Cyclohexanone monooxygenase and application thereof

A technology of cyclohexanone monooxygenase and cyclohexanone mono, which is applied in the field of enzyme engineering and can solve the problems of low concentration of omeprazole sulfide and the like

Active Publication Date: 2018-06-05
ZHEJIANG JINGXIN PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defect that the reaction substrate omeprazole sulfide concentration is too low in the existing bio-enzyme synthesis method, the present invention uses enzyme genetic engineering technology to transform a

Method used

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  • Cyclohexanone monooxygenase and application thereof
  • Cyclohexanone monooxygenase and application thereof
  • Cyclohexanone monooxygenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1: Site-directed mutation of cyclohexanone monooxygenase gene and construction of bacterial strain 2#

[0117] Using the QuickChange Site-directed Mutagenesis (Stratagene) method (Agilent Technologies), the cyclohexanone monooxygenase gene of the above comparative example 1 was subjected to protein engineering, specifically the cyclohexanone monooxygenase amino acid sequence SEQ ID NO: The serine (S) at the 386th position of 1 was subjected to site-directed mutation transformation, and the following primers were designed (the underline corresponds to the corresponding mutation site):

[0118] F4: GATGCGGGCGATGGC AAC TACAAGCGCATCG

[0119] R4: CGATGCGCTTGTA GTT GCCATCGCCCGCATC

[0120] PCR program setting:

[0121]Pre-denaturation at 95°C for 3 minutes;

[0122] 95°C, 45s; 55°C, 45s; 72°C, 2min 18 cycles;

[0123] 72°C, 7min.

[0124] The cyclohexanone monooxygenase gene of the present invention is obtained, its amino acid sequence is shown in SEQ ID NO:3...

Embodiment 2

[0133] Embodiment 2: Site-directed mutation of cyclohexanone monooxygenase gene and construction of bacterial strain 3#

[0134] Using the QuickChange Site-directed Mutagenesis (Stratagene) method (Agilent Technologies), the cyclohexanone monooxygenase gene of the above comparative example 1 was subjected to protein engineering, specifically the cyclohexanone monooxygenase amino acid sequence SEQ ID NO: The serine (S) at the 435th position of 1 was subjected to site-directed mutation transformation, and the following primers were designed (the underline corresponds to the corresponding mutation site):

[0135] F5: GGTCCACTGGCCAAT ACT CCTCCTATCATCG

[0136] R5: CGATGATAGGAGG AGT ATTGGCCAGTGGACC

[0137] PCR program setting:

[0138] Pre-denaturation at 95°C for 3 minutes;

[0139] 95°C, 45s; 55°C, 45s; 72°C, 2min 18 cycles;

[0140] 72°C, 7min.

[0141] The cyclohexanone monooxygenase mutant gene of the present invention is obtained, the amino acid sequence is shown in...

Embodiment 3

[0143] Embodiment 3: Site-directed mutation of cyclohexanone monooxygenase gene and construction of bacterial strain 4#

[0144] Using the QuickChange Site-directed Mutagenesis (Stratagene) method (Agilent Technologies), the cyclohexanone monooxygenase gene obtained by mutation in the above-mentioned embodiment 1 was subjected to protein engineering, specifically the cyclohexanone monooxygenase amino acid sequence SEQ ID The serine (S) at the 435th position of NO:3 was transformed by site-directed mutagenesis, and the following primers were designed (the underline corresponds to the corresponding mutation site):

[0145] F5: GGTCCACTGGCCAAT ACT CCTCCTATCATCG

[0146] R5: CGATGATAGGAGG AGT ATTGGCCAGTGGACC

[0147] PCR program setting:

[0148] Pre-denaturation at 95°C for 3 minutes;

[0149] 95°C, 45s; 55°C, 45s; 72°C, 2min 18 cycles;

[0150] 72°C, 7min.

[0151] The cyclohexanone monooxygenase mutant gene of the present invention is obtained, its amino acid sequence ...

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Abstract

The invention discloses cyclohexanone monooxygenase and an application thereof, in particular cyclohexanone monooxygenase obtained by site-specific mutagenesis and an application thereof. Compared with a SEQ ID NO: 1, the amino acid sequence of the cyclohexanone monooxygenase has gene mutation in at least one site as follows: serine Ser at the 386th site is mutated to asparagines Asn, and serine Ser at the 435th is mutated to threonine Thr. Experiments show that the cyclohexanone monooxygenase disclosed by the invention can catalytically convert a high concentration omeprazole thioether primerinto esomeprazole.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a cyclohexanone monooxygenase obtained through gene site-directed mutation and its application, in particular to the use of the cyclohexanone monooxygenase in the treatment of high-concentration omeprazole sulfide Applications in the catalytic conversion of substrates to esomeprazole. Background technique [0002] Both esomeprazole and omeprazole are proton pump inhibitors (Proton pumpinhibitors, PPIs) used to treat gastric ulcers, wherein esomeprazole is omeprazole in S configuration, and its chemical name is 5 -Methoxy-2-((S)-((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzimidazole, CAS accession number It is 119141-88-7, and its chemical structure is shown in Formula I below. The chemical name of omeprazole is 5-methoxy-2-(((4-methoxy-3,5-dimethyl-2-pyridyl)methyl)sulfinyl)-1H-benzo Imidazole has a CAS accession number of 73590-58-6, and its chemical st...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/16
CPCC12N9/0073C12P17/165C12Y114/13022
Inventor 金志平金圣芳郑飞张敏洁黄悦
Owner ZHEJIANG JINGXIN PHARMA
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