CN107056861B - Process and complete equipment for separating and obtaining ademetionine from fermentation system - Google Patents
Process and complete equipment for separating and obtaining ademetionine from fermentation system Download PDFInfo
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- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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Abstract
The invention discloses a process for separating and obtaining ademetionine from a fermentation system, which adopts a brand new process of regulating pH by two steps and separating by a membrane for three times to realize the separation of the ademetionine. According to the process disclosed by the invention, when the concentration of the SAM in the fermentation liquor is 50-140 g/L, the concentration of the SAM can reach 300g/L, the purity of the SAM is 99.80%, and the highest extraction rate of the SAM can reach 99.80%.
Description
Technical Field
The invention belongs to the field of biological pharmacy, relates to a process and complete equipment for separating and obtaining ademetionine from a fermentation system, and particularly relates to a process for regulating pH and a membrane separation method for separating and obtaining ademetionine from fermentation liquor.
Background
S-adenosylmethionine, also known as S-adenosylmethionine (SAM), is the active form of methionine.
S-adenosylmethionine contains an active methyl group, and is also called active methionine, and is widely present in various tissues and body fluids, and is involved in the transmethylation, polyamine synthesis, and transsulfuration of the body. S-adenosylmethionine is an important methyl donor in vivo, and is involved in methylation of amino acids, proteins, carbohydrates, polysaccharides, phospholipids and nucleic acids in vivo, thereby regulating physiological activities in vivo, such as information transmission, protein processing and modification, etc., and various diseases are caused if the content of S-adenosylmethionine is too low.
At present, SAM is mainly produced by biological fermentation. However, for many years, SAM separation and purification from biological fermentation liquid has been a technical problem which is not well solved. Because SAM and residual L-methionine (Met) have similar physicochemical properties, and the concentration of SAM products in the biological fermentation liquid is low, SAM is difficult to separate and purify by adopting the traditional method.
In the prior art, the Chinese patent ZL 200510019206.X of the invention provides a cation exchange resin chromatography extraction method which has high cost, long separation process and low production efficiency, and SAM is easy to degrade in the long-time separation process. In the Chinese patent ZL 20081002015.4, a trichloroacetic acid solvent precipitation method is adopted to separate SAM. However, trichloroacetic acid has high toxicity, strong irritation, complex subsequent treatment and higher cost. In the invention patent CN 102617681A, SAM is separated and concentrated by adopting a liquid film and an organic solvent. However, the organic solvent n-butyl alcohol, n-hexyl alcohol, n-octyl alcohol or decyl alcohol has great pollution to the environment, and the preparation of the liquid film is more complicated.
Research and development of a new separation and purification technology for high-efficiency and low-cost SAM separation and purification from biological fermentation broth are problems to be solved in SAM production at present.
Disclosure of Invention
In order to overcome the defects of the traditional separation method, the invention provides a novel separation and purification technology, which is used for efficiently separating and purifying SAM from biological fermentation liquor at low cost.
One of the technical purposes of the invention is to provide a process for separating and obtaining ademetionine from a fermentation system, wherein a method of regulating pH value in two steps and separating and concentrating by a membrane for three times is adopted in the process.
In the process, the technical problem to be solved by the first pH value adjustment is to solve the problem of excessive impurities in a fermentation system, and cell debris, foreign proteins and pigments in the feed liquid can be effectively removed by adjusting the pH value to be 1.5-2.0 and filtering through an ultrafiltration membrane.
In the process, the technical problem to be solved by the secondary pH value adjustment is that the SAM and the residual L-methionine (Met) have similar physicochemical properties, membrane separation is difficult to realize in the prior art, and the hydration radius of the chiral SAM in the solution can be effectively changed by adjusting the pH value to be 5.0-6.0, so that the membrane separation of the SAM is realized.
Another technical purpose of the invention is to provide complete equipment for separating and obtaining the ademetionine from a fermentation system, and the complete equipment can realize the high-efficiency separation of the fermentation liquor containing the SAM. Wherein, the complete equipment adopts the process used in the invention, namely a method of adjusting pH value in two steps and separating and concentrating by a membrane for three times.
Specifically, the technical scheme of the invention is as follows:
a process for separating and obtaining ademetionine from a fermentation system comprises the following steps in sequence:
(1) adding acid into the fermentation liquor containing SAM, adjusting the pH value to 1.5-2, and stirring until precipitate is generated;
(2) ultrafiltering the mixed fermentation liquor by a tubular ultrafiltration membrane with a wide flow passage;
(3) adding acid or alkali into the material after ultrafiltration, and adjusting the pH value to 5.0-6.0;
(4) filtering the pH-adjusted ultrafiltration permeate through a primary nanofiltration membrane, washing the materials with pure water and further concentrating;
(5) and the concentrated solution of the first-stage nanofiltration membrane is connected in series and concentrated by the second-stage nanofiltration membrane.
