Preparation method of L-cysteine hydrochloride

Abstract

The invention provides a preparation method of L-cysteine hydrochloride, and relates to the field of chemical processes. According to the preparation method of the L-cysteine hydrochloride, escherichia coli BL21 is adopted as an original host strain, and site-directed mutagenesis is performed on serine transacetylase in the escherichia coli original strain through feedback inhibition mutation, threonine synthetase gene deletion and serine transacetylase gene overexpression, so that the L-cysteine hydrochloride is obtained. By overexpressing the O-acetylserine sulfydryl lyase gene, promoting the combination of hydrogen sulfide and OAS to generate L-cysteine and removing the feedback inhibition of threonine, the method overcomes the defects that a large amount of hydrochloric acid is used and acidic waste is discharged in the preparation of a traditional process, reduces the discharge of waste acid by 80% or more, is renewable in raw materials, meets the requirements of green chemistry, and is suitable for industrial production. The future trend of a green and environment-friendly preparation process is achieved.

Description

TECHNICAL FIELD

[0001] The application relates to the chemical process field and particularly relates to a preparation method of L-cysteine hydrochloride. BACKGROUND

[0002] As a key amino acid derivative, L-cysteine hydrochloride has extremely important applications in many fields such as food, medicine and cosmetics. In the food industry, it is a high-efficiency food additive capable of playing an antioxidation role, effectively preventing food oxidation and deterioration and prolonging the shelf life of food. In the medical field, it has good detoxification effect and can effectively remove harmful substances in the body, reduce the burden on the liver and kidney and has a significant treatment effect on diseases such as viral hepatitis. At present, the preparation process of L-cysteine hydrochloride mainly includes a hair hydrolysis method. The hair hydrolysis method uses hair as raw material and obtains L-cysteine hydrochloride through a series of complex processes such as hydrolysis, separation and purification. The hair source is unstable and is greatly limited by raw material supply, so it is difficult to meet the demand of large-scale production. On the other hand, the process produces a large amount of wastewater, waste gas and waste residue, seriously pollutes the environment and has high treatment cost. Meanwhile, the production process of the hair hydrolysis method is complex, the energy consumption is high and the product quality is unstable, so it is difficult to meet the strict requirements of high-end markets on product quality.

[0003] Therefore, the application provides a preparation method of L-cysteine hydrochloride. Through modern biological technology means such as genetic engineering, the fermentation strain can be directionally reformed and optimized to improve the acid production capacity and production efficiency of the strain, so that the L-cysteine hydrochloride can be efficiently produced. SUMMARY

[0004] (I) Technical problems solved

[0005] In view of the defects of the prior art, the application provides a preparation method of L-cysteine hydrochloride. Compared with the traditional hair hydrolysis process and chemical synthesis process, the preparation method adopts a fermentation method to genetically modify and cultivate Escherichia coli, so that the key enzyme in the cysteine synthesis pathway can be directly fermented to produce L-cysteine, and then the L-cysteine is salted with hydrochloric acid. The preparation method overcomes the defects of the traditional process, such as the large use of hydrochloric acid and the discharge of acidic waste, reduces the discharge of waste acid by more than 80%, and the raw material is renewable, which meets the requirements of green chemistry and achieves the future trend of green and environmentally-friendly preparation process.

[0006] (II) Technical scheme

[0007] To achieve the above object, the application is implemented by the following technical scheme: a preparation method of L-cysteine hydrochloride, which adopts Escherichia coli BL21 as the original host strain, and the process steps of the preparation method are as follows:

[0008] Step 1: genetic modification of the strain, by feedback inhibition mutation, deletion of threonine synthetase gene, overexpression of serine transacetylase gene, site-directed mutagenesis of serine transacetylase in the original strain of Escherichia coli, overexpression of O-acetylserine thiol-lyase gene to promote the combination of hydrogen sulfide and OAS to generate L-cysteine, and relieve the feedback inhibition of threonine;

