Preparation and purification process of glycine
Glycine is prepared by bioconversion method and resin column technology of Fusarium oxysporus, which solves the problems of high temperature and high pressure and environmental pollution in the existing glycine production process, and achieves high yield, high yield and high purity glycine preparation.
Patent Information
- Application Number
- CN202411516946.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2024-12-31
AI Technical Summary
The existing glycine production process has problems such as long reaction time, high temperature and high pressure, low yield, serious environmental pollution and waste of resources. There are no research reports on the production of glycine in China on bioconversion methods.
Glycine was prepared by bioconversion method by Fusarium oxysporus. The bacteria were cultured in LB solid culture medium, inoculated into liquid culture medium for fermentation, and glycosylnitrile was added for conversion. The cationic and anionic resin columns were concentrated and elution. Finally, the glycine product is obtained by activated carbon treatment.
It improves the yield, yield and purity of glycine, reduces environmental pollution and resource waste, and achieves efficient glycine preparation and purification.
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Figure CN119220616A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of glycosylamine production, and in particular to a preparation and purification process of glycine. Background Art
[0002] Glycine, also known as aminoacetic acid, is a white, odorless crystal that is non-toxic, soluble in water, and insoluble in organic solvents. It is the simplest of the 20 common amino acids, a neutral aliphatic amino acid, and a non-essential amino acid for the human body. Glycine is also the main raw material for the synthesis of glyphosate, the world's largest pesticide, and its demand is very large. It can also be used as a flavoring agent and flavor enhancer in food, as well as an intermediate in the preparation of drugs for hypertension, liver damage, etc. It is widely used in the fields of food, medicine, and chemical industry.
[0003] At present, the production of glycine is all done by chemical methods, such as chloroacetic acid ammonolysis, Strecker method, etc. These methods have the characteristics of long reaction time, high temperature and high pressure, low product yield, large amount of acid or alkali needs to be added for product recovery and purification, complex process, and environmental unfriendly. For example, although the chloroacetic acid ammonolysis method is simple in process and does not require high equipment, it is difficult to separate products such as ammonium chloride, resulting in a low yield of glycine, and refining increases the production cost. In addition, hexamethylenetetramine, which is a reaction catalyst, cannot be recovered, resulting in serious waste of resources; the patent US5258550 of Japan MITSUITOATSU Chemical Company discloses a new method for chemical synthesis of glycine using hydroxyacetonitrile as raw material, first pre-reacting with carbon dioxide and ammonia at 80-120°C for 0.5-1h in the presence of water, and then carrying out the main reaction at 150-200°C, but this method requires high temperature and high pressure, high energy consumption, high requirements for equipment, and serious environmental pollution.
[0004] With the rapid development of biotechnology, the use of biotransformation to prepare organic acids or amino acids is attracting much attention. The biotransformation method mainly uses catalytic carriers such as free microbial cells or purified enzymes to convert the substrate glycine nitrile into glycine. At present, many patents have been applied for the preparation of glycine by biotransformation abroad, but there is no relevant research report on the production of glycine by biotransformation in China. Abroad, the patent US5238827 of Japan's NITTO Chemical Company discloses a method for using bacterial strains of the genera of Rhodococcus, Arthrobacter, Lactobacillus, Enterobacter and other bacteria as catalytic enzymes to convert glycine nitrile into glycine, and the patent W00148234 of ASAHI Chemical Company discloses a method for preparing glycine by hydrolyzing glycine nitrile using bacteria and microorganisms of the genera of yeast such as Acinetobacter, Corynebacterium, Mycobacterium, Rhodopseudomonas and Candida, as well as a method for recovering and purifying glycine, such as EP1243657, US20030040085, CN1433479, N1840522. These have laid the foundation for further research on the production of glycine by biotransformation. However, the yield and purity of glycine are both low. Therefore, in view of the above problems, it is necessary to develop a preparation and purification process of glycine. Summary of the invention
[0005] The technical problem to be solved by the present invention is: in view of the deficiencies of the prior art, a preparation and purification process of glycine is provided, by which a glycine product with high yield, high yield and high purity can be obtained.
