A kind of preparation method of d-amino acid

A technology of amino acid and acetyl amino acid, applied in biochemical equipment and methods, racemase/epimerase, fermentation, etc., can solve the problems of high production cost and low product concentration

Active Publication Date: 2021-09-10
浙江正硕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And no report adopts immobilized N-acetyl-D-aminoacylase and immobilized N-acetylamino acid racemase to produce D-amino acid
And there is the shortcoming that the by-product sodium acetate inhibits racemase, resulting in low product concentration and high production cost

Method used

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  • A kind of preparation method of d-amino acid
  • A kind of preparation method of d-amino acid
  • A kind of preparation method of d-amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Construction of recombinant bacteria

[0032] clone derived from Aspergillus oryzae The N-acetyl-D-aminoacylase gene was connected into pET41a vector (Novagen) or pKK223-3 vector, transformed into BL21(DE3) host bacteria, and D-acylase recombinant bacteria (NLase) were constructed.

[0033] clone derived from Sebekia Benihana The N-acetyl-amino acid racemase gene was connected to the pET41a vector (Novagen Company), and transformed into BL21(DE3) host bacteria to construct N-acetyl amino acid racemase recombinant bacteria (NAAR).

[0034] 2. Fermentation of recombinant bacteria to prepare enzymes

[0035] The recombinant bacteria NLase and NAAR were fermented with TB.

[0036] 1) culture medium

[0037] (1) LB:

[0038]

[0039] (2) Agar LB-Kan plate (g / L):

[0040]

[0041] Note: The culture medium is sterilized and cooled to 50-60°C, add 100μl Kan solution (100mg / ml) to 100ml LB, mix well, and pour it onto a plate (25-30ml / 6cm dish).

[0042] (3) TB ...

Embodiment 2

[0060] 1. Construction of recombinant bacteria

[0061] N-acetyl-D-aminoacylase derived from Alcaligenes denitrificans was cloned, and the gene was connected to pKK223-3 vector (Novagen Company) or transformed into BL21(DE3) host bacteria to construct D-acylase recombinant bacteria (NLase).

[0062] The N-acetyl-amino acid racemase derived from Amycolatopsis azurea was cloned, the gene was connected to pET28a vector (Novagen), and transformed into BL21(DE3) host bacteria to construct N-acetyl amino acid racemase recombinant bacteria (NAAR).

[0063] 2. Fermentation of recombinant bacteria to prepare enzymes

[0064] The recombinant bacteria NLase and NAAR were fermented with TB.

[0065] 1) culture medium

[0066] With embodiment 1.

[0067] 2) Fermentation process

[0068] (1) Strain activation: inoculate the recombinant bacteria NLase and NAAR on Kan LB agar plates respectively, and culture them at 37°C for 12-14 hours.

[0069] (2) First-class seeds: Pick a single bact...

Embodiment 3

[0083] 1. Construction of recombinant bacteria

[0084] The N-acetyl-D-aminoacylase derived from Alcaligenes denitrificans was cloned, the gene was connected into pET28a vector (Novagen), and transformed into BL21(DE3) host bacteria to construct D-acylase recombinant bacteria (NLase).

[0085] The N-acetyl-amino acid racemase derived from Deinococcus radiodurans was cloned, the gene was connected into pET28a vector (Novagen), and transformed into BL21(DE3) host bacteria to construct N-acetyl amino acid racemase recombinant bacteria (NAAR).

[0086] 2. Fermentation of recombinant bacteria to prepare enzymes

[0087] The recombinant bacteria NLase and NAAR were fermented with TB.

[0088] 1) culture medium

[0089] With embodiment 1.

[0090] 2) Fermentation process

[0091](1) Strain activation: Inoculate the recombinant bacteria NLase and NAAR on Kan LB agar plates respectively, and culture them at 37°C for 14 hours.

[0092] (2) First-class seeds: Pick a single bacterium...

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Abstract

The present invention relates to a preparation method of D-amino acid, comprising the following steps: 1. Construction of recombinant bacteria: cloning N-acetyl-D-aminoacylase gene, connecting the gene into pET series vector or pKK223-3 vector, and transforming it into BL21 (DE3) host bacteria, construct D-acylase recombinant bacteria (NLase); clone N-acetyl-amino acid racemase gene, connect the gene into pET series vector or pKK223-3 vector, transform into BL21(DE3) host bacteria, construct N-acetylamino acid racemase recombinant bacteria (NAAR); 2. Fermentation of recombinant bacteria; 3. Preparation of immobilized enzyme; 4. Transformation of immobilized enzyme combined with membrane separation; 5. Product crystallization. The invention reduces the cost of the enzyme and reduces the inhibition of the by-product acetic acid on the racemase. At the same time, because the conversion liquid has few impurities, the separation and purification are simple, the yield is high, and the product quality is good. The obtained D-amino acid has an e.e. value of more than 99.9%, a content of more than 99%, and a molar yield of more than 85%.

Description

technical field [0001] The invention relates to a preparation method of D-amino acid. Background technique [0002] There are 20 kinds of amino acids in nature, all of which are L-type amino acids, which are the basic structural units of proteins. Therefore, it is generally believed that D-amino acids rarely exist in nature. In recent years, with the deepening of scientific research, it has been found that although D-amino acids are not the basic structural nitrogen source of proteins, they are found in many plants, microorganisms and higher plants. Presence of D-amino acids. The research results show that D-amino acid has an irreplaceable position of L-amino acid in life activities and drug preparation. It is an important drug synthesis intermediate and food additive, and is widely used in the production of semi-synthetic antibodies, hormones, biologically active peptides and chemical pesticides. Especially as the side chain raw material of semi-synthetic antibiotics, D-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04C12N9/80C12N9/90C12N11/00
CPCC12N9/80C12N9/90C12N11/00C12P13/04C12Y305/01014C12Y501/00
Inventor 吴黎诚郭小雷章权
Owner 浙江正硕生物科技有限公司
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