Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine

A thioether monooxygenase and mutant technology, which is applied in the field of bioengineering, can solve the problems of low catalytic activity of biocatalysts, low substrate loading, poor substrate specificity, etc., and achieves a wide range of catalytic substrates and thermal stability. Good performance and strong substrate specificity

Pending Publication Date: 2022-08-02
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problems of low biocatalyst catalytic activity, poor thermal stability, poor substrate specificity and low substrate loading capacity in the biosynthesis of chiral sulfoxide drug dexlansoprazole sulfoxide, the invention selects the patent CN 112725297 A CbSMO with higher activity to lansoprazole sulfide G266D / L313P (i.e. in the amino acid sequence shown in SEQ ID No.3 in the patent CN 112725297 A, the 266th amino acid Gly is replaced by Asp, and the 313th amino acid Leu is replaced by Pro) as a target, it is further carried out by means of protein engineering Molecular transformation of lansoprazole provides a thioether monooxygenase mutant that significantly improves lansoprazole thioether catalytic activity, substrate specificity and thermal stability, and encodes a nucleic acid for the thioether monooxygenase mutant , a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a recombinant thioether monooxygenase mutant catalyst, and an application of the recombinant thioether monooxygenase catalyst in the preparation of chiral prazole

Method used

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  • Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine
  • Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine
  • Thioether monooxygenase mutant and application thereof in preparation of chiral prazole medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1: Random mutation screening of CbSMO mutants with improved lansoprazole thioether activity

[0090] According to the open reading frame of CbSMO, the upstream and downstream primers were designed as follows:

[0091] The upstream primer is shown in SEQ ID No.3.

[0092] Downstream primers are shown in SEQ ID No.4.

[0093] The underlined sequence of the upstream primer is the restriction site of Nde I, and the underlined sequence of the downstream primer is the restriction site of Hind III.

[0094] with recombinant plasmid pET28a-CbSMO G266D / L313P As a template, a random mutant library was established according to the method described in Example 7 of Patent CN 112725297 A.

[0095] Among them, CbSMO G266D / L313P The amino acid sequence is shown in SEQ ID No.2, and the corresponding nucleotide sequence is shown in SEQ ID No.1.

[0096] Briefly, error-prone PCR was performed with rTaq DNA polymerase to construct random mutation libraries. After PCR amplific...

Embodiment 2

[0097] Example 2: Random mutagenesis screening of CbSMO mutants with improved substrate specificity for lansoprazole sulfide

[0098] Due to the maternal CbSMO G266D / L313P In the process of catalyzing the oxidation of lansoprazole sulfide, the oxidized product dexlansoprazole sulfoxide will be recognized as a substrate and subjected to peroxidation to generate a by-product lansoprazole sulfone, which shows that the lansoprazole sulfide bottom The phenomenon of poor specificity. To address this issue, CbSMO mutants with improved specificity for lansoprazole sulfide substrates were screened using random mutagenesis.

[0099] Screening of CbSMO mutants with improved lansoprazole thioether substrate specificity can be achieved by making minor modifications to the operation steps of Example 1. Specifically, the substrate lansoprazole sulfide in the activity detection reaction in Example 1 was replaced with D-lansoprazole sulfoxide, and a high absorbance value indicated that more ...

Embodiment 3

[0100] Example 3: Screening of CbSMO mutants with improved thermal stability by random mutagenesis

[0101] Screening of CbSMO mutants with improved thermal stability can be achieved by making minor modifications to the operation steps of Example 1. Specifically, the crude enzyme solution after cleavage in Example 1 was allowed to stand at 40°C for 5 hours, and then the activity detection reaction was performed. The high absorbance value indicated that the residual activity of the CbSMO mutant was high, that is, the thermal stability of the CbSMO mutant. powerful.

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Abstract

The invention belongs to the technical field of bioengineering, and relates to a thioether monooxygenase mutant. Nucleic acid for coding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, and a recombinant expression transformant containing the recombinant expression vector; the invention also discloses an application of the recombinant thioether monooxygenase mutant in preparation of chiral prazole drugs. Compared with other biocatalysts for preparing optical pure prazole drugs, the thioether monooxygenase mutant provided by the invention has the advantages of high catalytic activity, strong substrate specificity, good thermal stability, wide catalytic substrate range and high stereoselectivity, and shows a wide application prospect in industrial application.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a thioether monooxygenase mutant, a nucleic acid encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, and a recombinant expression vector containing the recombinant expression vector. Recombinant expression transformants, preparation of mutant enzyme preparations, and application of mutant enzyme preparations in the preparation of chiral prazol drugs. Background technique [0002] Proton pump inhibitors (PPIs) are a class of drugs of choice widely used in the treatment of gastric acid digestive diseases. PPIs mainly include omeprazole which was launched in Sweden in 1988, lansoprazole which was launched in Japan in 1995, pantoprazole which was launched in Germany in 1997 and rabeprazole which was launched in the United States in 1999. Drugs were initially marketed in the form of racemates, which played a piv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P17/16C12Q1/26C12R1/19
CPCC12N9/0071C12N15/70C12P17/165C12Q1/26G01N2333/90245Y02P20/584
Inventor 郁惠蕾刘峰许建和潘江耿强赵晨
Owner EAST CHINA UNIV OF SCI & TECH
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