[0006] In order to solve the above technical problems, the technical solution of the present invention is:
[0007] A preparation and purification process of glycine comprises the following steps:
[0008] (1) Preparation of bacteria: Spread Fusarium oxysporum on LB solid medium and culture at 30°C for 7 days. 2 The bacterial moss was inoculated into 200 ml of seed liquid culture medium, and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 10-30, and then the cultured bacterial solution was inoculated into the fermentation medium, and cultured until the OD600 value of the bacterial solution was 200-300, centrifuged, and the collected bacterial bodies were washed and resuspended in a buffer solution, and the obtained bacterial suspension was used for standby;
[0009] (2) taking the bacterial suspension in step (1), adding the glycine nitrile solution, controlling the reaction temperature, the pH of the feed liquid system and the reaction time, and the conversion liquid obtained after the reaction is completed is used for standby;
[0010] (3) taking the conversion solution in step (2), filtering it once and twice, and collecting the filtrate for later use;
[0011] (4) taking the filtrate in step (3), entering it into a cationic resin column, and concentrating the collected effluent to obtain a concentrated solution for later use;
[0012] (5) taking the concentrated solution in step (4), adjusting the temperature and pH, and then entering the concentrated solution into an anion resin column, using an eluent to elute the anion resin column, and collecting the eluate for later use;
[0013] (6) taking the eluate in step (5), concentrating, cooling and crystallizing, filtering and washing to obtain a crude glycine product;
[0014] (7) Take the crude glycine product described in step (6), add purified water and stir to dissolve, then add activated carbon and continue stirring, filter and collect the decolorized liquid, cool and crystallize, filter, wash and dry to obtain the glycine product.
[0015] As an improved technical solution, the components of the LB solid culture medium in step (1) include 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest is deionized water, and the pH value of the LB solid culture medium is 7.0-7.2.
[0016] As an improved technical solution, the components of the seed liquid culture medium in step (1) include 10.0 g / L beef extract, 10.0 g / L peptone, 10.0 g / L yeast extract, 10.0 g / L glucose, 5.0 g / L sodium chloride, 1 g / L toluene, 1 g / L glycine and 1 g / L glycine nitrile, and the rest is water; and the pH of the seed liquid culture medium is 7.0-7.2.
[0017] As an improved technical solution, the concentration of the bacterial suspension in step (2) is 20-50 g / L, the concentration of the glycine nitrile solution is 50-150 g / L, the reaction temperature is controlled at 30-40°C, the pH is 6-8, the reaction time is 5-8h, and the amount of the bacterial suspension added to the reaction system is such that the final concentration is 20-40 OD.
[0018] As an improved technical solution, in step (3), the first filtration is performed using a ceramic membrane with a pore size of 20-50 nm, and the second filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da.
[0019] As an improved technical solution, in step (4), the filtrate enters the cationic resin column at a flow rate of 1-2 BV / h, and the solid content of the concentrated solution is 5-10 wt%.
[0020] As an improved technical solution, in step (5), the temperature of the concentrated solution is adjusted to 30-40°C and the pH is 6-8, and then it enters the anion resin column at a flow rate of 1-2BV / h; the eluent is a phosphate buffer solution with a pH of 4-6 and 0.1-0.5M, and the flow rate of the eluent is 0.5-1.5BV / h.
[0021] As an improved technical solution, the eluent in step (6) is concentrated to a solid content of 50-60 wt%, and then cooled to 10-15°C for crystallization.
[0022] As an improved technical solution, the decolorizing solution in step (7) is cooled to 5-10°C for crystallization.
