CN117821537A - Method for synthesizing butyric acid sustained release agent FBA by enzyme method - Google Patents

Method for synthesizing butyric acid sustained release agent FBA by enzyme method Download PDF

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CN117821537A
CN117821537A CN202311803051.3A CN202311803051A CN117821537A CN 117821537 A CN117821537 A CN 117821537A CN 202311803051 A CN202311803051 A CN 202311803051A CN 117821537 A CN117821537 A CN 117821537A
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coa ligase
ligase
fba
butyramide
butyric acid
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郑璞
樊昕雨
陈鹏程
吴丹
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Jiangnan University
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    • C12N9/93—Ligases (6)
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    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
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    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01002—Butyrate-CoA ligase (6.2.1.2)
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    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01003—Long-chain-fatty-acid-CoA ligase (6.2.1.3)
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

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Abstract

The invention discloses a novel method for synthesizing N- (1-carbamoyl-2-phenylethyl) butyramide by enzyme catalysis, belonging to the technical field of bioengineering. The invention establishes a path for synthesizing phenylalanine butyramide by enzyme catalysis for the first time, takes butyric acid and L-phenylpropionamide as substrates, and utilizes COA ligase to catalyze and synthesize the phenylalanine butyramide, thereby providing a new thought for synthesizing the phenylalanine butyramide. Compared with chemical synthesis: the reaction condition is an aqueous phase system, no toxic solvent is needed, the cost is low, the operation is safe and simple, the conversion rate of butyric acid is 100%, and the concentration of generated FBA is 0.52mg/mL.

