CN113862117B - Device and method for separating chiral substances by multi-liquid-phase system continuous enzyme method - Google Patents

Device and method for separating chiral substances by multi-liquid-phase system continuous enzyme method Download PDF

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CN113862117B
CN113862117B CN202111247760.9A CN202111247760A CN113862117B CN 113862117 B CN113862117 B CN 113862117B CN 202111247760 A CN202111247760 A CN 202111247760A CN 113862117 B CN113862117 B CN 113862117B
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CN113862117A (en
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李志刚
杨嘉威
苏金芬
杨博
王永华
陈华勇
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South China University of Technology SCUT
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Abstract

The invention belongs to the field of bioengineering, and discloses a device and a method for separating chiral substances by a multi-liquid-phase system continuous enzyme method, wherein the device comprises a reaction device and an evaporator; the reaction device is divided into a standing area, a reaction area and a dispersion buffer area from top to bottom, wherein the reaction area and the standing area and the reaction area and the dispersion buffer area are separated by a baffle plate, and the baffle plate is provided with a plurality of small holes communicated with the areas; the standing area is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with an upper phase solution outlet which is communicated with the evaporator, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower phase solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution inlet and outlet, and the inside of the reaction zone is provided with a stirrer; the bottom of the dispersion buffer zone is provided with an ester or amide compound sample inlet and a lower phase solution outlet. The method has the advantages of high raw material recycling rate, mild and controllable reaction conditions, continuous resolution, easy separation and recovery of products and the like.

Description

Device and method for separating chiral substances by multi-liquid-phase system continuous enzyme method
Technical Field
The invention belongs to the technical field of bioengineering and food, relates to separation of chiral substances and application of enzymes, and in particular relates to a device and a method for separating chiral substances by a multi-liquid-phase system continuous enzyme method.
Background
Chirality (Chirality) is one of the essential attributes of nature. Biological macromolecules, such as proteins, polysaccharides, nucleic acids, enzymes, etc., which are important bases for vital activities are almost entirely chiral. The "reaction arrest" drug occurring in 1960 resulted in the birth of a malformed fetus after the pregnant woman took it, because of the strong teratogenic effect of the S-configuration enantiomer of this drug. Therefore, the preparation of single chiral medicine with higher medicine effect and smaller side effect is a necessary trend to replace the traditional mixed raceme medicine.
At present, chiral substance resolution methods with wider application range mainly comprise a chemical resolution method, an induced crystallization method, a chromatographic method, an enzyme resolution method and the like. The chemical separation method has the advantages of simple operation and good stability, but also has the defects of serious pollution of chemical reagents, high price of chiral reagents, low recovery rate of products and the like. The induced crystallization method has the advantages of low cost, simple and convenient operation and high product purity, but the seed crystal is required to be continuously supplemented in the reaction and racemization reaction is required to be continuously carried out in the solution, so that the operation complexity and the production cost are increased. The chromatographic method directly uses the chiral chromatographic column for resolution, has the advantages of high efficiency, rapidness, simple operation, high purity and the like, but has small preparation amount, cannot meet the requirements of industrial production, and has the advantages that the chromatographic column is relatively expensive, a large amount of organic solvent is consumed, and the production cost is increased. Therefore, in recent years, an enzymatic resolution method having advantages of mild conditions, high safety, high product purity, low energy consumption, and the like has been attracting attention. However, in the preparation of most chiral products, they are at a relatively long distance from industrial applications. On the one hand, although the physicochemical properties of chiral products and enantiomer byproducts are different, in the traditional enzyme catalysis systems such as micro-water phase reaction systems, organic solvent-water reaction systems and the like, the products and the enzymes are difficult to separate synchronously, additional separation steps such as chromatography and the like are needed to separate, and the addition of the additional necessary steps greatly increases the difficulty of continuous production. On the other hand, in conventional reaction systems such as micro-aqueous phase and organic solvent-water reaction systems, there are a series of problems that the system is seriously emulsified and/or the product distribution condition is complicated, so that the product is difficult to recover, and the enzyme is difficult to recycle. In addition, the key factors affecting chiral resolution, namely the product concentration and the pH value of the system, are changed continuously along with the progress of the reaction, the forward reaction is greatly slowed down by the product inhibition effect when the product concentration is increased continuously, and the enzyme is deactivated due to the change of the pH value of the system, so that the reaction cannot be continued. These reasons have led to the fact that most of the research is currently in the laboratory research stage and has not been used for continuous industrial production.