[0023] After adopting the above technical solution, the beneficial effects of the present invention are:
[0024] In the present invention, the fungus Fusarium oxysporum is spread on LB solid culture medium, and after one week of culture, 1-2 cm 2 The bacterial moss is inoculated into a liquid culture medium for continued cultivation, and then the cultured bacterial liquid is inoculated into a fermentation medium again. After fermentation and centrifugation, the obtained bacterial bodies are resuspended in a buffer solution, and then glycine nitrile is added to control the reaction temperature, pH and reaction time. After the reaction is completed, the liquid is filtered twice, and the collected filtrate enters a cationic resin column. The collected effluent is concentrated, and the obtained concentrated liquid is adjusted in temperature and pH, and then enters an anionic resin column, and eluted with an eluent. The collected eluate is concentrated, cooled and crystallized, filtered, and washed. The obtained glycine crude product is then added to water for stirring and dissolving, and activated carbon is added. The collected decolorized liquid is filtered, and then cooled and crystallized, filtered, washed, and dried to obtain a glycine product. The above preparation and purification process greatly improves the output, yield and purity of glycine. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the liquid phase detection spectrum of glycine in Example 3. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0027] Example 1
[0028] A preparation and purification process of glycine comprises the following steps:
[0029] (1) Preparation of fungi: Fusarium oxysporum was spread on LB solid medium (components included 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest was deionized water, and the pH value of the LB solid medium was 7.0), and cultured at 30° C. for 7 days. 1 cm 2 The bacterial lawn was inoculated into 200 ml of seed liquid culture medium (components included beef extract 10.0 g / L, peptone 10.0 g / L, yeast extract 10.0 g / L, glucose 10.0 g / L, sodium chloride 5.0 g / L, toluene 1 g / L, glycine 1 g / L and glycine nitrile 1 g / L, and the rest was water; and the pH of the seed liquid culture medium was 7.0), and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 10, and then the cultured bacterial solution was inoculated into the fermentation medium (components included: glycerol 10.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, potassium dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium chloride 1.0 g / L, ferrous sulfate 0.02 g / L, caprolactam 1.0 g / L, pH 7.0) at an inoculum amount of 5% v / v. 7.50; the specific operation during the fermentation process is: the initial pH value of the fermentation medium is 7.20, the initial air flow rate is 1.5m 3 / L, maintaining the pressure at 0.05 MPa, the fermentation temperature at 25 ° C, and the rotation speed at 180 rpm), culturing until the OD600 value of the bacterial solution is 200, centrifuging, and the collected bacteria are washed with 0.5 M phosphate buffer and then resuspended in the buffer to obtain a 20 g / L bacterial suspension for use;
[0030] (2) Take 8.0 L of the 20 g / L bacterial suspension in step (1) (the amount of bacterial suspension added is such that the final concentration of the reaction system is 20 OD), add 30.0 L of 50 g / L glycine nitrile solution, control the reaction temperature to 30°C and the pH to 6, react for 5 h to terminate the reaction, and obtain 32.0 L of the conversion solution for use;
[0031] (3) 32.0 L of the conversion solution in step (2) was filtered once (ceramic membrane with a pore size of 20-50 nm) and then filtered twice (ultrafiltration membrane with a molecular weight cutoff of 5000 Da), and 43.5 L of the filtrate was collected for later use;
[0032] (4) 43.5 L of the filtrate from step (3) was taken and passed into a 10 L cationic resin column (filler was macroporous strongly acidic styrene-based cation exchange resin--732) at a flow rate of 1 BV / h. The collected effluent was concentrated (nanofiltration membrane with a molecular weight cutoff of 100-150 Da) to obtain 25.4 L of a concentrated solution with a solid content of 5 wt%;
[0033] (5) Take 25.4 L of the concentrated solution in step (4), adjust the temperature to 30° C. and pH to 6, and then enter a 20 L anion resin column (filler is a macroporous weakly basic styrene anion exchange resin--709) at a flow rate of 1 BV / h. Use 40 L of eluent (0.1 M, pH 4.0 phosphate buffer solution) at a flow rate of 0.5 BV / h to elute the anion resin column, and collect 42.0 L of eluate for use;
[0034] (6) 42.0 L of the eluate in step (5) was concentrated (concentrated at 50° C. and vacuum degree <-0.090 MPa) to obtain 3.2 L of a concentrate having a solid content of 50 wt %, cooled to 10° C. for crystallization, filtered, and washed to obtain 1484 g of crude glycine;
[0035] (7) Take 1484 g of the crude glycine product in step (6), add 1.4 L of purified water and stir to dissolve, then add 29.0 g of activated carbon and continue stirring, filter and collect the decolorized liquid, cool it to 5° C., crystallize it, filter it with suction, wash it, and dry it to obtain the glycine product.