Description

Method for synthesizing butyric acid sustained release agent FBA by enzyme method
Technical Field
The invention relates to a novel method for synthesizing N- (1-carbamoyl-2-phenylethyl) butyramide by enzyme catalysis, belonging to the technical field of bioengineering.
Background
Butyric acid is an important C4 chemical, which regulates the expression of various inflammatory factors by acting on immune cells in the intestinal tract, such as down-regulating the expression of inflammatory factors such as TNFaIL-17, nitric oxide, IL-6, IL-12, etc., inhibiting Histone Deacetylase (HDAC) activity to promote antibody production, etc., thereby reducing the inflammatory level of the intestinal tract, enhancing the protection of intestinal mucosa, and maintaining the intestinal homeostasis, and a great deal of evidence shows that butyric acid has a resistance effect on various human diseases (colon cancer, acute chronic diarrhea, obesity, hypercholesterolemia, ischemic stroke, etc.), and its derivatives have wide application in the industries of chemical industry, food, pharmacy, perfume, animal feed, etc. However, butyric acid is still very limited in its clinical application due to its poor organoleptic properties, such as unpleasant smell. Although its butyrate form has relatively good organoleptic properties, the salt form exhibits disadvantages of hygroscopicity, poor water solubility, deliquescence, etc.
FBA, the alias name is phenylalanine butyramide, and the molecular formula is C 13 H 18 N 2 O 2 Is an important derivative of butyric acid. As a novel butyric acid slow-release agent, the FBA has good organoleptic and physical and chemical properties. Research shows that the FBA can effectively inhibit intestinal mucosa inflammation, inflammatory diarrhea and acute visceral pain, and is even more effective than natural substances; and has effects of soothing and relieving skin, resisting red, treating alcoholic fatty liver, preventing insulin resistance, remodelling intestinal microbiota, and preventing severe acute respiratory syndrome coronavirus type 2 infection. In Ames and micronucleus assays, FBA does not show any in vitro genotoxicity, has the same pharmacokinetic properties and safety as butyrate, and has potential medical value.
The currently reported methods for synthesizing FBA are mainly chemical synthesis methods. For example, WO2009130735A1 discloses a method for synthesizing FBA by reacting phenylalanyl amide and butyryl chloride in chloroform solution, wherein the acyl chloride is extremely easy to hydrolyze, the reaction is required to be carried out in chloroform which is a harmful substance, and the post-treatment process is complicated. In another example, a method for producing FBA without chlorinated solvents is disclosed in the publication CN116438158A, where the FBA is synthesized using phenylalaninamide and butyric anhydride as substrates. The method does not need any chlorinated solvent, but needs high-concentration butyric anhydride, and has strong pungent and pungent smell. Therefore, there is a need to develop a safe and simple FBA synthesis method.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synthesizing FBA by biological enzyme catalysis, which aims to solve the technical problems that in the existing FBA chemical synthesis method, toxic solvents or strong-irritation substrate solutions are relied on, the operation is complicated, and the safe production is not facilitated.
The first technical scheme provided by the invention is a method for synthesizing phenylalanine butyramide by biological enzyme catalysis, which takes butyric acid and L-phenylpropionamide as substrates, utilizes CoA ligase as a catalyst to form a catalytic system, and catalyzes and synthesizes phenylalanine butyramide. CoA ligases in the context of the present invention are EC 6.2.1 acid-thio-ligases acid-thiol ligases.
In certain embodiments, the catalytic system further comprises ATP.
Further, in the above-mentioned catalytic system, the addition amount of ATP is 0.1 to 10mmol/L. CoA ligases require consumption of ATP cofactors in the catalytic process, activating carboxylic acid substrates to form acyl AMP intermediates.
In certain embodiments, the CoA ligase is one or more of 4-hydroxybutyrate CoA ligase (4-hydroxybutyrate CoA ligase), butyrate CoA ligase (butyrate CoA ligase), long-chain fatty acid CoA ligase, medium-chain fatty acid CoA ligase, or acetyl-coenzyme A synthetase (acetyl CoA synthetase).
Further, the gene encoding the 4-hydroxybutyrate CoA ligase (4-hydroxybutyrate-coa ligase) is derived from Metallosphaera sedula or Thermodesulforhabdus norvegica; the gene encoding the Butyrate CoA ligase (butyrate-coa ligase) is derived from Desulfotomaculum nigrificans, candidatus Terasakiella magnetica; the gene encoding the Long-chain fatty acid CoA ligase is derived from Geobacillus thermodenitrificans.
In certain embodiments, the amino acid sequence of the CoA ligase is as set forth in any one of SEQ ID NO. 1-5.
In certain embodiments, the concentration of butyric acid in the catalytic system is 0.1 to 10mmol/L.
In certain embodiments, the concentration of L-phenylpropionamide in the catalytic system is 2 to 10mmol/L.
In certain embodiments, the concentration ratio of butyric acid to phenylalanyl amide in the catalytic system is 1:4.
In certain embodiments, the amount of CoA ligase added in the catalytic system is 0.0010 to 0.1U/ml.
In some embodiments, 0.1-10 mmol/L MgCl is also added to the catalytic system 2 Or MnCl 2 。
In certain embodiments, the catalytic reaction is carried out for a period of time ranging from 0.5 to 16 hours, the catalytic reaction is carried out at a temperature ranging from 30 to 75 ℃, and the catalytic reaction has a pH ranging from 6 to 9.
The second technical scheme provided by the invention is a genetically engineered bacterium, which takes escherichia coli as a host cell to overexpress CoA ligase genes derived from Metallosphaera sedula, thermodesulforhabdus norvegica, desulfotomaculum nigrificans, candidatus Terasakiella magnetica or Geobacillus thermodenitrificans.
In certain embodiments, the vector plasmid used for overexpression by the genetically engineered bacterium is pET-28a (+), pET Duet1 or pRSFDuet.
In certain embodiments, the E.coli is E.coli BL21 (DE 3), E.coli JM109, or E.coli DH 5. Alpha.
The third technical scheme provided by the invention is a preparation method of CoA ligase, wherein the method is to culture the genetically engineered bacterium of the second technical scheme, induce the genetically engineered bacterium to express the CoA ligase, and crush thalli to obtain crude enzyme liquid containing the CoA ligase.
In certain embodiments, the genetically engineered bacterium is cultured in LB liquid medium at 37℃to OD at 220rpm 600 =0.6-0.8, adding 40. Mu.L of 0.5M IPTG, inducing at 20℃and 220rpm for 20h, centrifuging 8000 Xg to remove supernatant, and eluting with 50mmol/L Tris-solution at pH 7.5The HCl buffer solution redissolves the thalli, ultrasonic crushing is carried out, and 8000 Xg of supernatant is collected centrifugally to obtain crude enzyme solution.
The fourth technical scheme provided by the invention is the method of the first technical scheme, and the application of the genetically engineered bacterium of the second technical scheme or the method of the third technical scheme in the production of the FBA.
