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Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase

A technology of methylthiobutyric acid and nitrilase, applied in the direction of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the difficulties of crystallization and separation of methionine, incomplete conversion, and low activity of methioninase And other issues

Inactive Publication Date: 2013-02-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported use of wild bacteria containing nitrilase to catalyze the preparation of methioninase has relatively low activity and incomplete conversion, which brings difficulties to the subsequent crystallization and separation of methionine

Method used

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  • Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase
  • Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase
  • Method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1: Construction of prokaryotic expression plasmid of nitrilase gene

[0074] Using PCR technology, using the genomic DNA in CCTCC NO.M209044 of Acidovorax facilis (Acidovorax facilis) as a template (genome extraction kit from MP Company in the United States) as a template, and using the designed positive and negative primers for PCR amplification, the amplification system is shown in Table 1 As shown, the primers are as follows:

[0075] P1:5'-ATGGTTTCGTATAACAGCAAG-3'

[0076] P2:5'-CTACTTTGCTGGGACCGG-3'.

[0077] The PCR reaction system is:

[0078] Table 1 PCR amplification reaction system

[0079]

[0080]PCR reaction process: 94°C for 5min; 94°C for 45s, 55°C for 45s, 35 cycles; 72°C for 2min; 72°C for 10min.

[0081] The amplified product is a 1121bp nitrilase gene sequence (SEQ ID NO.1).

[0082] Take 5 μL of PCR reaction solution and use 0.9% agarose gel electrophoresis for detection. First, heat the prepared 0.9% agarose gel in a microwave ove...

Embodiment 2

[0083] Embodiment 2 Contains the construction of the recombinant genetically engineered bacterium of nitrilase gene

[0084] The SEQ ID NO.1 sequence that obtains according to embodiment 1, and introduce BamHI and SalI restriction endonuclease cut site, utilize restriction endonuclease BamHI and SalI to process SEQ ID NO.1 sequence; Then with the same restriction endonuclease Dicer-digested expression vector pET-28b was ligated to obtain the recombinant expression plasmid pET-28b-NIT, and it was electrotransformed into Escherichia coli BL21 (DE3) to obtain recombinant genetically engineered bacteria E.coli BL21 (DE3) / pET -28b-NIT.

Embodiment 3

[0085] Example 3: Expression of the nitrilase gene

[0086] (1) Cells containing the nitrilase gene: Inoculate the recombinant engineering bacteria E.coli BL21(DE3) / pET-28b-NIT constructed by the method in Example 2 and containing the recombinant plasmid pET-28b-NIT into LB liquid medium (Containing 50 μg / ml kanamycin), cultured at 37°C for 12 hours to obtain seed liquid, and inoculated the seed liquid into fresh LB liquid medium containing 50 μg / ml kanamycin at an inoculation volume of 3% by volume Medium, cultivated at 37°C to the cell concentration (i.e. OD 600 ) is about 0.6~0.8, then add the inducer IPTG (final concentration is 0.2mM) to the above LB liquid medium, induce culture at 37°C for 8h, obtain the induction culture medium, and then centrifuge the induction culture medium at 4°C, 10000rpm After 10 minutes, the wet cells were collected to obtain wet cells containing nitrilase (that is, whole cells of recombinant E. coli), which were used as an enzyme source for th...

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Abstract

The invention discloses a method for preparing 2-amino-4-methylthio butyric acid by using recombinant nitrilase, which comprises the following steps: performing water bath shaker reaction (25-40 DEG C, 150-200rpm) on an enzyme source and a substrate in a reaction system of a buffer solution with a pH value of 7.0-9.0, wherein the enzyme source is a wet bacterium obtained through the induced culture of a recombinant engineering bacterium containing nitrilase, and the substrate is 2-amino-4-methylthio butyronitrile; and after the reaction is finished, performing post treatment on the reaction liquid to obtain 2-amino-4-methylthio butyric acid. According to the invention, the gene engineering bacterium is used for experiment of methionine production through biological catalysis and can biologically catalyze methionine production effectively, and the 2-amino-4-methylthio butyronitrile conversion rate within 2 hours is up to 80-100%. Thus, the bacterium has wide application prospects in the field of methionine production through biological catalysis.

Description

(1) Technical field [0001] The present invention relates to the enzymatic production process of 2-amino-4-methylthiobutyric acid, in particular to an enzyme-catalyzed technology developed on the basis of enzymatic synthesis to produce 2-amino-4-methanol Thiobutyric acid method. (2) Background technology [0002] 2-Amino-4-methylthiobutyric acid (methionine) is widely used in the fields of medicine, food, feed and cosmetics, among which the amount of feed additive is the largest. As the first limiting essential amino acid for poultry, methionine is an essential additive in animal feed. Animal feed with methionine can help animals grow rapidly in a short time, increase lean meat mass and shorten feeding cycle, saving about 40 % feed. In the pharmaceutical industry, methionine is one of the main components of amino acid infusion and compound amino acids. In addition to participating in the transfer of methyl groups in animals, the metabolism of phosphorus and the synthesis o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12N15/60C12N1/21C12R1/19
Inventor 郑裕国金利群柳志强薛亚平沈寅初
Owner ZHEJIANG UNIV OF TECH
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