WO2020088510A1 - 一种(r)-邻氯扁桃酸的制备方法和用于该制备方法的一种循环式生物酶催化反应系统 - Google Patents

一种(r)-邻氯扁桃酸的制备方法和用于该制备方法的一种循环式生物酶催化反应系统 Download PDF

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WO2020088510A1
WO2020088510A1 PCT/CN2019/114309 CN2019114309W WO2020088510A1 WO 2020088510 A1 WO2020088510 A1 WO 2020088510A1 CN 2019114309 W CN2019114309 W CN 2019114309W WO 2020088510 A1 WO2020088510 A1 WO 2020088510A1
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reaction
resin
liquid
tank
preparation
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PCT/CN2019/114309
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English (en)
French (fr)
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赵涛涛
韩瑞
汪兆谱
张伟
王成
张琦
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武汉武药制药有限公司
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Priority claimed from CN201811277584.1A external-priority patent/CN110172401B/zh
Priority claimed from CN201811281559.0A external-priority patent/CN110184308A/zh
Priority claimed from CN201811415397.5A external-priority patent/CN110172021B/zh
Application filed by 武汉武药制药有限公司 filed Critical 武汉武药制药有限公司
Priority to EP19880056.7A priority Critical patent/EP3875594A4/en
Publication of WO2020088510A1 publication Critical patent/WO2020088510A1/zh

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    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/36—Means for collection or storage of gas; Gas holders
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02—Stirrer or mobile mixing elements
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/05—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in nitriles (3.5.5)
    • C12Y305/05001—Nitrilase (3.5.5.1)

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to a method for preparing (R) -o-chloromandelic acid.
  • the invention also relates to the field of biological enzyme catalysis equipment, in particular to a circulating biological enzyme catalytic reaction system used in the preparation method.
  • (R) -O-chloromandelic acid is a white crystalline powder with a chemical formula of C 8 H 7 ClO 3 , a molecular weight of 186.59, and a melting point of 86-92 ° C. It is an important pharmaceutical intermediate and fine chemical product.
  • Clopidogrel as a global blockbuster drug, is currently the best-selling drug on the market. With the expiration of the clopidogrel patent, there has been a wave of imitations around the world. Therefore, the demand for (R) -o-chloromandelic acid, which is an important intermediate, is growing at home and abroad. Therefore, how to efficiently and inexpensively prepare a large amount of (R) -o-chloromandelic acid with high purity will have important market value.
  • the ion exchange method uses a specific organic polymer resin to directly separate organic substances. It has the advantages of simple equipment, easy operation, short process flow, high selectivity, and easy resin regeneration.
  • the ion exchange resin is used to adsorb and separate o-chloromandelic acid.
  • the direct extraction of o-chloromandelic acid from the catalytic reaction system is mainly based on the different affinity of ion exchange resins for mandelic acid and impurities.
  • the affinity of ion exchange resin for o-chloromandelic acid depends on the physical and chemical properties of mandelic acid, the functional group characteristics of the resin, and the influence of other impurities and ions in the solution. Since o-mandelic acid is a weakly acidic organic acid, according to ion exchange theory It should be separated and extracted using alkaline resin.
  • the preparation method of the present invention adopts the step of separating and extracting (R) -o-chloromandelic acid by ion exchange method.
  • the present invention provides a circulating biological enzyme catalytic reaction system for the preparation method.
  • the present invention provides a method for preparing (R) -o-chloromandelic acid using the circulating biological enzyme catalytic reaction system.
  • the present invention relates to the following general inventive concept: after recycling (R) -o-chloromandelic acid using a reaction buffer, (R) -o-chloromandelic acid is extracted by using an ion exchange resin. Both the preparation and extraction processes are carried out in the circulating biological enzyme catalytic reaction system of the present invention.
  • the technical problem to be solved by the present invention is to significantly increase the utilization rate of the reaction buffer, thereby reducing the production cost of (R) -o-chloromandelic acid, and thus the production cost of clopidogrel.
  • the technical problem to be solved by the present invention is to improve the separation effect of o-chloromandelic acid, increase the product yield, increase the service life of strongly basic anion resin, reduce the production cost and reduce the discharge of waste water.
  • the object of the present invention is to provide a circulating biological enzyme catalytic reaction system to solve the problems in the prior art, simplify operation, and improve work efficiency.
  • a method for preparing (R) -o-chloromandelic acid which includes at least the following steps:
  • step (2) Extracting (R) -o-chloromandelic acid in the supernatant to obtain a flow-through liquid, optionally the flow-through liquid is recycled as the reaction buffer at least once in step (2), It is preferably 5 to 20 times, more preferably 6 to 18 times, and still more preferably 8 to 15 times.
  • the pH of the reaction buffer is 7.0-9.0;
  • the reaction buffer is selected from at least one of KH 2 PO 4 / K 2 HPO 4 buffer, NaH 2 PO 4 / Na 2 HPO 4 buffer or tris-hydroxymethylaminomethane-HCl buffer;
  • the pH of the reaction system in step (2) is 7.5-8.5.
  • the reaction temperature of the conversion reaction is 25-50 ° C, preferably 25-45 ° C, and more preferably 30 ° C.
  • the concentration of the engineered bacterial cells is 10 g / L to 40 g / L;
  • a co-solvent is further added to the reaction system in step (2); the co-solvent is preferably at least one of an alcohol having 1 to 8 carbon atoms and an alkane having 6 to 12 carbon atoms; preferably, a co-solvent
  • the amount added is 1% to 10% of the total volume of the reaction system.
  • the substrate is fed in batches 5 to 7 times, preferably 6 times; the concentration of the substrate in each feed is 10 to 50 mM, preferably 20 to 45 mM, more Preferably it is 25-30 mM.
  • the separation method is selected from at least one of centrifugation, suction filtration, and pressure filtration;
  • the centrifugation adopts 6000-10000rpm, preferably 6000-8000rpm, more preferably 7000rpm.