[0009] Step 2: strain screening and preservation, placing the strain on a plate containing 50% concentration of cysteine analog ethionine, screening the anti-feedback inhibition strain by high concentration of ethionine, then placing the screened strain in a shaking flask for fermentation screening, detecting the L-cysteine content in the fermentation broth by HPLC, and screening high-yield strains;

[0010] Step 3: preparation of culture medium, configuring the Escherichia coli culture medium according to the ratio of 30:5:3:1:5:2 of glucose, threonine, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, yeast, and sodium sulfide, adding trace metal element solution with a concentration of 0.1% to the culture medium, and placing the culture medium in a sterilization box at a temperature above 120 degrees Celsius and an atmospheric pressure of 0.1 MPa for sterilization for 30 minutes;

[0011] Step 4: strain fermentation control, inoculating the frozen strain into the liquid medium and adding 50 μg / mL of ampicillin, placing it in a 35-degree Celsius, 200 rpm shaking incubator for 12 hours, so that the OD 600 reaches 1.0-1.5, so that the bacterial cells are in the logarithmic growth phase, and then adjusting the temperature to 30 degrees Celsius for 24 hours of incubation;

[0012] Step 5: fermentation product extraction, placing the fermentation strain suspension in the culture medium into a centrifuge, centrifuging at 8000 rpm for 30 minutes, filtering the supernatant of the centrifuged strain through a 0.5 μm ceramic membrane to remove protein impurities, removing pigment and lipid-soluble impurities and removing small bacterial cell fragments, adding 2% (w / v) activated carbon powder to the filtered supernatant of the strain, placing it in a stirring device, setting the environment to 50 degrees Celsius and stirring for 1 hour to adsorb the pigment and lipid-soluble impurities, and then removing the activated carbon powder through a filter membrane after the impurities are adsorbed;

[0013] Step 6: fermentation product purification, using a strong acid cation exchange resin, adjusting the pH of the filtrate to 2.5, making the strain liquid pass through the resin column at a flow rate of 5-10 BV / h, making the L-cysteine adsorbed on the resin, and then eluting with 5 mol / L hydrochloric acid solution, collecting the elution peak with pH <1.0, so that the L-cysteine exists in the form of hydrochloride;

[0014] Step 7: The fermentation concentrate is crystallized, the eluent is concentrated to 1 / 3 of the original volume at 50℃ under a vacuum degree of less than or equal to -0.08 MPa to form a supersaturated solution, and then the supersaturated solution is cooled to 10℃ and left to stand for 48 hours to precipitate L-cysteine hydrochloride crystals, the L-cysteine hydrochloride crystals are dried at 65℃ under a vacuum degree of less than or equal to -0.06 MPa for 8 hours until the water content is less than or equal to 0.3%, and finally the L-cysteine hydrochloride crystals are crushed to obtain powdered L-cysteine hydrochloride;

[0015] By the above technical solution, L-cysteine hydrochloride is prepared by fermentation, and Escherichia coli is used as the original strain. The Escherichia coli microorganism is metabolized under suitable environmental conditions to convert inexpensive carbon sources, nitrogen sources and other nutrients into L-cysteine hydrochloride. By using genetic engineering technology, the Escherichia coli is overexpressed by genetic engineering means, and the genes of serine acetyltransferase and O-acetylserine thiol-lyase are overexpressed, and some genes competing with L-cysteine for substrates are knocked out.

[0016] Preferably, in the strain screening and preservation step, a strain liquid with a composite detection wavelength of 210 nm and a mobile phase of 0.1% phosphoric acid aqueous solution-acetonitrile=95:5 is screened, and the screened strain is placed in a test tube containing 35% glycerol and stored in a freezer at -90℃ for freeze-drying preservation.

[0017] By the above technical solution, glycerol tube-90℃ freezing, and freeze-drying method can achieve a survival rate of up to 95% for long-term preservation.

