Preparation method of boscalid
A technology of boscalid and boscalid, which is applied in the field of broad-spectrum fungicide boscalid and its preparation, can solve the problems of increasing the difficulty of mass production, high price, and increasing production costs, and achieve production The effect of low cost, simple post-processing and simple process
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[0043] Example 1 Strain construction of polypeptide enzyme recombinant transformant
[0044] The peptide enzyme sequences derived from the Jannaschia CCS1 strain, Jannaschia sp.EhC01 strain, Jannaschia aquimarina strain, and Litoreibacter ponti strain were searched on NCBI, and the codons were optimized in the E. coli expression system respectively (using the regular online website http: / / www.jcat.de / optimize), the optimized gene will be fully synthesized. A BamHI restriction site is added to the N end of each gene, and a XhoI restriction site is added to the C end, which is digested and connected to the BamHI and XhoI restriction sites of plasmid pET-28a. The N end has a His tag. The process is as follows:
[0045] The digestion system is as follows:
[0046] Target gene / plasmid 1ug
[0047] BamHI 1ul
[0048] XhoI 1ul
[0049] 10×buffer 3ul
[0050] ddH 2 O fill up to 30ul
[0051] The reaction conditions were digested overnight at 37°C, and then the target gene and plasmid were reco...
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[0079] Example 2 Preparation of catalytic bacteria and peptide enzyme protein
[0080] The E.coli RD01 transformant was inoculated into LB medium (yeast extract 0.5%, peptone 1%, sodium chloride 1%, pH 7.0) 50ml / 250ml containing 50μg / ml kanamycin at 37°C, Shake culture at 220 rpm, and add 0.5 mM IPTG for induction when it grows to an OD600 of 0.6-0.8. The induction conditions are 20 degrees and 200 rpm. The cells were collected by centrifugation and suspended in 50 mM phosphate buffer (pH 7.0).
[0081] The bacterial cells were disrupted by ultrasound and centrifuged, and the supernatant was passed through pre-filled and balanced Ni-NTA (purchased from Solarbio|Cat. No.: P2010) to collect the protein in the supernatant. Impurities were eluted with 20 mM imidazole and proteins were eluted with 250 mM imidazole. Then use a 30KDa protein concentration tube to concentrate and concentrate the protein to 2 mg / mL to obtain a protein concentrate, which is the peptide enzyme protein.
[00...
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[0087] Example 3 Preparation of boscalid
[0088] Mix 27.5g of 4'-chloro-2-aminobiphenyl and 66g of 2-chloronicotinoyl chloride into the reaction flask, and then add 54g of the crude protein extract of RD01 polypeptide enzyme of Example 2, and then remove 1.38 under the protection of nitrogen. g of activated carbon is added to the reaction system. The system was stirred at 28°C for 24h. After the reaction is over, the activated carbon is filtered out for reuse. The reaction solution was washed once with 200 mL of water, and the excess 2-chloronicotinyl chloride was recovered and 26.7 g of boscalid particles were obtained. The product was a white solid with a purity of 99.83%, a content of 99.31%, and a yield of 96.3%. 1H NMR (400MHz, CDCl3) δ8.47(dd,J=4.5,1.8Hz,1H),8.37(d,J=8.0Hz,1H),8.17(s,1H),8.14-8.11(m,1H),7.58-7.46(m, 3H), 7.39-7.33 (m, 3H), 7.26 (d, J=3.2 Hz, 2H).
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