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

Transaminase and application thereof in preparation of optical pure chiral amine

A technology of transaminase and enzyme preparation, applied in the field of biochemistry, can solve the problems of low specificity of enzyme substrate, poor enantiomeric selectivity, low conversion rate, etc., and achieve high reaction efficiency, high enantiomeric selectivity and high conversion rate. Effect

Pending Publication Date: 2022-04-12
SHANGHAI STA PHARMA R&D CO LTD +1
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that the enzyme substrate specificity in the prior art is low, the enantioselectivity is poor and the conversion rate is low, does not meet the demand of industrialized production, thereby provides a kind of can with high reaction efficiency, stereoselective Reagents and methods for the preparation of optically pure chiral amines based on their properties and yields

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
  • Transaminase and application thereof in preparation of optical pure chiral amine
  • Transaminase and application thereof in preparation of optical pure chiral amine
  • Transaminase and application thereof in preparation of optical pure chiral amine

Examples

Experimental program
Comparison scheme
Effect test

preparation example

[0084] Using the sequence of SEQ ID NO: 1 (coding sequence as shown in SEQ ID NO: 3) as the parent, adopting strategies such as rolling PCR, iterative saturation mutation, and combined mutation to carry out directed evolution transformation, and then transform the mutant into Escherichia coli BL21 (DE3) competent cells were evenly spread on LB agar plates with 50 μg / ml kanamycin, and placed in a 37°C incubator for static culture for 18 hours. The mutants on the transformed plate were picked with a toothpick into a 96-well plate, and cultured overnight at 37° C. in a shaker at 220 rpm. Take 50 microliters of bacterial liquid from the hole of the first plate and insert it into the corresponding hole of the second plate, incubate at 37°C and 220rpm for 2-3h, add IPTG with a final concentration of 0.2mM, and incubate at 30°C for 20h to obtain the corresponding Mutants were subjected to high-throughput screening. Combined with SFC detection and re-screening, mutants with significa...

Embodiment 1

[0089] Prepare basic phosphate buffer: weigh 27.8g of dipotassium hydrogen phosphate trihydrate and 10.6g of potassium dihydrogen phosphate, add 200mL of purified water, stir at room temperature until the solid dissolves, adjust the pH to 6.9-7.1, add distilled water to make up to 2L.

[0090] Prepare basic isopropylamine buffer solution: add 280 ml basic phosphate buffer solution, 84 ml isopropylamine and 40 ml 85% phosphoric acid into a 500 ml glass bottle to obtain basic isopropylamine buffer solution.

[0091] Add 3 ml of basic isopropylamine buffer solution to the 8 ml reaction bottle, and adjust the pH between 8.8 and 9.1. Add 20 mg of transaminase (amino acid sequence as shown in SEQ ID NO:1) lyophilized powder and 2 mg of pyridoxal 5-phosphate (PLP) to the reaction solution, stir until the solid is completely dissolved, add 50 mg of substrate and 100 microliters Dimethyl sulfoxide was stirred at 28-32°C for 16 hours to fully react.

[0092] The conversion rate detecte...

Embodiment 2

[0096] Add 28 ml of basic phosphate buffer solution, 9.8 ml of sec-butylamine and 2.2 to 3.0 ml of 85% phosphoric acid in a 100 ml jacket to adjust the pH between 8.5 and 9.0. Add 400 mg of transaminase transaminase (amino acid sequence shown in SEQID NO: 2) freeze-dried powder and 20 mg of PLP to the reaction solution, stir until the solid is completely dissolved, add 1 gram of substrate, and react in a closed manner at 28 to 32°C for 64 hours. It's fully responsive.

[0097] The conversion rate detected by SFC was 69.4%, and the ee value was >99%. Such as figure 2 Table 2 and Table 2 show that the peak at t=4.9min in the spectrum is the target compound (R)-1-(3-fluorophenyl)ethylamine.

[0098] Table 2

[0099]

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

PropertyMeasurementUnit
enantiomeric excessaaaaaaaaaa
Login to View More

Abstract

The invention discloses transaminase and application thereof in preparation of optical pure chiral amine. The amino acid sequence of the transaminase disclosed by the invention is shown as SEQ ID NO: 1, or compared with the SEQ ID NO: 1, the amino acid sequence of the transaminase has one or more of 65th, 94th, 132th, 300th and 327th amino acid mutations, and the mutations are increase, deletion or substitution of amino acid residues. The invention also discloses a nucleic acid molecule for coding the transaminase, a nucleic acid construct containing the nucleic acid molecule, a recombinant vector and a host cell. The invention further discloses an enzyme preparation, and the enzyme preparation contains the transaminase disclosed by the invention. The invention also discloses a method for preparing chiral amine by using the transaminase. The enzyme provided by the invention has the advantages of substrate specificity, enantioselectivity, high conversion rate and the like. The preparation method of the optically pure chiral amine provided by the invention has high reaction efficiency, stereoselectivity and yield.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to transaminase and its application, especially the application in biocatalysis for preparing optically pure chiral amine. Background technique [0002] Optically pure chiral amines are a class of important pharmaceutical and fine chemical intermediates. At present, more than 70% of the synthesis of drugs and their derivatives use chiral amines as intermediates. (R)-1-(3-fluorophenyl)ethylamine represented by the following formula II is a very important chiral amine, which is used in the synthesis of many pharmaceutical intermediates. [0003] [0004] At present, the methods for preparing (R)-1-(3-fluorophenyl)ethylamine mainly include chemical methods and biocatalytic methods. The general process of the chemical method is as follows, usually using racemic 1-(3-fluorophenyl)ethylamine as the raw material, using 3-methyl-2,4-acetylacetone to induce chirality, and then performing chemi...

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 Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P41/00C12P13/00
Inventor 周丹李杰郑晨抗叶家捷孙丰来朱景仰
Owner SHANGHAI STA PHARMA R&D CO LTD
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