A process for the preparation of an n-acyl-homoserine lactone

Abstract

The present application belongs to the technical field of culture additive of bacteria, and particularly relates to a preparation method of N-acyl-homoserine lactone. The present application can obtain homoserine and homoserine lactone by lysing methionine through cyanogen bromide after extracting protein from soybean, although other proteins exist in the lysing mixture, but do not have influence on the subsequent ring formation (dehydration). The homoserine lactone salt obtained after ring formation is substituted by alkyl chlorides with different carbon chain lengths, and different AHLs signal molecules can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of microbial culture additives, and particularly relates to a method for preparing an N-acyl-homoserine lactone. Background Technology

[0002] Quorum sensing is a ubiquitous communication mechanism in microorganisms. Microorganisms sense the density of surrounding cells by secreting autoinducers (AIs), thereby regulating gene expression, such as biofilm formation, virulence factor expression, and bioluminescence. Autoinducers, as key substances in microbial communication, play a crucial role in both intra- and inter-population communication. The AIs secreted vary among different bacterial species; for example, Gram-negative bacteria secrete N-acyl-homoserine lactones (AHLs), while Gram-positive bacteria secrete oligopeptides as autoinducers. The regulatory role of quorum sensing is closely related to the secretion of autoinducers and their interactions with other signaling molecules. For instance, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a ubiquitous second signaling molecule in bacteria, interacts with autoinducers to form a complex signal transduction network, receiving extracellular signals and participating in the regulation of various physiological functions, thus forming a complex communication mechanism. The communication mechanism formed by Gram-negative bacteria through quorum sensing is called acyl-homoserine lactone-mediated quorum sensing (AHLs-QS). This mechanism is currently used to investigate the formation of granular sludge and the simultaneous nitrification and denitrification processes. This mechanism can regulate biofilm accumulation by modulating the expression of related genes, and can also control EPS secretion.

[0003] Anaerobic ammonia oxidation (AAM) and sulfur autotrophic denitrification (SAD) are both caused by Gram-negative bacteria, hence the autoinducing agents they secrete are primarily AHLs. Current reports classify AHLs associated with AAM into two main categories: AHLs with substituents at the 3-carbon position (3-oxo-C6-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL) and AHLs without substituents at the 3-carbon position (C4-HSL, C6-HSL, C8-HSL, C12-HSL). Different bacterial species secrete significantly different types of AHLs, and these different AHLs have significantly different effects on microorganisms. Regarding heterotrophic microorganisms, researchers have detailed the roles of different signaling molecules in pollutant removal processes. C6-HSL and C8-HSL are mainly secreted by nitrifying bacteria and anaerobic ammonia oxidizing bacteria, and are associated with NH4+. +The removal of -N is closely related; C10-HSL and C12-HSL are mainly secreted by denitrifying bacteria and are related to NO3. - The removal of -N is closely related. In addition, AHL-type signaling molecules also reduce electron transfer resistance, thereby improving bacterial activity and pollutant removal efficiency.

[0004] Anaerobic ammonia oxidation (AHA) and sulfur autotrophic denitrification (SAD) are both popular research directions in the field of nitrogen removal in water treatment. They are complementary in principle and have similar operating environments, so coupling them can achieve relatively ideal nitrogen removal results. However, coupling them is not a simple mixing process; it requires long-term cultivation and adjustment to ensure stable operation. Current reports indicate that adding AHLs can regulate the interspecific relationship between the two, which is beneficial for their coupling.

[0005] Currently, the biggest obstacle to the application of AHLs in practical engineering culture is the lack of research on them. Apart from AHLs secreted by bacteria, there are no reports of preparing AHLs. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for preparing N-acyl-homoserine lactone. The preparation method provided by this invention can successfully prepare N-acyl-homoserine lactone, providing a basis for its application in the cultivation and coupling of anaerobic ammonia oxidation and sulfur autotrophic denitrifying bacteria, and for regulating the interspecies relationship between the two bacterial communities through quorum sensing mechanism.

