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Biological preparation method for R-3-aminopiperidine

A technology of aminopiperidine and biological preparation, which is applied in the direction of introducing foreign genetic material by using carrier, recombinant DNA technology, fermentation, etc., to achieve the effect of reducing separation cost, easy separation and cheap price

Active Publication Date: 2014-12-03
洛阳华荣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the synthesis of R-3-aminopiperidine from 3-ketopiperidine directly catalyzed by D-transaminase

Method used

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  • Biological preparation method for R-3-aminopiperidine
  • Biological preparation method for R-3-aminopiperidine

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Experimental program
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preparation example Construction

[0019] A biological preparation method of R-3-aminopiperidine, characterized in that: its preparation method comprises the following steps:

[0020] Step 1, preparation of recombinant D-transaminase genetically engineered bacteria

[0021] Choose from Arthrobacter. sp. The wild sequence of D-transaminase was artificially designed, and the designed gene sequence is shown as SEQ ID NO: 1; the sequence was cloned into the Nde I and Hind III sites of pET24a through whole gene synthesis; transformed Escherichia coli DH5α state cells; after picking positive transformants and identifying them by sequencing, the recombinant expression vector was obtained; the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) strain to obtain recombinant D-transaminase genetically engineered bacteria that could induce the expression of recombinant D-transaminase;

[0022] Step 2, preparation of recombinant D-transaminase

[0023] Inoculate recombinant D-transaminase genet...

Embodiment 1

[0029] A biological preparation method of R-3-aminopiperidine, characterized in that: its preparation method comprises the following steps:

[0030] Step 1, preparation of recombinant D-transaminase genetically engineered bacteria

[0031] Choose from Arthrobacter. sp. The wild sequence of D-transaminase was artificially designed, and the designed gene sequence is shown as SEQ ID NO: 1; the sequence was cloned into the Nde I and Hind III sites of pET24a through whole gene synthesis; transformed Escherichia coli DH5α state cells; after picking positive transformants and identifying them by sequencing, the recombinant expression vector was obtained; the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) strain to obtain recombinant D-transaminase genetically engineered bacteria that could induce the expression of recombinant D-transaminase;

[0032] Step 2, preparation of recombinant D-transaminase

[0033] Inoculate recombinant D-transaminase genet...

Embodiment 2

[0039] A biological preparation method of R-3-aminopiperidine, characterized in that: its preparation method comprises the following steps:

[0040] Step 1, preparation of recombinant D-transaminase genetically engineered bacteria

[0041] Choose from Arthrobacter. sp. The wild sequence of D-transaminase was artificially designed, and the designed gene sequence is shown as SEQ ID NO: 1; the sequence was cloned into the Nde I and Hind III sites of pET24a through whole gene synthesis; transformed Escherichia coli DH5α state cells; after picking positive transformants and identifying them by sequencing, the recombinant expression vector was obtained; the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) strain to obtain recombinant D-transaminase genetically engineered bacteria that could induce the expression of recombinant D-transaminase;

[0042] Step 2, preparation of recombinant D-transaminase

[0043] Inoculate recombinant D-transaminase genet...

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Abstract

The invention discloses a biological preparation method for R-3-aminopiperidine. The biological preparation method comprises the following steps: carrying out gene synthesis according to a sequence as shown in SEQ ID NO:1, cloning the gene into an expression vector pET22a to prepare a recombinant expression vector, and transferring the vector into escherichia coli to prepare a genetically engineered bacterium of recombinant D-transaminase; culturing the bacterium to prepare recombinant D-transaminase genetically engineered bacterium; adding 3-ketopiperidine with final concentration of 10-300mmol / L, 2-15wt% dimethyl sulfoxide or acetonitrile, phosphopyridoxal with final concentration of 0.1-1mmol / L, isopropylamine or D-alanine with final concentration of 0.02-1mol / L and 0.2-0.4wt% recombinant D-transaminase to form a reaction system to carry out reaction; and extracting the R-3-aminopiperidine in the reaction liquor after the reaction is ended. According to the biological preparation method, the cost is low, the conversion ratio is over 97%, the product yield is greater than 85% and byproducts are not produced, so that the biological preparation method is more suitable for industrial application.

Description

Technical field [0001] The present invention involves the preparation method of R-3-aminoprimidine. Specifically, it is a biological preparation method of R-3-amino pyrine. Background technique [0002] Bio -catalytic has the characteristics of mild response conditions, high efficiency, high three -dimensional and regional selectivity, and environmental friendly. Therefore, biocatalysis has been widely used in the research and development of new drugs.In addition, the use of rational design and directional evolution to transform and optimize the biocatalyst, such as improving the stability and substrate selectivity of the catalyst, which will enable it to better apply to the production process.Woronase is mainly used as a biocity agent to synthesize hand ammonia, hand amino acids, and hand amino alcohol. [0003] R-3-Aminozimidine is widely used in organic synthetic and drug production. The structure is as follows: [0004] [0005] There are currently useful chemistry methods,...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N15/70
Inventor 范文超丁鹏
Owner 洛阳华荣生物技术有限公司
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