[0036] Example 2
[0037] A preparation and purification process of glycine comprises the following steps:
[0038] (1) Preparation of fungi: Fusarium oxysporum was spread on LB solid medium (components included 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest was deionized water, and the pH value of the LB solid medium was 7.0), and cultured at 30° C. for 7 days. 1 cm 2 The bacterial lawn was inoculated into 200 ml of seed liquid culture medium (components included beef extract 10.0 g / L, peptone 10.0 g / L, yeast extract 10.0 g / L, glucose 10.0 g / L, sodium chloride 5.0 g / L, toluene 1 g / L, glycine 1 g / L and glycine nitrile 1 g, and the rest was water; and the pH of the seed liquid culture medium was 7.0), and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 15, and then the cultured bacterial solution was inoculated into the fermentation medium (components included: glycerol 10.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, potassium dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium chloride 1.0 g / L, ferrous sulfate 0.02 g / L, caprolactam 1.0 g / L, pH 7.0) at an inoculum amount of 5% v / v. 7.50; the specific operation during the fermentation process is: the initial pH value of the fermentation medium is 7.20, the initial air flow rate is 1.5m 3 / L, maintaining the pressure at 0.05 MPa, the fermentation temperature at 28 ° C, and the rotation speed at 200 rpm), culturing until the OD600 value of the bacterial solution is 230, centrifuging, and the collected bacteria are washed with 0.5 M phosphate buffer and then resuspended in the buffer to obtain a 20 g / L bacterial suspension for use;
[0039] (2) Take 12.5 L of the 20 g / L bacterial suspension in step (1) (the amount of bacterial suspension added is such that the final concentration of the reaction system is 30 OD), add 30.0 L of 50 g / L glycine nitrile solution, control the reaction temperature to 32° C., pH to 6.5, react for 6 h to terminate the reaction, and obtain 33.0 L of the conversion solution for use;
[0040] (3) 33.0 L of the conversion solution in step (2) was filtered once (ceramic membrane with a pore size of 20-50 nm) and then filtered twice (ultrafiltration membrane with a molecular weight cutoff of 8000 Da), and 46.0 L of the filtrate was collected for later use;
[0041] (4) 46.0 L of the filtrate in step (3) was taken and passed into a 10 L cationic resin column (filler was macroporous strongly acidic styrene-based cation exchange resin--732) at a flow rate of 1.2 BV / h. The collected effluent was concentrated (nanofiltration membrane with a molecular weight cutoff of 100-150 Da) to obtain 33.0 L of a concentrated solution with a solid content of 5 wt%;
[0042] (5) Take 33.0 L of the concentrated solution in step (4), adjust the temperature to 32° C. and the pH to 6.5, and then enter a 20 L anion resin column (filler is a macroporous weakly basic styrene anion exchange resin--709) at a flow rate of 1.2 BV / h, use 40 L of eluent (0.2 M, pH 4.5 phosphate buffer solution) at a flow rate of 0.8 BV / h to elute the anion resin column, and collect 42.5 L of the eluate for use;
[0043] (6) 42.5 L of the eluate in step (5) was concentrated (concentrated at 55° C. and vacuum degree <-0.090 MPa) to obtain 3.2 L of a concentrate having a solid content of 52 wt %, cooled to 10° C. for crystallization, filtered, and washed to obtain 1492 g of crude glycine;
[0044] (7) Take 1492 g of the crude glycine product in step (6), add 1.5 L of purified water and stir to dissolve, then add 29.8 g of activated carbon and continue stirring, filter and collect the decolorized liquid, cool it to 5°C for crystallization, filter it with suction, wash it, and dry it to obtain the glycine product.