[0007] This invention provides a method for preparing N-acyl-homoserine lactone, comprising the following steps:

[0008] Soybeans were subjected to protein extraction to obtain a methionine extract;

[0009] The methionine extract was mixed with a trifluoroacetic acid solution and a cyanogen bromide acetonitrile solution, and then methionine was cleaved under light-protected conditions to obtain a mixture of homoserine and homoserine lactone.

[0010] The homoserine and homoserine lactone mixture were mixed with an inorganic acid, and homoserine dehydration and salt formation reactions were carried out to obtain homoserine lactone salts.

[0011] Alkyl acid and oxalyl chloride were subjected to an acyl chloride reaction. The resulting product was mixed with homoserine lactone salt and then subjected to a substitution reaction under catalytic conditions to obtain N-acyl-homoserine lactone.

[0012] Preferably, the extractant comprises a strong alkaline solution; the pH value of the strong alkaline solution is 12-14.

[0013] Preferably, the extraction temperature is 100–130°C and the extraction time is 2–3 hours.

[0014] Preferably, the particle size of the methionine extract is ≤74μm.

[0015] Preferably, the concentration of the methionine extract in the trifluoroacetic acid solution is 3–5 g·L. -1 .

[0016] Preferably, the pyrolysis temperature is 4°C and the time is 24 hours.

[0017] Preferably, the dehydration reaction is carried out at a temperature of 40–60°C for 1–2 hours.

[0018] Preferably, the alkyl acid includes one or more of dodecanoic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid.

[0019] Preferably, the molar ratio of the alkyl acid to oxalyl chloride is 1:3 to 5; and the molar ratio of the acyl chloride product to homoserine lactone salt is 1.2 to 1.4:1.

[0020] Preferably, the substitution reaction is carried out at a temperature of 50–60°C for a time of 12–24 hours.

[0021] This invention provides a method for preparing N-acyl-homoserine lactone, comprising the following steps: extracting protein from soybeans to obtain a methionine extract; mixing the methionine extract in a trifluoroacetic acid solution and a cyanogen bromide in an acetonitrile solution, and then cleaving the methionine under light-protected conditions to obtain a mixture of homoserine and homoserine lactone; mixing the homoserine and homoserine lactone mixture with an inorganic acid to perform a homoserine dehydration and salt formation reaction to obtain a homoserine lactone salt; performing an acyl chloride reaction with an alkyl acid and oxalyl chloride, and then mixing the resulting product with the homoserine lactone salt, followed by a substitution reaction under catalytic conditions to obtain N-acyl-homoserine lactone. This invention first extracts protein from soybeans, and then cleaves methionine with cyanogen bromide to obtain homoserine and homoserine lactone. Although other proteins are present in the cleavage mixture, they do not affect the subsequent cyclization (dehydration) process. Different AHL signaling molecules can be obtained by substituting homoserine lactone salts with alkyl chloride compounds of different carbon chain lengths. Attached Figure Description

[0022] Figure 1 The pollutant removal status for Group A;

[0023] Figure 2 This shows the removal status of pollutants in Group B. Detailed Implementation

[0024] This invention provides a method for preparing N-acyl-homoserine lactone, comprising the following steps:

[0025] Soybeans were subjected to protein extraction to obtain a methionine extract;

[0026] The methionine extract was mixed with a trifluoroacetic acid solution and a cyanogen bromide acetonitrile solution, and then methionine was cleaved under light-protected conditions to obtain a mixture of homoserine and homoserine lactone.

[0027] The homoserine and homoserine lactone mixture were mixed with an inorganic acid, and homoserine dehydration and salt formation reactions were carried out to obtain homoserine lactone salts.

[0028] Alkyl acid and oxalyl chloride were subjected to acyl chloride reaction, and the resulting product was mixed with homoserine lactone salt. The mixture was then subjected to a substitution reaction under catalytic conditions to obtain N-acyl-homoserine lactone.