2. The process according to claim 1, wherein the ultrafiltration membrane is made of PVDF (polyvinylidene fluoride), has an inner diameter of 12mm, a working temperature of 5-50 ℃, a working pressure of less than or equal to 0.5MPa and a membrane pore diameter of 0.1 μm; the nanofiltration membrane is made of PVDF, the cut-off molecular weight is 70-150 Da, the working temperature is 5-50 ℃, and the working pressure is 4.1 MPa.
The process provided by the invention is characterized in that the concentration of SAM in the fermentation liquor is 50-140 g/L.
The process of the invention, wherein the acid is one of sulfuric acid and hydrochloric acid; the alkali is one of sodium hydroxide and ammonia water.
The complete set of automatic equipment for separating and obtaining the ademetionine from the fermentation system at least comprises two acid-base tanks, two feeding tanks, three sets of membrane equipment, power equipment, connecting pipelines and valves among the equipment and an automatic control system.
The complete automatic equipment comprises a modularized charging bucket device, a pump, an ultrafiltration membrane, a membrane component, an acid-base tank device, a charging bucket device, a pump, a nanofiltration membrane, a membrane component, a booster pump, a nanofiltration membrane, a membrane component, a connecting pipeline and an automatic control system.
The complete set of automation equipment provided by the invention is characterized in that the charging bucket device (i) at least comprises a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are positioned at the top; and the discharge through hole is positioned at the bottom.
The complete set of automation equipment of the invention is characterized in that the charging bucket device at least comprises a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are positioned at the top; and the discharge through hole is positioned at the bottom.
The complete set of automation equipment is characterized in that a discharge through hole of a charging bucket device is communicated with a pump II, and the pump II is communicated with an ultrafiltration membrane and a membrane component and a feeding hole; the material tank device is communicated with the ultrafiltration membrane and the membrane component and the discharge port end; the discharge port of the charging bucket device is communicated with a pump; the pump is communicated with a nanofiltration membrane and a membrane component feeding hole; the discharge hole of the nanofiltration membrane and the membrane component is communicated with the booster pump, and the discharge hole end of the booster pump is communicated with the feed hole end of the nanofiltration membrane and the membrane component.
The automatic control system comprises an online pH value detection and control system, and the online pH value detection and control system realizes the online pH value control by detecting the pH values of a charging tank I and a charging tank II on line and controlling the opening and closing of an acid-base port valve of the charging tank I and the charging tank II; the pump is provided with flow sensors ⑪ - ⑬, pressure sensors and a controller, the flow sensors and the pressure sensors collect pressure and flow data of an inlet and an outlet in the pump and transmit the pressure and flow data to the controller ⑭, the controller is internally provided with a data receiving module, a processing module and a communication module, the data receiving module receives data information transmitted by the two sensors, transmits the data information to the processing module, and finally transmits the data information to a service terminal through the communication module; the server receives the data message from the controller and displays it on its display ⑯.
In a preferred embodiment of the invention, hydrochloric acid solution is added into ademetionine feed liquid with the concentration of 50-140 g/L, the mixture is mixed, the pH value is adjusted to 1.5-2.0, precipitation is generated, the feed liquid passes through an ultrafiltration membrane with the membrane material PVDF, the inner diameter is 12mm, the working temperature is 5-50 ℃, the working pressure is less than or equal to 0.5MPa, the membrane pore diameter is 0.1 mu m, and the pH value of filtrate is adjusted to 5.0-6.0 by hydrochloric acid; performing nanofiltration through a primary nanofiltration membrane which is made of PVDF (polyvinylidene fluoride), has the molecular weight cutoff of 70-150 Da, the working temperature of 5-50 ℃ and the working pressure of 4.1 MPa until the SAM concentration is 200g/L and the purity is 99%; and (3) carrying out secondary nanofiltration on the filtrate, wherein the concentration of SAM is 300g/L, and the purity is 99.8%.
