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A kind of thioether monooxygenase derived from Pseudomonas and its synthetic application

A technology of genetically engineered bacteria and coding genes, which is applied in the fields of chiral drug synthesis, monooxygenase gene cloning and recombinant protein expression, can solve the problem of low catalytic efficiency, lack of high enantioselective thioether monooxygenase, Unable to meet the problems of green industrial production

Active Publication Date: 2017-11-10
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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  • A kind of thioether monooxygenase derived from Pseudomonas and its synthetic application
  • A kind of thioether monooxygenase derived from Pseudomonas and its synthetic application
  • A kind of thioether monooxygenase derived from Pseudomonas and its synthetic application

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1. Acquisition of monooxygenase encoding gene

[0029] The genome sequence of Pseudomonas strain was analyzed, and a monooxygenase sequence with a length of 972 bp was determined. A pair of specific primers were designed to be amplified by PCR technology, and the full-length coding frame sequence of the gene was finally obtained. The specific method is as follows: using the extracted genomic DNA as a template, using primers F: 5'-actcGGATCCatgcagaccgttgaacacgaat-3' and R: 5'-accgAAGCTT

[0030] PCR amplification of gtaggctttgtcccagct-3'. The PCR reaction system is as follows: 12.5 ul of 2×TaqPCR MasterMix, 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; 98 °C for 10 s, 56 °C for 30 s, 72 °C for 90 s; extension at 72 °C for 10 min, 30 cycles. PCR products were tested by 1% agarose gel electrophoresis to verify whether fragments of the size of the target gene were obtained.

Embodiment 2

[0031] Example 2. Construction of monooxygenase gene expression vector

[0032]Using the pET32a(+) vector (purchased from Clonetech) as the backbone, a prokaryotic expression vector of the monooxygenase gene pmo-6814 was constructed. The specific method is as follows: the PCR product of pmo-6814 was recovered by DNA gel recovery kit, the PCR product and the vector pET32a(+) were double digested with the restriction enzymes BamHI and HindⅢ, and the recovered target fragment and vector were digested with restriction enzymes. . After ligation with T4 DNA ligase at 16 °C for 4 h, the ligation product was transformed into E. coli DH5α. After overnight culture, pick out the grown monoclonal colony and shake the bacteria and extract the plasmid. The recombinant plasmid was detected by double enzyme digestion with BamHI and HindIII. The positive recombinant plasmid pET32a-pmo-6814 was selected for DNA sequencing to ensure the sequence was accurate. Finally, the positive recombinant...

Embodiment 3

[0033] Example 3. Monooxygenase protein recombinant expression and detection

[0034] After the genetically engineered bacteria BL21(DE3) containing pET32a-pmo-6814 plasmid stored in glycerol was plated and activated, single colonies were picked in 3 ml of liquid LB medium containing corresponding antibiotics, and cultured at 37 °C with shaking for 12 h, and the next day. Transfer to 50 mL of fresh LB liquid medium containing antibiotics with 1% inoculum, shake and culture at 37 °C at 250 rpm with an OD600 of 0.6 (about 3 h), add IPTG with a final concentration of 0.5 mM, and induce culture at 25 °C at 160 rpm 8h. After induction, the cells were collected by centrifugation at 8000 rpm / min for 5 min, the cells were resuspended in PBS buffer, the cells were disrupted by ultrasonic, and the cell debris was removed by centrifugation at 15,000 rpm for 5 min. After heating at 100 °C for 5 min in a constant temperature metal bath, SDS-PAGE electrophoresis was performed.

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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

[0001] A thioether monooxygenase derived from Pseudomonas and its synthetic application technical field [0002] 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 field of biocatalysis and biosynthesis. The present invention also relates to a method for monooxygenase gene cloning and recombinant protein expression. Background technique [0003] Chiral sulfoxide compounds are key intermediates in the synthesis of many drugs such as the antitumor drug Ustiloxin A, the anti-inflammatory drug sulindac, the gastric ulcer drug omeprazole, and the antibiotic sparoxomycin. has a very wide range of applications. At present, except for some transition metal catalysts such as titanium and vanadium, which can effectively catalyze the asymmetric oxidation of thioether substrates to generate chiral sulfoxide, most chem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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