CN105969785B - A kind of synthetic method of 4-hydroxyisoleucine - Google Patents

Abstract

The present invention relates to a kind of synthetic method of 4-hydroxyisoleucine, this method first constructs recombinant expression plasmid, expresses L-isoleucine-4-hydroxylase and L-glutamic acid oxidase simultaneously, then constructs Add L-glutamic acid and L-isoleucine two kinds of substrates in the catalytic reaction system of synthetic 4-hydroxyisoleucine as the precursor of catalytic reaction, through the catalytic reaction of 4h, L-glutamic acid and L ‑ The conversion rates of isoleucine were 97.01% and 98.45%, respectively. The present invention uses L-glutamic acid instead of α-ketoglutarate as a precursor for synthesizing 4-hydroxyisoleucine, can efficiently produce 4-hydroxyisoleucine, has low production cost, and is convenient for industrialized production.

Description

A kind of synthetic method of 4-hydroxyisoleucine

technical field

The invention relates to the field of compound biotechnology production, in particular to a method for synthesizing 4-hydroxyisoleucine.

Background technique

In the field of biotechnology, although the related cloning and expression technology using Escherichia coli as the host is very mature, the recombinant expression of heterologous proteins is still an important research content. In order to improve the solubility and expression of recombinant proteins, it takes a lot of time With a lot of laboratory work, the final success can only be achieved through repeated experiments and re-experimental efforts.

4-Hydroxyisoleucine (4-Hydroxyisoleucine, 4-HIL) is a kind of L-isoleucine hydroxylate mainly present in the seeds of fenugreek plants. The molecular formula of 4-HIL is C6H13NO3, the molecular weight is 147.17, the melting point is greater than 200°C, the boiling point is 332°C, and the density is 1.181g/cm3. Studies have shown that 4-hydroxyisoleucine has a glucose concentration-dependent activity of promoting insulin secretion and promoting the absorption of blood sugar by muscle cells, accelerating fat metabolism, lowering blood fat and protecting liver function. Therefore, as an ideal drug for effectively preventing and treating diabetes and obesity, 4-hydroxyisoleucine has broad application prospects and market demands.

The production methods of 4-hydroxyisoleucine include extraction method, chemical synthesis method, enzymatic method and biological method. At present, the technology of industrial production of 4-hydroxyisoleucine is relatively mature, but it is mainly extracted from heavy fenugreek. Its product separation and purification is difficult, difficult to prepare, low product purity, serious environmental pollution, high cost of mass production, and more configurations of 4-hydroxyisoleucine obtained by this method, but only (2S, 3R, 4S)-4 -Hydroxyisoleucine has the above-mentioned biological activity, so that the method has a low extraction rate (only 0.091-0.6%).

With the development of biotechnology, try to introduce enzyme-catalyzed reaction to simplify some steps in the process of chemical synthesis of 4-HIL. Enzymatic synthesis of 4-hydroxyisoleucine is a new method for synthesizing 4-hydroxyisoleucine, but it needs to add expensive α-ketoglutarate as a substrate. Wang et al. invented an eight-step method to synthesize 4-HIL in 2002. The method uses ethyl 2-methylacetoacetate as a raw material to obtain (2S, 3S)-3-hydroxyl-2-methylbutanol under the action of Geotrichum spp. Acetate ethyl ester, and then convert it into 4-HIL through 7-step chemical synthesis. Among them, the conversion of ethyl 2-methylacetoacetate by Geotrichum is the most critical step. The total yield of this method is 39%, and the reaction conditions are harsh, there are many steps, and it is easy to cause environmental pollution, so it still stays in the laboratory stage.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for synthesizing 4-hydroxyisoleucine.

In order to solve the problems of the technologies described above, the technical solution of the present invention is:

A method for synthesizing 4-hydroxyisoleucine, using L-glutamic acid as a catalytic precursor to enzymatically synthesize 4-hydroxyisoleucine, the specific steps are as follows:

(1) Constructing a recombinant expression vector comprising L-isoleucine-4-hydroxylase and L-glutamic acid oxidase genes;

(2) transforming the recombinant expression vector in step (1) into the desired bacterial strain to obtain the genetically engineered recombinant bacterial strain;

(3) cultivating the recombinant bacterial strain in step (2), thereby obtaining a bacterium expressing L-isoleucine-4-hydroxylase and L-glutamic acid oxidase;

(4) crushing the thalline in step (3) to obtain crude enzyme;

(5) adding the crude enzyme obtained in step (4) into the catalytic solution containing L-glutamic acid and L-isoleucine, and synthesizing 4-hydroxyisoleucine by enzymatic method.