In another embodiment of the invention, ademetionine feed liquid with the concentration of 50-140 g/L automatically flows into a charging bucket through an automatic valve, then the pH value of the feed liquid is automatically adjusted to 1.5-2.0 by acid of an acid tank through an automatic system, finally impurities in the feed liquid are removed by pumping an ultrafiltration membrane through a pump, meanwhile, pure water is automatically supplemented through the automatic valve to clean the material and then the material enters a ultrafiltration membrane, the permeate of the ultrafiltration membrane automatically flows into the charging bucket, then the pH value of the feed liquid is automatically adjusted to 5.0-6.0 by acid and alkali of the acid tank and a fifth through the automatic system, then the nanofiltration membrane is pumped into the acid tank through a pump to clean and concentrate the material for the first stage, the concentration of the SAM for the first stage concentration can reach 200g/L and 99%, and the effluent of the nanofiltration membrane is pumped into a second stage specially-made nanofiltration membrane through a booster pump to concentrate the SAM concentration of 300g/L, the purity was 99.80%.
The invention has the beneficial effects that
(1) The process belongs to a physical process, avoids the pollution of chemical reagents to products, has simple and reliable operation and strong usability, greatly reduces the production cost and is easy to realize industrial amplification.
(2) All the operations of the invention are integrated and modularized, and are centrally controlled by a computer, so that the product quality is stable, and the labor cost is saved. The pump is provided with a flow sensor, a pressure sensor and a controller, the flow sensor and the pressure sensor acquire pressure and flow data of an inlet and an outlet in the pump and transmit the pressure and flow data to the controller, the controller is internally provided with a data receiving module, a processing module and a communication module, the data receiving module receives data information transmitted by the two sensors and transmits the data information to the processing module, and finally the data information is transmitted to the service terminal through the communication module; the service terminal receives the data information sent by the controller and displays the data information on a display screen of the service terminal.
(3) Compared with the conventional roll-type or narrow-flow-channel ultrafiltration membrane, the membrane equipment adopted by the invention has the characteristics of strong pollution resistance and easy cleaning.
Drawings
FIG. 1: complete equipment for separating and obtaining ademetionine from fermentation system
Wherein, the first step is a charging bucket device; ② a pump; ③ the ultrafiltration membrane and the membrane component; fourthly, an acid-base tank device; fifthly, an acid-base tank device; sixthly, the device is a charging bucket; seventhly, a pump; eighthly, the nanofiltration membrane and the membrane component are used; ninthly, a booster pump; the red is nanofiltration membrane and membrane component; PG is a pressure gauge; AIR is a pneumatic valve; ⑪ - ⑬ are flow meters, ⑭ is a control box, ⑮ is a control host, and ⑯ is a display screen.
FIG. 2 chemical structure of S-adenosylmethionine.
Detailed Description
EXAMPLE 1 this example illustrates the composition and connection of a complete plant for isolating ademetionine from a fermentation System
A schematic of the plant is shown in figure 1. The equipment includes modular material tank, pump, ultrafiltering membrane, membrane assembly, acid and alkali tank, material tank, pump, nanofiltration membrane, membrane assembly, booster pump, nanofiltration membrane, membrane assembly and automatic control system.
The charging bucket device comprises a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are positioned at the top; and the discharge through hole is positioned at the bottom. The charging bucket device comprises a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are positioned at the top; and the discharge through hole is positioned at the bottom.
The material tank device is characterized in that a discharge through hole is communicated with a pump II, and the pump II is communicated with an ultrafiltration membrane and a membrane component and a feeding hole; the material tank device is communicated with the ultrafiltration membrane and the membrane component and the discharge port end; the discharge port of the charging bucket device is communicated with a pump; the pump is communicated with a nanofiltration membrane and a membrane component feeding hole; the discharge hole of the nanofiltration membrane and the membrane component is communicated with the booster pump, and the discharge hole end of the booster pump is communicated with the feed hole end of the nanofiltration membrane and the membrane component.
The automatic control device comprises an online pH value detection and control system, and realizes online pH value control by online detecting the pH values of a charging tank I and a charging tank II and controlling the opening and closing of valves of acid and alkali ports of the charging tank I and the charging tank II; the pump is provided with flow sensors ⑪ - ⑬, pressure sensors and a controller, the flow sensors and the pressure sensors collect pressure and flow data of an inlet and an outlet in the pump and transmit the pressure and flow data to the controller ⑭, the controller is internally provided with a data receiving module, a processing module and a communication module, the data receiving module receives data information transmitted by the two sensors, transmits the data information to the processing module, and finally transmits the data information to a service terminal through the communication module; the server receives the data message from the controller and displays it on its display ⑯.