[0029] This invention involves extracting protein from soybeans to obtain a methionine extract.

[0030] In this invention, prior to extraction, the process preferably includes peeling and pressing the soybeans sequentially, followed by grinding to obtain soybean powder. In this invention, the particle size of the soybean powder is preferably 10–200 μm, more preferably 10 μm.

[0031] In this invention, the extractant preferably comprises a strong alkaline solution. Preferably, the strong alkaline solution is a sodium hydroxide solution. Preferably, the pH value of the strong alkaline solution is 12-14, more preferably 13.

[0032] In this invention, the extraction temperature is preferably 100–130°C, more preferably 110–120°C; the extraction time is preferably 2–3 hours, more preferably 2.5 hours. In this invention, the extraction is preferably carried out under stirring conditions, and the stirring speed is preferably 200–500 rpm, more preferably 300 rpm.

[0033] In this invention, after extraction, the extraction liquid is preferably further subjected to centrifugation and filtration in sequence, and the protein extract obtained by filtration is concentrated to dryness to obtain methionine extract.

[0034] In this invention, the centrifugation speed is preferably 1000-2000 rpm, more preferably 1500 rpm. In this invention, the centrifugation and filtration operations are preferably repeated three times.

[0035] In this invention, the filtration preferably also yields solid residue.

[0036] After obtaining the methionine extract, the present invention mixes the trifluoroacetic acid solution of the methionine extract with the acetonitrile solution of cyanogen bromide, and then performs methionine cleavage under light-protected conditions to obtain a mixture of homoserine and homoserine lactone.

[0037] In this invention, the concentration of the methionine extract in the trifluoroacetic acid solution is preferably 3-5 g·L. -1 More preferably 4g·L -1 In this invention, the preparation of the trifluoroacetic acid solution of the methionine extract is preferably achieved by mixing the methionine extract and an aqueous trifluoroacetic acid solution. In this invention, the mass concentration of the aqueous trifluoroacetic acid solution is preferably 75%. In embodiments of this invention, it is preferable to mix both the methionine extract and the solid residue in the aqueous trifluoroacetic acid solution to utilize the residual protein in the solid residue.

[0038] In this invention, the acetonitrile solution of cyanogen bromide is preferably a saturated acetonitrile solution of cyanogen bromide.

[0039] In this invention, the volume ratio of the trifluoroacetic acid solution of the methionine extract to the acetonitrile solution of cyanogen bromide is preferably 10:3 to 5, more preferably 5:2.

[0040] In this invention, prior to the pyrolysis, the methionine extract is preferably subjected to freeze-drying and grinding sequentially. In this invention, the freeze-drying is preferably a first freeze-drying and a second freeze-drying performed sequentially. In this invention, the temperature of the first freeze-drying is preferably -20°C, and the time is preferably 12 hours. In this invention, the temperature of the second freeze-drying is preferably -40°C, and the time is preferably 3 days. In this invention, the grinding is preferably performed to a particle size ≤74 μm.

[0041] In this invention, the pyrolysis temperature is preferably 4°C and the pyrolysis time is preferably 24h.

[0042] In this invention, after pyrolysis, the process preferably further includes concentrating the pyrolysis liquid to dryness. In this invention, the concentration temperature is preferably 80–90°C, more preferably 85°C.

[0043] After obtaining a mixture of homoserine and homoserine lactone, the present invention mixes the homoserine and homoserine lactone mixture with an inorganic acid to carry out a homoserine dehydration reaction and a salt formation reaction to obtain homoserine lactone salt.

[0044] In this invention, the inorganic acid includes hydrochloric acid; the concentration of the hydrochloric acid is preferably 6 mol / L.

[0045] In this invention, the mass ratio of the homoserine and homoserine lactone mixture to the inorganic acid is preferably 1:45-55, more preferably 1:50.