[0045] Example 3
[0046] A preparation and purification process of glycine comprises the following steps:
[0047] (1) Preparation of fungi: Fusarium oxysporum was spread on LB solid medium (components included 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest was deionized water, and the pH value of the LB solid medium was 7.2), and cultured at 30°C for 7 days. 2 The bacterial lawn was inoculated into 200 ml of seed liquid culture medium (components included beef extract 10.0 g / L, peptone 10.0 g / L, yeast extract 10.0 g / L, glucose 10.0 g / L, sodium chloride 5.0 g / L, toluene 1 g / L, glycine 1 g / L and glycine nitrile 1 g, and the rest was water; and the pH of the seed liquid culture medium was 7.2), and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 20, and then the cultured bacterial solution was inoculated into the fermentation medium (components included: glycerol 10.0 g / L, yeast extract 5.0 g / L, peptone 10.0 g / L, potassium dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 0.1 g / L, sodium chloride 1.0 g / L, ferrous sulfate 0.02 g / L, caprolactam 1.0 g / L, pH 7.2) at an inoculum amount of 8% v / v. 7.50; the specific operation during the fermentation process is: the initial pH value of the fermentation medium is 7.20, the initial air flow rate is 1.5m 3 / L, maintain the pressure at 0.05 MPa, the fermentation temperature at 30 ° C, and the rotation speed at 240 rpm), culture until the OD600 value of the bacterial solution is 250, centrifuge, and the collected bacteria are washed with 0.5 M phosphate buffer and then resuspended in the buffer to obtain a 35 g / L bacterial suspension for use;
[0048] (2) Take 7.2 L of the 35 g / L bacterial suspension in step (1) (the amount of bacterial suspension added is such that the final concentration of the reaction system is 30 OD), add 30 L of 100 g / L glycine nitrile solution, control the reaction temperature to 35 ° C, pH to 7, react for 6.5 h to terminate the reaction, and obtain 33.5 L of the conversion solution for use;
[0049] (3) 33.5 L of the conversion solution in step (2) was taken and filtered once (ceramic membrane with a pore size of 20-50 nm) and then filtered twice (ultrafiltration membrane with a molecular weight cutoff of 8000 Da), and 63.5 L of the filtrate was collected for later use;
[0050] (4) 63.5 L of the filtrate from step (3) was taken and passed into a 15 L cationic resin column (filler was macroporous strongly acidic styrene-based cation exchange resin--732) at a flow rate of 1.5 BV / h. The collected effluent was concentrated (nanofiltration membrane with a molecular weight cutoff of 100-150 Da) to obtain 41.0 L of a concentrated solution with a solid content of 8 wt%;
[0051] (5) Take 41.0 L of the concentrated solution in step (4), adjust the temperature to 35° C. and the pH to 7, and then enter a 40 L anion resin column (filler is a macroporous weakly basic styrene anion exchange resin--709) at a flow rate of 1.5 BV / h, use 80 L of eluent (0.3 M, pH 5.0 phosphate buffer solution) at a flow rate of 1 BV / h to elute the anion resin column, and collect 83.5 L of the eluate for use;
[0052] (6) 83.5 L of the eluate in step (5) was concentrated (concentrated at 60° C. and vacuum degree <-0.090 MPa) to obtain 6.09 L of a concentrate having a solid content of 55 wt %, cooled to 12° C. for crystallization, filtered, and washed to obtain 2986 g of crude glycine;
[0053] (7) Take 2986 g of the crude glycine product in step (6), add 2.9 L of purified water and stir to dissolve, then add 60.0 g of activated carbon and continue stirring, filter and collect the decolorized liquid, cool it to 8° C., crystallize it, filter it with suction, wash it, and dry it to obtain the glycine product.
[0054] Example 4
[0055] A preparation and purification process of glycine comprises the following steps:
[0056] (1) Preparation of fungi: Fusarium oxysporum was spread on LB solid medium (components included 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest was deionized water, and the pH value of the LB solid medium was 7.1), and cultured at 30°C for 7 days. 2 The bacterial lawn was inoculated into 200 ml of seed liquid culture medium (components included 10.0 g / L beef extract, 10.0 g / L peptone, 10.0 g / L yeast extract, 10.0 g / L glucose, 5.0 g / L sodium chloride, 1 g / L toluene, 1 g / L glycine and 1 g / L glycine nitrile, and the rest was water; and the pH of the seed liquid culture medium was 7.1), and the culture was cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 25, and then the cultured bacterial solution was inoculated into the fermentation