Preferably, in the synthesis method of the above-mentioned 4-hydroxyisoleucine, in step (1), the sequences shown in the sequence listing <400>1 and <400>2 are known ido and lgox original nucleotide sequences, and the sequence listing < The sequences shown in 400>3 and <400>4 are the codon-optimized nucleotide sequences of idol and lgox for E.coli BL21 (DE3), which are constructed to contain L-isoleucine-4-hydroxylase (ido) and the recombinant expression vector of L-glutamic acid oxidase (lgox) gene—plasmid pETduet-ido-lgox, the sequence of the obtained plasmid pETduet-ido-lgox is the sequence shown in Sequence Listing <400>5.

Preferably, in the synthetic method of the above-mentioned 4-hydroxyisoleucine, the bacterial strain described in step (2) is Corynebacterium gultamicum, Escherichia coli, Serratia marcescens , Mycobacterium album, Bacillus subtilis, Bacillus amyloliquefaciens or Bacillus thuringiensis, Bacillus licheniformis, Bacillus sphaericus or wax Bacillus cereus and Bacillus weihenstephanensis.

Preferably, in the above-mentioned synthetic method of 4-hydroxyisoleucine, L-glutamic acid and L-isoleucine are used as substrates in the step (5), and the recombinant L - The whole cell of isoleucine-4-hydroxylase and L-glutamic acid oxidase is used as a catalyst to react, and the pH is adjusted to 2.0-10.0 at the initial stage of the reaction, and the total reaction time is 2-10h.

Preferably, in the above-mentioned synthesis method of 4-hydroxyisoleucine, the concentrations of the substrates L-glutamic acid and L-isoleucine as catalytic substrates are respectively 5-30 g/L, and the addition of resuspended cells The amount is 5-30g/L, and the reaction temperature is 20°C-35°C. The crude enzyme uses these two substrates to synthesize 4-hydroxyisoleucine.

The beneficial effects of the present invention are:

The above-mentioned synthesis method of 4-hydroxyisoleucine is simple to operate, uses L-glutamic acid instead of α-ketoglutarate as a precursor to synthesize 4-hydroxyisoleucine, and can achieve high density in a simple synthetic medium After culturing, the conversion rates of L-glutamic acid and L-isoleucine can reach 91.2%-97.01% and 93.5%-98.45%, respectively.

Description of drawings

Figure 1 shows the expression plasmid pETduet-ido-lgox with 6Xhis.

Figure 2 is the pETduet-ido-lgox enzyme digestion verification diagram, in which, M: Marker; 1: ido fragment; 2: lgox fragment; 3: pETduet BamHI single enzyme digestion; 4: pETduet-ido-lgox BamHI single enzyme digestion; : pETduet-ido-lgox BamHI, Hind III double digestion; 6: pETduet-ido-lgox Bjl II, Xho I double digestion; 7: pETduet-ido-lgox BamHI, Hind III, Bjl II, Xho I four digestion.

Fig. 3 is 4-HIL standard substance, sample high performance liquid chromatography.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with specific embodiments.

Example 1

Construct strain E.coli BL21(DE3)/pETduet-ido-lgox and express L-isoleucine-4-hydroxylase and L-glutamic acid oxidase, using substrates L-glutamic acid and L-iso Enzymatic Synthesis of 4-Hydroxyisoleucine from Leucine

(1) Obtain ido and lgox nucleotide sequences

According to the nucleotide sequences of ido and lgox shown in Sequence Listing <400>1 and <400>2, the nucleotide sequences shown in Sequence Listing <400>3 and <400>4 were obtained by performing codon optimization on E.coli BL21(DE3) acid sequence. ido and lgox are exogenous genes, in order to increase their expression in the host E.coli BL21(DE3), it is necessary to optimize their codons to the most frequently used sequences in the host, wherein,

ido optimized nucleotide sequence for E.coli BL21(DE3), primers:

Upstream primer: 5'TAT GGATCC ATGAAAATGAGCGGTTTTAGCA 3'

Downstream primer: 5' CCC AAGCTT TTATTTGGTTTCTTTATAGCTAAAGGTC 3'

lgox optimized nucleotide sequence for E.coli BL21(DE3), primers:

Upstream primer: 5'TAA AGATCT CTGCCGGCACCGGC 3'

Downstream primer: 5'ACT CTCGAG GGCGGTATGAATCTCTAATGCG 3'

(2) Construction of plasmid

pETduet was digested with BamH I, Hind III, Bgl II, and Xho I respectively, and the digested products were purified after 1.0% agarose electrophoresis, and then purified with a gel cutting recovery kit. Then, in the ligation system containing SolutionI, ligation reaction was carried out with the ido and lgox nucleotide sequences recovered by the same digestion and purification at 16°C for 12 hours; the reaction product after the ligation was transformed into host cell E. The positive clones were cultured overnight at 37°C in LB medium, and a small amount of plasmids were extracted for sequencing verification, and the plasmid pETduet- ido-lgox (see Figure 1, Figure 2), the sequence of the obtained plasmid pETduet-ido-lgox is the sequence shown in Sequence Listing <400>5.

(3) Construction of genetically engineered bacteria E.coli BL21(DE3)/pETduet-ido-lgox

The plasmid pETduet-ido-lgox was used to transform the host E.coli BL21(DE3) competent cells (the pETduet is a commercial plasmid), and the positive colonies were picked and preserved. The bacterial name was named E.coli BL21(DE3)/pETduet- ido-lgox.

(4) Obtain the crude enzyme L-isoleucine-4-hydroxylase and L-glutamic acid oxidase required for catalysis

The obtained genetically engineered bacteria E.coli BL21(DE3)/pETduet-ido-lgox was inoculated into 100 mL of LB liquid medium containing ampicillin (100 μg/mL) with a 0.1% inoculum of glycerol bacteria, and cultivated at 37° C. at 200 rpm for 12 hours, Then transfer 500 mL of LB liquid medium containing ampicillin (100 μg/mL) (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.0-7.2, sterilized at 121 °C for 20 min at a 2% inoculation rate) ), cultured at 200 rpm at 37°C until OD600=0.6±0.1, adding a final concentration of 0.1mmol/L IPTG, and induced culture at 32°C for 7h. After the end, centrifuge at 7000rpm for 5min to remove the supernatant, wash the cells twice with 0.75% NaCl solution to remove the supernatant, and freeze at -80°C for 12h to obtain the required crude enzymes L-isoleucine-4-hydroxylase and L- Glutamate oxidase.

(5) Enzyme-catalyzed synthesis of 4-hydroxyisoleucine

Add the obtained crude enzyme into the prepared reaction buffer, the final concentration is 25g/L; catalyze the reaction at 32°C and 150rpm in a water-bath shaker for 4 hours, centrifuge to take the supernatant, and obtain the product 4-hydroxyisoleucine acid. As detected by HPLC, after 4 hours of catalytic reaction, the conversion rates of L-glutamic acid and L-isoleucine were 95.42% and 96.71%, respectively.

The above reaction buffer preparation method: L-glutamic acid 20g/L, L-isoleucine 15g/L, Fe ²⁺ 20mmol/L, Vc5mmol/L, Hepes 50mmol/L, pH 7.0.

(6) Detection of 4-hydroxyisoleucine

Centrifuge the catalytic reaction solution at 8000g for 5min and take the supernatant, derivatize it with 0.8% (V/V) 2,4-dinitrofluorobenzene and measure the content of 4-HIL by high performance liquid chromatography. The detection condition is: Agilent C18 (15mm × 4.6mm, 5 μm), using acetonitrile/sodium acetate binary gradient elution, column temperature 33 ℃, detection wavelength 360nm (high performance liquid chromatography see Figure 3), according to the measurement results of high performance liquid phase method, according to the Comparing the peak eluting times of standard products, it was determined that the target product was 4-hydroxyisoleucine.

Embodiment 2 Enzyme Catalytic Synthesis of 4-Hydroxyisoleucine

Add the crude enzyme obtained in Example 1(4) into the prepared reaction buffer, the final concentration is 20g/L; catalyze the reaction at 30°C and 150rpm in a water bath shaker for 4h, centrifuge and take the supernatant to obtain the product 4-Hydroxyisoleucine. As detected by HPLC, after 4 hours of catalytic reaction, the conversion rates of L-glutamic acid and L-isoleucine were 91.2% and 93.5%, respectively.

