CN113862117A - Device and method for splitting chiral substance by multi-liquid-phase system continuous enzymatic method - Google Patents
Device and method for splitting chiral substance by multi-liquid-phase system continuous enzymatic method Download PDFInfo
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- CN113862117A CN113862117A CN202111247760.9A CN202111247760A CN113862117A CN 113862117 A CN113862117 A CN 113862117A CN 202111247760 A CN202111247760 A CN 202111247760A CN 113862117 A CN113862117 A CN 113862117A
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Abstract
The invention belongs to the field of bioengineering, and discloses a device and a method for splitting chiral substances by a multi-liquid-phase system continuous enzymatic method, wherein the device comprises a reaction device and an evaporator; the reaction device is divided into a standing zone, a reaction zone and a dispersing buffer zone from top to bottom, the reaction zone and the standing zone and the reaction zone and the dispersing buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; 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 dispersing buffer zone is provided with an ester or amide compound injection port and a lower phase solution outlet. The method has the advantages of high raw material repeated utilization rate, mild and controllable reaction conditions, continuous resolution, easy separation and recovery of products and the like.
Description
Technical Field
The invention belongs to the technical field of bioengineering and food, relates to separation of chiral substances and application of enzymes, and particularly relates to a device and a method for separating chiral substances by a multi-liquid-phase system continuous enzymatic method.
Background
Chirality (Chirality) is one of the essential attributes of nature. Biological macromolecules, such as proteins, polysaccharides, nucleic acids and enzymes, which are important bases for life activities, are almost all chiral. "Ready" drugs, which occurred in 1960, led to the development of malformed fetuses after administration to pregnant women, because the S-enantiomer of the drug had a strong teratogenic effect. Therefore, the preparation of single chiral drugs with higher drug effect and less side effect to replace the traditional mixed racemic drugs is a necessary trend.
At present, chiral substance resolution methods with wide application range mainly comprise chemical resolution methods, induced crystallization methods, chromatography methods, enzyme resolution methods and the like. The chemical resolution 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 product recovery rate and the like. The induced crystallization method has the advantages of low cost, simple operation and high product purity, but needs to continuously supplement seed crystals in the reaction and continuously carry out racemization reaction in the solution, thereby increasing the complexity of the operation and the production cost. The chromatography directly utilizes the chiral chromatographic column for resolution, has the advantages of high efficiency, rapidness, simple and convenient operation, high purity and the like, but has small preparation amount, cannot meet the requirement of industrial production, and is expensive, consumes a large amount of organic solvent and increases the production cost. Therefore, in recent years, an enzymatic resolution method having advantages such as mild conditions, high safety, high product purity, and low energy consumption has been attracting attention. However, in most chiral products, they are produced at a great distance from industrial use. On the one hand, although the chiral product and the enantiomer by-product thereof have different physicochemical properties, in traditional enzyme catalysis systems such as a micro-aqueous phase reaction system and an organic solvent-water reaction system, the product and the enzyme are difficult to be separated synchronously, extra separation steps such as chromatography are needed to separate the product and the enzyme, and the addition of the extra necessary steps greatly increases the difficulty of continuous production of the product. On the other hand, in the traditional reaction system such as micro-water phase and organic solvent-water, a series of problems exist, such as that the system emulsification is serious and/or the product distribution is complex, so that the product recovery is difficult, and the enzyme is difficult to recycle. In addition, the concentration of the product and the pH value of the system, which are key factors influencing chiral resolution, can be constantly changed along with the progress of the reaction, the forward reaction can be greatly slowed down by the product inhibition effect when the concentration of the product is constantly increased, and the enzyme can be inactivated due to the change of the pH value of the system, so that the reaction cannot be continued. Due to the reasons, most of the research is in the laboratory research stage at present and is not used for continuous industrial production.