medium (components included: 10.0 g / L glycerol, 5.0 g / L yeast extract, 10.0 g / L peptone, 4.0 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 1.0 g / L sodium chloride, 0.02 g / L ferrous sulfate, 1.0 g / L caprolactam, pH 7.1) at an inoculum amount of 8% v / v. 7.20; the specific operation during the fermentation process is: the initial pH value of the fermentation medium is 7.20, the initial air flow rate is 1.5m 3 / L, maintain the pressure at 0.05 MPa, the fermentation temperature at 32 ° C, and the rotation speed at 275 rpm), culture until the OD600 value of the bacterial solution is 280, centrifuge, and the collected bacteria are washed with 0.5 M phosphate buffer and then resuspended in the buffer to obtain a 35 g / L bacterial suspension for use;
[0057] (2) Take 8.3 L of the 35 g / L bacterial suspension in step (1) (the amount of bacterial suspension added is such that the final concentration of the reaction system is 35 OD), add 30.0 L of 100 g / L glycine nitrile solution, control the reaction temperature to 38°C and the pH to 7.5, react for 7 h to terminate the reaction, and obtain 32.0 L of the conversion solution for use;
[0058] (3) 32.0 L of the conversion solution in step (2) was taken, first filtered (ceramic membrane with a pore size of 20-50 nm), then filtered twice (ultrafiltration membrane with a molecular weight cutoff of 10000 Da), and 61.0 L of the filtrate was collected for later use;
[0059] (4) 61.0 L of the filtrate from step (3) was taken and passed into a 15 L cationic resin column (filler was macroporous strongly acidic styrene-based cation exchange resin--732) at a flow rate of 1.8 BV / h. The collected effluent was concentrated (nanofiltration membrane with a molecular weight cutoff of 100-150 Da) to obtain 40.0 L of a concentrated solution with a solid content of 8 wt%;
[0060] (5) Take 40.0 L of the concentrated solution in step (4), adjust the temperature to 38° C. and the pH to 7.5, and then enter a 40 L anion resin column (filler is a macroporous weakly basic styrene anion exchange resin--709) at a flow rate of 1.8 BV / h, use 80 L of eluent (0.35 M, pH 5.5 phosphate buffer solution) at a flow rate of 1.2 BV / h to elute the anion resin column, and collect 84.0 L of the eluate for use;
[0061] (6) 84.0 L of the eluate in step (5) was concentrated (concentrated at 65° C. and vacuum degree <-0.090 MPa) to obtain 5.3 L of a concentrated solution with a solid content of 60 wt %, cooled to 13° C. for crystallization, filtered, and washed to obtain 2969 g of crude glycine;
[0062] (7) Take 2969 g of the crude glycine product in step (6), add 2.9 L of purified water and stir to dissolve, then add 59.0 g of activated carbon and continue stirring, filter and collect the decolorized liquid, cool it to 8° C., crystallize it, filter it with suction, wash it, and dry it to obtain the glycine product.
[0063] Example 5
[0064] A preparation and purification process of glycine comprises the following steps:
[0065] (1) Preparation of fungi: Fusarium oxysporum was spread on LB solid medium (components included 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest was deionized water, and the pH value of the LB solid medium was 7.2), and cultured at 30° C. for 7 days. 2 cm 2 The bacterial lawn was inoculated into 200 ml of seed liquid culture medium (components included 10.0 g / L beef extract, 10.0 g / L peptone, 10.0 g / L yeast extract, 10.0 g / L glucose, 5.0 g / L sodium chloride, 1 g / L toluene, 1 g / L glycine and 1 g / L glycine nitrile, and the rest was water; and the pH of the seed liquid culture medium was 7.2), and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 30, and then the cultured bacterial solution was inoculated into the fermentation medium (components included: 10.0 g / L glycerol, 5.0 g / L yeast extract, 10.0 g / L peptone, 4.0 g / L potassium dihydrogen phosphate, 0.1 g / L magnesium sulfate heptahydrate, 1.0 g / L sodium chloride, 0.02 g / L ferrous sulfate, 1.0 g / L caprolactam, pH 7.2) at an inoculum amount of 10% v / v. 7.20; the specific operation during the fermentation process is: the initial pH value of the fermentation medium is 7.20, the initial air flow rate is 1.5m 3 / L, maintaining the pressure at 0.05 MPa, the fermentation temperature at 35 ° C, and the rotation speed at 300 rpm), culturing until the OD600 value of the bacterial solution is 300, centrifuging, and the collected bacteria are washed with 0.5 M phosphate buffer and then resuspended in the buffer to obtain a 50 g / L bacterial suspension for use;