Currently, few examples of enantioselective organic catalytic reactions are carried out by using a continuous flow process in enzymatic chiral resolution, most of industrial applications are batch type resolution production, large-sized ground tanks or tanks are adopted, racemized substrates are prepared into a solution with a certain concentration, a certain amount of resolving enzymes are added, stirring production is carried out under proper reaction conditions, and after a mixed solution is obtained, the mixed solution is taken out and then a new substrate is re-injected. Intermittent resolution production has the defects of low resolution efficiency, high cost, high raw material energy consumption, complicated subsequent separation and purification steps, low recovery rate of enzyme and products, difficulty in controlling product inhibition, system pH value and the like. For example, alja Westerbeek et al studied the use of a two-step single enzyme tandem process in the preparation of chiral halohydrins by halodehalogenase catalyzed asymmetric and tandem kinetic resolution, but yields were only 24% and 52%, resulting in relatively large costs of production and more energy consumption (Tetrahedron, 2012,68 (37): 7645-7650.). Ayelet Fishman developed an efficient two-step enzymatic process for the production of (R) and (S) -ethyl-3-hydroxybutyrate, on the order of thousands of kilograms, but required fractionation downstream of the reaction to separate the product, which is prone to side reactions and significant product losses (Biotechnology and bioengineering,2001,74 (3): 256-263.).
The subject group developed a novel multi-liquid phase system in the previous study, under which not only the resolution efficiency of the enzyme can be promoted, but also the enzyme-rich phase can be efficiently recycled (CN 201811500754.8), but the process is extremely easy to be interfered by a pumping device and the like to generate an emulsification phenomenon, thereby influencing the separation of the enzyme-rich phase, and the inhibition effect of the product and the pH value of the system are difficult to control, so that the process is difficult to continuously operate all the time. Therefore, it is highly desirable to develop an apparatus and method for resolution of chiral materials that is efficient, controllable, continuously catalyzed and separable.
Disclosure of Invention
The invention aims to solve the problems of high cost, complex reaction operation, difficult continuous production, difficult control of reaction conditions, product inhibition and the like in the existing industrial process for separating chiral substances by an enzyme method, and provides a device and a method for separating chiral substances by a multi-liquid-phase system continuous enzyme method.
The aim of the invention is achieved by the following technical scheme:
a device for separating chiral substances by a multi-liquid-phase system continuous enzyme method comprises a reaction device and an evaporator; the reaction device is divided into a standing area, a reaction area and a dispersion buffer area from top to bottom, wherein the reaction area and the standing area and the reaction area and the dispersion buffer area are separated by a baffle plate, and the baffle plate is provided with a plurality of small holes communicated with the areas; the standing area is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with an upper phase solution outlet which is communicated with the evaporator, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower phase solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution inlet and outlet, and the inside of the reaction zone is provided with a stirrer; the bottom of the dispersion buffer zone is provided with an ester or amide compound sample inlet and a lower phase solution outlet.
Preferably, a circulating water heating sleeve is arranged outside the reaction zone.
Preferably, an automatic potentiometric titrator buret is arranged in the lower liquid phase zone of the standing zone, and an automatic potentiometric titrator electrode tip is arranged in the reaction zone.
Preferably, the lower phase solution outlet communicates with the second evaporator via an ultrafilter.
A method for separating chiral substances by utilizing the device continuous enzyme method comprises the following steps:
(1) Preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing for layering to obtain an upper-phase solution and a lower-phase solution, continuously introducing the lower-phase solution into a standing area, passing through a reaction area, and entering and filling a dispersion buffer area; dissolving enzyme in the upper phase solution, and introducing the solution into a reaction zone; introducing an ester or amide compound formed by racemization or single enantiomer chiral matters containing a hydrophobic solvent into a dispersion buffer area, adjusting the flow rate of feed liquid to enable the ester or amide compound to enter a reaction area, and carrying out enzyme catalytic splitting reaction; meanwhile, the pH value of the lower phase is changed by using an automatic potentiometric titrator so as to adjust the pH value of the reaction system. The dispersion buffer zone can avoid the direct mixing of esters or amides composed of racemized or single enantiomer chiral compounds containing hydrophobic solvent with enzyme-containing solution to prevent emulsification.
(2) The product after the catalytic reaction is automatically extracted to a standing area, the standing area plays a role in standing and layering, and after standing and layering, the upper phase is communicated with a first evaporator to evaporate and remove the hydrophobic solvent, so as to obtain an ester or amide product of the single optically active chiral product; wherein the other single optically active chiral product is predominantly enriched in the middle or lower liquid layer.
Preferably, the enzyme-containing medium phase extracted in the step (2) is returned to the reaction zone through a return pipe to continue the reaction. When another single optically active chiral product is present in the mesophase, the reaction is inhibited by accumulating more product in the mesophase over a period of time. Thus, the intermediate phase extracted into the rest area can be periodically discharged to release the inhibition; and obtaining another single optically active chiral product through simple operations such as filtration, rotary evaporation and the like.
Preferably, step (2) when the lower phase is in the presence of another single optically active chiral product, the lower phase is passed through a second evaporator to evaporate water after ultrafiltration of the enzyme to give the other single optically active chiral product.
Preferably, the feeding and the reaction process in the step (1) are carried out under the conditions of continuous stirring and heating, wherein the heating temperature is 30-70 ℃, and the stirring rotating speed is 100-1000rpm; regulating the pH value of the reaction system to 3-13; the mass ratio of the soluble salt, the hydrophilic solvent and the water is 0.1-1 and 0.1-5 respectively.
Preferably, the soluble salt is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate, potassium phosphate and potassium dihydrogen phosphate.
