Method for extracting and purifying benzylamine in fermentation liquor

Abstract

The invention discloses a method for extracting and purifying benzylamine in fermentation liquor, which comprises the following steps: adjusting the pH value of the fermentation liquor, then centrifuging the fermentation liquor, taking supernate, adding an extracting agent, carrying out oscillation extraction, layering after extraction, taking an extraction phase, and then carrying out reduced pressure distillation on the extraction phase to remove the extracting agent, thereby obtaining purified benzylamine. According to the technical scheme, an environment-friendly green solvent is selected for extraction, so that potential hazards of chemicals to the environment and human beings are reduced; benzylamine can be separated at a low temperature through reduced pressure distillation, and the recovery rate and purity of benzylamine are improved. The developed purification process is relatively simple, is suitable for large-scale industrial production, and is also beneficial to the development of green biosynthesis.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical extraction and purification, and more particularly to a method for extracting and purifying benzylamine in a fermentation broth. Background Art

[0002] Benzylamine, also known as benzylamine, is an important organic synthesis intermediate. It is widely used in medicine, military industry, agriculture and other fields. It is often used as a buffer, curing agent, and precursor compound of antifungal drugs and high-energy propellants. Biosynthesis is emerging as a more environmentally friendly method, and many useful compounds have been synthesized, such as artemisinin, lignans, alkaloids, hyaluronic acid, etc. At present, the synthesis of benzylamine is mainly based on chemical methods, mainly using benzaldehyde, benzyl chloride, benzonitrile, etc. as substrates, through amination or hydrogenation. The traditional chemical synthesis of benzylamine will produce toxic waste and high cost, while the recent biosynthesis method can overcome this defect and has the advantages of low energy consumption and green sustainability. Exploring the efficient recovery of benzylamine from microbial fermentation broth is an important part of the research on biosynthesis of benzylamine.

[0003] At present, the research on the biosynthesis of benzylamine is still in its preliminary stage, and further research is needed in terms of separation and purification. Summary of the invention

[0004] In view of this, the present invention provides a method for extracting and purifying benzylamine in a fermentation broth, comprising the following steps:

[0005] Step 1: Adjust the pH of the fermentation liquid, then centrifuge the fermentation liquid, take the supernatant and add the extractant for oscillation extraction, and take the extraction phase by stratification after extraction;

[0006] Step 2: The extract phase is subjected to reduced pressure distillation to remove the extractant to obtain purified benzylamine.

[0007] Preferably, in step 1, 1 mol / L dilute hydrochloric acid and / or 10 mol / L sodium hydroxide are used to adjust the pH of the fermentation liquid to 11.

[0008] Preferably, in step 1, the centrifugation speed is 4000 rpm and the time is 10 min.

[0009] Preferably, the amount of the extractant added in step 1 is 5 g / L based on the weight of benzylamine.

[0010] Preferably, the extractant is dichloromethane.

[0011] Preferably, the number of extractions in step 1 is 3 times or more.

[0012] Preferably, in step 1, static stratification or centrifuge at 4000 rpm for 2 min is used to assist stratification.

[0013] Preferably, the vacuum distillation in step 2 is carried out at a vacuum degree of 0.07 MPa and a temperature of 30°C.

[0014] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for extracting and purifying benzylamine in a fermentation broth, which has the following beneficial effects:

[0015] The technical solution of the present invention selects an environmentally friendly green solvent for extraction to reduce the potential harm of chemicals to the environment and humans; the benzylamine can be separated at a lower temperature by vacuum distillation, thereby improving the recovery rate and purity of the benzylamine. The developed purification process is relatively simple, suitable for large-scale industrial production, and also contributes to the development of green biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 is the effect of the extractant on the extraction rate of benzylamine (4 mL system, single extraction, pH 8.6);

[0018] Figure 2 The effect of feed solution pH on benzylamine extraction (4 mL system, CH2Cl2);

[0019] Figure 3 The effect of benzylamine extraction at pH 8.6 and pH 11 (4 mL system);