  • the extraction method uses an anionic resin for adsorption, preferably a strongly basic anionic resin.
  • the preparation method further includes the following steps:
  • the organic solvent is preferably ethyl acetate or chloroform; the separation method is preferably rotary evaporation.
  • the step of extracting (R) -o-chloromandelic acid includes at least the following steps:
  • the pretreatment includes the steps of alkali washing and acid washing in sequence;
  • the strongly basic anion resin is preferably a quaternary ammonium salt strongly basic anion resin;
  • the eluent is used to elute the strongly basic anion resin of step (ii) to obtain an eluent, and the eluent is extracted and concentrated to obtain the (R) -o-chloromandelic acid.
  • the step of alkaline washing includes: soaking the strongly alkaline anion resin with an alkaline solution with a concentration of 0.5 to 5 mol / L for 10 to 60 minutes, and then using the alkaline solution to 1.0 to Wash at a speed of 5.0ml / min;
  • the ratio of the volume of the alkaline solution to the mass of the strongly basic anion resin during soaking is 2 to 5: 1; Calculated in Kg, or preferably, the volume of the alkaline solution is measured in mL, and the mass of the strongly basic anion resin is calculated in g;
  • the base is selected from monobasic bases.
  • the acid washing step includes first soaking the strongly basic anion resin with an acid solution having a concentration of 0.5 to 5 mol / L for 15 to 30 minutes, and then using the acid solution to 1.0 to Wash at a speed of 5.0ml / min;
  • the ratio of the volume of the acid solution to the mass of the strongly basic anion resin during soaking is 2 to 5: 1; preferably, the volume of the acid solution is measured in L, and the mass of the strongly basic anion resin Calculated in Kg, or preferably, the volume of the acid solution is measured in mL, and the mass of the strongly basic anion resin is calculated in g;
  • the acid is selected from monoacids.
  • the acid washing further includes a step of washing to neutrality; preferably, the alkali washing further includes a step of washing away mechanical impurities with water;
  • the water is selected from purified water.
  • the step of infiltration further includes a step of infiltration.
  • the step of infiltration includes: adding a buffer solution to the strongly basic anion resin to infiltrate for 10 to 30 minutes.
  • the volume of the buffer solution is 2 to 5 times the mass of the strongly basic anion resin; the pH value of the buffer solution is preferably 7 to 9.
  • the volume of the buffer solution is counted in L, and the mass of the strong basic anion resin is calculated in Kg, or preferably, the volume of the buffer solution is measured in mL, and the mass of the strong basic anion resin is in g meter.
  • the eluted strong basic anion resin is washed to neutrality and then enters step (ii) for recycling.
  • the strong basic anion resin is regenerated; preferably , The regeneration process includes at least one of the following ways:
  • Method 1 Soak the mixed alkaline solution for 10 to 60 minutes, and then wash at a rate of 1.0 to 5.0 ml / min; the mixed alkaline solution contains a monobasic alkali with a concentration of 2 to 5 mol / L and a mass percentage concentration of 10% to 30% sodium chloride; preferably, the ratio of the volume of the mixed alkaline solution to the mass of the strongly basic anion resin is 2 to 5: 1; preferably, the volume of the mixed alkaline solution is measured by L, the The mass of the strong basic anion resin is Kg, or preferably, the volume of the mixed alkaline solution is measured in mL, and the mass of the strong basic anion resin is g.
  • Method 2 First soak in mixed acid solution for 10 to 60 minutes, and then wash at a rate of 1.0 to 5.0 ml / min; the mixed acid solution contains 2 to 5 mol / L of monobasic acid and the volume percentage concentration is 10% to 50% Lower alcohol; preferably, the lower alcohol is selected from alcohols having 1 to 6 carbon atoms; more preferably, the ratio of the volume of the mixed acid solution to the mass of the strongly basic anion resin is 2 to 5: 1; Preferably, the volume of the mixed acid solution is counted in L, and the mass of the strong basic anion resin is calculated in Kg, or preferably, the volume of the mixed acid solution is measured in mL. The mass is in g.
  • the regeneration treatment further includes a step of washing to neutrality with water, and the water is preferably purified water.
  • the adsorption temperature is 10-60 ° C;
  • the elution temperature is 10-60 ° C; preferably, the organic solvent used for the extraction is at least one selected from ester organic solvents, ether organic solvents, and alcohol organic solvents ; More preferably, ester organic solvents having 4 to 8 carbon atoms, ether organic solvents having 2 to 6 carbon atoms, and alcohol organic solvents having 1 to 8 carbon atoms; more preferably ethyl acetate, ether, At least one of ethanol.
  • step (ii) the flow rate of the adsorption is 0.1-10 ml / min; in step (iii), the flow rate of the elution is 0.1-10 ml / min.
  • the liquid to be treated is an aqueous phase reaction liquid or an aqueous phase reaction liquid containing an organic phase miscible with water;
  • the liquid to be treated includes a reaction liquid obtained by chemical synthesis, biocatalytic synthesis, and resolution synthesis;
  • the pH of the liquid to be treated is 2-8.
  • the reaction liquid after the adsorption in step (ii) is recycled as the reaction liquid of the biocatalytic reaction system.
  • a circulating biological enzyme catalytic reaction system used in the preparation method of the present invention which includes a circulating fixed immobilized reaction bed and a resin column connected in sequence Upstream temporary storage tank, resin column body, resin column downstream temporary storage tank;
  • the circulating fixed bed includes:
  • the pre-stirring device set above the reaction tank, the temporary storage tank downstream of the resin column is also connected to the pre-stirring device;
  • a spraying device provided between the pre-stirring device and the reaction tank, the spraying device extending into the top of the reaction tank for spraying the reaction liquid to the reaction tank;
  • At least one reaction plate arranged inside the reaction tank at intervals to divide the inside of the reaction tank into a plurality of reaction spaces, and the inside of the reaction plate is filled with a catalyst;
  • the bottom stirring device provided at the bottom of the reaction tank is connected to the temporary storage tank upstream of the resin column, and the bottom of the reaction tank is also connected to the pre-stirring device.