Preferably, the hydrophilic solvent is a polymer and/or an ionic liquid, wherein the polymer comprises one or two of polyethylene glycol and polypropylene glycol; the ionic liquid comprises [ BMIM ]]Br、[BMIM]BF 4 、[EMIM]ETSO 4 、[OMIM]Cl、[BMIM]PF 6 One or two or more of them.
Preferably, the enzyme comprises one or more than two of Lipase AYS, lipase AY30, lipase MAS1H108A, lipase G Amano 50, lipase CALB and Novozyme 435, and the enzyme concentration is 5-2000U/mL.
Preferably, the ester or amide compound formed by the racemization or single enantiomer chiral substance accounts for 0.1% -10%, preferably 1% -5% of the mass of the hydrophobic solvent.
Preferably, the hydrophobic solvent is one or more of n-hexane, isopropyl ether, ethyl acetate, isooctane, petroleum ether, diethyl ether, benzene and toluene.
Preferably, the ester or amide compound formed by the racemized or single enantiomer chiral substance comprises one or more than two of racemic methyl mandelate, racemic ibuprofen methyl ester, racemic naproxen methyl ester, racemic 1- (4-methoxyphenyl) ethanol acetate, R-methyl mandelate and S-methyl mandelate.
Preferably, the device for separating chiral substances by using the multi-liquid-phase system continuous enzyme method further comprises the following technical characteristics:
a. the device is made of one or more than two of glass materials, metal materials, organic polymer materials and ceramic materials;
b. the connecting pipeline is one or more than two of a silicone tube, a fluororubber tube and a stainless steel tube;
c. the stirrer is one of paddle type, tooth type, bent blade opening turbine type, anchor type, frame type, spiral belt type, screw type, cloth Lu Majin type, bent blade opening turbine type, bent blade disc turbine type, propelling type and straight blade disc turbine type stirrers;
d. the pump is one or more than two of a volumetric pump, a vane pump, a fluid power pump and an electromagnetic pump;
e. the evaporator is one of a central circulation pipe evaporator, a suspension basket evaporator, an external heating type evaporator, a column evaporator, a forced circulation evaporator, a climbing film evaporator, a falling film evaporator, a lifting-falling film evaporator, a scraper film evaporator and a direct contact heat transfer evaporator;
f. the ultrafilter is one of plate ultrafiltration, hollow fiber ultrafiltration, capillary ultrafiltration and tubular ultrafiltration.
Preferably, the device for separating chiral substances by using the multi-liquid-phase system continuous enzyme method further comprises the following technical characteristics:
a. the baffle between the reaction zone and the standing zone and the baffle between the reaction zone and the dispersion buffer zone are made of polytetrafluoroethylene materials, the diameters of the baffle and the baffle are consistent, and a plurality of small holes are formed for blocking the reaction liquid and extracting and separating the products. Rubber rings with the same size as the inner diameter of the upper, middle and lower part pipes are arranged on both sides of the baffle plate so as to achieve the sealing effect;
b. the middle phase in the reaction zone is extracted into the standing zone in a small part, and the first reflux valve and the second reflux valve are used for refluxing the middle phase in the standing zone into the reaction zone for continuous reaction;
c. the byproduct generated in the resolution reaction process is enriched in a lower phase rich in soluble salt, and a lower phase solution outlet is used for replacing a lower phase solution to relieve the inhibition effect;
d. the activity of the enzyme is gradually reduced after long-time reaction, so that the enzyme-containing solution inlet and outlet are used for entering a neutral phase and replacing the neutral phase in order to maintain the reaction efficiency; the return pipe is a detachable hose, and the medium phase can be discharged through the return pipe;
e. the automatic potentiometric titrator buret is used for adjusting the pH value of a multi-liquid-phase system together with an electrode head of the automatic potentiometric titrator by dripping acid or alkali into the downward-phase solution;
f. the circulating water heating sleeve is used for controlling the temperature and heating and/or refrigerating the reaction device. Circulating water is pumped in from a water inlet, and flows back to the circulating water heating pump from a water outlet after being filled with the circulating water heating sleeve;
g. the stirrer is used for stirring, so that reactants are more fully and uniformly mixed, and the reaction rate is quickened.
Preferably, in the device for separating chiral substances by using the multi-liquid-phase system continuous enzyme method, the middle phase inlet and the middle phase flowing out of the replacement port and the lower phase flowing out of the lower phase replacement port may each contain a single optical rotation chiral product after the separation reaction, and the middle phase or the lower phase may be extracted by adopting modes of simple centrifugation, standing, direct filtration, ultra-filtration of enzyme by an ultra-filter, rotary evaporation, extraction and the like.