At present, the enzyme method chiral resolution adopts a continuous flow process to carry out enantioselective organic catalytic reaction, the examples are few, most of the industrial applications are intermittent resolution production, a large-scale ground tank or tank is adopted, a racemized substrate is prepared into a solution with a certain concentration, a certain amount of resolving enzyme is added, stirring production is carried out under a proper reaction condition, a mixed solution is obtained and then taken out, and a new substrate is injected again. The intermittent resolution production has the defects of low resolution efficiency, high cost, high energy consumption of raw materials, complex and 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 Westerbek et al studied the use of a two-step single-enzyme tandem process in the preparation of chiral halohydrins by the enzymatic asymmetric and tandem kinetic resolution of haloalkane dehalogenases, but at yields of only 24% and 52%, resulting in relatively high production costs and high energy consumption (Tetrahedron,2012,68(37): 7645-. Ayelet Fishman developed an efficient two-step enzymatic process for the production of (R) and (S) -ethyl-3-hydroxybutyrate on a scale of several thousand kilograms, but required fractionation downstream of the reaction to separate the product, which was prone to side reactions and high product loss (Biotechnology and bioengineering,2001,74(3): 256-263).
In the earlier research, a novel multi-liquid-phase system is developed in the subject group, the multi-liquid-phase system can promote the enzyme resolution efficiency and efficiently recycle the enzyme-rich phase (CN201811500754.8), but the process is easily interfered by a pump and other motive equipment to generate an emulsification phenomenon, so that the separation is influenced, the inhibition effect of a product and the pH value of the system are difficult to control, and the process is always difficult to continuously operate. Therefore, it is necessary to develop an apparatus and a method for resolving chiral substances with high efficiency, controllability, continuous catalysis and separation.
Disclosure of Invention
The invention aims to provide a device and a method for resolving chiral substances by a multi-liquid-phase system continuous enzymatic method, aiming at the problems of high cost, complex reaction operation, difficult continuous production, difficult control of reaction conditions, product inhibition and the like in the industrial process of resolving chiral substances by the existing enzymatic method.
The purpose of the invention is realized by the following technical scheme:
a device for splitting chiral substances by a multi-liquid-phase system continuous enzymatic method comprises a reaction device and an evaporator; the reaction device is divided into a standing zone, a reaction zone and a dispersing buffer zone from top to bottom, the reaction zone and the standing zone and the reaction zone and the dispersing buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; 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 dispersing buffer zone is provided with an ester or amide compound injection port and a lower phase solution outlet.
Preferably, a circulating water heating sleeve is arranged outside the reaction zone.
Preferably, the lower liquid phase zone of the standing zone is provided with an automatic potentiometric titrator burette, and the reaction zone is provided with an automatic potentiometric titrator electrode tip.
Preferably, the lower phase solution outlet is in communication with the second evaporator via an ultrafilter.
A method for separating chiral substances by using the continuous enzymatic method of the device comprises the following steps:
(1) preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing and layering to obtain an upper-phase solution and a lower-phase solution, continuously introducing the lower-phase solution into a standing zone, passing through a reaction zone, and entering and filling a dispersion buffer zone; dissolving enzyme in the upper phase solution, and introducing the solution into a reaction zone; ester or amide compounds composed of racemic or single enantiomer chiral substances containing hydrophobic solvents are introduced into a dispersion buffer zone, the flow rate of feed liquid is adjusted to enable the ester or amide compounds to enter a reaction zone, and enzyme catalysis resolution reaction is carried out; meanwhile, the pH value of the lower phase is changed by utilizing an automatic potentiometric titrator so as to adjust the pH value of the reaction system. The dispersing buffer zone can prevent ester or amide compounds formed by racemic or single enantiomer chiral substances containing hydrophobic solvents from being directly mixed with the enzyme-containing solution, so as to prevent emulsification.
(2) The product after catalytic reaction is automatically extracted to a standing zone, the standing zone plays a role in standing and layering, and after standing and layering, the upper phase is introduced into a first evaporator to be evaporated and remove a hydrophobic solvent, so that an ester or amide product of a single optical rotation chiral product is obtained; wherein the other single optical chiral product is mainly enriched in the middle liquid layer or the lower liquid layer.