  • the pre-mixing device includes:
  • the mixing paddle set inside the mixing tank and the mixing motor set outside the mixing tank are connected to the mixing paddle;
  • the addition funnel set on the mixing tank.
  • the bottom stirring device includes a stirring paddle provided inside the reaction tank and a stirring motor provided outside the reaction tank, and the stirring motor is connected to the stirring paddle.
  • the circulating biological enzyme catalytic reaction system further includes an exhaust gas collection device, a power pump, and a negative-pressure extraction pipe.
  • the reaction tank at each reaction space is provided with an exhaust hole, and the exhaust gas collection device passes through the power pump,
  • the negative pressure suction pipe is connected to the suction hole of each reaction space.
  • a filling cavity for accommodating a catalyst is provided inside the reaction plate, and a removable sand core screen is provided on the upper and lower surfaces of the filling cavity.
  • the reaction plate is detachably connected to the side wall of the stirring tank.
  • the temporary storage tank upstream of the resin column, the main body of the resin column, and the temporary storage tank downstream of the resin column are connected with a vacuum tube, a compressed air tube, and a temperature probe.
  • the reaction tank at each reaction space is provided with an observation window.
  • a temperature control jacket is provided outside the stirring tank, the temporary storage tank upstream of the resin column, the main body of the resin column, and the temporary storage tank downstream of the resin column.
  • a method for preparing (R) -o-chloromandelic acid using the circulating biological enzyme catalytic reaction system according to the present invention includes at least The following steps:
  • reaction liquid reaches the bottom layer of the reaction tank (1), is transported to the pre-stirring device (2) under the action of the stirring blade (21), and then continues to be sprayed into the reaction tank (1); preferably, the above (The steps a) to (c) are performed in order 1 to 20 times; preferably 5 to 20 times, more preferably 6 to 18 times, and more preferably 8 to 15 times;
  • step (e) extracting (R) -o-chloromandelic acid in the supernatant to obtain a flow-through liquid, optionally the flow-through liquid is recycled as the reaction buffer at least once in step (b), It is preferably 5 to 20 times, more preferably 6 to 18 times, and even more preferably 8 to 15 times.
  • the reaction liquid is introduced into the temporary storage tank (9) upstream of the resin column, and the ion exchange resin pretreatment is performed in advance in the resin column body (6), and then the upstream of the resin column is temporarily
  • the reaction liquid in the storage tank (9) is sent to the resin column body (6) for adsorption, and flows out of the resin column body (6) into the downstream storage tank (7) of the resin column to become a flow-through liquid; the flow-through The liquid is sent to the pre-stirring device (2) as a buffer solution.
  • the eluent is used to enter the resin column body (6) to elute the resin, the eluent is collected, and then extracted and concentrated to obtain (R ) -O-chloromandelic acid finished product.
  • the product in the supernatant is separated to obtain a flow-through liquid containing a very small amount of product.
  • the present invention significantly improves the utilization of the reaction buffer by directly using the resulting flow-through liquid as a buffer solution in the conversion reaction. Rate, thereby reducing the production cost of (R) -o-chloromandelic acid, and thus reducing the production cost of clopidogrel, will produce good economic and social benefits.
  • the method of the invention significantly reduces the discharge of waste liquid, and realizes the green and environmentally friendly production of (R) -o-chloromandelic acid, which is of great significance for the industrialization of (R) -o-chloromandelic acid, and can also improve the biological and chemical industries in China.
  • the production level of chiral drugs in the fields of medicine, medicine and other fields is conducive to promoting the development of green production and industrial biotechnology in China, and is of great significance to the sustainable development of our economy.
  • the separation effect is good, the equipment is simple, the operation is simple, and the yield of the product obtained is high; and the strong basic anion resin used has a long service life (it can continue to be used after regeneration treatment).
  • the water phase system can continue to provide a reaction system for the next biocatalysis, greatly reducing production costs and reducing waste water discharge.
  • the circulating biological enzyme catalytic reaction system provided by the present invention has the following beneficial effects:
  • the reaction plate of the embodiment of the present invention is filled with a catalyst, so that the reaction plate filled with the enzyme catalyst can be prepared in advance, and the reaction plate and the reaction tank of the circulating fixed reaction bed are detachably connected to make the catalyst It is easy to replace, and the reaction plates of the previous batch are taken out and quickly replaced, which greatly saves time and simplifies the operation;
  • Circulating fixed reaction bed, resin column upstream storage tank, resin column body, resin column temporary storage tank can be equipped with multiple groups, which can quickly switch the reaction liquid pipeline when replacing the catalyst or ion exchange resin To the new circulating fixed reaction bed or resin column, the catalytic reaction will not be interrupted;
  • the present invention adopts the "dual reaction liquid system", which can ensure that the reaction liquid enters the product separation and purification device composed of the upstream storage tank of the resin column, the main body of the resin column, and the temporary storage tank downstream of the resin column from the circulating fixed reaction bed.
  • “Substitute reaction liquid” enters the circulating fixed reaction bed to avoid equipment idle, effectively improve production efficiency;
  • the reaction liquid enters the circulating fixed reaction bed from the product separation and purification device, there are also “substitute reaction liquid” entering the product Separation and purification device to avoid idle equipment and improve the space-time yield;
  • the present invention is provided with an exhaust gas collection device for volatile substances, which is treated according to relevant technical standards after collection to meet the needs of enterprises for clean production and green catalysis;
  • the present invention is easy to operate, and the related equipment has a simple structure without dead corners, which is convenient for cleaning after production, which greatly reduces the labor intensity of the operator and is conducive to improving production efficiency.
  • FIG. 1 is a front view of a circulating biological enzyme catalytic reaction system provided by an embodiment of the present invention.
