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Preparation method of D-amino acid

An amino acid and acetyl amino acid technology, applied in biochemical equipment and methods, enzymes, hydrolase and other directions, can solve the problems of low product concentration and high production cost, achieve high yield, good product quality, and reduce the cost of enzymes.

Active Publication Date: 2019-01-04
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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  • Preparation method of D-amino acid
  • Preparation method of D-amino acid
  • 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 plate).

[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 into 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 ba...

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 invention relates to a preparation method of D-amino acid. The method comprises the steps of 1, the constructing of recombinant bacteria: an N-acetyl-D-aminoacylase gene is cloned, and the gene isconnected to a pET series carrier or a pKK 223-3 carrier and transformed to BL21 (DE3) host bacteria to construct D acylase recombinant bacteria (NLase); an N-acetyl-amino acid racemase gene is cloned, the gene is connected to the pET series carrier of the pKK223-3 carrier, transformed to the BL21(DE3) host bacteria to construct N-acetylamino acid racemase recombinant bacteria (NAAR); 2, recombinant bacteria fermentation; 3, immobilized enzyme preparation; 4, immobilized enzyme transforming combining with membrane separation; 5, product crystallization. By means of the method, the cost of theenzyme is reduced, the inhibition on the racemase of byproduct acetic acid is reduced, meanwhile, the transformation solution has little impurity, the separation and purification are simple, the yield is high, the product quality is good, the e.e. value of the obtained D-amino acid reaches 99.9% or above, the content is 99% or above, and the mole yield reaches 85% or above.

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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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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