Preferably, the enzyme-containing phase extracted to the standing zone in step (2) is returned to the reaction zone via a return pipe to continue the reaction. When another single optically active chiral product is present in the middle phase, the accumulation of more product in the middle phase inhibits the reaction over time. Thus, the middle phase extracted into the rest zone can be periodically drained to relieve the inhibition; and the other single optical rotation chiral product is obtained through simple operations such as filtration, rotary evaporation and the like.
Preferably, in the step (2), when the other single optical chiral product exists in the lower phase, the lower phase is subjected to ultrafiltration to remove the enzyme and then is introduced into a second evaporator to remove water by evaporation, so that the other single optical chiral product is obtained.
Preferably, the feeding and reaction processes in the step (1) are carried out under the conditions of continuous stirring and heating, the heating temperature is 30-70 ℃, and the stirring speed is 100-1000 rpm; adjusting the pH value of the reaction system to 3-13; the mass ratio of the soluble salt to the hydrophilic solvent to 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]BF4、[EMIM]ETSO4、[OMIM]Cl、[BMIM]PF6One or more than two 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 racemic or single enantiomer chiral substance accounts for 0.1-10% of the mass of the hydrophobic solvent, and preferably accounts for 1-5%.
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 racemic or single enantiomer chiral compound comprises one or more 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 the continuous enzymatic resolution of chiral substances by the multi-liquid-phase system 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, toothed sheet type, bent blade opening turbine type, anchor type, frame type, helical belt type, screw type, Brumakin type, folded blade opening turbine type, bent blade disc turbine type, push type and straight blade disc turbine type stirrers;
d. the pump is one or more than two of a displacement pump, a vane pump, a fluid power pump and an electromagnetic pump;
e. the evaporator is one of a central circulating tube evaporator, a hanging basket evaporator, an external heating type evaporator, a Levenu evaporator, a forced circulation evaporator, a climbing-film evaporator, a falling-film evaporator, a rising-falling-film evaporator, a scraper film evaporator and a direct contact heat transfer evaporator;
f. the ultrafilter is plate type ultrafiltration, hollow fiber ultrafiltration, capillary ultrafiltration, or tubular ultrafiltration.
Preferably, the device for the continuous enzymatic resolution of chiral substances by the multi-liquid-phase system 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 reaction zone are consistent, and the baffle is provided with a plurality of small holes for blocking reaction liquid and extracting and separating products. Rubber rings with the same size as the inner diameter of the upper, middle and lower part pipes are arranged on the two surfaces of the baffle plate so as to achieve the sealing effect;
b. because a small part of the middle phase in the reaction zone can be extracted into the standing zone, 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 by-products generated in the splitting reaction process can be enriched in the lower phase rich in soluble salts, and the lower phase solution outlet is used for replacing the lower phase solution to remove the inhibition effect;
d. because the activity of the enzyme is gradually reduced after the enzyme reacts for a long time, in order to maintain the reaction efficiency, the enzyme-containing solution inlet and outlet are used for the entry of the middle phase and the replacement of the middle phase; the return pipe is a detachable hose and can discharge the middle phase through the return pipe;
e. the titrating tube of the automatic potentiometric titrator is used for regulating the pH value of a multi-liquid-phase system together with the electrode head of the automatic potentiometric titrator by dripping acid or alkali into the lower-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 accelerated.
Preferably, in the device for the multi-liquid-phase system continuous enzymatic resolution of chiral substances, both the middle phase inlet and the middle phase flowing out from the exchange port and the lower phase flowing out from the exchange port can contain single optical chiral products after resolution reaction, and the middle phase or the lower phase can be extracted by simple centrifugation, standing, direct filtration, ultrafiltration by an ultrafilter, rotary evaporation, extraction and other modes.
