Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of ketoreductase mutant used for producing darunavir intermediate

A technology of darunavir and reductase, applied in biocatalysis methods and application fields, can solve the problems of low enzyme activity and low conversion efficiency, and achieve the effects of high cycle times, low three-waste emissions, and high alcohol dehydrogenase activity

Active Publication Date: 2022-03-04
NANJING LANGEN BIOLOGICAL SCI & TECH
View PDF5 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of ketoreductase mutant used for producing darunavir intermediate
  • A kind of ketoreductase mutant used for producing darunavir intermediate
  • A kind of ketoreductase mutant used for producing darunavir 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The embodiment of the present invention discloses a ketoreductase mutant used to produce darunavir intermediates, which belongs to the field of biocatalytic methods and application technologies, and specifically relates to a method for producing (2S,3S)-1-chloro-3- tert-butoxycarbonylamino-4-phenyl-2-butanol converts 3S-1-chloro-3 ‑tert-butoxycarbonylamino‑4‑phenyl‑2‑butanone is converted to (2S,3S)‑1‑chloro‑3‑tert‑butoxycarbonylamino‑4‑phenyl‑2‑butanol, as compared to wild-type Compared with the sequence, it has higher alcohol dehydrogenase activity, has more than 90% similarity with SEQ ID NO.8 and has one or more mutations in the following characteristics: I54E, S85A, K182V, the ketoreductase mutant The sequence is SEQ ID NO.2. The present invention uses alcohols for coenzyme circulation, the substrate concentration is as high as 110g / L, the amount of substrate / NADP is as high as 1100:1, the number of coenzyme cycles is high, and the downstream application range is effectively expanded.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com