EXAMPLE 2 this example illustrates the isolation of ademetionine from a fermentation System
Adding a hydrochloric acid solution into an ademetionine feed liquid with the concentration of 50-140 g/L, mixing, adjusting the pH value to 1.5-2.0, generating a precipitate, passing the feed liquid through an ultrafiltration membrane made of PVDF (polyvinylidene fluoride) and having the inner diameter of 12mm, the working temperature of 5-50 ℃, the working pressure of less than or equal to 0.5MPa and the membrane pore diameter of 0.1 mu m, and adjusting the pH value of the filtrate to 5.0-6.0 by using hydrochloric acid; performing nanofiltration through a primary nanofiltration membrane which is made of PVDF (polyvinylidene fluoride), has the molecular weight cutoff of 70-150 Da, the working temperature of 5-50 ℃ and the working pressure of 4.1 MPa until the SAM concentration is 200g/L and the purity is 99%; and (3) carrying out secondary nanofiltration on the filtrate, wherein the concentration of SAM is 300g/L, and the purity is 99.8%.
Example 3 this example illustrates the practice of the process of the invention in the apparatus of the example
Take 20m3The SAM fermentation treatment liquid has the concentration of 80g/L of adenosylmethionine, automatically flows into a charging bucket ① through an automatic valve, automatically adjusts the pH value of the liquid to 1.5-2.0 by using acid of an acid tank ④ through an automatic system, finally is pumped into a special ultrafiltration membrane ③ through a pump ② to remove cell fragments, foreign proteins and pigments in the liquid, and is automatically supplemented with pure water through the automatic valve to clean the material, and the permeate of the special ultrafiltration membrane ③ is automatically purifiedThe obtained product flows into a charging tank ⑥, the pH value of the feed liquid is automatically adjusted to 5.0-6.0 by acid through the acid and alkali of acid tanks ④ and ⑤ by an automatic system, then the obtained product is pumped into a special nanofiltration membrane ⑧ by a pump ⑦, the effluent of the nanofiltration membrane ⑧ is pumped into a second-stage special nanofiltration membrane ⑩ by a booster pump ⑨, and the obtained product has SAM concentration of 280g/L, purity of 99.50% and yield of 89%.
Example 4 this example illustrates the practice of the process of the invention in the apparatus of the example
Take 20m3The SAM fermentation treatment liquid has the concentration of 120g/L of adenosylmethionine, automatically flows into a material tank ① through an automatic valve, automatically adjusts the pH value of the material liquid to 1.5-2.0 by using acid of an acid tank ④ through an automatic system, finally is pumped into a special ultrafiltration membrane ③ through a pump ② to remove cell fragments, foreign proteins and pigments in the material liquid, and is automatically supplemented with pure water through the automatic valve to clean the material, the permeation liquid of the special ultrafiltration membrane ③ automatically flows into the material tank ⑥, then the pH value of the material liquid is automatically adjusted to 5.0-6.0 by using acid of acid and alkali of the acid tanks ④ and ⑤ through the automatic system, and is pumped into a special effluent liquid ④ 0 through a pump ⑦, and the effluent liquid of a nanofiltration membrane ⑧ is pumped into a second-stage special nanofiltration membrane ④ 2 through a booster pump ④ 1, so that the concentration of the SAM in the material is 298g/L, the purity of 99.60%, and the yield of 89..
Example 5 this example illustrates the practice of the process of the invention in the apparatus of the example
Take 20m3The SAM fermentation treatment liquid has the concentration of 140g/L of adenosylmethionine, automatically flows into a material tank ① through an automatic valve, automatically adjusts the pH value of the material liquid to 1.5-2.0 by using acid of an acid tank ④ through an automatic system, finally is pumped into a special ultrafiltration membrane ③ through a pump ② to remove cell fragments, foreign proteins and pigments in the material liquid, is automatically supplemented with pure water through the automatic valve to clean the material, automatically flows into the material tank ⑥ through permeate liquid of the special ultrafiltration membrane ③, automatically adjusts the pH value of the material liquid to 5.0-6.0 by using acid of acid and alkali of the acid tanks ④ and ⑤ through the automatic system, then is pumped into a special nanofiltration membrane ④ 0 through a pump ⑦, is pumped into a nanofiltration membrane ⑧ through a booster pump, and is pumped into a second-stage special nanofiltration membrane ④ 2 through a booster pump ④ 1 to obtain the SAM fermentation treatment liquid with the concentration of 300g/L, the purity of SAM 99.60The content was 90%.
