CN107988131B

CN107988131B - Method for high-yield production of α -ketone-gamma-methylthiobutyric acid

Info

Publication number: CN107988131B
Application number: CN201810035749.8A
Authority: CN
Inventors: 刘立明; 张灿; 刘佳; 陈修来; 罗秋玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-01-15
Filing date: 2018-01-15
Publication date: 2020-06-09
Anticipated expiration: 2038-01-15
Also published as: CN107988131A

Abstract

The invention discloses a method for producing α -keto-gamma-methylthiobutyric acid with high yield, which belongs to the technical field of bioengineering.A gene derived from Rhodococcus erythropolis encoding L-amino acid oxidase is artificially synthesized and codon-optimized by a molecular biology hand and then is connected with an expression vector.A constructed expression plasmid is introduced into E.coli BL21(DE3), an L-amino acid oxidase engineering bacterium containing high copy recombination of the expression vector is obtained by screening a kanamycin resistant plate, the L-amino acid oxidase engineering bacterium is used for converting L-methionine to produce α -keto-gamma-methylthiobutyric acid, the efficient production of α -keto-gamma-methylthiobutyric acid is realized by controlling the bacterium amount, the temperature and the pH value, 30g/L wet bacterium is put into the bacterium under the conditions of 20 ℃ and pH7.5 for reaction for 24h, the yield of α -keto-gamma-methylthiobutyric acid can reach 95.18g/L, the conversion rate is 95.84%, and the space-time yield is 3.97 g/L/h.

Description

Method for high-yield production of α -ketone-gamma-methylthiobutyric acid

Technical Field

The invention relates to a method for producing α -keto-gamma-methylthiobutyric acid with high yield, belonging to the technical field of biological engineering.

Background

The amount of L-methionine entering blood circulation is very small because L-methionine is rapidly decomposed in the body, α -keto-gamma-methylthiobutyric acid, which is a derivative of L-methionine, can increase the utilization efficiency of the body, furthermore, α -keto-gamma-methylthiobutyric acid, which is safe and non-toxic, can inhibit the growth of tumor cells, and is also used to treat uremia patients.

α -Keto-gamma-methylthiobutyric acid (α -Keto-gamma-methylthiobutyric acid, KMTB for short) is a Keto acid containing bifunctional groups,molecular formula C₅H₈O₃The method for producing the α -keto-gamma-methylthiobutyric acid by the enzyme catalysis method is characterized in that the enzyme catalysis method is used for producing the α -keto-gamma-methylthiobutyric acid by converting methionine into enzyme produced by microorganisms, and the enzyme catalysis method is used for producing the α -keto-gamma-methylthiobutyric acid by converting methionine into the methionine, is basically a one-step enzymatic reaction, can avoid feedback regulation in a plurality of metabolic pathways, achieves the aim of accumulating a large amount of amino acid hydrogen peroxide, improves the safety of the large-scale preparation of the large-scale keto-gamma-methylthiobutyric acid, is high in safety, is an enzymatic reaction for producing the α -keto-gamma-methylthiobutyric acid by using a biotechnology method, is an economic application method for producing the large-scale keto-gamma-methylthiobutyric acid by applying a biological technology method, is an economic application method for producing the L-keto-gamma-methylthiobutyric acid by using a hydrogen peroxide substrate D, and has an influence on the production of L-amino acid hydrogen peroxide substrate D, and the production of the L-amino acid hydrogen peroxide, and the L-amino acid hydrogen peroxide are more attractive, and the production cost of the enzyme catalysis method for producing the L-amino acid hydrogen peroxide is increased.

Disclosure of Invention

The first purpose of the invention is to provide a recombinant bacterium for high yield of L-amino acid oxidase, which takes escherichia coli as a host and pET series plasmids as a vector to express the L-amino acid oxidase, wherein the L-amino acid oxidase contains an amino acid sequence shown as SEQ ID No. 1.

In one embodiment of the invention, the vector is a pET series vector.

In one embodiment of the invention, the vector is pET28 a.

In one embodiment of the invention, the e.coli comprises e.coli BL21, e.coli jm109, e.coli DH5 α or e.coli top 10.

The second purpose of the invention is to provide a construction method of a recombinant bacterium for high yield of L-amino acid oxidase, which comprises the steps of connecting a vector with a gene for coding the L-amino acid oxidase, and transforming the gene into a host cell; the gene contains a nucleotide sequence shown as SEQID NO. 2.