Recent researches show that the polymer and ionic multi-liquid phase system is convenient for separating products and adjusting the pH value of a lower phase, and the products can be separated only by standing or low-speed centrifugation, thus being particularly suitable for developing a set of continuous device based on the system. The reaction zone of the device ensures that three phases are fully mixed by regulating and controlling the stirring speed, thereby accelerating the resolution rate, and the pH value of a reaction system can be monitored in real time through an electrode head of an automatic potentiometric titrator; the dispersion buffer zone prevents the upper phase and the middle phase (containing enzyme solution) from being directly contacted with each other to generate emulsification phenomenon, which is unfavorable for enzyme catalytic resolution reaction and subsequent separation. The reaction zone can further extract, separate and take away the by-products in the lower phase after resolution by a countercurrent extraction mode; the rest area changes the pH value of the lower phase through an automatic potentiometric titrator burette so as to adjust the pH value of the system, and the upper phase product and the enzyme can be separated through rest layering. Finally, the device can recover the resolved product after simple operations such as filtration, rotary evaporation and the like, has the advantages of simplified industrial flow, high catalytic efficiency, energy conservation and consumption reduction, repeatable enzyme recycling, product inhibition removal, real-time system pH value regulation and control and the like, and can be applied to continuous industrial production of chiral substances.
Compared with the prior art, the invention has the beneficial effects that:
the invention solves the problems of high cost, complex reaction operation, difficult continuous production, low reaction efficiency, difficult recovery of products and the like in the existing continuous chiral material splitting process, and provides a device and a method for splitting chiral materials by a multi-liquid-phase system continuous enzyme method, which have the following specific advantages:
(1) The ester or amide substrate of one single optical rotation chiral product can be distributed in a hydrophobic phase by utilizing a polymer and/or ionic liquid type multi-liquid phase system continuous enzyme method to split chiral substances, the hydrolyzed other single optical rotation chiral product is mainly enriched in the other phase, and the product can be recycled by simple centrifugation, standing, filtration, ultrafiltration or rotary evaporation and other modes; meanwhile, by establishing a dispersion buffer zone, the phenomenon of direct emulsification caused by direct contact of an upper phase and a middle phase is avoided, and the split reaction and the separation are facilitated;
(2) The reaction system is further extracted and separated in a countercurrent extraction mode, byproducts of the reaction system are concentrated in the lower phase of the system, and the inhibition effect of reaction negative products can be relieved after the lower phase is replaced; meanwhile, the pH value of the reaction system is stabilized by regulating and controlling the pH value of the lower phase on line by an automatic potentiometric titrator, so that the reaction can be continuously and stably carried out for a long time;
(3) The stirring speed of the reaction zone of the device is intelligently regulated, so that the problems that the system is unfavorable for extraction layering due to too high stirring speed or incomplete mixing due to too low stirring speed are avoided, three phases are fully mixed, the resolution rate is accelerated, and the pH value of the reaction system can be monitored in real time;
(4) Under this system, the enzyme is mainly concentrated in the mesophase. The enzyme can be recycled through extraction layering, medium phase reflux and other modes in the standing area, so that the enzyme loss and the cost are reduced;
(5) The continuous device has the advantages of less material consumption, low cost, energy conservation and environmental protection when being heated by using circulating water; the method has the advantages of low energy consumption, high raw material utilization rate, mild and controllable reaction conditions, continuous reaction and the like, and solves the technical problem that the existing method is difficult to split and produce chiral substances by a long-time continuous enzyme method.
Drawings
FIG. 1 is a schematic structural diagram of a device for separating chiral substances by a multi-liquid-phase system continuous enzyme method.
In the figure, 1, a reaction zone, 2, a dispersion buffer zone, 3, a standing zone, 4, a stirrer, 5, an upper phase product outlet, 6, a first reflux valve, 7, an automatic potentiometric titrator tube, 8, a circulating water outlet, 9, a second reflux valve, 10, a first baffle plate, 11, a lower phase solution inlet, 12, a second baffle plate, 13, an automatic potentiometric titrator electrode tip, 14, a circulating water heating sleeve, 15, a circulating water inlet, 16, an enzyme-containing solution inlet and outlet, 17, an ester or amide compound inlet, 18, a lower phase solution outlet, 19, an ultrafilter, 20, a first evaporator, 21, a second evaporator, 22, a circulating water adding heat pump, 23, an upper phase product delivery pump, 24, a first lower phase delivery pump, 25, an enzyme-containing solution delivery pump, 26, an ester or amide compound delivery pump, 27, a second lower phase delivery pump, 28, an ultrafiltration delivery pump, 29, an ultrafiltered product delivery pump, 30, a first lower phase storage tank, 31, an enzyme-containing solution storage tank 32, an amide compound storage tank, and a second storage tank 33.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples, but embodiments of the present invention are not limited thereto, and may be performed with reference to conventional techniques for process parameters that are not specifically noted.
Lipase AY30 (Candida rugosa lipases) used in this example was purchased from Japanese Kogyo Co., ltd., lipase CALB (Candida antarctica Lipase B) and Novozym 435 were purchased from NoveXin (China) biological Co., ltd. A mutant of lipase MAS1H108A, which is a source of Streptomyces sp.strain W007 (Gene number: AY 260764), has been disclosed in Chinese patent No. CN111944798A, and belongs to the prior art.
