Bioconversion method for anti-AIDS-medicine-reyataz midbody

A biotransformation and anti-AIDS technology, applied in the field of medicine and biology, can solve the problems of too many yeast cells, low enzyme activity, and a large proportion of NAD cost.

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...

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  • Bioconversion method for anti-AIDS-medicine-reyataz midbody
  • Bioconversion method for anti-AIDS-medicine-reyataz midbody
  • Bioconversion method for anti-AIDS-medicine-reyataz midbody

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Experimental program
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Effect test

Embodiment 1

[0118] The biotransformation method of the anti-AIDS drug atazanavir intermediate provided in the present embodiment 1 comprises the following steps:

[0119] 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.

[0120] Use Primer Premier (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 , resulting in the following primers:

[0121] 1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

[0122] CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATA

[0123] 2 TGTATATCTCCCTT

[0124] GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTG

[0125] 3 AACTGGTTAAAG

[0126] GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCA

[0127] 4 GTTCACGACCGA

[0128] ACGCTATCGTTATCGCTGCTGAAATGGCTCCGTCTGCTGACAA

[0129] 5 AGAAGGTGCTGA

[0130] CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAG...

Embodiment 2

[0172] The biotransformation method of the anti-AIDS drug atazanavir intermediate provided in this embodiment 2 comprises the following steps:

[0173] 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.

[0174] Use Primer Premier (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 , resulting in the following primers:

[0175] 1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCG

[0176] CCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCATA

[0177] 2 TGTATATCTCCCTT

[0178] GTTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTG

[0179] 3 AACTGGTTAAAG

[0180] GCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAACCA

[0181] 4 GTTCACGACCGA

[0182] ACGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTGACAA

[0183] 5 AGAAGGTGCTGA

[0184] CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCA

[01...

Embodiment 3

[0226] The biotransformation method of the anti-AIDS drug atazanavir intermediate provided in this embodiment 3 comprises the following steps:

[0227] 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.

[0228] Use Primer Premier (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 , resulting in the following primers:

[0229] 1 TGTTTAACTTTAAGAAGGAGATATACATATGACCATCGC

[0230] ACCGGTAACAACAGCAACAACGTTGTTCAGAGCGATGGTCAT

[0231] 2 ATGTATATCTCCT

[0232] TTGTTGCTGTTGTTACCGGTGCTGCTGGTGGTATCGGTCGTGA

[0233] 3 ACTGGTTAAAGC

[0234] CAGCAGCGATAACGATAGCGTTAGCAGCTTTCATAGCTTTAAC

[0235] 4 CAGTTCACGACC

[0236] CGCTATCGTTATCGCTGCTGAAATGGCTAAATCTGCTAACAAA

[0237] 5GAAGGTGCTGAC

[0238] CAGCTTCAGAGGTAACGTCGTGCTGCAGGTAGTGGTCAGCA

[0...

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Abstract

The invention discloses a bioconversion method for anti-AIDS-medicine-reyataz midbody, and belongs to the technical field of medical biology. Secondary structures of genes and codon usage biases are adjusted, the length of primers is controlled within 60 base, the primers are obtained, the obtained primers are dissolved with double distilled water, then the dissolved primers are added into a reaction system, a prepared PCR reaction system is amplified, obtained DNA fragments are subjected to gel cutting purification, and then are cloned into NdeI/XhoI loci of pET30a with a homologous recombination method, a successfully-sequenced DNA sequence is SEQ ID NO.3, and is named as Sst-2, and the corresponding amino acid sequence of the Sst-2 is SEQ ID NO.4. The bioconversion method is mild in reaction condition, and has the low requirement for equipment, the high temperature or cooling is not required in the production process, energy consumption is low, purification is convenient, single enzyme catalysis is used in the whole system, the cycle number of coenzyme is high, and the reaction condition is mild.

Description

technical field [0001] The invention relates to a preparation method of an atazanavir intermediate, in particular to a biotransformation method of an anti-AIDS drug atazanavir intermediate, belonging 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 methods, and...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N9/04C12P13/02C12R1/19
CPCY02A50/30
Inventor 丁雪峰钱明
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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