In one embodiment of the invention, the gene encoding the L-amino acid oxidase is as shown in SEQ ID NO.1 and the E.coli is E.coli BL21(DE 3).

In one embodiment of the invention, the host cell includes, but is not limited to, E.coli.

In one embodiment of the present invention, the construction of the recombinant bacterium for producing L-amino acid oxidase with high yield specifically comprises the following steps: pET28a is used as a vector, the gene shown in SEQ ID NO.2 is connected with the vector, and the L-amino acid oxidase shown in SEQ ID NO.1 is recombined and expressed in Escherichia coli E.coli BL21(DE 3).

The third object of the present invention is to provide a method for producing L-amino acid oxidase using the recombinant bacterium, which comprises culturing the recombinant bacterium to OD₆₀₀When the expression level is 0.6-0.8, IPTG is added to induce the expression of L-amino acid oxidase.

In one embodiment of the invention, the method is to culture the recombinant bacterium to OD₆₀₀0.6-0.8, and after addition of 0.4mM IPTG at a final concentration, induction was carried out at 25 ℃ for 12 h.

The fourth purpose of the invention is to provide a method for producing α -keto-gamma-methylthiobutyric acid by using the recombinant bacterium, which is characterized in that L-methionine is used as a substrate, and the recombinant bacterium is used for converting the substrate to produce α -keto-gamma-methylthiobutyric acid.

In one embodiment of the invention, the conversion conditions are: the pH value is 7-9, the conversion temperature is 15-37 ℃, and the conversion time is 20-24 h.

In one embodiment of the invention, the conversion conditions are that the pH is 7-8, the conversion temperature is 20 ℃, and the conversion time is 24 h.

In one embodiment of the invention, the conversion is carried out in a shaker at a shaker speed of 200 rpm.

In one embodiment of the present invention, wet cells of the recombinant bacterium are used for transformation, and the addition amount of the wet cells is 25-35 g/L.

In one embodiment of the present invention, the transformation system is used for whole-cell transformation by adding wet cells of the recombinant bacterium to a reaction system containing methionine at a final concentration of 80-200g/L L.

In one embodiment of the invention, the transformation is carried out by adding 30g/L of whole cells to a solution of L-methionine at a concentration of 100g/L and transforming for 24h at 20 ℃.

In one embodiment of the present invention, the conversion system uses L-methionine as a substrate, and the amount of the L-amino acid oxidase whole-cell catalyst added is 30 g/L.

The invention also provides application of the recombinant bacterium in the fields of food, medicine and chemical industry.

The method has the beneficial effects that 1, the L-amino acid oxidase from the rhodococcus erythropolis is used for converting the L-methionine to produce α -ketone-gamma-methylthio butyric acid, the enzyme shows high activity after being expressed in escherichia coli, the requirement of industrial scale production can be better met, the yield of α -ketone-gamma-methylthio butyric acid can reach 95.18g/L to the maximum under the conditions of 2, 20 ℃ and pH7.5, the conversion period needs 24 hours, and the production efficiency is greatly improved.

Drawings

FIG. 1 is an HPLC analysis spectrum of a conversion product, (A) α -ketone-gamma-methylmercapto sodium butyrate standard substance, (B) conversion liquid reacted for 24 hours;

FIG. 2 shows the effect of different bacterial amounts on the production of α -keto-gamma-methylthiobutyric acid;

FIG. 3 shows the effect of conversion at different temperatures on the production of α -keto-gamma-methylthiobutyric acid;

FIG. 4 is a graph showing the effect of conversion at different pH conditions on the production of α -keto-gamma-methylthiobutyric acid;

FIG. 5 shows the effect of L-amino acid oxidase derived from other sources on the production of α -keto-gamma-methylthiobutyric acid.

Detailed Description

And (3) pretreating a sample to be detected, namely centrifuging the conversion solution at 10000rpm for 2min, collecting supernatant, preparing standard solution by using α -ketone-gamma-methylthio butyric acid sodium salt as a standard substance, filtering the supernatant and the standard solution which are diluted moderately by 0.22 mu m microporous filter membranes respectively, and determining the content of α -ketone-gamma-methylthio butyric acid and the content of residual L-methionine by using high performance liquid chromatography.

α -keto-gamma-methylthiobutyric acid content is determined by high performance liquid chromatography, column C18 ODSYPERSIL (250 mm. times.4.6 mm, 5 μm), mobile phase 0.0275% dilute sulfuric acid, filtration through 0.22 μm filter membrane, column temperature 40 deg.C, detection wavelength 225nm, sample injection amount 10 μ L, and flow rate 1.0 mL/min.

