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

Active Publication Date: 2019-06-28
NANJING NUOYUN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the process of international patent WO2011005527, due to the low enzyme activity of the enzyme used, 0.3g NAD is needed to catalyze 90 grams of substrate, and the ratio of substrate: NAD is only 300, resulting in an excessive proportion of NAD cost per kilogram of product produced , so that the production cost is high. In the Chinese patent CN102732579, Saccharomyces cerevisiae is used for whole-cell catalysis, but too many yeast cells are required (10g dry cells), and the catalytic substrate can only be completely converted when it is not greater than 0.1mM. This process production efficiency is low

Method used

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  • Alcohol dehydrogenase for producing Atazanavir intermediate
  • Alcohol dehydrogenase for producing Atazanavir intermediate
  • Alcohol dehydrogenase for producing Atazanavir intermediate

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an alcohol dehydrogenase for producing an Atazanavir intermediate and belongs to the technical field of medical biology. The alcohol dehydrogenase shows stronger catalytic activity than a wild alcohol dehydrogenase shown as SEQ ID NO.8 and can be obtained by means of in-vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, directional evolution methods and the like. The alcohol dehydrogenase has one or more mutations of following characteristics including T37A, D38E, P41K, D44N, V45K. The alcohol dehydrogenase has the advantages that, the reaction conditions are mild, the requirement for equipment is low, high temperature or cooling in a production process is not required, and the energy consumption is low; since enzyme catalysis has highefficiency and exclusive selectivity, no by-products are generated when a method is utilized to produce key intermediates of Atazanavir, and purification is convenient. In addition, a reaction solventmainly consists of water, the discharge amount of three wastes is small, and the alcohol dehydrogenase is environmentally friendly.

Description

technical field [0001] The invention relates to a preparation method of an atazanavir intermediate, in particular to an alcohol dehydrogenase used for the production of an atazanavir intermediate, and belongs to the field of medical biotechnology. Background technique [0002] Atazanavir, sold under the trade name Reyataz, is an antiretroviral drug used for the treatment and prevention of HIV / AIDS. It is currently one of the main anti-AIDS drugs in the world. On the list, it is the most important drug needed by the basic health system. Bristol-Myers Squibb first developed atazanavir, which was publicly introduced in Chinese patent CN10282508C. The US FDA approved it for marketing in 2003, and China also in 2007 Approved for its listing. [0003] (2R,3S)-1-Chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol is used as a key intermediate for the preparation of atazanavir. At present, the main production processes include chemical synthesis and biosynthesis There are two met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04
CPCY02E50/10
Inventor 丁雪峰钱明
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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