Example 1
A device for separating chiral substances by a multi-liquid-phase system continuous enzyme method comprises a reaction device and an evaporator; the reaction device is divided into a standing area 3, a reaction area 1 and a dispersion buffer area 2 from top to bottom, wherein the reaction area 1 and the standing area 3 and the reaction area 1 and the dispersion buffer area 2 are separated by a baffle plate, and the baffle plate is provided with a plurality of small holes communicated with the areas; the standing area 3 is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with an upper phase solution outlet 5, the outlet is communicated with the first evaporator 20, the middle liquid phase area is communicated with the reaction area through a return pipe, and the enzyme-containing middle phase extracted into the standing area can flow back to the reaction area 1 through a first return valve 6 and a second return valve 9. The lower liquid phase zone is provided with a lower phase solution sample inlet 11 and an automatic potentiometric titrator burette 7; the lower part of the reaction zone is provided with an enzyme-containing solution inlet and outlet 16, and the inside of the reaction zone is provided with a stirrer 4 and an electrode head 13 of an automatic potentiometric titrator; the bottom of the dispersion buffer zone 2 is provided with an ester or amide compound sample inlet 17 and a lower phase solution outlet 18. The outside of the reaction zone is provided with a circulating water heating sleeve 14.
The method for separating chiral substances by utilizing the device continuous enzyme method comprises the following steps:
step (1): firstly preparing a 3L double water phase system, namely 16 percent of Na according to mass fraction 2 SO 4 After 19% of PEG600 and 65% of water are dissolved, the mixture is uniformly mixed and prepared into a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ for standing for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): the circulating water was turned on, 1g of Lipase CALB enzyme was added to 100mL of the upper phase PEG600, and 300mL of the lower phase Na was added 2 SO 4 Introducing the mixture into a continuous device under the conditions of heating and stirring to fill the dispersion buffer zone 2, introducing the PEG600 solution containing the enzyme into the reaction zone 1, wherein the stirring speed is 500rpm, and the temperature of circulating water is 45 ℃.
Step (3): and dissolving the mixed type 1- (4-methoxyphenyl) ethanol acetate with a proper amount of isooctane solution to prepare the mixed type 1- (4-methoxyphenyl) ethanol acetate isooctane solution with the substrate amount of 0.5% (v/v). Introducing the solution into the reaction zone 1 of the device from the sample inlet 17 of the ester or amide compound at a flow rate of 0.5mL/min, and simultaneously introducing the lower phase Na at a flow rate of 0.2mL/min 2 SO 4 The solution is introduced into the reaction zone 1 of the device from the lower phase solution sample inlet 11, and enzyme catalytic reaction is carried out in the reaction zone 1. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in a standing area from top to bottom, and the S-1- (4-methoxyphenyl) ethanol acetate after the resolution of the 1- (4-methoxyphenyl) ethanol acetate is mainly enrichedIn the upper liquid layer, the resolved R-1- (4-methoxyphenyl) ethanol is mainly concentrated in the lower liquid layer, the resolved acetic acid is mainly concentrated in the lower liquid layer, the enzyme-containing lower liquid layer can reflow back to the reaction zone again to participate in the reaction, and the pH value of the lower phase is regulated by an automatic potentiometric titrator to neutralize the resolved acetic acid. Finally, the reaction had a conversion of 46% at 5 minutes and 48% at 5 hours, and remained at 48% ee at 48 hours p The value was kept at 99%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution mixed-rotation type 1- (4-methoxyphenyl) ethanol acetate can only reach 50 percent at most.
Example 2
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): firstly, preparing a 3L double water phase system, namely (NH) according to 15 percent of mass 4 ) 2 SO 4 15% of PEG600 and 70% of water are dissolved and then mixed uniformly to prepare a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ to stand for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): circulating water was started, 5g Lipase MAS1H108A was added to 100mL of the upper phase PEG600, and 300mL of the lower phase (NH 4 ) 2 SO 4 Introducing the mixture into a continuous device under the conditions of heating and stirring to fill the dispersion buffer zone 2, introducing the PEG600 solution containing the enzyme into the reaction zone 1, wherein the stirring speed is 500rpm, and the temperature of circulating water is 30 ℃.
Step (3): and (3) dissolving the mixed methyl mandelate with a proper amount of isopropyl ether solution to prepare the isopropyl ether solution of the mixed methyl mandelate with the substrate amount of 4% (w/v). The solution was introduced into the reaction zone 1 of the apparatus from the sample inlet 17 for the ester or amide compound at a flow rate of 1mL/min, while the lower phase (NH) 4 ) 2 SO 4 The solution is introduced into the reaction zone 1 from the lower phase solution sample inlet 11, and enzyme catalytic reaction is carried out in the reaction zone 1. The pH value of the lower phase is regulated by an automatic potentiometric titrator, and the pH value of a stable system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, and the spiral almond is mixedThe S-methyl mandelate after methyl ester resolution is mainly enriched in the upper liquid layer, the R-mandelic acid after resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can reflow back to the reaction zone again to continuously participate in the reaction. Finally, the conversion of the reaction reaches 38% at 5h and remains at 40% at 30h, ee p The value was kept at 75%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution mixed-rotation methyl mandelate can reach 50 percent at most.