Calculation of the space-time yield: space-time yield (g/L/h) ═ KMTB yield (g/L)/conversion time (h)

Example 1: construction of L-amino acid oxidase gene-containing engineering bacteria

(1) An artificially synthesized L-AAO deaminase gene which is optimized by codons and contains BamHI and XhoI enzyme cutting sites is shown as SEQ ID NO. 2.

(2) The target gene and an expression vector pET28a 37 are cut by restriction enzymes BamH I and XhoI for 2h at 37 ℃;

(3) connecting the target gene after enzyme digestion and gel recovery with plasmid pET28a 16 ℃ for 10h by using T4 ligase respectively;

(4) introducing the constructed expression plasmid into E.coli BL21(DE3), and culturing in LB plate containing kanamycin for 12 h;

(5) and carrying out PCR and enzyme digestion verification on colonies growing out of the plate, carrying out sequencing verification on plasmids containing target genes, and selecting a strain with completely correct target genes, namely the L-amino acid oxidase gene engineering bacteria E.

Example 2: induced expression of genetically engineered bacteria

(1) Inoculating the constructed genetically engineered bacterium E.coli BL21-L-AAO into LB slant culture medium for culture for 12 h;

(2) inoculating a ring of slant seeds into an LB culture medium, and culturing for 6 h;

(3) inoculating E.coli BL21-L-AAO seed liquid into TB fermentation medium, and culturing to OD₆₀₀At a concentration of 0.6, IPTG was added to the cells at a final concentration of 0.4mM for induction, and after 12 hours of induction at 25 ℃ the cells were collected and washed with sterile physiological saline.

Example 3 Effect of different bacterial amounts on the production of α -keto-gamma-methylthiobutyric acid

Taking the wet thalli obtained in example 2, performing whole-cell transformation by taking the wet thalli as a catalyst after centrifugally collecting the thalli, controlling the pH to be 8.0 and the temperature to be 30 ℃, adjusting the adding amount of the thalli (30g/L,40g/L,50g/L,60g/L), measuring the concentration of α -ketone-gamma-methylthiobutyric acid in a supernatant after reaction by using high performance liquid chromatography (shown in figure 2) after converting the thalli with a final concentration of 100g/L and 20mM Tris-HCL for 24h, wherein the concentration of α -ketone-gamma-methylthiobutyric acid is 77.45g/L when the thalli is 30g/L, the concentration of α -ketone-gamma-methylthiobutyric acid is 94.46g/L when the thalli is 40g/L, the concentration of α -ketone-gamma-methylthiobutyric acid is 96.84g/L when the thalli is 50g/L, and the concentration of methylthiobutyric acid is 29.4625 g/L when the thalli is 60 g/L.

Example 4 Effect of conversion temperature ratio on production of α -keto-gamma-methylthiobutyric acid

Taking wet thalli obtained in example 2, performing whole-cell transformation by taking the wet thalli as a catalyst after centrifugally collecting the thalli, controlling the pH to be 8.0, adjusting the reaction temperature to be 15-37 ℃, and measuring the optimal temperature, wherein the reaction liquid comprises L-methionine with the final concentration of 100g/L, 20mM Tris-HCL and 30g/L wet thalli, and after 24h of transformation, the concentration of α -keto-gamma-methylthiobutyric acid in a supernatant obtained after the reaction is measured by high performance liquid chromatography (as shown in figure 3), and the result shows that the concentration of α -keto-gamma-methylthiobutyric acid is 80.29g/L when the temperature is 15 ℃, the concentration of α -keto-gamma-methylthiobutyric acid is 87.35g/L when the temperature is 20 ℃, the concentration of α -keto-gamma-methylthiobutyric acid is 83.10g/L when the temperature is 25 ℃, the concentration of α -keto-gamma-methylthiobutyric acid is 77.34g/L when the temperature is 30 ℃, the concentration of 867-gamma-methylthiobutyric acid is 35g/L when the temperature is 37 ℃, the yield of the methylthiobutyric acid is increased gradually at 35-35 g/L and the yield is increased when the temperature is 30 ℃.

