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A kind of cyclohexanone monooxygenase and its application

A technology of cyclohexanone monooxygenase, which is applied in the field of enzyme engineering, can solve the problem of low concentration of omeprazole sulfide, and achieve the effects of low price, superior industrial application value, and reduced production cost

Active Publication Date: 2021-09-17
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 and screen the cyclohexanone monooxygenase of the prior art, Construction of cyclohexanone monooxygenase with high enzymatic activity, which can catalyze the conversion of high concentration omeprazole sulfide substrate to esomeprazole

Method used

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  • A kind of cyclohexanone monooxygenase and its application
  • A kind of cyclohexanone monooxygenase and its application
  • A kind of cyclohexanone monooxygenase and its application

Examples

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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 a cyclohexanone monooxygenase and its application, especially discloses a cyclohexanone monooxygenase obtained by site-directed mutation and its application. Compared with SEQ ID NO: 1, the The amino acid sequence of cyclohexanone monooxygenase has a genetic mutation at least one of the following sites: the 386th serine Ser is mutated into asparagine Asn, and the 435th serine Ser is mutated into threonine Thr. Experiments show that the cyclohexanone monooxygenase of the invention can catalyze the conversion of high-concentration omeprazole sulfide substrate into 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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Patent Type & Authority Patents(China)
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