Alcohol dehydrogenase for producing Atazanavir intermediate
A technology of alcohol dehydrogenase and intermediates, which is applied in the field of medicine and biology, can solve the problems of large proportion of NAD cost, excessive yeast cells, and low production efficiency, and achieve low equipment requirements, low energy consumption, and low discharge of three wastes Effect
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Embodiment 1
[0036] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.
[0037] Use PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base. The following primers were obtained:
[0038]
[0039]
[0040] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.
[0041] 2mM dNTP mix (2mM each dNTP)
[0042] The prepared PCR reaction system was placed in the Bioer XPcycler gene amplification instrument, and the amplification was carried out according to the following procedures: 98°C for 30s, 55°C for 45...
Embodiment 2
[0044] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.
[0045] Use PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base. The following primers were obtained:
[0046]
[0047]
[0048] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.
[0049] 2mM dNTP mix (2mM each dNTP)
[0050] The prepared PCR reaction system was placed in the Bioer XP cycler gene amplification instrument, and the amplification was carried out according to the following procedures: 98°C for 30s, 55°C for 4...
Embodiment 3
[0052] Through the method of whole gene synthesis, the secondary structure and codon bias of the gene are adjusted to achieve high expression in Escherichia coli.
[0053] Use PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) to design, and ensure that the Tm difference is controlled within 3°C, and the primer length is controlled within 60base. The following primers were obtained:
[0054]
[0055]
[0056] The above primers were synthesized, and the obtained primers were dissolved in double-distilled water, and then added to the following reaction system, so that the final concentration of each primer was 30 nM, and the final concentration of the first and last primers was 0.6 μM.
[0057] 2mM dNTP mix (2mM each dNTP)
5μl
10×Pfu buffer
5μl
Pfu DNA polymerase (10U / μl)
0.5μl
ddH2O
Bring the total volume of the reaction system to 50 μl
[0058] The prepared PCR reaction system was pla...
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