[0046] In this invention, the temperature of the dehydration reaction is preferably 40-60°C, more preferably 50°C, and the time is preferably 1-2 hours, more preferably 1.5 hours.

[0047] In this invention, the dehydration reaction is preferably carried out by mixing homoserine with a homoserine lactone mixture and an inorganic acid, followed by heating under reflux. In this invention, the reflux temperature is the temperature of the dehydration reaction. In this invention, the dehydration reaction involves dehydrating the homoserine in the homoserine and homoserine lactone mixture to obtain homoserine lactone.

[0048] In this invention, the salt-forming reaction is preferably a dehydration reaction followed by a reaction between homoserine lactone and inorganic acid to form a salt.

[0049] In this invention, after the salt formation reaction, it is preferable to further include evaporating and concentrating the liquid obtained from the dehydration reaction to dryness, washing and filtering it sequentially.

[0050] In this invention, the evaporation and concentration temperature is preferably 80–90°C, more preferably 85°C. In this invention, the washing is preferably performed with anhydrous ethanol. In this invention, the washing is preferably performed 3–4 times.

[0051] After obtaining homoserine lactone salt, the present invention performs an acyl chloride reaction on alkyl acid and oxalyl chloride, and mixes the resulting product with homoserine lactone salt, and then performs a substitution reaction sequentially under catalytic conditions to obtain N-acyl-homoserine lactone.

[0052] In this invention, the alkyl acid preferably includes one or more of dodecanoic acid, tetradecanoic acid, hexadecanoic acid, and octadecanoic acid, more preferably dodecanoic acid. In this invention, the solvent in the mixture of the alkyl acid and oxaloyl chloride is preferably...

[0053] In this invention, the medium for the acyl chloride reaction is preferably dichloromethane. The preferred molar ratio of the alkyl acid to dichloromethane is 0.1–0.3 mol:1 L, more preferably 0.2 mol:1 L. The preferred molar ratio of the alkyl acid to oxalyl chloride is 1:3–5, more preferably 1:4.

[0054] In this invention, the temperature of the acyl chloride reaction is preferably 80-100°C, more preferably 80°C, and the time is preferably 4-8 hours, more preferably 6 hours.

[0055] In this invention, after the acyl chloride reaction, it is preferable to further concentrate the system obtained from the acyl chloride reaction to dryness.

[0056] In this invention, the catalyst is preferably triethylamine. In this invention, the medium for the substitution reaction is preferably a mixture of dichloromethane and dimethylformamide; the volume ratio of dichloromethane to dimethylformamide in the mixture is preferably 1:1.

[0057] In this invention, the molar ratio of the acyl chloride product to the homoserine lactone salt is preferably 1.2 to 1.4:1, more preferably 1.3:1.

[0058] In this invention, the temperature of the substitution reaction is preferably 50-60°C, more preferably 55°C, and the time is preferably 12-24h, more preferably 24h.

[0059] In this invention, after the substitution reaction, it is preferable to further concentrate and wash the resulting liquid from the substitution reaction in sequence.

[0060] In this invention, the concentration temperature is preferably 60–80°C, more preferably 70°C. In this invention, the washing reagent is preferably dichloromethane.

[0061] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] Weigh 1 kg of mature soybeans, remove the skins, extract the oil, grind and sieve. Add the soybean powder to a 0.1 mol / L sodium hydroxide solution, heat at 100℃ and stir at 300 rpm for 2.5 h, and centrifuge at 1500 rpm. Repeat the above steps 3 times to obtain solid residue and protein solution.

[0064] The protein supernatant was evaporated and concentrated to near dryness at 120°C, and then frozen together with the solid residue at -20°C and then refrigerated at -40°C for 3 days. Subsequently, it was ground in a ball mill at 4500 rpm for 10 minutes and passed through a 200-mesh sieve.