We have recently found that polymer and ionic multi-liquid phase system is convenient for separating product and adjusting lower phase pH value, and only needs to be stood or centrifuged at low speed to separate product, and is especially suitable for developing a set of continuous device based on the system. The reaction zone of the device can fully mix three phases by regulating and controlling the stirring speed, thereby accelerating the splitting speed, and the pH value of a reaction system can be monitored in real time through an electrode tip of an automatic potentiometric titrator; the dispersion buffer zone prevents the direct contact of the upper phase and the middle phase (containing enzyme solution) to avoid emulsification, which is not beneficial to the enzyme catalysis resolution reaction and the subsequent separation. The reaction zone can further extract, separate and take away the split by-products in the lower phase by a countercurrent extraction mode; the standing zone changes the pH value of the lower phase through a burette of an automatic potentiometric titrator so as to adjust the pH value of the system, and the upper phase product and the enzyme can be separated through standing and layering. Finally, the device can recover the split product after simple operations such as filtration, rotary evaporation and the like, has the advantages of simplified industrial process, high catalytic efficiency, energy conservation, consumption reduction, enzyme recycling, product inhibition removal, real-time regulation and control of the pH value of the system 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 overcomes the problems of high cost, complex reaction operation, difficult continuous production, low reaction efficiency, difficult product recovery and the like in the current process of continuously splitting chiral substances, provides a device and a method for splitting chiral substances by a multi-liquid-phase system continuous enzymatic method, and has the specific advantages that:
(1) the chiral substance is resolved by a polymer and/or ionic liquid type multi-liquid-phase system continuous enzymatic method, an ester or amide substrate of a single optical chiral product can be distributed in a hydrophobic phase, another single optical chiral product after hydrolysis 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, a dispersion buffer zone is established, so that the phenomenon of direct emulsification caused by direct contact between an upper phase and a middle phase is avoided, and the resolution reaction and separation are facilitated;
(2) the reaction system is further extracted and separated in a countercurrent extraction mode, so that 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 online regulating and controlling the pH value of the lower phase by an automatic potentiometric titrator, so that the reaction can be continuously, stably and for a long time;
(3) the reaction zone of the device intelligently regulates and controls the stirring speed, so that the condition that the system is not beneficial to extraction layering due to overhigh stirring speed or incomplete mixing due to overlow stirring speed is avoided, three phases are fully mixed, the splitting speed 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 middle phase. The enzyme can be recycled in a standing area by extraction layering, medium phase reflux and other modes, so that the loss of the enzyme is reduced, and the cost is reduced;
(5) the continuous device has the advantages of less material consumption, low cost, energy conservation and environmental protection by using circulating water for heating; the method has the advantages of low energy consumption, high utilization rate of raw materials, mild and controllable reaction conditions, continuous reaction and the like, and solves the technical problem that the chiral substances are difficult to be produced by long-time continuous enzymatic resolution.
Drawings
FIG. 1 is a schematic structural diagram of a device for splitting chiral substances by a multi-liquid-phase system continuous enzymatic 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 burette, 8, a circulating water outlet, 9, a second reflux valve, 10, a first baffle, 11, a lower phase solution inlet, 12, a second baffle, 13, an automatic potentiometric titrator electrode head, 14, a circulating water heating sleeve, 15, a circulating water inlet, 16, an enzyme-containing solution inlet, 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 heating pump, 23, an upper phase product delivery pump, 24, a first lower phase delivery pump, 25, an enzyme-containing solution delivery pump, 26, a delivery pump for ester or amide compounds, 27, a second lower phase delivery pump, 28, a delivery pump, 29. a product delivery pump after ultrafiltration, 30, a first lower phase material storage tank, 31, an enzyme-containing solution material storage tank, 32, an ester or amide compound material storage tank, 33 and a second lower phase material storage tank.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.
Lipase AY30(Candida rugosa lipases) used in this example was purchased from Japan Tianye, Lipase CALB (Candida antarctica Lipase B) and Novozym 435 were purchased from Novoxin (China) BioLimited. Lipase MAS1H108A is a mutant derived from Streptomyces sp.strain W007 (gene number: AY260764), which is disclosed in Chinese patent CN111944798A and belongs to the prior art.