[0020] Figure 4 The effect of adding NaCl to the feed solution on the extraction of benzylamine with dichloromethane (4 mL system, pH 11);

[0021] Figure 5 The effects of three extractions and biotransformation system on benzylamine extraction (4 mL system, pH 11);

[0022] Figure 6 is the chromatogram of the extract;

[0023] Figure 7 The effect of temperature and vacuum degree on the loss rate of benzylamine during distillation;

[0024] Figure 8 is the liquid chromatogram of benzylamine sample;

[0025] Fig. 9 This is the positive ion mass spectrum of the benzylamine sample. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Raw materials and instruments

[0028] Raw materials: dichloromethane, benzylamine and other reagents are all analytically pure. Escherichia coli fermentation broth (LB medium).

[0029] Instruments: Agilent 1260 InfinityⅡ high performance liquid chromatograph (Agilent), 2-16PK high-speed refrigerated centrifuge (Sigma), RV10 rotary evaporator (IKA), SHZ-D vacuum water pump (Shanghai Xiniu Lab Instrument), TSQFortis triple quadrupole mass spectrometer (THermoFisher, USA).

[0030] Extraction method: After preparing 10 g / L benzylamine solution with the fermentation broth, centrifuge it to obtain clarified fermentation broth, centrifuge at 4000 rpm for 10 min, and measure the pH to be 8.6.

[0031] In a centrifuge tube, add the selected extractant at a ratio of feed liquid: extractant = 2:1 (V:V) for the first extraction and shake for 2 minutes. This process may produce emulsification. A centrifuge can be used at 4000 rpm for 2 minutes to assist in stratification. If there is no emulsification, let it stand for 20 minutes. Perform three parallel experiments.

[0032] Purification of benzylamine by vacuum distillation

[0033] Effect of vacuum distillation on the distillation of pure benzylamine: Take 100μL of benzylamine and place it in a distillation bottle. Add 10mL of dichloromethane and remove the dichloromethane by vacuum distillation after it is fully dissolved. The temperature is set in the range of 20-30℃ and the vacuum degree is controlled in the range of 0.05-0.1MPa.

[0034] The processing of actual samples refers to the above operation.

[0035] Benzylamine detection analysis: The concentration and purity of benzylamine were detected by Agilent 1260 InfinityⅡ high performance liquid chromatography. The chromatographic column was a GL Sciences InertSustain C18 column (4.6 mm×250 mm, particle size 5 μm), the mobile phase was 0.1% trifluoroacetic acid (TFA): acetonitrile (80:20, V / V), the flow rate was 0.5 mL / min, the column temperature was 32°C, the detection wavelength was 210 nm, and the injection volume was 10 μL.

[0036] The purity of benzylamine was detected by a triple quadrupole mass spectrometer (TSQ Fortis Thermo Fisher), the ion source was ESI+, and the sample was diluted with deionized water before testing.

[0037] 1. Research on the selection of extraction agent

[0038] In order to explore the extraction effect of different extractants on benzylamine in fermentation broth, the extraction of five organic solvents such as dichloromethane was studied. According to the structural characteristics and physicochemical properties of benzylamine, water solubility, boiling point and toxicity were used as reference indicators for extractant screening. Ethyl acetate, dichloromethane, n-butanol, cyclohexane and benzene were initially selected for the experiment. The results are as follows Figure 1 The results show that all five organic solvents can extract benzylamine from the fermentation broth, with dichloromethane having the highest extraction rate of 20.6%, followed by ethyl acetate at 14.2%, benzene at 12.2%, n-butanol at 8.5%, and cyclohexane at the lowest extraction rate of 3.9%. Since benzene is toxic and the extraction rate of cyclohexane is too low, these two solvents were excluded, and dichloromethane, n-butanol and ethyl acetate were initially selected as extractants for subsequent research.

