R-type omega-aminotransferase and application thereof

A technology of transaminase and configuration, which is applied in the field of synthesis of chiral compounds and can solve the problems such as the inability to meet the needs of industrial production of chiral amines

Inactive Publication Date: 2015-02-04
ASYMCHEM LAB TIANJIN +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide an R-type ω-transaminase and its application, so as to improve th...

Method used

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  • R-type omega-aminotransferase and application thereof
  • R-type omega-aminotransferase and application thereof
  • R-type omega-aminotransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Preparation R type omega-transaminase

[0064] The concrete steps of the preparation method of R type ω-transaminase of the present invention are as follows:

[0065] (1) Construction of the template:

[0066] Under the premise of not changing the amino acid sequence, the optimized transaminase gene taA (sequence As shown in SEQ ID NO.: 4, the corresponding amino acid sequence is shown in SEQ ID NO.: 5), the synthetic taA gene was connected to the vector pUC57 to obtain the recombinant plasmid pUC-taA, and then restricted by Nde Ⅰ and Xho Ⅰ Recombinant plasmid pUC-taA and vector pET-22b(+) were simultaneously digested with endonuclease, and the digested product was ligated with T4DNA ligase, and the ligated product was transformed into competent cells of Escherichia coli DH5α strain. After revived on a shaker, spread on LB petri dishes containing ampicillin with a final concentration of 50 μg / ml, and culture overnight in a 37°C incubator. Pick a single ...

Embodiment 2

[0082] Embodiment 2: Activity experiment 1 of R type ω-transaminase

[0083] Add 1 g of the main raw material (N-BOC-piperidone, CAS: 79099-07-3) and 1 mL of dimethyl sulfoxide into the reaction flask. Triethanolamine buffer solution, 0.765g isopropylamine, 0.01g pyridoxal phosphate and 0.01g above-mentioned amino acid sequence as shown in SEQ ID NO.: 2 triethanolamine buffer solution, 0.765g isopropylamine, 0.01g above-mentioned amino acid sequence as shown in SEQ ID NO.: 2, the pH of the system is 9.5 , 30 ℃ constant temperature stirring for 12h. The pH of the system was adjusted to above 10 with 2N NaOH, extracted twice with ethyl acetate, the organic phase was dried, filtered, and concentrated to obtain the crude product (Chinese name: (R)-1-N-Boc-3-aminopiperidine, CAS: 188111-79-7), detected by gas chromatography (GC), the conversion rate was 90.8%, and the e.e value was 100%.

[0084] The nuclear magnetic data of gained product is as follows: 1H-NMR (300MHz, CDCl3) de...

Embodiment 3

[0085] Embodiment 3: Activity experiment 2 of R type ω-transaminase

[0086] Add 0.1g of the main raw material (2,4-dichloroacetophenone, CAS: 2234-16-4) and 1.5mL of polyethylene glycol PEG-400 into the reaction bottle, after the raw material is dispersed, add 23.5ml of phosphate buffer (pH8.0), 0.031g of isopropylamine, 0.0075g of pyridoxal phosphate and 0.02g of the mutant R-type ω-transaminase whose amino acid sequence is shown in SEQ ID NO.: 2, the pH of the system is 8.0, and the temperature is stirred at 45°C 20h. The pH of the system was adjusted to above 10 with 2N NaOH, extracted twice with ethyl acetate, the organic phase was dried, filtered, and concentrated to obtain the crude product ((R)-2,4-dichlorophenethylamine), detected by GC, with a conversion rate of 95 %, e.e. value 100%.

[0087] The nuclear magnetic data of gained product is as follows: 1H NMR (400MHz, DMSO D6): δ=7.67 (d 1H), 7.60 (d, 1H), 7.47 (dd, 1H), 7.34 (dd, 4H), 7.23-7.12 (m , 6H), 4.84 (s, ...

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PUM

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Abstract

The invention discloses an R-type aminotransferase and application thereof. The R-type omega-aminotransferase contains the amino acid sequence disclosed as SEQ ID NO:2, or the omega-aminotransferase-active protein amino acid sequence with high stereoselectivity-R configuration catalytic activity, which has at least 80% of homogeneity with the amino acid sequence disclosed as SEQ ID NO:2 or is subjected to substitution, deficiency or addition of one or more amino acids, wherein the high stereoselectivity means that the content of one stereomer is at least 1.1 times or so of the other stereomer. The R-type omega-aminotransferase has high stereoselectivity, can efficiently synthesize R configuration chiral amines with higher chiral purity, and is suitable for industrial production of chiral amines.

Description

technical field [0001] The invention relates to the field of synthesis of chiral compounds, in particular to an R-type ω-transaminase and its application. Background technique [0002] Chiral amines widely exist in nature and are structural units of many important biologically active molecules. They are important intermediates for the synthesis of natural products and chiral drugs. Many chiral amines can also be important chiral auxiliary agents and chiral resolution reagent. Therefore, the preparation of chiral amine compounds has very important economic significance. [0003] At present, the preparation of chiral amines mainly adopts the method of chemical reduction, using prochiral ketones to prepare optically active amines. Under the catalysis of Pd / C and quinine, prochiral ketones react with formic acid and inorganic ammonia / organic primary amines to form chiral amines; another researcher uses ruthenium complexes as catalysts to asymmetrically Amination reduction to ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P13/00
CPCC12N9/1096C12N15/70C12P13/00C12P13/001C12P41/006C12Y206/01C12P17/12
Inventor 洪浩高峰李艳君张艳李少贺
Owner ASYMCHEM LAB TIANJIN
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