  • reaction tank 11- reaction plate, 12- observation window, 13- circulation pipeline, 2- pre-mixing device, 21- stirring blade, 22- stirring motor, 23- feeding funnel, 3- spray device, 4- control Warm jacket, 5-negative pressure extraction pipeline, 51-power pump, 6-resin column body, 7-resin column temporary storage tank, 81-vacuum tube, 82-compressed air tube, 83-temperature probe, 9-resin Temporary storage tank upstream of the column.
  • An embodiment of the present invention relates to a method for preparing (R) -o-chloromandelic acid.
  • the engineered bacteria obtained through cultivation serve as a catalyst, o-chloromandenitrile as a substrate, and reaction buffer as a reaction medium for reaction; after the reaction is completed , Separate and collect the supernatant; extract (R) -o-chloromandelic acid in the supernatant to obtain the flow-through liquid, which contains a very small amount of product.
  • the reaction buffer is used to recycle the reaction cycle The number of times can reach more than 10 times, so the buffer utilization rate of the embodiment of the present invention is increased by more than 10 times compared with the same amount of reaction buffer.
  • the preparation method of the embodiment of the present invention includes at least the following steps:
  • the E. coli engineering bacteria expressing nitrilase used in step (1) may use commercially available strains, and preferably adopt the engineering bacteria constructed in the method disclosed in application number CN201510978973.7.
  • the culturing in step (1) includes shaking flask culture or fermentor culture, and centrifuging to collect engineered bacterial cells.
  • a co-solvent may be added to the reaction system in step (2); specifically, the co-solvent may be selected from at least one of an alcohol having 1 to 8 carbon atoms and an alkane having 6 to 12 carbon atoms Specific; can be selected from ethanol, methanol, n-hexane, n-propanol, isopropanol, n-butanol, acetonitrile, n-hexanol, n-pentanol, n-octanol and other organic solvents, co-solvent The amount added is 1% to 10% of the total volume of the reaction system.
  • the flow-through liquid is used as the reaction buffer in step (2) for 5 to 20 times. If the number of cycles is too high, the ionic strength and other parameters in the buffer change significantly, which is not conducive to the normal reaction; if the number of cycles Too little, the reutilization rate of the reaction buffer is low, the product output ratio of the buffer volume per unit volume is low, and the advantage of reducing wastewater discharge is not obvious.
  • the flow-through liquid is used for 6 to 18 times, and more preferably, the flow-through liquid is used for 8 to 15 times.
  • Steps (3) and (4) in the embodiment of the present invention will cause a reduction in the volume of the reaction system, and a new reaction buffer is added to make up for the lost volume, thereby further improving the yield of the product.
  • step (2) the pH of the reaction buffer is 7.0-9.0.
  • the reaction buffer may be selected from at least one of KH 2 PO 4 / K 2 HPO 4 buffer, NaH 2 PO 4 / Na 2 HPO 4 buffer or tris-hydroxymethylaminomethane-HCl buffer, and Not limited to this.
  • the pH of the reaction system in step (2) is 7.5-8.5.
  • 1 ⁇ 2M HCl and 1 ⁇ 2M NaOH can be used to adjust the pH value of the reaction system during the reaction.
  • the reaction temperature of the conversion reaction is 25-50 ° C
  • the upper limit of the reaction temperature of the conversion reaction may be 50 ° C, 48 ° C, 45 ° C, 42 ° C, 40 ° C, 38 ° C
  • the lower limit of the reaction temperature of the reaction may be 25 ° C, 28 ° C, 30 ° C, and 35 ° C, and the temperature range may be composed of any value in the upper limit and the lower limit.
  • the reaction temperature of the conversion reaction is preferably 25 to 45 ° C, and more preferably 30 ° C.
  • the concentration of the engineered bacterial cells is 10 g / L to 40 g / L.
  • the upper limit of the concentration of engineered bacterial cells can be 40g / L, 35g / L, 30g / L, 25g / L, 22g / L, and the lower limit of the concentration of engineered bacterial cells can be 10g / L, 12g / L, 15g / L, 18g / L, 20g / L; the range of the concentration of engineered bacterial cells can be composed of any value in the upper and lower limits.
  • step (2) the substrate is fed in batches 5 to 7 times, preferably 6 times. If the number of substrate additions is too many, the reaction rate will decrease significantly, and a large amount of impurities such as aldehyde and substrate will accumulate, reducing the reaction yield; if the substrate additions are too low, the product concentration accumulated by the system will be low.
  • the catalytic potential of the enzyme catalyst in the system has not been fully realized, resulting in economic waste.
  • the substrate concentration is 10-50mM for each feeding. If the substrate concentration is too high, it will cause irreversible deactivation of the enzyme catalyst; if the substrate concentration is too low, the product enantiomeric excess will be low, and the system product concentration accumulation rate is too slow, resulting in waste of post-treatment costs.
  • the upper limit of the substrate concentration in each feeding can be 50 mM, 48 mM, 45 mM, 40 mM, 35 mM, 30 mM, and the lower limit of the substrate concentration in each feeding can be 10 mM, 12 mM, 15 mM, 20 mM, 22 mM, 25 mM.
  • the range of substrate concentration for each feeding can be composed of any value in the upper and lower limits.
  • the substrate concentration per feed is 20-45 mM, more preferably 25-30 mM.
  • the substrate concentration when the substrate concentration is 25 mM for each feeding, when the substrate is added 6 times for each conventional reaction flow, the substrate catalytic concentration can reach 1500 mM, which can significantly improve the product output ratio of the buffer.
  • step (2) the catalyst, the substrate, the reaction buffer and the co-solvent are uniformly mixed by magnetic stirring.
  • the separation method is selected from at least one of centrifugation, suction filtration, and pressure filtration; and preferably centrifugation.
  • 6000 to 10000 rpm is used for centrifugation, preferably 6000 to 8000 rpm, and more preferably 7000 rpm.
  • the extraction method is to use an anionic resin for adsorption, preferably a strongly basic anionic resin.