[0039] 2. Optimization of extraction conditions

[0040] (1) Effect of pH on benzylamine extraction

[0041] The factor affecting benzylamine extraction is mainly the pH value of the aqueous phase, which determines the existence state of the extracted substance, that is, whether it exists in a dissociated state or a non-dissociated state. In order to improve the extraction rate of benzylamine, this experiment conducted an extraction study on benzylamine fermentation broth under the conditions of pH 5, 7, 9, 10 and 11 with dichloromethane as the extractant. Before extraction, the pH of the feed solution was adjusted with 1 mol / L dilute hydrochloric acid and 10 mol / L sodium hydroxide, and then clarified with a centrifuge.

[0042] The results are as follows Figure 2As shown, the results show that under pH=5, benzylamine was not detected in the extraction phase, and it is inferred that the benzylamine extraction rate under acidic conditions is 0; benzylamine can be detected under neutral and alkaline conditions, indicating that benzylamine is extracted; under pH=7, the extraction rate is only 4.3%, and as the pH value increases, the benzylamine extraction rate also increases. The highest extraction rate is 81.3% under pH=11. In summary, pH has a significant effect on the extraction of benzylamine, and the best extraction rate is obtained under pH=11. Subsequent studies are also conducted under pH=11.

[0043] Based on the analysis of the existence forms of benzylamine under different pH conditions, it was determined that benzylamine should be extracted under alkaline conditions. The three organic solvents preliminarily screened were used as extractants, and extraction was performed once at pH = 8.6 (initial pH) and pH = 11. The results are as follows: Figure 3 As shown. When pH=11, the extraction rates of the three extractants were much higher than those under the initial pH conditions, and under pH=11, the extraction rate of dichloromethane was the highest, followed by n-butanol, and the extraction rate of ethyl acetate was significantly lower than the first two solvents. Considering that the boiling point and price of dichloromethane were lower than n-butanol, dichloromethane was finally determined as the extractant for subsequent research.

[0044] (2) Effect of salt on benzylamine extraction

[0045] Since benzylamine has strong hydrophilicity, adding salt can usually change the solubility of the solute in the feed solution, reducing the solubility of benzylamine. Therefore, in this study, when exploring the effect of different amounts of NaCl on extraction, the feed solution (mL): salt (mg) ratio was 40:0-40:30. Figure 4 As shown, Figure 4 It shows that under the condition of pH = 11, the addition of salt has no obvious effect on the extraction of benzylamine. The main reason is that at pH = 11, benzylamine is completely in a molecular state. At this time, the interaction between benzylamine and water is the weakest, and it is easier to distribute into the organic solvent.

[0046] (3) Effects of extraction times and biotransformation system on benzylamine extraction

[0047] In this laboratory study, the biosynthesis of benzylamine uses L-phenylalanine as a substrate to explore the effect of L-phenylalanine on the extraction rate of benzylamine. In order to recover as much benzylamine as possible, the benzylamine fermentation broth was subjected to three extraction studies. During the three extractions, the extraction was performed at a ratio of feed: extractant = 2:1, 2:1, and 4:1 (V:V).

[0048] The results are as follows Figure 5 As shown, Figure 5It shows that after increasing the number of extractions to 3 times, the extraction rate gradually increases, and the total extraction rate of 3 extractions can reach 98.3%. After each extraction, the analysis of the reaction substrate L-phenylalanine in the raffinate showed that its concentration did not change at all, indicating that L-phenylalanine was not extracted into dichloromethane. The chromatogram of the extract showed a single peak (such as Figure 6 ), indicating that L-phenylalanine has no effect on benzylamine extraction.

[0049] 3. Extraction of benzylamine by vacuum distillation

[0050] When the extract was distilled under reduced pressure at 25°C and a commonly used vacuum degree of 0.1 MPa (water pump), a large loss of benzylamine was observed. Therefore, the evaporation of benzylamine at different temperatures and vacuum degrees was studied. Figure 7 The loss rate of benzylamine at different temperatures and vacuum degrees is given. When the distillation temperature is 20℃, 25℃ and 30℃, benzylamine is lost, but the loss rate is not much different, and the distillation time will increase with the decrease of temperature. The change of vacuum degree has a significant effect on the loss rate of benzylamine in the distillation process. Too high or too low vacuum degree will increase the loss rate of benzylamine. Overall, the loss rate of benzylamine is relatively small at 0.07MPa, so the subsequent operation is selected at 30℃ and 0.07MPa.