Example 3
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): firstly preparing a 3L double water phase system, namely 15 percent of Na according to mass fraction 2 SO 4 15% of PEG400 and 70% of water are dissolved and then uniformly mixed into a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ for standing for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): circulating water was started, 7.5g Lipase AY30 enzyme was added to 150mL of the upper phase PEG400, and 250mL of the lower phase Na was added 2 SO 4 Introducing the mixture into a continuous device under the conditions of heating and stirring to fill the dispersion buffer zone 2, introducing the PEG400 solution containing the enzyme into the reaction zone 1, wherein the stirring speed is 600rpm, and the temperature of circulating water is 37 ℃.
Step (3): and (3) dissolving the mixed naproxen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the mixed naproxen methyl ester with the substrate amount of 2% (w/v). Introducing the solution into the reaction zone 1 from the sample inlet 17 of the ester or amide compound at a flow rate of 0.5mL/min, and simultaneously introducing the lower phase Na at a flow rate of 0.1mL/min 2 SO 4 The solution is introduced into the reaction zone 1 from the lower phase solution sample inlet 11, and enzyme catalytic reaction is carried out in the reaction zone 1. The pH value of the lower phase is regulated by an automatic potentiometric titrator, and the pH value of a stable system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, R-naproxen methyl ester after resolution of the mixed naproxen methyl ester is mainly enriched in the upper liquid layer, S-naproxen after resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can reflow back to the reaction area again to continuously participate in the reactionShould be. Finally, the conversion of the reaction was maintained at 48%, ee, at 30h p The value was kept at 95%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution mixed spinning naproxen methyl ester can only reach 50 percent at most.
Example 4
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): firstly preparing a 3L double water phase system, namely 17 percent of Na according to mass fraction 2 SO 4 15% of PEG400 and 68% of water are dissolved and then uniformly mixed into a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ for standing for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): the circulating water was turned on, 5g of Lipase AY30 enzyme was added to 150mL of the upper phase PEG400, and 250mL of the lower phase Na was added 2 SO 4 Under the conditions of heating and stirring, the mixture is introduced into a continuous device from a sample inlet 11 to fill a dispersion buffer zone 2, and then the PEG400 solution containing enzyme is introduced into a reaction zone 1, wherein the stirring speed is 600rpm, and the temperature of circulating water is 37 ℃.
Step (3): and (3) dissolving the mixed-type ibuprofen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the mixed-type ibuprofen methyl ester with the substrate amount of 1% (v/v). Introducing the solution from the sample inlet 17 of the ester or amide compound into the reaction zone 1 at a flow rate of 0.2mL/min, and simultaneously introducing the lower phase Na at a flow rate of 0.05mL/min 2 SO 4 The solution is introduced into the reaction zone 1 from the lower phase solution sample inlet 11, and enzyme catalytic reaction is carried out in the reaction zone 1. The pH value of the lower phase is regulated by an automatic potentiometric titrator, and the pH value of a stable system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, R-ibuprofen methyl ester after resolution of the mixed rotation type ibuprofen methyl ester is mainly enriched in the upper liquid layer, S-ibuprofen after resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can flow back to the reaction area again to continuously participate in the reaction. Finally, the conversion of the reaction reaches 42% at 25h, ee p The value was kept at 80%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution mixed rotation ibuprofen methyl ester can only reach 50 percent at most.
Example 5
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): 200mg of Novozym 435 enzyme was weighed and added to 10mL of a 20g/L isopropyl mandelate ether solution to carry out the reaction, methanol was added in a molar ratio of 4:1 with mandelic acid at a temperature of 30℃for a reaction time of 6 hours. To the reaction mixture was added 1mol/L sodium bicarbonate solution equimolar to the unreacted mandelic acid, and the mixture was allowed to stand for delamination, and the upper phase was an isopropyl ether solution rich in methyl R-mandelate, the E.e. value of which was 67%.
Step (2): firstly, preparing a 3L double water phase system, namely (NH) according to 15 percent of mass 4 ) 2 SO 4 15% of PEG600 and 70% of water are dissolved and then mixed uniformly to prepare a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ to stand for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (3): the circulating water was turned on, 6g Lipase MAS1H108A was added to 150mL of the upper phase PEG600, and 250mL of the lower phase (NH 4 ) 2 SO 4 Introducing the mixture into a continuous device under the conditions of heating and stirring to fill the dispersion buffer zone 2, introducing the PEG600 solution containing the enzyme into the reaction zone 1, wherein the stirring speed is 500rpm, and the temperature of circulating water is 30 ℃.
Step (4): isopropyl ether solution rich in methyl R-mandelate was introduced into reaction zone 1 from sample inlet 17 for the ester or amide compound at a flow rate of 0.2mL/min while the lower phase (NH) 4 ) 2 SO 4 The solution is introduced into the reaction zone 1 from the lower phase solution sample inlet 11, and enzyme catalytic resolution reaction is carried out in the reaction zone 1. The pH value of the lower phase is regulated by an automatic potentiometric titrator, and the pH value of a stable system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, residual R-methyl mandelate and S-methyl mandelate after the resolution reaction are mainly enriched in the upper liquid layer, R-mandelic acid is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can reflow back to the reaction area again to continuously participate in the reaction. Finally, the inverseThe conversion should reach 98% at 5h and remain at 90% at 25 h. The theoretical value of the conversion rate of the single methyl mandelate enantiomer of the chiral resolution of the esterification and hydrolysis double enzyme cascade reaction can reach 100 percent.