  • an anionic resin for adsorption preferably a strongly basic anionic resin.
  • Specific options include HZ-202 or 711 resin.
  • the preparation method of the embodiment of the present invention further includes the following steps:
  • the eluent is 1 ⁇ 2M hydrochloric acid
  • the organic solvent is selected from ethyl acetate or chloroform;
  • the method of separation is rotary evaporation.
  • Another embodiment of the present invention provides a method for separating and extracting (R) -o-chloromandelic acid in the preparation method of (R) -o-chloromandelic acid of the present invention.
  • Strongly basic anion resin with strong affinity and strong adsorption capacity for mandelic acid adsorbs (R) -o-chloromandelic acid.
  • the separation and extraction method according to the embodiment of the present invention includes the following steps:
  • Pretreatment of strongly basic anion resin includes the steps of washing mechanical impurities with water, alkaline washing, acid washing and water washing to neutral, preferably:
  • the steps of alkaline washing are as follows: firstly soak the strong alkaline anion resin with an alkaline solution with a concentration of 0.5 to 5 mol / L for 10 to 60 min, and then wash with the alkaline solution at a rate of 1.0 to 5.0 ml / min until all the liquid flows;
  • the base is selected from monobasic bases, and more preferably, the base is selected from NaOH solution and KOH solution;
  • the concentration of the lye used is too low, the resin will not be converted to OH type. If it is too high, the amount of lye will increase, resulting in an increase in cost. If it is not fully converted, if the time is too long, it will increase the time of the process and is not suitable for industrial production.
  • the acid washing step includes: soaking the strongly basic anion resin with an acid solution with a concentration of 0.5 to 5 mol / L for 15 to 30 minutes, and then washing with the acid solution at a rate of 1.0 to 5.0 ml / min until all the liquid flows down ;
  • the acid is selected from monobasic acids, more preferably, the acid solution is selected from dilute hydrochloric acid;
  • the concentration of the acid solution used is too low, the resin will not be converted to Cl type. If it is too high, the amount of acid solution will increase and the cost will increase. If it is not fully converted, if the time is too long, it will increase the time of the process and is not suitable for industrial production.
  • step (ii) Adopting the strongly basic anion resin treated in step (i) to adsorb (R) -o-chloromandelic acid to be treated by dynamic adsorption; wherein, the (R) -o-chloromandelic acid
  • the liquid to be treated is the supernatant containing (R) -o-chloromandelic acid;
  • step (3) The eluent is used to elute the strongly basic anion resin of step (2) to obtain an eluent. After the eluent is extracted and concentrated, the crude product of (R) -o-chloromandelic acid is obtained. After purification, (R) -o-chloromandelic acid is obtained.
  • the strongly basic anion resin is selected from quaternary ammonium salt strongly basic anion resins, and preferably HZ-202 type resins.
  • the ratio of the volume of the alkali solution to the mass of the strongly basic anion resin during soaking is 2 to 5: 1
  • the ratio of the volume of the acid solution to the mass of the strongly basic anion resin during soaking is 2 to 5: 1.
  • the unit of the volume of the lye or acid solution is L
  • the unit of the mass of the strongly basic anion resin is Kg
  • the unit of the mass of the strongly basic anion resin is g.
  • the step of water washing uses purified water for washing.
  • the step of infiltration is further included before adsorption.
  • the step of infiltration includes: adding a buffer solution to the strongly basic anion resin to infiltrate for 10 to 30 minutes, and the volume of the buffer solution is strongly alkaline 2 to 5 times the mass of anionic resin.
  • the volume of the buffer solution is counted in L, and the mass of the strong basic anion resin is calculated in Kg, or preferably, the volume of the buffer solution is measured in mL, and the mass of the strong basic anion resin is in g meter.
  • the pH value of the buffer solution is 7-9.
  • the buffer solution has the same pH value as the buffer solution in the to-be-treated solution.
  • the buffer can be a biocatalyzed buffer, such as KH 2 PO 4 / K 2 HPO 4 buffer, NaH 2 PO 4 / Na 2 HPO 4 buffer.
  • the eluted strong basic anion resin is washed to neutrality and then enters step (ii) for recycling, thereby greatly reducing production costs.
  • the absorption efficiency of the strongly basic ion exchange resin decreases significantly after 15-20 cycles of recycling, the resin should be regenerated.
  • the regeneration includes at least one of the following methods:
  • Method 1 Soak the mixed alkaline solution for 10 to 60 minutes, and then wash at a rate of 1.0 to 5.0 ml / min until all the liquid flows down; the mixed alkaline solution contains a monobasic alkali with a concentration of 2 to 5 mol / L and a mass percentage concentration of 10 % To 30% sodium chloride; preferably, the ratio of the volume of the mixed alkaline solution to the mass of the strongly basic anion resin is 2 to 5: 1; when the unit of the mixed alkaline solution volume is L, the unit of the resin mass is Kg , Or when the unit of the volume of the mixed lye is mL, the unit of the resin mass is g.
  • Method 2 First soak in mixed acid solution for 10 to 60 minutes, then wash at 1.0 to 5.0 ml / min until all liquid flows down; mixed acid solution contains 2 to 5 mol / L of monobasic acid and volume percentage concentration is 10% ⁇ 50% lower alcohol; preferably, the lower alcohol is selected from alcohols having 1 to 6 carbon atoms; more preferably, the ratio of the volume of the mixed acid solution to the mass of the strongly basic anion resin is 2 to 5: 1; the mixed acid When the unit of liquid volume is L, the unit of resin mass is Kg; or when the unit of mixed acid liquid volume is mL, the unit of resin mass is g.
  • the regeneration treatment further includes a step of washing to neutrality, and the water is preferably purified water.
  • either of the method 1 and method 2 may be used, or the method 1 may be used first, and then the method 2 may be used. Treating twice is equivalent to regenerating twice, washing away the residual organic impurities in the resin, which is more conducive to improving the performance of the resin.