[0066] (2) Take 7.5 L of the 50 g / L bacterial suspension in step (1) (the amount of bacterial suspension added is such that the final concentration of the reaction system is 40 OD), add 30.0 L of 150 g / L glycine nitrile solution, control the reaction temperature to 40°C and the pH to 8, and react for 8 h to terminate the reaction. The resulting conversion solution 35.0 L is set aside;
[0067] (3) 35.0 L of the conversion solution in step (2) was taken, first filtered (ceramic membrane with a pore size of 20-50 nm), then filtered twice (ultrafiltration membrane with a molecular weight cutoff of 10000 Da), and 65.0 L of the filtrate was collected for later use;
[0068] (4) 65.0 L of the filtrate from step (3) was taken and passed into a 15 L cationic resin column (filler was macroporous strongly acidic styrene-based cation exchange resin--732) at a flow rate of 2 BV / h. The collected effluent was concentrated (nanofiltration membrane with a molecular weight cutoff of 100-150 Da) to obtain 34.5 L of a concentrated solution with a solid content of 10 wt%;
[0069] (5) Take 34.5 L of the concentrated solution in step (4), adjust the temperature to 40° C. and pH to 8, and then enter a 50 L anion resin column (filler is a macroporous weakly basic styrene anion exchange resin--709) at a flow rate of 2 BV / h, use 100 L of eluent (0.5 M, pH 6.0 phosphate buffer solution) at a flow rate of 1.5 BV / h to elute the anion resin column, and collect 102 L of eluate for later use;
[0070] (6) 102 L of the eluate in step (5) was concentrated (concentrated at 70° C. and vacuum degree <-0.090 MPa) to obtain 8.75 L of a concentrated solution with a solid content of 60 wt %, cooled to 15° C. for crystallization, filtered, and washed to obtain 4408 g of crude glycine;
[0071] (7) Take 4408 g of the crude glycine product in step (6), add 4.4 L of purified water, stir and dissolve, then add 88 g of activated carbon and continue stirring. After filtering, the decolorized liquid collected is cooled to 10° C. for crystallization, filtered, washed and dried to obtain the glycine product.
[0072] In order to better demonstrate that the process of the present invention can obtain a glycine product with high yield, high yield and high purity, 13 comparative examples are given with reference to Example 3, and the details are shown in the following.
[0073] Comparative Example 1
[0074] The difference from Example 3 is that the LB solid culture medium in step (1) does not contain toluene, and the remaining operations are the same.
[0075] Comparative Example 2
[0076] The difference from Example 3 is that the reaction temperature in step (2) is controlled to be 30° C., and the rest of the operations are the same.
[0077] Comparative Example 3
[0078] The difference from Example 3 is that the reaction temperature in step (2) is controlled to 40° C., and the rest of the operations are the same.
[0079] Comparative Example 4
[0080] The difference from Example 3 is that the filler in the cationic resin column in step (4) is a macroporous weakly acidic acrylic acid cation exchange resin---D113, and the rest of the operations are the same.
[0081] Comparative Example 5
[0082] The difference from Example 3 is that the filler in the anion resin column in step (4) is a macroporous strong alkaline styrene-based anion exchange resin---717, and the rest of the operations are the same.
[0083] Comparative Example 6
[0084] The difference from Example 3 is that in step (5), the flow rate of the concentrated solution entering the anion resin column is 2.5 BV / h, and the other operations are the same.
[0085] Comparative Example 7
[0086] The difference from Example 3 is that in step (5), the flow rate of the eluent entering the anion resin column is 2.5 BV / h, and the rest of the operations are the same.
[0087] Comparative Example 8
[0088] The difference from Example 3 is that the temperature of the concentrated solution in step (5) is 25° C., and the remaining operations are the same.
[0089] Comparative Example 9
[0090] The difference from Example 3 is that the temperature of the concentrated solution in step (5) is 45° C., and the remaining operations are the same.
[0091] Comparative Example 10
[0092] The difference from Example 3 is that the pH of the concentrated solution in step (5) is 5, and the remaining operations are the same.
[0093] Comparative Example 11
[0094] The difference from Example 3 is that the pH of the concentrated solution in step (5) is 9, and the remaining operations are the same.
[0095] Comparative Example 12
[0096] The difference from Example 3 is that the pH of the eluent in step (5) is 3, and the rest of the operations are the same.
