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Ketoreductase mutant for producing prezista intermediate

A technology of darunavir and reductase, applied in biocatalysis methods and application fields, can solve the problems of low conversion efficiency, low enzyme activity, etc., and achieve the effects of low three-waste discharge, high cycle times, and widening application scope.

Active Publication Date: 2020-09-15
NANJING LANGEN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to solve the problem of low enzyme activity and low conversion efficiency in the prior art (2S, 3S)-1-chloro-3-tert-butoxycarbonylamino-4-phenyl-2-butanol enzymatic preparation process technical problem

Method used

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  • Ketoreductase mutant for producing prezista intermediate
  • Ketoreductase mutant for producing prezista intermediate
  • Ketoreductase mutant for producing prezista intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 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. 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 , to get splicing primers.

[0027] 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.

[0028]

[0029] 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 45s, 72°C for 120s, 35x. The DNA fragment obtained by PCR was gel-cut and purif...

Embodiment 2

[0030] Synthesis of embodiment 2 control protein CKSm gene sequence

[0031] According to the sequence shown in SEQ ID NO.7, Nanjing GenScript Biology was entrusted to carry out the whole gene synthesis of the coding sequence of the protein, and cloned it into pET30a to obtain the control protein expression plasmid NYK-CKSm. Its corresponding amino acid sequence is SEQID NO.8.

Embodiment 3

[0032] Embodiment 3 shake flask expression test

[0033]Pick a single colony of Escherichia coli containing the expression vector and inoculate it in 10ml of high-pressure sterilized medium: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, Ammonium Chloride 2.68g / L, Sodium Sulfate 0.71g / L, Magnesium Sulfate Heptahydrate 0.493g / L, Ferric Chloride Hexahydrate 0.027g / L, Glycerin 5g / L, Glucose 0.8g / L, Add Calcium Namycin to 50mg / L. Cultivate overnight at 30°C, 250rpm. The next day, take a 1L Erlenmeyer flask and insert it into 100ml of high-pressure sterilized medium at an inoculation ratio of 1:100: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, phosphoric acid Potassium dihydrogen 3.4g / L, Ammonium chloride 2.68g / L, Sodium sulfate 0.71g / L, Magnesium sulfate heptahydrate 0.493g / L, Ferric chloride hexahydrate 0.027g / L, Glycerin 5g / L, Glucose 0.3g / L, add kanamycin to 50mg / L. Cultivate at 3...

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Abstract

The embodiment of the invention discloses a ketoreductase mutant for producing a prezista intermediate, belongs to the technical field of biological catalysis methods and application, and particularlyrelates to a method for producing (2S, 3S)-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol through the ketoreductase mutant. The ketoreductase mutant is derived from wild type ketoreductaseof Starmerellamagnoliae and can convert 3S-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol into (2S, 3S)-1-chloro-3-tert-butyloxycarbonylamino-4-phenyl-2-butanol; compared with a wild sequence, the ketoreductase mutant has higher alcohol dehydrogenase activity, has more than 90% similarity with SEQ ID NO.8 and has one or more of the following characteristics: I54E, S85A and K182V, and thesequence of the ketoreductase mutant is SEQ ID NO.2. Alcohols are used for coenzyme circulation, the substrate concentration is up to 110 g / L, the ratio of the substrate dosage to the NADP dosage isup to 1100: 1, the number of times of coenzyme circulation is high, and the downstream application range is effectively widened.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis methods and applications, and relates to a ketoreductase mutant used to produce darunavir intermediates, in particular to a ketoreductase mutant used to produce (2S,3S)-1-chloro-3-tert - butoxycarbonylamino-4-phenyl-2-butanol method. Background technique [0002] Ketoreductases are versatile catalysts for the enantioselective reduction of aldehydes or ketones to their corresponding alcohols. (R)-specific ketoreductases have different properties from (S)-specific ketoreductases, and these catalysts are being used more and more frequently in the industrial synthesis of optically active alcohols. Optical activity is a necessary condition for the selective action of many pharmaceutically and agrochemically active compounds, and in some cases one enantiomer has beneficial pharmaceutical activity while the other has genotoxic effects. Therefore, in the synthesis of pharmaceutically and agricultur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P41/00C12P13/02C12R1/19
CPCC12N9/0006C12N15/70C12P41/002C12P13/02C12Y101/01002C12N2800/22
Inventor 丁雪峰王乾李佳松
Owner NANJING LANGEN BIOLOGICAL SCI & TECH
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