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Method for efficiently producing KMTB (alpha-keto-gamma-methylthiobutyric acid)

A high-yield, amino acid technology, applied in the field of bioengineering, can solve the problems of increasing cost, protein denaturation, affecting the utilization of D-amino acid oxidase, etc., and achieve the effect of improving production efficiency and high vitality

Active Publication Date: 2018-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But using this method will be accompanied by the generation of hydrogen peroxide
Hydrogen peroxide can cause protein denaturation, thereby affecting the availability of D-amino acid oxidase
In order to eliminate the influence of hydrogen peroxide, it is necessary to add catalase, which additionally increases the cost of production

Method used

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  • Method for efficiently producing KMTB (alpha-keto-gamma-methylthiobutyric acid)
  • Method for efficiently producing KMTB (alpha-keto-gamma-methylthiobutyric acid)
  • Method for efficiently producing KMTB (alpha-keto-gamma-methylthiobutyric acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Containing the construction of L-amino acid oxidase genetically engineered bacteria

[0033] (1) The codon-optimized artificially synthesized L-AAO deaminase gene containing BamHI and XhoI restriction sites, as shown in SEQ ID NO.2.

[0034] (2) Digest the target gene and expression vector pET28a at 37°C for 2 hours with restriction endonucleases BamHI and XhoI;

[0035] (3) Ligate the target gene and plasmid pET28a after digestion and gel recovery with T4 ligase at 16°C for 10 h;

[0036] (4) Introduce the constructed expression plasmid into E.coli BL21(DE3), and culture it on the LB plate containing kanamycin for 12h;

[0037] (5) Perform PCR and enzyme digestion verification on the colonies grown on the plate, and perform sequencing verification on the plasmid containing the target gene, and select the strain with the correct target gene, which is the genetically engineered strain expressing L-amino acid oxidase E.coli BL21-L-AAO.

Embodiment 2

[0038] Embodiment 2: Induced expression of genetically engineered bacteria

[0039] (1) Insert the constructed genetically engineered bacteria E.coli BL21-L-AAO into LB slant medium and cultivate for 12 hours;

[0040] (2) Put a ring of slanted seeds into the LB medium and cultivate for 6 hours;

[0041] (3) Put the E.coli BL21-L-AAO seed liquid into the TB fermentation medium and cultivate it to OD 600 0.6, adding IPTG with a final concentration of 0.4mM for induction, after induction at 25°C for 12 hours, the cells were collected, and the cells were washed with sterile normal saline.

Embodiment 3

[0042] Embodiment 3: the influence of different bacterial body weights on the production of α-keto-γ-methylthiobutyric acid

[0043]The wet thallus obtained in Example 2 was taken, collected by centrifugation and used as a catalyst for whole cell transformation. The pH is controlled to be 8.0, the temperature is 30°C, and the addition amount of bacteria is adjusted (30g / L, 40g / L, 50g / L, 60g / L). The reaction solution included L-methionine with a final concentration of 100 g / L and 20 mM Tris-HCL. After converting 24h, the situation of the concentration of α-keto-γ-methylthiobutyric acid in the supernatant liquid after adopting high performance liquid chromatography to measure reaction (such as figure 2 shown), when the final concentration of bacterium was 30g / L, the concentration of α-keto-γ-methylthiobutyric acid was 77.45g / L; when the bacterium was 40g / L, the concentration of α-keto-γ -The concentration of methylthiobutyric acid is 94.46g / L; when the final concentration of ...

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Abstract

The invention discloses a method for efficiently producing KMTB (alpha-keto-gamma-methylthiobutyric acid) and belongs to the technical field of bioengineering. A gene derived from rhodococcus erythropolis encoded L-amino acid oxidase is subjected to artificial synthesis and codon optimization with a molecular biology method and then connected with an expression vector. A constructed expression plasmid is introduced into E. coli BL21 (DE3), and expression vector high-copy recombinant engineering bacteria containing L-amino acid oxidase are obtained through kanamycin resistance plate screening.The engineering bacteria are used for converting L-methionine to produce KMTB. Efficient production of KMTB is realized by controlling bacterium quantity, temperature and pH. 30 g / L of wet bacteria are put under the conditions of 20 DEG C and pH of 7.5 to react for 24 h, the yield of KMTB can reach 95.18 g / L, the conversion rate is 95.84%, and the space time yield is 3.97 g / L / h.

Description

technical field [0001] The invention relates to a method for high-yielding α-keto-γ-methylthiobutyric acid, which belongs to the technical field of bioengineering. Background technique [0002] As an essential amino acid, L-methionine can only be obtained through diet. Important for human and poultry health. It also plays an important role in the function of cells. Because L-methionine is rapidly decomposed in the body, the amount entering the blood circulation is very small. As a derivative of L-methionine, α-keto-γ-methylthiobutyric acid can increase the utilization efficiency of the body. In addition, α-keto-γ-methylthiobutyric acid is safe and non-toxic, can inhibit the growth of tumor cells, and is also used to treat uremia patients. [0003] α-Keto-γ-methylthiobutyric acid (α-Keto-γ-methylthiobutyric acid, referred to as KMTB) is a keto acid containing two functional groups, the molecular formula is C 5 h 8 o 3 S. The production methods of α-keto-γ-methylthiobu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/06C12P7/40C12R1/19
CPCC12N9/0022C12P7/40C12Y104/03002
Inventor 刘立明张灿刘佳陈修来罗秋玲
Owner JIANGNAN UNIV
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