[0097] Comparative Example 13
[0098] The difference from Example 3 is that the pH of the eluent in step (5) is 7, and the rest of the operations are the same.
[0099] Table 1
[0100]
[0101]
[0102] It can be found from the data in Table 1 that, compared with other embodiments and comparative examples 1-13, the production, yield and purity of glycine in Example 3 are the best.
[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A process for preparing and purifying glycine, characterized in that: The following steps are involved: (1) Preparation of bacteria: Spread Fusarium oxysporum on LB solid medium and culture at 30°C for 7 days. 2 The bacterial moss was inoculated into 200 ml of seed liquid culture medium, and cultured at 30°C and 120 rpm until the OD600 value of the bacterial solution was 10-30, and then the cultured bacterial solution was inoculated into the fermentation medium, and cultured until the OD600 value of the bacterial solution was 200-300, centrifuged, and the collected bacterial bodies were washed and resuspended in a buffer solution, and the obtained bacterial suspension was used for standby; (2) taking the bacterial suspension in step (1), adding the glycine nitrile solution, controlling the reaction temperature, the pH of the feed liquid system and the reaction time, and the conversion liquid obtained after the reaction is completed is used for standby; (3) taking the conversion solution in step (2), filtering it once and twice, and collecting the filtrate for later use; (4) taking the filtrate in step (3), entering it into a cationic resin column, and concentrating the collected effluent to obtain a concentrated solution for later use; (5) taking the concentrated solution in step (4), adjusting the temperature and pH, and then entering the concentrated solution into an anion resin column, using an eluent to elute the anion resin column, and collecting the eluate for later use; (6) taking the eluate in step (5), concentrating, cooling and crystallizing, filtering and washing to obtain a crude glycine product; (7) Take the crude glycine product described in step (6), add purified water and stir to dissolve, then add activated carbon and continue stirring, filter and collect the decolorized liquid, cool and crystallize, filter, wash and dry to obtain the glycine product.
2. A process for preparing and purifying glycine according to claim 1, characterized in that: The components of the LB solid culture medium in step (1) include 5 g / L yeast powder, 10 g / L agar, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L glycine nitrile, 1 g / L toluene and 1 g / L glycine, and the rest is deionized water. The pH value of the LB solid culture medium is 7.0-7.
2.
3. A process for preparing and purifying glycine according to claim 1, characterized in that: The components of the seed liquid culture medium in step (1) include 10.0 g / L beef extract, 10.0 g / L peptone, 10.0 g / L yeast extract, 10.0 g / L glucose, 5.0 g / L sodium chloride, 1 g / L toluene, 1 g / L glycine and 1 g / L glycine nitrile, and the rest is water; and the pH of the seed liquid culture medium is 7.0-7.
2.
4. A process for preparing and purifying glycine according to claim 1, characterized in that: In step (2), the concentration of the bacterial suspension is 20-50 g / L, the concentration of the glycine nitrile solution is 50-150 g / L, the reaction temperature is controlled to be 30-40° C., the pH is 6-8, the reaction time is 5-8 h, and the amount of the bacterial suspension added to the reaction system is such that the final concentration is 20-40 OD.
5. A process for preparing and purifying glycine according to claim 1, characterized in that: In step (3), the first filtration is performed using a ceramic membrane with a pore size of 20-50 nm, and the second filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000-10000 Da.
6. A process for preparing and purifying glycine according to claim 1, characterized in that: In step (4), the filtrate enters the cationic resin column at a flow rate of 1-2 BV / h, and the solid content of the concentrated solution is 5-10 wt%.
7. A process for preparing and purifying glycine according to claim 1, characterized in that: In step (5), the concentrated solution is adjusted to a temperature of 30-40°C and a pH of 6-8, and then enters the anion resin column at a flow rate of 1-2 BV / h; the eluent is a phosphate buffer solution of pH 4-6 and 0.1-0.5 M, and the flow rate of the eluent is 0.5-1.5 BV / h.
8. A process for preparing and purifying glycine according to claim 1, characterized in that: The eluate in step (6) is concentrated to a solid content of 50-60 wt%, and then cooled to 10-15°C for crystallization.
9. A process for preparing and purifying glycine according to claim 1, characterized in that: In step (7), the decolorizing solution is cooled to 5-10° C. for crystallization.