Example 1
A device for splitting chiral substances by a multi-liquid-phase system continuous enzymatic method comprises a reaction device and an evaporator; the reaction device is divided into a standing zone 3, a reaction zone 1 and a dispersing buffer zone 2 from top to bottom, the reaction zone 1 and the standing zone 3 and the reaction zone 1 and the dispersing buffer zone 2 are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; the standing zone 3 is divided into an upper liquid phase zone, a middle liquid phase zone and a lower liquid phase zone, the upper liquid phase zone is provided with an upper phase solution outlet 5, the outlet is communicated with a first evaporator 20, the middle liquid phase zone is communicated with the reaction zone through a return pipe, and the enzyme-containing phase extracted to the standing zone can return to the reaction zone 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 injection port 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 tip 13 of an automatic potentiometric titrator; the bottom of the dispersing buffer zone 2 is provided with an ester or amide compound injection port 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 using the continuous enzymatic method of the device comprises the following steps:
step (1): firstly, 3L of aqueous two-phase system is prepared, namely Na with the mass fraction of 16 percent2SO419% PEG600 and 65% water were dissolved and mixed in a 3L separatory funnel, and the separatory funnel was placed in an incubator at 37 ℃ and left to stand for 24 hours. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): circulating water is started, 1g of Lipase CALB enzyme is added into 100mL of PEG600 at the upper phase, and 300mL of Na at the lower phase2SO4Introducing into a continuous device under heating and stirring conditions to fill the dispersion buffer 2, and introducing enzyme-containing PEG600The solution was introduced into the reaction zone 1 at a stirring rate of 500rpm and a circulating water temperature of 45 ℃.
And (3): dissolving the mixed rotary type 1- (4-methoxyphenyl) ethanol acetate by using a proper amount of isooctane solution to prepare the mixed rotary type 1- (4-methoxyphenyl) ethanol acetate isooctane solution with the substrate amount of 0.5 percent (v/v). Introducing the solution into the reaction zone 1 of the device from the ester or amide compound injection port 17 at a flow rate of 0.5mL/min, and simultaneously introducing the lower phase Na at a flow rate of 0.2mL/min2SO4The solution is introduced into the reaction area 1 of the device from the lower phase solution injection port 11, and the enzyme catalysis reaction is carried out in the reaction area 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, the S-1- (4-methoxyphenyl) ethanol acetate after the 1- (4-methoxyphenyl) ethanol acetate is split is mainly enriched in the upper liquid layer, the R-1- (4-methoxyphenyl) ethanol after the split is mainly enriched in the middle liquid layer, the acetic acid after the split is mainly enriched in the lower liquid layer, the middle liquid layer containing the enzyme can be refluxed back into the reaction area again to continuously participate in the reaction, and an automatic potentiometric titrator adjusts the pH value of the lower phase so as to neutralize the acetic acid after the split. Finally, the reaction reached 46% conversion at 5 minutes, 48% conversion at 5 hours and remained 48% at 48 hours, eepThe value remained at 99%. The theoretical value of the conversion rate of the traditional kinetic chiral resolution racemic 1- (4-methoxyphenyl) ethanol acetate can only reach 50 percent at most.
Example 2
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing 3L of aqueous two-phase system, namely 15 percent (NH) by mass4)2SO415% PEG600 and 70% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): the circulating water was turned on, 5g Lipase MAS1H108A was added to 100mL of the upper phase PEG600, and 300mL of the lower phase (NH)4)2SO4Introducing into a continuous device under heating and stirring conditions to make it full of componentsDisperse buffer 2, then introduce the enzyme-containing PEG600 solution into the reaction zone 1, with stirring speed of 500rpm and circulating water temperature of 30 ℃.
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). Introducing the solution into the reaction zone 1 of the device from the ester or amide compound injection port 17 at a flow rate of 1mL/min, and simultaneously introducing the lower phase (NH) at a flow rate of 0.3mL/min4)2SO4The solution is introduced into the reaction area 1 from the lower phase solution injection port 11, and the enzyme catalysis reaction is carried out in the reaction area 1. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing 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 a standing area from top to bottom, S-methyl mandelate after the mixed rotation type methyl mandelate is split is mainly enriched in the upper liquid layer, R-methyl mandelate after the split is mainly enriched in the middle liquid layer, and the middle liquid layer containing the enzyme can flow back to the reaction area again to continuously participate in the reaction. Finally, the reaction reached 38% conversion at 5h and was maintained at 40% ee at 30hpThe value remained at 75%. The theoretical value of conversion rate of racemic methyl mandelate for traditional kinetic chiral resolution can only reach 50%.