[0018] Preferably, in the medium preparation step, the metal elements in the trace metal element solution include Fe, Mn and Zn, and the sodium sulfide needs to be filtered separately through a 0.22μm filter membrane before being configured to avoid the generation of H2S volatilization at high temperature.

[0019] By the above technical solution, glucose in the culture medium is responsible for providing energy for the cultivation of the strain, sulfathiazole is used for the synthesis of amino acids, potassium dihydrogen phosphate maintains the stability of pH, magnesium sulfate heptahydrate activates enzyme activity as a catalyst, sodium sulfide is the source of cysteine thiol, and the trace metal element solution is responsible for promoting the rapid growth of the bacterial body.

[0020] Preferably, in the strain fermentation control step, the initial pH of the culture medium is adjusted to 7.0, and the pH is maintained at 6.5-6.8 by adding hydrochloric acid during the cultivation process. The dissolved oxygen is controlled at DO≥30% air saturation during the cultivation process, and the DO is reduced to 15% after 12 hours to promote anaerobic metabolism to generate cysteine.

[0021] By the above technical solution, when the dissolved oxygen concentration is too low, the growth rate of microorganisms is significantly slowed down, and the yield of L-cysteine hydrochloride is also reduced, so the dissolved oxygen control needs to be in the optimal value area to make the fermentation efficiency of the strain highest.

[0022] Preferably, in the strain fermentation control step, 1% Na2S solution needs to be added every 12 hours to maintain the S 2- concentration ≤ 0.3 mmol / L, and the cultivated strain is determined after 48 hours to have residual sugar < 1 g / L and L-cysteine concentration ≥ 15 g / L.

[0023] By the above technical solution, the addition of Na2S needs to be monitored in real time by an ion electromotor.

[0024] Preferably, in the fermentation concentrate crystallization step, crystal seeds are added to adjust the pH to 1.0-1.5 to promote crystallization, and the crystals are centrifuged at 5000 rpm for 10 minutes, washed with a small amount of cold ethanol for 3 times to remove surface adsorbed impurities.

[0025] By the above technical solution, the fermentation concentrate crystallization is realized by cooling crystallization, the solution containing L-cysteine hydrochloride is cooled to a certain temperature, the solubility of the solute is reduced, and high-purity L-cysteine hydrochloride is obtained by crystallization.

[0026] (III) Beneficial effects

[0027] The present application provides a preparation method of L-cysteine hydrochloride, which has the following beneficial effects:

[0028] The present application provides a preparation method of L-cysteine hydrochloride. Compared with the traditional hair hydrolysis process and chemical synthesis process, the present preparation method uses fermentation method to genetically modify and cultivate Escherichia coli, so that it can directly ferment L-cysteine by expressing key enzymes in the cysteine synthesis pathway, and then salt with hydrochloric acid. The present preparation method overcomes the shortcomings of traditional processes, such as the use of large amounts of hydrochloric acid and the discharge of acidic waste, reduces the discharge of waste acid by more than 80%, and is renewable, meets the requirements of green chemistry, and achieves the future trend of green and environmentally friendly preparation process. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The present application provides a preparation process flow chart of L-cysteine hydrochloride. DETAILED DESCRIPTION

[0030] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. It should be noted that the described embodiments are only some of the embodiments of the present invention, and are not intended to be exhaustive. Based on the embodiments of the present invention, all other embodiments derived by persons of ordinary skill in the art without inventive effort are within the scope of protection of the present invention. It should be noted that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the exemplary embodiments of the present invention. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to scale. Technologies, methods, and devices known to persons of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such technologies, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely illustrative and not limiting. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar reference numerals and letters denote similar items in the following figures. Therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0031] Example 1:

[0032] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing L-cysteine ​​hydrochloride, wherein the preparation method uses Escherichia coli BL21 as the original host strain, and the process steps of the preparation method are as follows:

[0033] Step 1: Genetic modification of the strain: By using feedback inhibition mutation, the threonine synthase gene is deleted, and the serine transacetylase gene is overexpressed. Site-directed mutagenesis of the serine transacetylase in the original E. coli strain is performed. By overexpressing the O-acetylserine sulfhydryl lyase gene, the binding of hydrogen sulfide to OAS is promoted to generate L-cysteine, thereby relieving the feedback inhibition of threonine.