Claims (6)
1. A process for separating and obtaining ademetionine from a fermentation system is characterized by comprising the following steps in sequence:
(1) adding acid into the fermentation liquor containing SAM, adjusting the pH value to 1.5-2, and stirring until precipitate is generated;
(2) ultrafiltering the mixed fermentation liquor by a tubular ultrafiltration membrane with a wide flow passage;
(3) adding acid or alkali into the material after ultrafiltration, and adjusting the pH value to 5.0-6.0;
(4) filtering the pH-adjusted ultrafiltration permeate through a primary nanofiltration membrane, washing the materials with pure water and further concentrating;
(5) the concentrated solution of the first-stage nanofiltration membrane is connected in series and concentrated by the second-stage nanofiltration membrane;
the ultrafiltration membrane is made of PVDF (polyvinylidene fluoride), the inner diameter of the ultrafiltration membrane is 12mm, the working temperature is 5-50 ℃, the working pressure is less than or equal to 0.5MPa, and the membrane aperture is 0.1 mu m; the nanofiltration membrane is made of PVDF (polyvinylidene fluoride), the cut-off molecular weight is 70-150 Da, the working temperature is 5-50 ℃, and the working pressure is 4.1 MPa;
the concentration of SAM in the fermentation liquor is 50-140 g/L;
the acid is one of sulfuric acid and hydrochloric acid; the alkali is one of sodium hydroxide and ammonia water;
the process adopts the following complete automatic equipment: at least comprises two acid-base tanks, two feeding tanks, three groups of membrane equipment, power equipment, connecting pipelines and valves among the equipment and an automatic control system.
2. The process as claimed in claim 1, wherein the equipment comprises a first modular material tank device (r), a first pump (r), an ultrafiltration membrane and membrane module (r), a first acid and alkali tank device (r), a second material tank device (c), a second pump (c), a first nanofiltration membrane and membrane module (r), a booster pump (c), a second nanofiltration membrane and membrane module (r), connecting pipelines and an automatic control system.
3. The process as claimed in claim 2, wherein the first material tank device (r) comprises at least a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are arranged at the top; and the discharge through hole is positioned at the bottom.
4. The process as claimed in claim 2, wherein the second charging bucket device comprises at least a feeding through hole, a water adding through hole, an acid adding through hole and an alkali adding through hole which are arranged at the top; and the discharge through hole is positioned at the bottom.
5. The process as claimed in claim 2, wherein the discharge through hole of the first material tank device (I) is communicated with a first-stage pump (II), and the first-stage pump (II) is communicated with the feed inlets of the ultrafiltration membrane and the membrane module (III); the material tank device II is communicated with the discharge port end of the ultrafiltration membrane and the membrane component I; the discharge hole of the charging bucket device II is communicated with a secondary pump; the secondary pump (c) is communicated with the feed inlet of the primary nanofiltration membrane and the membrane component (viii); the discharge port of the first nanofiltration membrane and membrane component (r) is communicated with a booster pump (ninu), and the discharge port of the booster pump is communicated with the feed port of the second nanofiltration membrane and membrane component (r).
6. The process as claimed in claim 2, wherein the automatic control system comprises an online pH value detection and control system, and the online pH value control is realized by online detecting the pH values of the first charging bucket device (I) and the second charging bucket device (II) and controlling the opening and closing of the acid-base port valve of the first charging bucket device (I) and the second charging bucket device (II); the pump is also provided with three flow sensors (⑪ - ⑬), a pressure sensor and a controller, wherein the flow sensors and the pressure sensor acquire pressure and flow data of an inlet and an outlet in the pump and transmit the pressure and flow data to the controller (⑭), the controller is internally provided with a data receiving module, a processing module and a communication module, the data receiving module receives data information transmitted by the two sensors and transmits the data information to the processing module, and finally the data information is transmitted to a service terminal through the communication module; the service terminal receives the data information sent by the controller and displays the data information on a display screen (⑯).
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| CN107722040A (en) * | 2017-10-10 | 2018-02-23 | 南京志坤环保科技有限公司 | A kind of membrane separating method and device for recycling ceftazidime mother liquor |
| CN110396120B (en) * | 2019-02-13 | 2020-05-12 | 山东惠仕莱生物科技有限公司 | Method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation liquor |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1935825A (en) * | 2006-10-13 | 2007-03-28 | 浙江大学 | Method for preparing S-adenosine-L-methionine sulfate |
| CN102747123A (en) * | 2012-07-31 | 2012-10-24 | 无锡福祈制药有限公司 | Process for preparing ademetionine butanedisulfonate |
| CN104178540A (en) * | 2014-07-23 | 2014-12-03 | 浙江震元制药有限公司 | Method for synthesizing ademetionine by biological catalytic process |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1935825A (en) * | 2006-10-13 | 2007-03-28 | 浙江大学 | Method for preparing S-adenosine-L-methionine sulfate |
| CN102747123A (en) * | 2012-07-31 | 2012-10-24 | 无锡福祈制药有限公司 | Process for preparing ademetionine butanedisulfonate |
| CN104178540A (en) * | 2014-07-23 | 2014-12-03 | 浙江震元制药有限公司 | Method for synthesizing ademetionine by biological catalytic process |
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