Example 3
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): firstly, 3L of aqueous two-phase system is prepared, namely 15 percent of Na by mass2SO415% PEG400 and 70% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): circulating water is started, 7.5g of Lipase AY30 enzyme is added into 150mL of PEG400 on the upper phase, and 250mL of Na on the lower phase2SO4The mixture was introduced into a continuous apparatus under heating and stirring conditions to fill the dispersion buffer 2, and then the enzyme-containing PEG400 solution was introduced into the reaction zone 1 at a stirring rate of 600rpm and a circulating water temperature of 37 ℃.
Step (3)): dissolving the mixed spinning naproxen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the mixed spinning naproxen methyl ester with the substrate amount of 2 percent (w/v). Introducing the solution into the reaction zone 1 from the ester or amide compound inlet 17 at a flow rate of 0.5mL/min, and simultaneously introducing lower phase Na at a flow rate of 0.1mL/min2SO4The solution is introduced into the reaction area 1 from the lower phase solution injection port 11, and the enzyme catalysis reaction is carried out in the reaction area 1. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing 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 a standing area from top to bottom, R-naproxen methyl ester after the separation of the mixed spinning naproxen methyl ester is mainly enriched in the upper liquid layer, S-naproxen after the separation is mainly enriched in the middle liquid layer, and the middle liquid layer containing the enzyme can be refluxed back to the reaction area to continuously participate in the reaction. Finally, the reaction maintains a conversion of 48% at 30h, eepThe value remained at 95%. The theoretical value of the conversion rate of the conventional kinetic chiral resolution racemic naproxen methyl ester can only reach 50 percent at most.
Example 4
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing 3L of aqueous two-phase system, namely Na with the mass fraction of 17%2SO415% PEG400 and 68% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): circulating water is started, 5g of Lipase AY30 enzyme is added into 150mL of PEG400 at the upper phase, and 250mL of Na at the lower phase2SO4The mixture was introduced into a continuous apparatus from an inlet 11 under heating and stirring conditions to fill a dispersion buffer 2, and then a PEG400 solution containing an enzyme was introduced into a reaction zone 1 at a stirring rate of 600rpm at a circulating water temperature of 37 ℃.
And (3): dissolving the racemic ibuprofen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the racemic ibuprofen methyl ester with the substrate amount of 1% (v/v). The solution is separated from the ester or the mixture at a flow rate of 0.2mL/minThe amide compound injection port 17 is introduced into the reaction zone 1, and simultaneously, the lower phase Na is injected at the flow rate of 0.05mL/min2SO4The solution is introduced into the reaction area 1 from the lower phase solution injection port 11, and the enzyme catalysis reaction is carried out in the reaction area 1. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing 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 a standing area from top to bottom, R-ibuprofen methyl ester after the mixed rotation type ibuprofen methyl ester is split is mainly enriched in the upper liquid layer, S-ibuprofen after the split 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 reaches 42% conversion at 25h, eepThe value remained at 80%. The theoretical value of conversion rate of the traditional kinetic chiral resolution racemic ibuprofen methyl ester can only reach 50 percent at most.
Example 5
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): 200mg of Novozym 435 enzyme is weighed and added into 10mL of 20g/L isopropyl mandelate solution for reaction, methanol with the molar ratio of 4:1 to the mandelic acid is added, the reaction temperature is 30 ℃, and the reaction time is 6 h. Adding 1mol/L sodium bicarbonate solution which is equimolar with the unreacted mandelic acid into the reaction mixture, standing and demixing, wherein the upper phase is isopropyl ether solution rich in R-methyl mandelate, and the e.e value of the R-methyl mandelate reaches 67%.
Step (2): firstly, preparing 3L of aqueous two-phase system, namely 15 percent (NH) by mass4)2SO415% PEG600 and 70% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
And (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)2SO4Introducing into a continuous device under heating and stirring conditions to fill the dispersion buffer region 2, introducing enzyme-containing PEG600 solution into the reaction region 1, stirring at 500rpm, and circulatingThe temperature of the circulating water is 30 ℃.