Example 6
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): firstly preparing a 3L double water phase system, namely, according to the mass fraction of 18 percent of K 2 HPO 4 10% of [ BMIM ]]PF 6 Dissolving 18% PEG600 and 54% water, mixing, adding into a 3L separating funnel, and standing in an incubator at 37deg.C for 24 hr. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): circulating water was turned on to phase PEG600 with [ BMIM ] on 150mL]PF 6 5g of Lipase AY30 enzyme was added to the mixture of (B) and 250mL of phase K was added 2 HPO 4 Introducing into a continuous device under heating and stirring to fill the dispersion buffer zone 2, and introducing enzyme-containing PEG600 and [ BMIM ]]PF 6 The solution was stirred in reaction zone 1 at a stirring rate of 700rpm and the circulating water temperature was 37 ℃.
Step (3): and (3) dissolving the mixed-type ibuprofen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the mixed-type ibuprofen methyl ester with the substrate amount of 1% (v/v). Introducing the solution into the reaction zone 1 of the device from the sample inlet 17 of the ester or amide compound at a flow rate of 0.5mL/min, and simultaneously introducing the lower phase K at a flow rate of 0.1mL/min 2 HPO 4 The solution is introduced into the reaction zone 1 of the device from the lower-phase solution sample inlet 11, and enzyme catalytic resolution reaction is carried out in the reaction zone 1. The pH value of the lower phase is regulated by an automatic potentiometric titrator, and the pH value of a stable system is kept at 7. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, R-ibuprofen methyl ester after resolution of the mixed rotation type ibuprofen methyl ester is mainly enriched in the upper liquid layer, S-ibuprofen after resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can flow back to the reaction area again to continuously participate in the reaction. Finally, the reaction was run at 4h ee p Up to 76.9% and the conversion rate was 20%. Ee at 20h p Stabilized at 74% conversion was 23%.
Example 7
A method for separating chiral substances by using the continuous enzyme method of the device of the embodiment 1 comprises the following steps:
step (1): firstly preparing a 3L double water phase system, namely Na with the mass fraction of 20 percent 2 SO 4 15% of PEG400 and 65% of water are dissolved and then uniformly mixed into a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ for standing for 24 hours. After significant delamination, the upper and lower phases were separately passed through 0.45 μm filters to remove impurities.
Step (2): 5g Lipase CALB enzyme was added to 100mL of the upper phase PEG400, and 300mL of the lower phase Na was added 2 SO 4 The continuous device was charged to fill the dispersion buffer zone 2, and then the enzyme-containing PEG400 solution was charged to the reaction zone 1 with stirring at 500rpm.
Step (3): and (3) dissolving the mixed methyl mandelate with a proper amount of isopropyl ether solution to prepare the isopropyl ether solution of the mixed methyl mandelate with the substrate amount of 1% (w/v). Introducing the solution into the reaction zone 1 of the device from the sample inlet 17 of the ester or amide compound at a flow rate of 5mL/min, and simultaneously introducing the lower phase Na at a flow rate of 1mL/min 2 SO 4 The solution is introduced into the reaction zone 1 from the lower phase solution sample inlet 11, and enzyme catalytic reaction is carried out in the reaction zone 1. In the reaction process, an upper liquid layer, a middle liquid layer and a lower liquid layer are sequentially arranged in the standing area from top to bottom, the S-methyl mandelate after the resolution of the mixed methyl mandelate is mainly enriched in the upper liquid layer, the R-mandelic acid after the resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing enzyme can reflow back to the reaction area again to continuously participate in the reaction. Finally, the reaction was maintained at 35% ee at 30h p The value was kept at 60%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution mixed-rotation methyl mandelate can reach 50 percent at most.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (4)

1. A method for separating chiral substances by a continuous enzyme method, which is characterized by comprising the following steps:
(1) Preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing for layering to obtain an upper-phase solution and a lower-phase solution, continuously introducing the lower-phase solution into a standing area, passing through a reaction area, and entering and filling a dispersion buffer area; dissolving enzyme in the upper phase solution, and introducing the solution into a reaction zone; introducing an ester or amide compound formed by racemization or single enantiomer chiral matters containing a hydrophobic solvent into a dispersion buffer area, adjusting the flow rate of feed liquid to enable the ester or amide compound to enter a reaction area, and carrying out enzyme catalytic splitting reaction; the hydrophilic solvent is a polymer and/or an ionic liquid;
(2) The product after the catalytic reaction is automatically extracted to a static area, after static layering, the upper phase is introduced into an evaporator to evaporate and remove the hydrophobic solvent, and the ester or amide products of the single optically active chiral product are obtained; the middle phase or the lower phase is filtered to remove enzyme by ultrafiltration and then is introduced into a second evaporator to remove water by evaporation, thus obtaining another single optically active chiral product; the enzyme-containing medium phase extracted in the step (2) to the standing area flows back to the reaction area through a return pipe to continue the reaction;
the device comprises a reaction device and an evaporator; the reaction device is divided into a standing area, a reaction area and a dispersion buffer area from top to bottom, wherein the reaction area and the standing area and the reaction area and the dispersion buffer area are separated by a baffle plate, and the baffle plate is provided with a plurality of small holes communicated with the areas; the standing area is divided into an upper liquid phase area, a middle liquid phase area and a lower liquid phase area, the upper liquid phase area is provided with an upper phase solution outlet which is communicated with the evaporator, the middle liquid phase area is communicated with the reaction area through a return pipe, and the lower liquid phase area is provided with a lower phase solution sample inlet; the lower part of the reaction zone is provided with an enzyme-containing solution inlet and outlet, and the inside of the reaction zone is provided with a stirrer; the bottom of the dispersion buffer zone is provided with an ester or amide compound sample inlet and a lower phase solution outlet; the lower phase solution outlet is communicated with the second evaporator through an ultrafilter; a circulating water heating sleeve is arranged outside the reaction zone; the lower liquid phase zone of the standing zone is provided with an automatic potentiometric titrator buret, and the reaction zone is provided with an automatic potentiometric titrator electrode head.
