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Monooxygenase complex and application thereof in chiral sulfoxide synthesis

A technology of monooxygenase and complex, which is applied in the field of molecular biology, can solve the problems of lack of chiral sulfoxide synthase and cannot meet the needs of green industrial production, and achieve the effects of environmental friendliness, easy operation and mild reaction conditions

Active Publication Date: 2018-09-28
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, existing studies have shown that chiral sulfoxide synthases with high activity and high enantioselectivity are still very scarce and cannot meet the needs of green industrial production. It is still a challenge to obtain biological enzymes with high catalytic efficiency and corresponding biocatalytic synthesis methods. Difficulties and bottlenecks in the development of chiral sulfoxide biocatalytic preparation

Method used

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  • Monooxygenase complex and application thereof in chiral sulfoxide synthesis
  • Monooxygenase complex and application thereof in chiral sulfoxide synthesis
  • Monooxygenase complex and application thereof in chiral sulfoxide synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: the acquisition of monooxygenase complex gene, comprises the following steps:

[0027] Using genomic DNA as a template, use primers 1: 5'-GGCCAGATCTCATGAATCAGACCGACACATC-3' and 2: 5'-AATTCTCGAGGCGTGTCGCCTTCAGCGCG-3' for PCR amplification to obtain the pmTodA gene; use primers 3: 5'-GGAAGGATCCGATGATTGATTCAGCCAACAG-3' and 4: 5 '-AACCGTCGACCTAGAAGAAGAAACTGAGG-3' was used for PCR amplification to obtain pmTodB gene; use primer 5: 5'-GGCCAGATCTATGGCGTTGCCCGGCAGCC-3' and 6: 5'-AATTCTCGAG ACGTTAGGTCTCCTTCATTCG-3' for PCR amplification to obtain pmTodC gene; use primer 7: 5' -GGAAGGATCCATGACTTGGACATACATATT-3' and 8:5'-AACCGTCGACCTTCAACTCCCCGTTGTCG-3' were amplified by PCR to obtain the pmTodD gene. The PCR reaction system was as follows: 10.0 μL of 2×Taq PCR Master Mix, 1 μL of genomic DNA, 0.5 μL of upstream and downstream primers, and 8.0 μL of ddH2O. PCR reaction conditions: 95°C for 10 min; 98°C for 10 s, 58°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 5 mi...

Embodiment 2

[0028] Embodiment 2: the construction of monooxygenase complex genetically engineered bacterium, comprises the following steps:

[0029] The DNA fragment containing the pmTodA gene sequence and the vector pETDute-1 were double digested with restriction endonucleases BamH I and Sal I, and the recovered target fragment and vector were digested. Using T4 DNA ligase, after ligation at 16°C for 4 hours, the ligation product was transformed into Escherichia coli DH5α. After overnight culture, the grown monoclonal colony was picked and shaken to extract the plasmid, and the recombinant plasmid pETDute1-pmTodA was detected by double enzyme digestion with BamH I and Sal I. Next, after performing the same operation on the pmTodB gene and the recombinant plasmid pETDute1-pmTodA with Bgl II and Xho I, the recombinant vector pETDute1-pmTodA-B was obtained. Next, the DNA fragment containing the pmTodC gene sequence and the vector pRSFDute-1 were double-digested with restriction enzymes Bam...

Embodiment 3

[0030] Embodiment 3: the acquisition of monooxygenase complex recombinant protein, comprises the following steps:

[0031] After activating the genetically engineered bacteria BL21 (DE3) stored in glycerol containing recombinant plasmids, pick a single colony in 2ml of liquid LB medium containing the corresponding antibiotics, shake and culture at 37°C for 12 hours, and transfer to 2% inoculum the next day. Inoculated in fresh 100mL LB liquid medium containing antibiotics, 37°C 250rpm shaking culture OD600 was 0.6 (about 3h), added IPTG with a final concentration of 0.2mM, 25°C 160rpm induction culture for 8h. After the induction, collect the cells by centrifugation at 5000rpm / min for 5min, resuspend the cells in PBS buffer, break up the cells by ultrasonic, and centrifuge at 15000rpm for 5min to remove cell debris, take the supernatant and mix it with 2x loading buffer, and put it in a constant temperature metal bath SDS-PAGE electrophoresis analysis was performed after heati...

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Abstract

The invention relates to a monooxygenase complex in the field of molecular biology. The monooxygenase protein complex consists of four subunits, and the sequence of an amino acid residue is shown as SEQ ID No.1. The (R)-type sulfoxide with high optical purity is prepared by using the monooxygenase protein complex. Compared with other existing preparation methods, the used preparation method is short in reaction time, mild in reaction conditions, environmentally-friendly and simple and convenient in operation.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a monooxygenase complex and its application in the synthesis of chiral sulfoxide. Background technique [0002] Chiral sulfoxides are a very important class of compounds, which can be used as chiral intermediates and auxiliary agents, chiral ligands, catalysts and clinical drugs, and are widely used in organic synthesis and drug synthesis. The existing chiral sulfoxide synthesis technology still has shortcomings such as excessive oxidation, many by-products, and harsh reaction conditions. Due to the high efficiency and high selectivity of biological enzymes, biocatalytic technology is expected to develop into a new way for industrial green preparation of chiral sulfoxide drugs. However, existing studies have shown that chiral sulfoxide synthases with high activity and high enantioselectivity are still very scarce and cannot meet the needs of green industrial production. It is st...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12P11/00
CPCC12N9/0071C12P11/00
Inventor 杨加伟
Owner ZUNYI MEDICAL UNIVERSITY
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