And (4): the isopropyl ether solution rich in R-methyl mandelate was introduced into reaction zone 1 from ester or amide compound inlet 17 at a flow rate of 0.2mL/min, while the lower phase (NH) was introduced at a flow rate of 0.05mL/min4)2SO4The solution is introduced into the reaction zone 1 from the lower phase solution injection port 11, and the enzyme catalysis resolution reaction is carried out in the reaction zone 1. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing 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 a standing area from top to bottom, the residual R-methyl mandelate and S-methyl mandelate after the resolution reaction are mainly enriched in the upper liquid layer, the R-methyl mandelate is mainly enriched in the middle liquid layer, and the middle liquid layer containing the enzyme can be returned to the reaction area again to continuously participate in the reaction. Finally, the reaction reached 98% conversion at 5h and was maintained at 90% at 25 h. The theoretical value of the conversion rate of a single methyl mandelate enantiomer by esterification and hydrolysis double-enzyme cascade reaction chiral resolution can reach 100 percent at most.
Example 6
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): firstly preparing 3L of aqueous two-phase system, namely 18 percent of K by mass2HPO410% of [ BMIM]PF618% PEG600 and 54% water are dissolved and mixed evenly to prepare a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): circulating water was turned on, and PEG600 and [ BMIM ] were added to 150mL of the solution]PF6Adding 5g of Lipase AY30 enzyme into the mixed solution, and adding 250mL of lower phase K2HPO4Introducing into a continuous device under heating and stirring conditions to fill the dispersion buffer 2, and introducing enzyme-containing PEG600 and [ BMIM ]]PF6The solution was introduced into the reaction zone 1 at a stirring rate of 700rpm and a circulating water temperature of 37 ℃.
And (3): dissolving the mixed-rotation ibuprofen methyl ester by using a proper amount of isooctane solution to prepare the isooctane solution of the mixed-rotation ibuprofen methyl ester with the substrate amount of 1 percent (v/v)And (4) liquid. Introducing the solution into the reaction zone 1 of the device from the ester or amide compound injection port 17 at the flow rate of 0.5mL/min, and simultaneously introducing the lower phase K at the flow rate of 0.1mL/min2HPO4The solution is introduced into the reaction zone 1 of the device from the lower phase solution injection port 11, and the enzyme catalysis resolution reaction is carried out in the reaction zone 1. The pH value of the lower phase is adjusted by an automatic potentiometric titrator, and the pH value of the stabilizing 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 a standing area from top to bottom, R-ibuprofen methyl ester after the mixed rotation type ibuprofen methyl ester is split is mainly enriched in the upper liquid layer, S-ibuprofen after the split 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 is ee at 4hp76.9 percent and the conversion rate is 20 percent. Ee at 20hpThe stabilization was at 74% and the conversion was 23%.
Example 7
The method for separating chiral substances by using the continuous enzymatic method of the device in the embodiment 1 comprises the following steps:
step (1): firstly, preparing 3L of aqueous two-phase system, namely 20 percent of Na by mass2SO415% PEG400 and 65% water are dissolved and mixed evenly in a 3L separating funnel, and the separating funnel is placed in an incubator at 37 ℃ and stands for 24 h. After the separation, the upper phase and the lower phase were passed through 0.45 μm filter membranes to remove impurities.
Step (2): 100mL of the upper PEG400 phase was added with 5g of Lipase CALB enzyme, and 300mL of the lower Na phase was added2SO4The reaction mixture was introduced into a continuous apparatus to fill up the dispersion buffer 2, and then the enzyme-containing PEG400 solution was introduced into the reaction zone 1 at a stirring rate of 500 rpm.