The invention establishes a path for synthesizing phenylalanine butyramide by enzyme catalysis for the first time, uses butyric acid and L-phenylpropionamide as substrates and uses COA ligase to catalyze and synthesize the phenylalanine butyramide, and provides a new thought for the synthesis of FBA. Compared with chemical synthesis: the reaction condition is an aqueous phase system, no toxic solvent is needed, the cost is low, the operation is safe and simple, the conversion rate of butyric acid is 100%, and the concentration of generated FBA is 0.52mg/mL.
Drawings
FIG. 1 is a diagram showing the enzymatic synthesis of FBA.
FIG. 2 is a protein gel electrophoresis; lane M: mark, lane 1: no-load BL21; lanes 2-3: a4YDT1 crude enzyme-pure enzyme; lanes 4-5: a4INB3 crude enzyme-pure enzyme; 6-7: F6B5W2 crude enzyme-pure enzyme; 8-9: a0A1I4VGJ2 crude enzyme-pure enzyme; 10-11: A0A1C3RK29 crude enzyme-pure enzyme.
FIG. 3 is a diagram of the HPLC detection of FBA generated by the enzyme catalyzed reaction.
FIG. 4 effect of butyric acid concentration on FBA synthesis.
FIG. 5 effect of concentration of phenylpropionamide on FBA synthesis.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
The plasmids and cells referred to in the following examples are as follows:
the strain E coli BL21 (DE 3), plasmid pET28a (+) is a commercial strain, plasmid.
The following examples relate to the following media:
LB medium: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast extract, and sterilizing at 121deg.C for 15min.
The following examples relate to the following methods:
1. butyric acid and FBA detection method: (1) sample treatment: after the reaction, 1mL of reaction solution is taken, an equal volume of methanol is added to denature the protein, the reaction is finished, the reaction solution is centrifuged at 12000rpm for 5min, and then a supernatant filter membrane is taken for HPLC detection. (2) HPLC detection conditions: detecting by using an Aglient high performance liquid chromatograph, amethylst C18-H,4.6x250mmol/L column, and the flow rate is 1mL/min; mobile phase a-water (0.1% tfa), mobile phase B-methanol (0.1% tfa); gradient elution, 0-5min,10-30% B;5-6min,30% B;6-15min,30-80% B;15-20min,80-90% B;20-24min,90-10% B; the detector is an ultraviolet-visible light detector, the detection wavelength is 217nm, and the detection temperature is 35 ℃.
2. Method of protein purification: the recombinant enzyme is fused with His tag and can be purified by Ni2+ affinity chromatography. The Ni-HisTrap HP column (GE Healthcare, shanghai, china) was equilibrated with a binding solution (20 mmol/L phosphate buffer, 500mmol/L NaCl,20mmol/L imidazole, pH 7.4). The eluate (20 mmol/L phosphate buffer, 500mmol/L NaCl,300mmol/L imidazole) was used for continuous gradient elution to collect the target protein, which was dialyzed against 50mmol/L Tris-HCl buffer to remove salts and imidazole.
3. The method for detecting the protein concentration comprises the following steps: bovine serum albumin standard solutions of different concentrations were prepared in microwell plates. When the concentration of the target protein is measured, the prepared sample is added into a micro-pore plate, then coomassie brilliant blue G250 solution is added, after uniform mixing, the reaction is carried out for 5min at normal temperature, and the absorbance at 595nm is detected by an enzyme-labeled instrument
4. Enzyme activity detection method of CoA ligase: the concentration of FBA formed after the reaction was checked by HPLC and the corresponding enzyme activity. At a certain temperature, pH, 1. Mu. Mol of FBA per minute was produced and was defined as an enzyme activity unit, denoted by U. The enzyme activity calculation formula:
wherein U is enzyme activity, U/mg; c is the concentration of FBA, mg/L; v is the total reaction volume, L;1 000 is the conversion coefficient of mg to μg; n is the dilution of the sample; 234.3 is the molar coefficient of FBA, g/mol; t is the reaction time, min; m is the amount of sample, mg.
Example 1: synthesis and preparation of coenzyme A ligase
The 4-hydroxybutyrate CoA ligase gene derived from Metallosphaera sedula, thermodesulforhabdus norvegica (Uniprot ID: A4YDT1, A0A1I4VGJ2, respectively, amino acid sequence: SEQ ID NO.1, SEQ ID NO. 2), the Butyrate CoA ligase gene derived from Desulfotomaculum nigrificans, candidatus Terasakiella magnetica (Uniprot ID: F6B5W2, A0A1C3RK29, respectively, amino acid sequence: SEQ ID NO.3, SEQ ID NO. 4), and the Long-chain fat-acid CoA ligase gene derived from Geobacillus thermodenitrificans (Uniprot ID: A4INB3, amino acid sequence: SEQ ID NO. 5) were obtained by gene synthesis, and a recombinant plasmid was obtained by insertion between BamHI and HindIII of pET-28a (+). Recombinant bacteria were inoculated into LB (3 mL, 50. Mu.g mL) -1 Kanamycin) and incubated for 18h (37 ℃,220 rpm). 1mL was then transferred to 50mL LB medium containing kanamycin and incubated to OD 600 =0.6 to 0.8 (37 ℃,220 rpm), followed by addition of IPTG (final concentration 0.4 mmol/L) at reduced temperature, induction for 20h (20 ℃,220 rpm). After induction, the cells were collected by centrifugation at 8000 Xg for 5min, washed once with sterile water, centrifuged, suspended with 10mL of buffer solution and sonicated, and the supernatant was collected by centrifugation at 8000 Xg for 5min to obtain a crude enzyme solution, which was subjected to SDS-PAGE after protein purification (see FIG. 2). The 5 target proteins were purified by nickel column affinity chromatography with protein concentrations shown in table 1, wherein Butyrate CoA ligase from c.terakiella magnetica was very low and inactive after purification. The reactions were then carried out at the optimum reaction temperature, respectively, and FBA was detected by HPLC (see fig. 3) and the corresponding enzyme activities were calculated, with the highest enzyme activity of 4-hydroxybutyrate CoA ligase of m.
TABLE 1
Example 2: influence of the reaction temperature on the enzyme catalysis
Enzyme catalyzed reverseThe reaction system is as follows: 50mmol/L Tris-HCl buffer is added with 4mmol/L butyric acid, 6mmol/L L-phenylalanyl amide, 10mmol/L ATP,10mmol/L MgCl 2 And 0.2ml of the enzyme solution purified in example 1, pH 8, at 30-75℃and at 100rpm in a water bath shaker for 16h. The optimum reaction temperatures for CoA ligases from M.sedula, T.norvegica, D.nigricans, C.terasakiella magnetica, G.thermodemitification are 75, 75, 50, 30, 60℃respectively.
Example 3: effect of butyric acid concentration on FBA Synthesis
As shown in FIG. 4, 0.2mL of crude enzyme solution of 4-hydroxybutyrate CoA ligase in M.sedula and 0.1-10 mmol/L of butyric acid were used for the enzyme-catalyzed reaction, and the concentration of FBA reached the highest value at 2mmol/L of butyric acid and 0.52mg/mL and the concentration of FBA began to decrease at a butyric acid concentration of more than 6 mmol/L. When the concentration of butyric acid is 2mmol/L, the concentration of FBA produced can reach 0.5mg/mL.
Example 4: effect of phenylalaninamide concentration on FBA Synthesis
As shown in FIG. 5, the concentration of L-phenylalanyl amide was 1 to 10mmol/L, and the concentration of FBA produced was 0.52mg/mL when the concentration of phenylalanyl amide was 8mmol/L, as the enzyme activity was gradually increased with the increase of the phenylalanyl amide concentration, as described in examples 2 and 3.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