  • the adsorption temperature is 10-60 ° C, preferably 15-50 ° C.
  • the elution temperature is 10-60 ° C, preferably 15-50 ° C.
  • the organic solvent used for the extraction is at least one selected from ester organic solvents, ether organic solvents, and alcohol organic solvents; preferably ester organic solvents having 4 to 8 carbon atoms and 2 carbon atoms
  • the adsorption flow rate is 0.1 to 10 ml / min, preferably 1 to 9 ml / min, and more preferably 2 to 8 ml / min.
  • the elution flow rate is 0.1 to 10 ml / min, preferably 1 to 9 ml / min, and more preferably 2 to 8 ml / min.
  • the liquid to be treated is an aqueous phase reaction liquid or an aqueous phase reaction liquid containing an organic phase miscible with water; specifically, the liquid to be treated may be selected from the reaction liquids obtained by chemical synthesis, biocatalytic synthesis, and resolution synthesis Not limited to this.
  • the liquid to be treated may be selected from the reaction liquid obtained by biocatalytic synthesis.
  • the reaction liquid after the adsorption in step (ii) can be recycled as the reaction liquid of the biocatalytic reaction system, thereby greatly reducing the discharge of waste water.
  • the pH of the strongly basic anion resin should be consistent with the most suitable pH for the resin adsorption (R) -o-chloromandelic acid, and the most suitable pH for the strongly basic anion resin adsorption (R) -o-chloromandelic acid is 2 ⁇ 8. Therefore, formulating the liquid to be adsorbed into a solution with a pH of 2 to 8 and then adsorbing it can further improve the adsorption efficiency.
  • the concentration method may be concentration under reduced pressure, and the specific conditions are: 45-55 ° C, 0.09 MPa; preferably 50 ° C, 0.09 MPa.
  • the maximum amount of (R) -o-chloromandelic acid in the liquid to be extracted should not be greater than the minimum exchange capacity of the strongly basic anion resin, that is, the required resin should be in excess.
  • the liquid to be extracted contains at least one of (R) -o-chloromandelic acid, (S) -o-chloromandelic acid, and (R, S) -o-chloromandelic acid.
  • the specific operation steps of the method for separating and extracting (R) -o-chloromandelic acid using a strong basic anion resin in the embodiments of the present invention are as follows:
  • Pretreatment of strong basic anion resin Take a certain amount of strong basic anion resin (aqueous) in the exchange column, wash away mechanical impurities with purified water, add 0.5 ⁇ 5M NaOH solution to soak for 15min, the volume and strength of NaOH solution The mass ratio of basic anion resin is 2 ⁇ 5: 1, washed with 0.5 ⁇ 5M NaOH solution at 1.0 ⁇ 5.0ml / min, then add 0.5 ⁇ 5M dilute hydrochloric acid to soak, the volume of dilute hydrochloric acid solution and strong alkaline anion resin The mass ratio is 2 ⁇ 5: 1, washed with 0.5 ⁇ 5M dilute hydrochloric acid at 1.0 ⁇ 5.0ml / min, and finally washed with purified water until neutral.
  • the adsorption temperature is 10 to 60 ° C, and the static adsorption time is 2 to 6 hours.
  • the dynamic adsorption time is 10-20min; among them, the static adsorption can obtain the maximum adsorption amount of the resin, the measurement is the most accurate, but the operation is difficult, the resin cannot be operated repeatedly, and is not suitable for industrial production; dynamic adsorption increases the possibility of experiment Operable, repeatable operation, but the amount of adsorption will be less than static adsorption.
  • the adsorbed reaction liquid flows down from the exchange column at 0.1-10 ml / min; the adsorbed reaction liquid is recycled as the reaction liquid of the biocatalytic reaction system;
  • Resin desorption and elution use 1M dilute hydrochloric acid as the eluent to elute the resin, the elution temperature is 10 ⁇ 60 °C, use 5 times the volume of the strong alkaline anion resin mass of the eluent to 0.1 ⁇ 10ml / min for elution and desorption, so that the product (R) -o-chloromandelic acid is desorbed by the eluent and enters the eluent;
  • an embodiment of the present invention provides a circulating biological enzyme catalytic reaction system used in the preparation method of the present invention, including a serially connected circulating immobilized reaction bed, a temporary storage tank 9 upstream of a resin column, and a resin column Body 6, temporary storage tank 7 downstream of the resin column;
  • the circulating fixed bed includes:
  • the pre-stirring device 2 provided above the reaction tank 1, the temporary storage tank 7 downstream of the resin column is also connected to the pre-stirring device 2;
  • a spraying device 3 provided between the pre-stirring device 2 and the reaction tank 1, the spraying device 3 extends to the inside of the top of the reaction tank 1 for spraying the reaction liquid to the reaction tank 1;
  • At least one reaction plate 11 disposed inside the reaction tank 1 at intervals to divide the inside of the reaction tank 1 into a plurality of reaction spaces, and the inside of the reaction plate 11 is filled with a catalyst;
  • the bottom stirring device provided at the bottom of the reaction tank 1 is connected to the temporary storage tank 9 upstream of the resin column, and the bottom of the reaction tank 1 is also connected to the pre-stirring device 2.
  • the pre-mixing device 2 includes:
  • a mixing tank connected to the spray device 3;
  • the stirring paddle 21 provided inside the stirring tank and the stirring motor 22 provided outside the stirring tank are connected to the stirring paddle 21;
  • the addition funnel 23 provided on the stirring tank.
  • the bottom stirring device includes a stirring paddle 21 provided inside the reaction tank 1 and a stirring motor 22 provided outside the reaction tank 1.
  • the stirring motor 22 is connected to the stirring paddle 21.
  • the circulating biological enzyme catalytic reaction system further includes an exhaust gas collection device, a power pump 51, and a negative-pressure extraction pipe 5.