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 ester or amide compound injection port 17 at a flow rate of 5mL/min, and simultaneously introducing the lower phase Na at a flow rate of 1mL/min2SO4The solution is introduced into the reaction area 1 from the lower phase solution injection port 11, and the enzyme catalysis reaction is carried out in the reaction area 1. During the reaction process, the reaction solution is from top to bottom in a standing zoneThe upper liquid layer, the middle liquid layer and the lower liquid layer are arranged next time, the S-methyl mandelate after the resolution of the methyl mandelate of the mixed rotation type is mainly enriched in the upper liquid layer, the R-mandelate after the resolution is mainly enriched in the middle liquid layer, and the middle liquid layer containing the enzyme can flow back to the reaction area again to continuously participate in the reaction. Finally, the reaction is maintained at 35% ee at 30hpThe value remained at 60%. The theoretical value of conversion rate of racemic methyl mandelate for traditional kinetic chiral resolution can only reach 50%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A device for splitting chiral substances by a multi-liquid-phase system continuous enzymatic method is characterized by comprising a reaction device and an evaporator; the reaction device is divided into a standing zone, a reaction zone and a dispersing buffer zone from top to bottom, the reaction zone and the standing zone and the reaction zone and the dispersing buffer zone are separated by a baffle, and the baffle is provided with a plurality of small holes communicated with the zones; 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 dispersing buffer zone is provided with an ester or amide compound injection port and a lower phase solution outlet.
2. The apparatus of claim 1, wherein a circulating water heating jacket is provided outside the reaction zone.
3. The apparatus of claim 2, wherein the lower liquid phase zone of the resting zone is provided with an autopotentiometric titrator burette and the reaction zone is provided with an autopotentiometric electrode tip.
4. The apparatus according to any one of claims 1 to 3, wherein the lower phase solution outlet is in communication with the second evaporator via an ultrafilter.
5. A method for continuously and enzymatically resolving chiral substances by using the device of any one of claims 1 to 4, which is characterized by comprising the following steps:
(1) preparing a double-liquid-phase solution from soluble salt, a hydrophilic solvent and water, standing and layering to obtain an upper-phase solution and a lower-phase solution, continuously introducing the lower-phase solution into a standing zone, passing through a reaction zone, and entering and filling a dispersion buffer zone; dissolving enzyme in the upper phase solution, and introducing the solution into a reaction zone; ester or amide compounds composed of racemic or single enantiomer chiral substances containing hydrophobic solvents are introduced into a dispersion buffer zone, the flow rate of feed liquid is adjusted to enable the ester or amide compounds to enter a reaction zone, and enzyme catalysis resolution reaction is carried out;
(2) and (3) automatically extracting the product after the catalytic reaction to a standing area, standing for layering, introducing the upper phase into an evaporator, and evaporating to remove the hydrophobic solvent to obtain the ester or amide product of the single optical rotation chiral product.
6. The method of claim 5, wherein the enzyme-containing phase extracted in step (2) into the standing zone is returned to the reaction zone via a return line to continue the reaction.
7. The method as claimed in claim 6, wherein the feeding and reaction processes in step (1) are carried out under continuous stirring and heating conditions, the heating temperature is 30-70 ℃, and the stirring speed is 100-1000 rpm; adjusting the pH value of the reaction system to 3-13; the mass ratio of the soluble salt to the hydrophilic solvent to the water is 0.1-1 and 0.1-5 respectively.
8. The method of claim 7, 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 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]BF4、[EMIM]ETSO4、[OMIM]Cl、[BMIM]PF6One or more than two of the above;
the enzyme comprises one or more than two of Lipase AYS, Lipase AY30, Lipase MAS1H108A, Lipase GAmano 50, Lipase CALB and Novozyme 435, and the enzyme concentration is 5-2000U/mL.
9. The process of claim 8, wherein the phase or lower phase of step (2) is ultrafiltered to remove the enzyme and passed into a second evaporator to remove water by evaporation to obtain another single optically active chiral product.
10. The method according to claim 9, wherein the ester or amide compound composed of racemic or single enantiomer chiral substances accounts for 0.1% -10% of the mass of the hydrophobic solvent;
the hydrophobic solvent is one or more than two of n-hexane, isopropyl ether, ethyl acetate, isooctane, petroleum ether, diethyl ether, benzene and toluene;
the ester or amide compound formed by the racemic or single enantiomer chiral substance comprises one or more than two of mixed methyl mandelate, mixed methyl ibuprofen, mixed naproxen methyl ester, mixed 1- (4-methoxyphenyl) ethanol acetate, R-methyl mandelate and S-methyl mandelate.
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