[0034] Step 2: strain screening and preservation: strains are plated on a plate containing 50% ethionine, a cysteine ​​analog, to screen for strains resistant to feedback inhibition. The screened strains are then shaken in a shake flask for fermentation screening. The L-cysteine ​​content in the fermentation broth is measured by HPLC to screen for high-yield strains.

[0035] Step 3: Prepare the culture medium. Prepare an E. coli culture medium by mixing glucose, methionine, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, yeast, and sodium sulfide in a ratio of 30:5:3:1:5:2. Add a 0.1% trace metal element solution to the culture medium. After the preparation is completed, place the culture medium in a sterilization box at a temperature above 120 degrees Celsius and an atmospheric pressure of 0.1 MPa for 30 minutes.

[0036] Step 4: Strain fermentation control, the frozen strain is inoculated into the liquid medium and 50 μg / mL ampicillin is added, and it is placed in a 35-degree Celsius, 200 rpm shaking incubator for 12 hours to make the OD 600 reach 1.0-1.5, so that the bacterial cells are in the logarithmic growth phase. The temperature is then adjusted to 30 degrees Celsius for 24 hours of incubation;

[0037] Step 5: Fermentation product extraction, the fermentation strain suspension in the culture medium is placed in a centrifuge, and centrifugation is performed at 8000 rpm for 30 minutes. The supernatant of the centrifuged strain is filtered through a 0.5 μm ceramic membrane to remove protein impurities. The color and fat-soluble impurities are removed, and the small bacterial cell fragments are removed. The filtered supernatant of the strain is added with 2% (w / v) activated carbon powder, and it is placed in a stirring device. The environment is set to 50 degrees Celsius, and stirring is performed for 1 hour to adsorb the color and fat-soluble impurities. After the impurities are adsorbed, the activated carbon powder is removed by a filter membrane.

[0038] Step 6: Fermentation product purification, a strong acid cation exchange resin is used, the pH of the filtrate is adjusted to 2.5, the strain liquid is passed through the resin column at a flow rate of 5-10 BV / h, and L-cysteine is adsorbed on the resin. Then, 5 mol / L hydrochloric acid solution is used for elution, and the elution peak with pH <1.0 is collected, so that L-cysteine exists in the form of hydrochloride.

[0039] Step 7: Fermentation product concentration and crystallization, the eluate is concentrated to 1 / 3 of the original volume under the condition of 50 degrees Celsius and a vacuum degree of ≤-0.08 MPa to form a supersaturated solution. Then, the supersaturated solution is cooled to 10 degrees Celsius and left to stand for 48 hours to precipitate L-cysteine hydrochloride crystals. The L-cysteine hydrochloride crystals are dried at 65 degrees Celsius and a vacuum degree of ≤-0.06 MPa for 8 hours until the water content is ≤0.3%. Finally, the L-cysteine hydrochloride is obtained in the form of powder after crushing.

[0040] Example 2:

[0041] A method for preparing L-cysteine hydrochloride, which uses a fermentation method to prepare L-cysteine hydrochloride. Escherichia coli is used as the original strain, and the microorganism is used to metabolize cheap carbon sources, nitrogen sources and other nutrients into L-cysteine hydrochloride under suitable environmental conditions. Genetic engineering technology is used to overexpress the genes of serine acetyltransferase and O-acetylserine thiol-lyase in Escherichia coli through genetic engineering methods, and some genes that compete with L-cysteine for substrates are knocked out.