  • the reaction tank 1 at each reaction space is provided with an extraction hole, and the exhaust gas collection device passes through
  • the power pump 51 and the negative-pressure suction pipe 5 are connected to the suction hole of each reaction space.
  • the reaction plate 11 is provided with a filling cavity for accommodating the catalyst, and the upper and lower surfaces of the filling cavity are provided with a removable sand core screen.
  • the reaction plate 11 is detachably connected to the side wall of the stirring tank.
  • a drawer-type detachable connection can be used.
  • the temporary storage tank 9 upstream of the resin column, the main body 6 of the resin column, and the temporary storage tank 7 downstream of the resin column are connected to a vacuum tube 81, a compressed air tube 82, and a temperature probe 83.
  • an observation window 12 is provided on the reaction tank 1 at each reaction space.
  • a temperature control jacket 4 is provided outside the stirring tank, the resin column upstream storage tank 9, the resin column body 6, and the resin column downstream storage tank 7.
  • the reaction tank 1 has a cylindrical tank structure, and the tank top and tank bottom have smooth curved hemispherical structures.
  • the top of the reaction tank 1 is provided with a pre-mixing device 2 for the feed liquid, the top side of the pre-mixing device 2 has an addition funnel 23 and a stirring motor 22, the top of the reaction tank 1 has a reaction liquid inlet and a spray device 3, and a control is provided outside the reaction tank 1
  • the temperature jacket 4 is provided with a negative-pressure suction pipe 5 and a suction hole outside the reaction tank 1, and the negative pressure is provided by the power pump 51.
  • the reaction tank 1 is a multi-stage "steamer" structure, and is connected by a mechanism between the tank bodies Connected in series, the reaction tank 1 has an observation window 12 on the side, a reaction plate 11 filled with immobilized enzyme is horizontally arranged inside the reaction tank 1, and a stirring paddle 21 is provided at the bottom center of the reaction tank 1
  • the stirring paddle 21 is powered by a stirring motor 22 placed outside the reaction tank 1.
  • a material sampling tube is provided at the bottom of the reaction tank 1.
  • the reaction tank 1 and the material-liquid pre-stirring device 2 are connected by an external circulation pipe of the material tank 13 and the power pump 51 are connected to form a circulation path, and the bottom of the reaction tank 1 is connected to the upstream storage tank 9 of the resin column by a material pipeline.
  • a vacuum tube 81, a compressed air tube 82, and a temperature are provided above the temporary storage tank 9 upstream of the resin column Probe 83, with temperature control jacket 4 on the side, bottom There is a sampling tube, and the bottom and the resin column body 6 are connected by a material pipe.
  • a vacuum tube 81, a compressed air tube 82, a temperature probe 83 are also arranged above the resin column body 6, a temperature control jacket 4 is provided on the side, and a sampling tube is provided on the bottom
  • the resin column body 6 and the resin column downstream temporary storage tank 7 are connected by a material pipeline.
  • the resin column downstream temporary storage tank 7 is provided with a vacuum tube 81, a compressed air tube 82, a temperature probe 83, and a temperature control side
  • the jacket 4 is provided with a sampling tube at the bottom, and a material communication pipe is provided between the temporary storage tank 7 downstream of the resin column and the material-liquid pre-stirring device 2 outside the top layer of the reaction tank 1.
  • the whole casing system is controlled by the valve to control the direction of the material.
  • the valve can be matched with the switch to make the material circulate in the catalytic reactor or enter the purification system for product purification.
  • the reaction tank 1 may adopt a detachable multi-stage series structure.
  • a connection mechanism between tank bodies is used to connect the units in series to form a multi-stage "steamer" structure.
  • the shell of the circulating fixed reaction bed that is, the reaction tank 1 is a cylinder, and the top and bottom of the tank are smooth curved hemispherical structures without dead angles, which is convenient for cleaning after the reaction.
  • the reaction tank 1 is The multi-level "steamer" structure is connected in series by the connecting mechanism, which is convenient for the rapid disassembly and maintenance of equipment.
  • reaction liquid pre-stirring device 2 outside the top layer of the reaction tank 1, which can mix the materials evenly.
  • the mixed materials are evenly distributed on the reaction plate 11 through the spray device 3 inside the top layer of the reaction tank 1, so that the catalyst can evenly contact the materials.
  • the bottom surface of the reaction plate 11 is a sand core screen structure, the reaction plate 11 can be filled with immobilized large particle enzyme catalyst (hereinafter referred to as immobilized enzyme), and the bottom surface of the reaction plate 11 is detachable structure, which is convenient for enzyme replacement and cleaning, etc.
  • the side of the reaction plate 11 is a water-tight and corrosion-resistant soft sealing mechanism, which can be attached to the reaction tank 1, and the reaction plate 11 is fixed by a locking device in the slot of the reaction tank 1.
  • the side of the reaction tank 1 has a negative-pressure suction pipe 5, through the negative-pressure power pump 51, the volatile substances generated by the catalytic reaction can be taken away from the reaction tank 1, which is beneficial to promote the catalytic reaction; the extracted volatile substances enter the special exhaust gas
  • the centralized processing of the collection device is in line with the concept of cleaner production.
  • the bottom of the reaction tank 1 has a bottom stirring device, which can mix the liquid after the catalytic reaction evenly, and introduce it into the pre-stirring device 2 on the top to participate in the next cycle reaction.
  • a circulation pipe 13 of the reaction liquid and a circulation pump on the circulation pipe 13 At the bottom of the reaction tank 1, there is a circulation pipe 13 of the reaction liquid and a circulation pump on the circulation pipe 13, and the reaction liquid ascends through the circulation pipe 13 into the pre-stirring device 2, and the reaction liquid can flow downstream through the spray device 3 to enter the next cycle
  • the benefit of the reaction liquid circulation reaction is that firstly it avoids the mechanical damage to the enzyme catalyst by the stirring device during the traditional stirring reaction process; secondly, it facilitates the separation of the enzyme catalyst from the reaction liquid; in addition, the reaction to volatile substances facilitates Collect volatile substances for centralized processing.