[0065] Weigh 4g of the sieved fraction and dissolve it in 1L of 75% trifluoroacetic acid aqueous solution. Add 400mL of saturated cyanobromide acetonitrile solution and let it stand for 24h at 4℃ in the dark. Then, evaporate and concentrate to dryness at 85℃ to obtain a mixture of homoserine and homoserine lactone. Dissolve the mixture in 200mL of 6mol / L hydrochloric acid and heat under reflux at 40℃ for 1.5h (dehydration and salt formation reaction). Then, evaporate and concentrate to dryness at 80℃. The obtained product is repeatedly washed with anhydrous ethanol and filtered four times to obtain homoserine lactone salt.

[0066] 0.25 mol of dodecanoic acid was added to 1 L of dichloromethane, followed by 0.8 mol of oxaloyl chloride. The reaction was carried out at 80 °C and 100 rpm, and the mixture was stirred and concentrated to dryness. The product was added to 500 mL of a 1:1 mixture of dichloromethane and dimethylformamide, along with 0.2 mol of homoserine lactone obtained by washing and filtration and 40 mL of triethylamine. The reaction was carried out overnight at 55 °C and then evaporated and concentrated to dryness at 80 °C. The concentrated product was washed with dichloromethane, and the undissolved portion was N-dodecanoyl-L-homoserine lactone, denoted as C12-HSL.

[0067] Example 2

[0068] Weigh 1 kg of mature soybeans, remove the skin, extract oil, grind, and sieve. Add the soybean powder to a 0.1 mol / L sodium hydroxide solution and heat at 100℃ with stirring at 300 rpm for 2.5 h. Centrifuge at 1500 rpm to separate the residue. Repeat the above steps three times to obtain solid residue and protein solution. Evaporate and concentrate the protein supernatant at 120℃ to near dryness, and freeze it together with the solid residue at -20℃. Then, use a refrigerated dryer to dry at -40℃ for 3 days. Subsequently, grind it in a ball mill at 4500 rpm for 10 min and pass it through a 200-mesh sieve. Weigh 5 g of the sieved fraction and dissolve it in 1 L of 75% trifluoroacetic acid aqueous solution. Add 500 mL of saturated cyanoacetonitrile bromide solution and let it stand at 4℃ in the dark for 24 h. Then, evaporate and concentrate it to dryness at 85℃ to obtain a mixture of homoserine and homoserine lactone. The mixture was dissolved in 200 mL of 6 mol / L hydrochloric acid, heated under reflux at 40 °C for 2 h, and then evaporated and concentrated to dryness at 80 °C. The product was repeatedly washed with anhydrous ethanol and filtered four times to obtain homoserine lactone salt.

[0069] 0.4 mol of dodecanoic acid was added to 1.5 L of dichloromethane, followed by 1.5 mol of oxaloyl chloride. The mixture was subjected to acyl chloride reaction at 80 °C and 100 rpm, and then stirred and concentrated to dryness. 500 mL of a 1:1 mixture of dichloromethane and dimethylformamide was added. 0.3 mol of homoserine lactone obtained by washing and filtration and 50 mL of triethylamine were added to the product. The mixture was reacted overnight at 55 °C and then evaporated and concentrated to dryness at 80 °C. The concentrated product was washed with dichloromethane, and the undissolved portion was N-dodecanoyl-L-homoserine lactone.

[0070] Example 3

[0071] The only difference from Example 1 is that dodecanoic acid is replaced with tetradecanoic acid, denoted as C4-HSL.

[0072] Example 4

[0073] The only difference from Example 1 is that dodecanoic acid is replaced with hexadecanoic acid, denoted as C6-HSL.