Claims (10)

1. A method for synthesizing phenylalanine butyramide by using biological enzyme as catalyst is characterized in that butyric acid and L-phenylpropionamide are used as substrates, coA ligase is used as catalyst to form a catalytic system, and phenylalanine butyramide is synthesized by catalysis.
2. The method of claim 1, wherein the catalytic system further comprises ATP.
3. The method according to claim 2, wherein the amount of ATP added in the catalytic system is 0.1 to 10mmol/L.
4. The method of any one of claims 1-3, wherein the CoA ligase is one or more of 4-hydroxybutyrate CoA ligase, butyrate CoA ligase, long-chain fatty acid CoA ligase, medium-chain fatty acid CoA ligase, and acetyl-CoA synthetase.
5. The method of any one of claims 1 to 4, wherein the amino acid sequence of the CoA ligase is set forth in any one of SEQ ID nos. 1 to 5.
6. The method according to any one of claims 1 to 5, wherein in the catalytic system, the concentration of butyric acid is 0.1 to 10mmol/L, the concentration of L-phenylpropionamide is 2 to 10mmol/L, and the addition amount of CoA ligase is 0.0010 to 0.1U/ml;
the catalyst system is also added with 0.1-10 mmol/L MgCl 2 Or MnCl 2 。
7. The method according to any one of claims 1 to 6, wherein the time of the catalytic reaction is 0.5 to 16 hours, the temperature of the catalytic reaction is 30 to 75 ℃, and the pH of the catalytic reaction is 6 to 9.
8. The genetically engineered bacterium is characterized in that the genetically engineered bacterium takes escherichia coli as a host cell and overexpresses 4-hydroxybutyrate coenzyme A ligase genes from Metallosphaera sedula or Thermodesulforhabdus norvegica, butyrate coenzyme A ligase genes from Desulfotomaculum nigrificans or Candidatus Terasakiella magnetica or long-chain fatty acid coenzyme A ligase genes from Geobacillus thermodenitrificans.
A method for producing a CoA ligase, comprising culturing the genetically engineered bacterium of claim 8, inducing the bacterium to express the CoA ligase, and disrupting the bacterium to obtain a crude enzyme solution containing the CoA ligase.
10. Use of the method of any one of claims 1 to 7, the genetically engineered bacterium of claim 8 or the method of claim 9 for the production of phenylalanine butyramide.
CN202311803051.3A 2023-12-25 2023-12-25 Method for synthesizing butyric acid sustained release agent FBA by enzyme method Pending CN117821537A (en)

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