[0051] 4. Establishment of the extraction process and purity characterization of the prepared benzylamine samples

[0052] After the separation and purification conditions were determined, the large volume of benzylamine fermentation broth was separated. 500 mL of fermentation broth was selected in the experiment, and dichloromethane was selected as the extractant. The extraction was performed three times at pH = 11. The results showed that the total extraction rate of benzylamine was 96%, and the final distillation recovery of benzylamine was 94%. The pure benzylamine after distillation was detected by chromatography and mass spectrometry. Figure 8 and Fig. 9 As shown, the chromatogram shows a single peak. Figure 6 ), which is the elution peak of benzylamine, indicating that the prepared sample is high-purity benzylamine; the mass spectrum shows that there is an ion fragment peak of 108.14, which is consistent with the molecular weight of benzylamine 107.15, also indicating that the prepared sample is high-purity benzylamine.

[0053] In summary, the extractant dichloromethane selected in the present invention has a stronger extraction ability for benzylamine than other extractants such as esters (ethyl acetate, etc.), alcohols (n-butanol), and aromatic hydrocarbons (benzene). After optimizing the pH and the number of extractions, the extraction rate can reach 98.3%.

[0054] Benzylamine has a high boiling point of 184-185°C at normal pressure, while dichloromethane has a boiling point of 39.8°C, so benzylamine should not evaporate from the extract even under rotary evaporation. However, in actual research, under the condition of a vacuum degree of 0.1MPa (water pump) in reduced pressure distillation, along with the dichloromethane evaporation, benzylamine that would not evaporate originally also has a significant loss, indicating that high-boiling-point substances also evaporate to a certain extent under reduced pressure conditions, and this point generally does not attract the attention of researchers; for a dichloromethane system with benzylamine as the main body, the present invention finally determines a temperature of 30°C and a vacuum degree of 0.07MPa, which not only ensures that dichloromethane evaporates more quickly, but also reduces the evaporation of benzylamine.

[0055] The liquid-liquid extraction and coupled vacuum distillation method established in this paper using fermentation broth as raw material and dichloromethane as extractant achieved efficient purification of benzylamine in fermentation broth, providing technical support for the scale-up of biological preparation of benzylamine; the ultra-low vacuum distillation idea adopted by the institute also provides a new idea for other vacuum distillation operations.

[0056] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown in this article, but will comply with the widest range consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting and purifying benzylamine in a fermentation broth, characterized in that: The following steps are involved: Step 1: Adjust the pH of the fermentation liquid, then centrifuge the fermentation liquid, take the supernatant and add the extractant for oscillation extraction, and take the extraction phase by stratification after extraction; Step 2: The extract phase is subjected to reduced pressure distillation to remove the extractant to obtain purified benzylamine.

2. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: In step 1, 1 mol / L dilute hydrochloric acid and / or 10 mol / L sodium hydroxide are used to adjust the pH of the fermentation liquid to 11.

3. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: In step 1, the centrifugation speed is 4000 rpm and the time is 10 min.

4. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: The amount of the extractant added in step 1 is 5 g / L based on the weight of benzylamine.

5. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1 or 4, characterized in that: The extractant is dichloromethane.

6. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: The number of extractions in step 1 is 3 times or more.

7. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: In step 1, static stratification or centrifuge at 4000 rpm for 2 min is used to assist stratification.

8. The method for extracting and purifying benzylamine in a fermentation broth according to claim 1, characterized in that: The vacuum degree of the reduced pressure distillation in step 2 is 0.07 MPa and the temperature is 30°C.

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