2. The process of claim 1, wherein the feeding and reacting in step (1) are carried out under continuous stirring and heating conditions, the heating temperature is 30-70 ℃, and the stirring speed is 100-1000rpm; regulating the pH value of the reaction system to 3-13; the mass ratio of the soluble salt, the hydrophilic solvent and the water is 0.1-1 and 0.1-5 respectively.
3. The method of claim 2, wherein the soluble salt is one or more of sodium citrate, sodium chloride, sodium sulfate, ammonium sulfate, sodium carbonate, dipotassium hydrogen phosphate, potassium phosphate, and potassium dihydrogen phosphate;
the polymer comprises one or two of polyethylene glycol and polypropylene glycol; the ionic liquid comprises [ BMIM ]]Br、[BMIM]BF 4 、[EMIM]ETSO 4 、[OMIM]Cl、[BMIM]PF 6 One or two or more of them;
the enzyme comprises one or more than two of Lipase AYS, lipase AY30, lipase MAS1H108A, lipase G Amano 50, lipase CALB and Novozyme 435, and the enzyme concentration is 5-2000U/mL.
4. A process according to claim 3, wherein the ester or amide compound of racemic or single enantiomer chiral form comprises 0.1% to 10% by mass of the hydrophobic solvent;
the hydrophobic solvent is one or more of n-hexane, isopropyl ether, ethyl acetate, isooctane, petroleum ether, diethyl ether, benzene and toluene;
the ester or amide compounds formed by racemized or single enantiomer chiral substances comprise one or more than two of racemized methyl mandelate, racemized ibuprofen methyl ester, racemized naproxen methyl ester, racemized 1- (4-methoxyphenyl) ethanol acetate, R-methyl mandelate and S-methyl mandelate.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2036208A1 (en) * 1969-07-21 1971-02-11 Sagami Chemical Research Center, Tokio: Device for collecting a solid substance from raw materials in a liquid or liquid gaseous phase
US5057427A (en) * 1988-04-07 1991-10-15 Sepracor, Inc. Method for resolution of stereoisomers
CN104971665A (en) * 2015-07-21 2015-10-14 宁波金海晨光化学股份有限公司 Synthesizer for 4,4-dimethyl-1,3-dioxane
CN107417662A (en) * 2017-07-03 2017-12-01 湖北三里枫香科技有限公司 A kind of liquid acid type metaformaldehyde manufacturing technique method and extractive reaction tower
CN109628508A (en) * 2018-12-10 2019-04-16 华南理工大学 A kind of method of Enzymatic Resolution chiral material
CN110819383A (en) * 2018-08-14 2020-02-21 何巨堂 Process for the upflow hydrogenation of poor quality hydrocarbons using reactors with internal parallel reaction zones

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2036208A1 (en) * 1969-07-21 1971-02-11 Sagami Chemical Research Center, Tokio: Device for collecting a solid substance from raw materials in a liquid or liquid gaseous phase
US5057427A (en) * 1988-04-07 1991-10-15 Sepracor, Inc. Method for resolution of stereoisomers
CN104971665A (en) * 2015-07-21 2015-10-14 宁波金海晨光化学股份有限公司 Synthesizer for 4,4-dimethyl-1,3-dioxane
CN107417662A (en) * 2017-07-03 2017-12-01 湖北三里枫香科技有限公司 A kind of liquid acid type metaformaldehyde manufacturing technique method and extractive reaction tower
CN110819383A (en) * 2018-08-14 2020-02-21 何巨堂 Process for the upflow hydrogenation of poor quality hydrocarbons using reactors with internal parallel reaction zones
CN109628508A (en) * 2018-12-10 2019-04-16 华南理工大学 A kind of method of Enzymatic Resolution chiral material

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
木瓜脂肪酶催化洛芬类药物的酶促拆分;尤朋永;邱健;蔡雯雯;苏二正;魏东芝;;华东理工大学学报(自然科学版)(06);第687-693页 *

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