Method for enzymatic synthesis of esomeprazole
A technology for esomeprazole and enzymatic synthesis, which is applied in directions such as fermentation, can solve problems such as complicated operation and many procedures, and achieve the effects of simplifying operation steps, reducing production costs and shortening time.
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Embodiment 1
[0047] Embodiment 1: Construction of cyclohexanone monooxygenase genetically engineered bacteria
[0048] Commissioned Shanghai Jierui Bioengineering Co., Ltd. to custom synthesize cyclohexanone monooxygenase gene fragments SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the corresponding encoded amino acid sequences are SEQ ID NO.2 and SEQ ID NO.2, respectively. ID NO.4 and SEQ ID NO.6. Then use the gene fragment as a template to amplify and expand by PCR (add Nde I and BamH I endonuclease fragments to both ends of the gene fragment), and insert the gene fragment into the pET28a plasmid using the Nde I and BamH I endonuclease sites At last, the vector obtained by ligation was transformed into Escherichia coli BL21 (DE3), and the recombinant Escherichia coli genetically engineered strains containing the cyclohexanone monooxygenase gene were constructed, respectively denoted as bacterial strain 1#, bacterial strain 2# and bacterial strain 3 #.
[0049] The primer sequences desi...
Embodiment 2
[0052] Embodiment 2: Construction of ketoreductase genetically engineered bacteria
[0053] Entrusted Shanghai Jierui Bioengineering Co., Ltd. to customize and synthesize the ketoreductase gene fragments SEQ ID NO.7, SEQ ID NO.9 and SEQ ID NO.11, and the corresponding encoded amino acid sequences are SEQ ID NO.8 and SEQ ID NO.10 respectively and SEQ ID NO.12. Then use the gene fragment as a template to amplify and expand by PCR (add Nde I and BamH I endonuclease fragments to both ends of the gene fragment), and insert the gene fragment into the pET28a plasmid using the Nde I and BamH I endonuclease sites , and finally the vector obtained by ligation was transformed into E. coli BL21(DE3), and recombinant E. coli genetically engineered strains containing the ketoreductase were constructed, which were respectively designated as strain 4#, strain 5# and strain 6#.
[0054] The primer sequences designed for strain 4# PCR amplification extension are as follows:
[0055] Forward p...
Embodiment 3
[0063] Embodiment 3: the construction of co-expression genetically engineered bacteria
[0064] With reference to "Molecular Cloning-A laboratory Manual" (third edition, 2001), the following enzyme digestion, connection, or preparation and transformation of competent cells were performed.
[0065] Design following primers F5 and R5, take the gene fragment of SEQ ID NO.1 cyclohexanone monooxygenase as template, expand the described cyclohexanone monooxygenase gene fragment by PCR amplification (add Nde I and BamH I endonuclease fragment); and utilize Nde I and BamH I endonuclease sites to insert the gene into the pET-21a plasmid, transfer the ligated vector into Escherichia coli Trans-T1, and construct the recombinant plasmid, named pETC. Colony PCR verification was performed with primer T7 / R3, the recombinant plasmid was extracted and sequenced, and the recombinant plasmid pETC with correct results was obtained.
[0066] Forward primer F5: GGCCATATGAATAATTTTGTTTAACTTTAAGAAGG...
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