EXAMPLE 5 Effect of pH conversion on the production of α -keto-gamma-methylthiobutyric acid

Taking the wet thalli obtained in example 2, performing whole-cell transformation by taking the wet thalli as a catalyst after centrifugally collecting the thalli, controlling the temperature to be 20 ℃, adjusting the reaction pH to be 7.0-9.0, measuring the concentration of α -keto-gamma-methylthiobutyric acid in a supernatant after the reaction by using high performance liquid chromatography (as shown in figure 4) after 24h of transformation, wherein the concentration of α -keto-gamma-methylthiobutyric acid in the supernatant after the reaction is measured by using high performance liquid chromatography, and the result shows that the concentration of the α -keto-gamma-methylthiobutyric acid is 88.90g/L when the pH is 7.0, the concentration of the α -keto-gamma-methylthiobutyric acid is 95.18g/L when the pH is 7.5, the concentration of the α -keto-gamma-methylthiobutyric acid is 87.87g/L when the pH is 8.0, the concentration of the α -keto-gamma-methylthiobutyric acid is 71.50g/L when the pH is 8.5, the yield of the methylthiobutyric acid is increased when the pH is 9.0, the yield of the methylthiobutyric acid is 95-7-gamma-methylthiobutyric acid is 95 g/L, and the yield is increased when the pH is 8.5.5, and the yield is 95-7.5.

Example 6

The wet bacterial cells obtained in example 2 were used as a whole cell catalyst for converting L-methionine to α -keto-gamma-methylthiobutanoic acid, a reaction system for preparing 20mL of a substrate and L-amino acid oxidase, and a reaction mixture was prepared from L-methionine at a final concentration of 100g/L, 20mM Tris-HCl and 30g/L whole cells, and after reacting at 20 ℃ for 24 hours, the reaction mixture was filtered through a 0.22 μm filter, and the results were analyzed by high performance liquid chromatography, at which time the KMTB yield reached 95.18g/L, the conversion rate was 95.84%, and the space-time yield was 3.97g/L/h, thus achieving high-efficiency production of α -keto-gamma-methylthiobutanoic acid.

Comparative example 1

According to the same strategy as in examples 1-2, L-amino acid oxidases derived from Corynebacterium glutamicum [ Genbank accession No. AJE67504], Bacillus subtilis [ Genbank accession No. AKN12962], Bacillus thuringiensis [ Genbank accession No. AEA14048], Bacillus megaterium [ Genbank accession No. WP-026679766 ] and Bacillus cereus [ Genbank accession No. AQQ62701] were expressed in Escherichia coli, respectively, and the results of comparison of the yields of α -keto- γ -methylthiobutyric acid under the same conditions as in example 6 are shown in FIG. 5, in which the yield of α -keto- γ -methylthiobutyric acid was 49.12g/L when L-amino acid oxidase derived from Corynebacterium glutamicum was expressed under the same transformation conditions, the yield of α -keto- γ -methylthiobutyric acid was 57.96g/L when L-amino acid oxidase derived from Bacillus subtilis was expressed, the yield of 4934-keto- γ -methylthiobutyric acid was 48340 g/L when L-amino acid oxidase derived from Bacillus thuringiensis was expressed, and the yield of 18-methylthiobutyric acid oxidase was 5835 g/L-methylthiobutyric acid oxidase derived from Bacillus thuringiensis was expressed under the same transformation conditions as shown in FIG. 5.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A method for producing α -ketone-gamma-methylthio butyric acid is characterized in that L-methionine is used as a substrate, a genetically engineered bacterium is used for converting the substrate to produce α -ketone-gamma-methylthio butyric acid, the genetically engineered bacterium is used for expressing L-amino acid oxidase by using escherichia coli as a host and pET series plasmids as a carrier, and the amino acid sequence of the L-amino acid oxidase is shown as SEQ ID No. 1.

2. The method of claim 1, wherein the vector is pET28a and the E.coli strain comprises E.coli BL21, E.coli JM109, E.coli DH5 α, or E.coli TOP 10.

3. The method according to claim 1, wherein the genetically engineered bacterium is constructed by linking a vector to a gene encoding an L-amino acid oxidase, transforming the linked vector into a host cell; the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

4. The method according to claim 1, wherein the conversion is carried out at a pH of 7-9, 15-37 ℃ for 20-24 h.

5. The method of claim 1, wherein the transformation is performed by wet cells of genetically engineered bacteria, and the addition amount of the wet cells is 25-35 g/L; the transformation system was transformed into whole cells in a reaction system containing methionine to a final concentration of 80-200g/L L.

6. Use of the method according to any one of claims 1 to 5 in the fields of food, medicine and chemical industry.