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Thioether monooxygenase from pseudomonas monteilii as well as synthesis and application thereof

A thioether monooxygenase and protein technology, which is applied in the fields of biocatalysis, biosynthesis and molecular biology, can solve the problems of green industrial production, low catalytic efficiency, and lack of highly enantioselective thioether monooxygenase And other issues

Active Publication Date: 2015-04-29
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing studies have shown that thioether monooxygenases with high activity, high enantioselectivity and high substrate tolerance are still very scarce
No matter using isolated monooxygenase, thioether monooxygenase-producing strains, or recombinantly expressed thioether monooxygenase genetically engineered bacteria, the catalytic efficiency is very low, which cannot meet the needs of green industrial production

Method used

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  • Thioether monooxygenase from pseudomonas monteilii as well as synthesis and application thereof
  • Thioether monooxygenase from pseudomonas monteilii as well as synthesis and application thereof
  • Thioether monooxygenase from pseudomonas monteilii as well as synthesis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Acquisition of monooxygenase-encoding genes

[0031] By analyzing the genome sequence of Pseudomonas strain, a 972 bp monooxygenase sequence was determined, a pair of specific primers were designed to amplify by PCR technology, and finally the full-length coding frame sequence of the gene was obtained. The specific method is as follows: using the extracted genomic DNA as a template, use primers F: 5’-actcGGATCCatgcagaccgttgaacacgaat-3’ and R: 5’-accgAAGCTT

[0032] gtaggctttgtcccagct-3' for PCR amplification. The PCR reaction system is as follows: 12.5 ul of 2×TaqPCR Master Mix, 1 ul of genomic DNA, 0.5 ul of upstream and downstream primers, and 10.5 ul of ddH2O. PCR reaction conditions: 95°C for 10 min; cycle of 98°C for 10 s, 56°C for 30 s, 72°C for 90 s; extension at 72°C for 10 min, 30 cycles. The PCR product was electrophoresed on a 1% agarose gel to verify whether a fragment conforming to the size of the target gene was obtained.

Embodiment 2

[0033] Example 2. Monooxygenase gene expression vector construction

[0034] The monooxygenase gene was constructed using the pET32a(+) vector (purchased from Clonetech) as the backbone pmo-6814 prokaryotic expression vector. The specific method is as follows: Use the DNA gel recovery kit to recover pmo-6814 The PCR product was digested with restriction endonucleases BamHI and HindIII on the PCR product and the vector pET32a(+), and the recovered target fragment and vector were digested. After ligation with T4 DNA ligase at 16 °C for 4 h, 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 was detected by double enzyme digestion with BamHI and HindIII. Select the positive recombinant plasmid pET32a- pmo-6814 DNA sequencing is performed to ensure the sequence is accurate. Finally, the positive recombinant plasmids with accurate sequ...

Embodiment 3

[0035] Example 3. Recombinant expression and detection of monooxygenase protein

[0036] Will be stored in glycerol containing pET32a- pmo-6814 After activation of the genetically engineered bacteria BL21(DE3) with the plasmid, pick a single colony in 3 ml of liquid LB medium containing the corresponding antibiotics, culture with shaking at 37 °C for 12 h, and transfer to a fresh medium containing 1% of the inoculum the next day. In 50 mL LB liquid medium of antibiotics, shake culture at 37°C 250 rpm with OD600 of 0.6 (about 3h), add IPTG with a final concentration of 0.5 mM, and induce culture at 25°C 160 rpm for 8h. After induction, collect the cells by centrifugation at 8,000 rpm / min for 5 min, resuspend the cells in PBS buffer, break up the cells by ultrasonic, and centrifuge at 15,000 rpm for 5 min to remove cell debris, take the supernatant and mix with 5x loading buffer, and place in SDS-PAGE electrophoresis analysis was performed after heating in a constant tempera...

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Abstract

The invention clones, expresses and discloses a monooxygenase PMO-6814 from pseudomonas monteilii as well as application thereof, and particularly relates to a genetically engineered bacterium which has the activity of a monooxygenase and is prepared by cloning the encoding gene of the monooxygenase PMO-6814 from a pseudomonas monteilii genome, constructing an expression vector of the encoding gene and guiding the expression vector into escherichia coli. The monooxygenase protein has the amino acid sequence as shown in SEQ ID No.1, and the corresponding encoding gene of the monooxygenase protein has the nucleotide sequence as shown in SEQ ID No.2. After being reconstructed and expressed, the monooxygenase protein is capable of effectively catalyzing a thioether substrate to generate sulfoxide, has relatively high conversion efficiency and stereoisomeride enantioselectivity, and can be used for the biosynthesis of chiral sulfoxide medicines.

Description

technical field [0001] Generally, the present invention relates to the fields of biocatalysis and biosynthesis and molecular biology. Specifically, the present invention relates to the synthesis of chiral drugs using monooxygenase genes in the fields of biocatalysis and biosynthesis. The invention also relates to a method for monooxygenase gene cloning and recombinant protein expression. Background technique [0002] Chiral sulfoxide compounds are key intermediates in the synthesis of many drugs such as the antineoplastic drug Ustiloxin A, the anti-inflammatory drug sulindac, the drug omeprazole for the treatment of gastric ulcer, and the antibiotic sparsomycin. has a very wide range of applications. At present, except for some transition metal catalysts such as titanium and vanadium that can effectively catalyze the asymmetric oxidation reaction of thioether substrates to generate chiral sulfoxides, most chemical catalysts still have excessive oxidation, many by-products ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P11/00C12R1/38
CPCC12N9/0069C12N15/70C12P11/00
Inventor 杨加伟陈永正张红燕崔宝东韩文勇
Owner ZUNYI MEDICAL UNIVERSITY
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