[0042] In the strain screening and preservation step, the strain liquid is screened at a detection wavelength of 210 nm and a mobile phase of 0.1% phosphoric acid aqueous solution-acetonitrile = 95:5. The screened strain is placed in a test tube containing 35% glycerol and stored in a freezer at -90°C for freeze-drying preservation. The glycerol tube is stored at -90°C, and the survival rate of the freeze-drying method is as high as 95%. In the culture medium preparation step, the metal elements in the trace metal element solution include Fe, Mn, and Zn. The sodium sulfide needs to be filtered separately through a 0.22 μm filter before being configured to avoid the generation of H2S volatilization at high temperatures. The glucose in the culture medium provides energy for the cultivation of the strain. The sulfanilic acid is used to synthesize amino acids. The potassium dihydrogen phosphate maintains the stability of pH. The magnesium sulfate heptahydrate serves as a catalyst to activate enzyme activity. The sodium sulfide is a source of cysteine thiol, and the trace metal element solution is responsible for promoting the rapid growth of the bacterial body.

[0043] Example 3

[0044] A method for preparing L-cysteine hydrochloride, in the strain fermentation control step, the initial pH of the culture medium needs to be adjusted to 7.0, and the pH is maintained at 6.5-6.8 by adding hydrochloric acid during the cultivation process. The dissolved oxygen is controlled at DO≥30% air saturation during the cultivation process. After 12 hours, the DO decreases to 15%, promoting anaerobic metabolism to generate cysteine. When the dissolved oxygen concentration is too low, the growth rate of the microorganism slows down significantly, and the yield of L-cysteine hydrochloride also decreases. Therefore, the dissolved oxygen control needs to be within the optimal value range to maximize the efficiency of strain fermentation. In the strain fermentation control step, 1% Na2S solution needs to be added every 12 hours to maintain the S 2- concentration in the fermentation broth at ≤0.3 mmol / L. After 48 hours, the cultivated strain is determined to have residual sugar <1 g / L and L-cysteine concentration ≥15 g / L. The addition of Na2S needs to be monitored in real time by an ion meter. In the fermentation concentrate crystallization step, crystal seeds are added to adjust the pH to 1.0-1.5 to promote crystallization and make the crystals centrifuge at 5000 rpm for 10 minutes. A small amount of cold ethanol is used to wash the crystals three times to remove the surface-adsorbed impurities. The fermentation concentrate crystallization is achieved by cooling crystallization. The solution containing L-cysteine hydrochloride is cooled to a certain temperature, the solubility of the solute is reduced, and high-purity L-cysteine hydrochloride is obtained by crystallization.

[0045] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing L-cysteine ​​hydrochloride, characterized in that: The preparation method uses Escherichia coli BL21 as the original host strain, and the process steps of the preparation method are: Step 1: Genetic modification of the strain: By using feedback inhibition mutation, the threonine synthase gene is deleted, and the serine transacetylase gene is overexpressed. Site-directed mutagenesis of the serine transacetylase in the original E. coli strain is performed. By overexpressing the O-acetylserine sulfhydryl lyase gene, the binding of hydrogen sulfide to OAS is promoted to generate L-cysteine, thereby relieving the feedback inhibition of threonine. Step 2: strain screening and preservation: strains are plated on a plate containing 50% ethionine, a cysteine ​​analog, to screen for strains resistant to feedback inhibition. The screened strains are then shaken in a shake flask for fermentation screening. The L-cysteine ​​content in the fermentation broth is measured by HPLC to screen for high-yield strains. Step 3: Prepare the culture medium. Prepare an E. coli culture medium by mixing glucose, methionine, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, yeast, and sodium sulfide in a ratio of 30:5:3:1:5:

2. Add a 0.1% trace metal element solution to the culture medium. After the preparation is completed, place the culture medium in a sterilization box at a temperature above 120 degrees Celsius and an atmospheric pressure of 0.1 MPa for 30 minutes. Step 4: Strain fermentation control: inoculate the frozen strain into liquid culture medium, add 50 μg / mL ampicillin, and culture in a shaking incubator at 35 degrees Celsius and 200 rpm for 12 hours until the OD 600 Reach 1.0-1.5, so that the bacteria are in the logarithmic growth phase and grow rapidly, then adjust the temperature to 30 degrees Celsius and incubate for 24 hours; Step 5: Extraction of fermentation products: The strain suspension that has reached the fermentation standard in the culture medium is placed in a centrifuge and centrifuged at 8000 rpm for 30 minutes. The supernatant of the strain after extraction and centrifugation is filtered through a 0.5 μm ceramic membrane to remove protein impurities, pigments and fat-soluble impurities, and microbial fragments. 2% (w / v) activated carbon powder is added to the filtered and extracted supernatant of the strain, and the mixture is placed in a stirring device and stirred at 50 degrees Celsius for 1 hour to adsorb pigments and fat-soluble impurities. After the impurities are adsorbed, the activated carbon powder is removed through the filter membrane; Step 6: Purify the fermentation product by using a strongly acidic cation exchange resin, adjusting the pH of the filtrate to 2.5, and passing the strain liquid through the resin column at a flow rate of 5-10 BV / h to allow L-cysteine ​​to adsorb to the resin. Then, elute with a 5 mol / L hydrochloric acid solution, and collect the elution peak with a pH < 1.0, so that L-cysteine ​​exists in the form of hydrochloride; Step 7: The fermented product is concentrated and crystallized. The eluate is concentrated to 1 / 3 of its original volume at 50°C and a vacuum degree of ≤-0.08 MPa to form a supersaturated solution. The supersaturated solution is then cooled to 10°C and allowed to stand for 48 hours to precipitate L-cysteine ​​hydrochloride crystals. The L-cysteine ​​hydrochloride crystals are dried at 65°C and a vacuum degree of ≤-0.06 MPa for 8 hours to a water content of ≤0.3%. Finally, the powdered L-cysteine ​​hydrochloride is obtained by pulverizing.

2. A method for preparing L-cysteine ​​hydrochloride according to claim 1, characterized in that: In the strain screening and preservation step, a strain solution with a composite detection wavelength of 210 nm and a mobile phase of 0.1% phosphoric acid aqueous solution-acetonitrile = 95:5 was screened out, and the screened strain was placed in a test tube with a 35% glycerol concentration and placed in a freezer at -90 degrees Celsius for freeze-drying and storage.

3. A method for preparing L-cysteine ​​hydrochloride according to claim 1, characterized in that: In the culture medium preparation step, the metal elements in the trace metal element solution include Fe, Mn, and Zn, and sodium sulfide needs to be separately filtered through a 0.22 μm filter membrane before preparation to avoid the volatilization of H2S generated by high temperature.

4. The method for preparing L-cysteine ​​hydrochloride according to claim 1, wherein: In the strain fermentation control step, the initial pH of the culture medium needs to be adjusted to 7.0, and hydrochloric acid is added during the cultivation process to maintain the pH at 6.5-6.

8. During the cultivation process, the dissolved oxygen is controlled at DO ≥ 30% of air saturation, and the DO is reduced to 15% after 12 hours to promote anaerobic metabolism to produce cysteine.

5. A method for preparing L-cysteine ​​hydrochloride according to claim 1, characterized in that: In the fermentation control step of the strain, it is necessary to add a 1% Na2S solution every 12 hours to keep the S in the fermentation liquid 2- The concentration was ≤0.3mmol / L. After 48 hours, the cultured strain was judged to have residual sugar <1g / L and L-cysteine ​​concentration ≥15g / L.

6. The method for preparing L-cysteine ​​hydrochloride according to claim 1, wherein: In the fermentation product concentration and crystallization step, seed crystals are added to adjust the pH to 1.0-1.5 to promote crystallization, and the crystals are centrifuged at 5000 rpm for 10 minutes and washed three times with a small amount of cold ethanol to remove impurities adsorbed on the surface.

Images