Above-mentioned reaction buffer preparation method: L-glutamic acid 20g/L, L-isoleucine 15g/L, Fe ²⁺ 20mmol/L, Vc5mmol/L, 0.1mol/L sodium carbonate-sodium bicarbonate buffer solution ( The configuration method is to dissolve 21.2g of sodium carbonate and 67.2g of sodium bicarbonate in water and adjust the volume to 1L), and the pH is 9.1.

Embodiment 3 Enzyme Catalytic Synthesis of 4-Hydroxyisoleucine

Add the crude enzyme obtained in Example 1(4) into the prepared reaction buffer, the final concentration is 30g/L; catalyze the reaction at 32°C and 150rpm in a water-bath shaker for 4h, centrifuge to take the supernatant, and obtain the product 4-Hydroxyisoleucine. As detected by HPLC, after 4 hours of catalytic reaction, the conversion rates of L-glutamic acid and L-isoleucine were 97.01% and 98.45%, respectively.

The above reaction buffer preparation method: L-glutamic acid 20g/L, L-isoleucine 15g/L, Fe ²⁺ 20mmol/L, Vc5mmol/L, Hepes 50mmol/L, pH 7.0.

Embodiment 4 Enzyme Catalytic Synthesis of 4-Hydroxyisoleucine

Add the crude enzyme obtained in Example 1(4) into the prepared reaction buffer, the final concentration is 20g/L; catalyze the reaction at 25°C and 150rpm water bath shaker for 4h, centrifuge to take the supernatant, and obtain the product 4-Hydroxyisoleucine. HPLC detection showed that after 4 hours of catalytic reaction, the conversion rates of L-glutamic acid and L-isoleucine were 92.47% and 96.15%, respectively.

The above reaction buffer preparation method: L-glutamic acid 20g/L, L-isoleucine 15g/L, Fe ²⁺ 20mmol/L, Vc5mmol/L, Hepes 50mmol/L, pH 7.0.

The detailed description of the above-mentioned synthetic method of 4-hydroxyisoleucine with reference to the specific embodiment is illustrative rather than limiting, and several examples can be listed according to the limited scope, so without departing from Changes and modifications under the general concept of the present invention shall fall within the protection scope of the present invention.

Claims (3)

1. a kind of synthetic method of 4-hydroxyisoleucine, it is characterised in that: Pidolidone is used to close as catalytic precursor enzyme process At 4-hydroxyisoleucine, the specific steps are as follows:

(1) building includes l-Isoleucine -4- hydroxylase and L-GLOD gene recombinant vectors, wherein with Sequence shown in sequence table SEQ ID NO.1 and sequence table SEQ ID NO.2 is known ido and lgox original nucleotide sequences, sequence Sequence shown in list SEQ ID NO.3 and sequence table SEQ ID NO.4 is that idol and lgox is directed toE. coli BL21(DE3) close The nucleotide sequence of numeral optimization, building obtain including l-Isoleucine -4- hydroxylase and L-GLOD gene Recombinant expression carrier --- plasmid pETduet-ido-lgox, gained plasmid pETduet-ido-lgoxSequence be sequence table Sequence shown in SEQ ID NO.5；

(2) bacterial strain needed for converting the recombinant expression carrier in step (1) obtains genetic engineering recombination strain；

(3) recombinant bacterial strain in incubation step (2), to obtain expression l-Isoleucine -4- hydroxylase and Pidolidone oxygen Change the thallus of enzyme；

(4) thallus in destruction step (3), obtains thick enzyme；

(5) the thick enzyme obtained in step (4) is added into the catalytic liquid containing Pidolidone and l-Isoleucine, utilizes enzyme process Synthesize 4-hydroxyisoleucine.

2. the synthetic method of 4-hydroxyisoleucine according to claim 1, it is characterised in that: in the step (5) with Pidolidone and l-Isoleucine are substrate, the recombination l-Isoleucine -4- hydroxylase and L- produced using the step (3) The whole cell of dglutamic oxidase is that catalyst is reacted, and initial reaction stage adjusts pH2.0-10.0, reacts total time 2-10 h.

3. the synthetic method of 4-hydroxyisoleucine according to claim 2, it is characterised in that: the substrate Pidolidone As catalysis substrate, its concentration is 5-30 g/L with l-Isoleucine, and the additional amount that cell is resuspended is 5-30 g/L, reaction temperature Degree is 20 DEG C -35 DEG C, and thick enzyme synthesizes 4-hydroxyisoleucine using both substrates.