazole drugs
  • Phenylacetone monooxygenase and application thereof in preparation of prazole drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Recombinant expression of phenylacetone monooxygenase

[0050] Screened by gene mining to a source of Limnobacter sp . A novel phenylacetone monooxygenase ( ln PAMO) gene (GenBank No. KYP10950.1), amino acid sequence and reported cyclohexanone propiophenone monooxygenase ( Ac CHMO and CHMO-NCIMB9871) have a low amino acid sequence similarity of 40%. phenylacetone monooxygenase ln PAMO amino acid sequence (SEQ ID NO: 2) was codon optimized to obtain ln The base optimized sequence of PAMO gene is shown as SEQ ID NO:1.

[0051] Synthetic codon-optimized ln The PAMO gene fragment was digested with BamHI and XhoI restriction endonucleases, and connected to the plasmid pET28a to construct the recombinant plasmid pET28a- ln PAMO, and transform the recombinant plasmid into Escherichia coli BL21(DE3) for heterologous expression.

[0052] Heterologous expression: Pick a single clone of the recombinant plasmid on the plate and inoculate it into 25 mL LB li...

Embodiment 2

[0053] Embodiment 2 Propiophenone monooxygenase ln Substrate profiling assays for PAMO whole-cell catalysis

[0054] Take 10 mg of the harvested bacterial cells after induction of expression in Example 1, resuspend with 390 μL of 100 mM Tris buffer at pH 9.0, add 50 μL of 100 mM substrate solution (propiophenone, acetophenone, cyclohexanone, benzyl thioether or omeprazole thioether, final concentration 10 mM, dissolved in methanol), 10 μL 10 mM NADP + (final concentration 0.2 mM), 10 μL glucose dehydrogenase, 10 μL 1 M glucose, placed at 30°C, 1000 rpm for 2 h. The amount of product generated was detected by HPLC, and the conversion rate was calculated. The liquid chromatography conditions for the thioether substrate reaction are: C18 reversed-phase column, mobile phase is acetonitrile: water = 53:47, flow rate 1 mL / min, column temperature 30°C, detection wavelength 254 nm, detection time 13 min . Gas phase detection conditions: 30 m*0.32 mm*0.5 μm C18 gas phase column, h...

Embodiment 3

[0058] Embodiment 3 Propiophenone monooxygenase ln Site-directed mutagenesis of PAMO

[0059] Rational design, protein homology modeling based on amino acid sequence (SEQ ID NO: 2), and molecular docking construction ln Structural model of the PAMO-coenzyme-substrate complex, examining phenylacetone monooxygenase separately ln Amino acid residues within 5 angstroms of the FAD and NADP ligand-binding regions of PAMO ( figure 2 ). Analyze each secondary force, and through site-directed mutagenesis, mutate amino acid residues at targeted positions to enhance ln Binding effect of PAMO on the coenzymes FAD and NADP. In addition, in the substrate channel region, the key amino acid residues that recognize and bind to the substrate omeprazole thioether are analyzed, and the amino acid residues are mutated at key positions through site-directed mutagenesis to promote the effective recognition of omeprazole thioether by the substrate channel , to increase the substrate binding...

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Abstract

The invention provides propiophenone monooxygenase of which the amino acid sequence is obtained by mutation of the amino acid sequence as shown in SEQ ID NO: 2, and discloses a technical method for asymmetric biocatalytic synthesis of sulfoxide drugs on the basis of the propiophenone monooxygenase. A propiophenone monooxygenase gene from Limnobacter sp. Is obtained through gene mining and screening, a recombinant escherichia coli expression strain is constructed by adopting a gene engineering technology, and enzyme activity and stereoselectivity are improved by adopting a molecular modification technology. Meanwhile, co-expression strains of a monooxygenase mutant and different dehydrogenases are constructed. Omeprazole thioether is used as a raw material, and a whole-cell or zymoprotein catalysis method is adopted for asymmetric catalytic synthesis of optically pure esomeprazole. A target product can be obtained through one-step reaction under a mild condition, no by-product omeprazole sulfone exists, and meanwhile, the invention is environmentally-friendly and is an environmentally-friendly biological catalytic synthesis approach.

Description

technical field [0001] The invention belongs to the technical field of biological pharmacy and biochemical industry, and in particular relates to a phenylacetone monooxygenase and its application in the preparation of prazoles. Background technique [0002] Esomeprazole, also known as esomeprazole or ( S )-omeprazole, the chemical name is 5-methoxy-2-(( S )-((4-methoxy-3, 5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole, the chemical structure is shown below. Esomeprazole is Omeprazole ( S )-single configuration isomer, mainly used in the treatment of duodenal ulcer, gastric ulcer, gastritis and gastrointestinal esophagitis, it has been clinically proven that the drug is more effective than racemate and (R)-omeprazole or Other prazole drugs have lower toxic and side effects and better curative effect. The chemical method for preparing esomeprazole is to use a metal catalyst to asymmetrically oxidize thioether to synthesize esomeprazole, but this method has disadvan...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N1/21C12P17/16C12P41/00C12R1/19
CPCC12N9/0073C12N15/70C12P17/165C12P41/002C12Y114/13092C12Y102/01002
Inventor 林娟许鑫琦张娅娇许炼苏冰梅
Owner FUZHOU UNIVERSITY
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