[0074] Example 5

[0075] The only difference from Example 1 is that dodecanoic acid is replaced with octadecanoic acid, denoted as C8-HSL. Application Example 1

[0076] Appropriate amounts of the prepared C4-HSL, C6-HSL, C8-HSL, and C12-HSL AHLs were respectively introduced into a reactor for a coupled anaerobic ammonia oxidation and sulfur autotrophic denitrification process. The reactor type was an SBR reactor. Simulated nitrogen-containing wastewater, prepared from sodium nitrate, ammonium sulfate, and tap water, was introduced into the reactor using a peristaltic pump. The simulated nitrogen-containing wastewater had a nitrate nitrogen concentration of 50 mg / L and an ammonia nitrogen concentration of 40 mg / L. The reactor has an effective volume of 6L. The entire reaction process is divided into three stages, designated as stages I, II, and III. Each stage runs for a period of time, with hydraulic retention times of 8h, 8h, and 4h, respectively. In stages II and III, the reactor load is increased by changing the concentration ratio of the simulated wastewater and reducing the hydraulic retention time, respectively. Group A (with added signaling molecules) and Group B (without added signaling molecules) serve as controls. The reactor's resistance to shock loads is tested by adjusting the concentration ratio and concentration of pollutants in the simulated wastewater. The reactor observes whether the signaling molecules can coordinate the relationship between bacterial species in the coupled system to better remove pollutants.

[0077] The removal results for pollutants in Group A and Group B are shown below. Figure 1 and Figure 2 .from Figures 1-2 It was observed that during the microbial culture and biofilm formation stage (Stage I, 0–16 days), the experimental group with added signaling molecules showed a significantly earlier increase in biofilm load compared to the control group without added signaling molecules. This is consistent with the literature report that AHLs can improve microbial growth rate, indicating that the AHLs signaling molecules prepared by this method can indeed promote microbial growth. In Stages II (16–32 days) and III (32–50 days), it was observed that the experimental group with added signaling molecules generally adapted more quickly to the biofilm load and restored the pollutant degradation rate to its original level. This is consistent with the literature report that AHLs can improve denitrification efficiency, and also indirectly demonstrates that AHLs can indeed regulate the synergistic symbiotic relationship between microbial species in complex systems.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing N-acyl-homoserine lactones, characterized in that, The method comprises the following steps: extracting protein from soybean to obtain methionine extract; mixing trifluoroacetic acid solution of the methionine extract and acetonitrile solution of cyanogen bromide, and then performing methionine cleavage under light-proof condition to obtain homoserine and homoserine lactone mixture; mixing the homoserine and homoserine lactone mixture with inorganic acid, and then performing homoserine dehydration reaction and salt formation to obtain homoserine lactone salt; performing acyl chloride reaction on alkanoic acid and oxalyl chloride, mixing the obtained acyl chloride product with the homoserine lactone salt, and then performing substitution reaction under the condition of catalyst to obtain N-acyl-homoserine lactone.

2. The production method according to claim 1, characterized by, The extraction agent for the extraction comprises strong alkali solution, and the pH value of the strong alkali solution is 12-14.

3. The production method according to claim 1 or 2, characterized by, The extraction temperature is 100-130℃, and the extraction time is 2-3h.

4. The method of claim 1, wherein, The particle size of the methionine extract is ≤74μm.

5. The production method according to claim 1 or 4, characterized by, The concentration of the methionine extract in the trifluoroacetic acid solution of the methionine extract is 3-5 g·L -1 .

6. The production method according to claim 1 or 4, characterized by, The cleavage temperature is 4℃, and the cleavage time is 24h.

7. The preparation method according to claim 1, characterized in that, The dehydration reaction temperature is 40-60℃, and the dehydration reaction time is 1-2h.

8. The method of claim 1, wherein, The alkanoic acid comprises one or more of dodecanoic acid, tetradecanoic acid, hexadecanoic acid and octadecanoic acid.

9. The production method according to claim 1 or 8, characterized by, The molar ratio of the alkanoic acid to oxalyl chloride is 1:3-5, and the molar ratio of the acyl chloride product to the homoserine lactone salt is 1.2-1.4:

1.

10. The method of claim 1, wherein, The substitution reaction temperature is 50-60℃, and the substitution reaction time is 12-24h.

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