  • the present invention also provides a continuous product separation device.
  • a resin separation system is used, that is, the resin separation system includes a temporary storage tank 9 upstream of the resin column, a resin column body 6, and a temporary storage tank 7 downstream of the resin column.
  • TLC, HPLC and other detection methods are used to ensure that the product concentration of the reaction solution reaches the established separation standard.
  • the upward valve of the circulation pipe 13 at the bottom of the reaction tank 1 can be closed, the pipe valve connected to the resin separation system can be opened, and the vacuum tube 81 of the temporary storage tank 9 upstream of the resin column can be used to draw the reaction liquid into the temporary storage tank 9 upstream of the resin column.
  • the pipeline valve connected to the resin separation system is closed, and the reaction hydraulic pressure in the temporary storage tank 9 upstream of the resin column is compressed into the resin column body 6 by using the compressed air pipe 82.
  • the reaction solution drops out of the resin column body 6 at a uniform rate and enters the temporary storage tank 7 downstream of the resin column.
  • the reaction liquid from which the product is separated in the temporary storage tank 7 downstream of the resin column can enter the reaction liquid pre-stirring device 2 outside the top layer of the reaction tank 1 through the pipeline again. After adding the reaction substrate, it can participate in the next batch of cyclic reaction after stirring.
  • the first full buffer is passed through the reaction liquid pre-stirring device 2, and the substrate is added to stir to form a substrate dispersion, which enters the reaction tank 1 (the reaction plate 11 contains presets Immobilized enzyme), using the reaction liquid circulation pipeline 13 at the bottom of the reaction tank 1 to achieve a closed-loop reaction.
  • the buffer enters the resin column Upstream temporary storage tank 9.
  • the second full amount of buffer solution enters the reaction tank 1 to participate in the catalytic reaction, and at this time, the immobilized enzyme in the reaction plate 11 is not replaced.
  • the first buffer solution entering the temporary storage tank 9 upstream of the resin column is purified by the resin column body 6 and then pumped into the temporary storage tank 7 downstream of the resin column.
  • the first purified buffer enters the reaction tank 1 relay reaction and circulates sequentially, thus achieving a seamless two-step process of catalysis and purification Convergence. In other words, the recycling of the reaction buffer is achieved.
  • the array reaction tank 1 and the array reaction plate 11 can be configured.
  • the enzyme activity in the first group of reaction tanks 1 is reduced to the extent that it cannot effectively promote the reaction process, there are two options available. Options: 1The pipeline on the previous reaction tank 1 can be quickly switched to the current reaction tank 1 to realize the quick connection of the catalytic reaction of the reaction tank 1; 2The newly prepared reaction plate 11 containing immobilized enzyme can also be quickly replaced to achieve catalysis The reaction is fast.
  • the working principle of the present invention In the actual operation process, two full buffers are set to react in sequence: the first full buffer is passed through the reaction liquid pre-stirring device 2 and the substrate is added to stir to form a substrate dispersion and enter the reaction tank 1 (reaction The plate 11 contains preset immobilized enzymes), the material passes through the circulation pipeline 13 and the power pump 51 is turned on to achieve a closed-loop reaction. The reaction starts the negative pressure power pump 51 on the negative-pressure suction pipe 5 in the whole process. The volatile substances are taken away from the reaction tank 1, which is beneficial to promote the catalytic reaction; the extracted volatile substances enter the special exhaust gas collection device for centralized treatment, which is in line with the concept of cleaner production.
  • the buffer solution After the product concentration of the reaction liquid reaches a predetermined separation standard, the buffer solution enters the temporary storage tank 9 upstream of the resin column. Immediately after that, a second portion of the entire buffer enters the reaction tank 1 to participate in the catalytic reaction.
  • the first buffer solution entering the temporary storage tank 9 upstream of the resin column is purified by the resin column body 6 and then pumped into the temporary storage tank 7 downstream of the resin column.
  • the first purified buffer enters the reaction tank 1 relay reaction and circulates sequentially, thus achieving a seamless two-step process of catalysis and purification Connection, that is, the recycling of the reaction buffer is realized.
  • the array reaction tank 1 and the array reaction plate 11 are also configured.
  • the enzyme activity in the first group of reaction tanks 1 decreases to the extent that it cannot effectively promote the reaction process, there are two options.
  • reaction tank 1 of the present invention by having a reaction liquid pre-stirring device 2 outside the top layer of the reaction tank 1, a uniform reaction system can be formed to ensure a uniform substrate concentration in the system.
  • the spraying device 3 on the top of the reaction tank 1 can evenly spray the reaction buffer on the catalyst, so that the catalyst can fully exert its catalytic effect.
  • the detachable multi-stage series "steamer" structure of the reaction tank 1 can reduce the labor intensity of the operator in the process of cleaning the reaction tank 1 and replacing the enzyme catalyst.
  • the negative pressure of the pipe is provided by the power pump 51, which can take the volatile substances generated by the catalytic reaction away from the reaction tank 1, which is beneficial to promote the catalytic reaction and can
  • the centralized treatment of the extracted volatile substances is in line with the concept of cleaner production.
  • Each series unit of the reaction tank 1 is provided with an independent temperature control jacket 4 to facilitate accurate temperature control of the reaction system.
  • the reaction liquid circulation channel is formed through the reaction liquid external circulation pipeline 13 of the reaction tank 1 and the power pump 51 of the reaction liquid external circulation pipeline 13 of the reaction tank 1, and the benefit of the reaction liquid circulation reaction is that it avoids the traditional stirring reaction process , The mechanical damage of the stirring device to the enzyme catalyst; Secondly, it is easy to separate the enzyme catalyst from the reaction liquid.
  • Each pipeline of the present invention is provided with a valve, which can control the direction of the material through the valve switch coordination of the pipeline system, so that the material can be circulated and reacted in the catalytic reaction tank 1 or enter the purification system to purify the product.
  • the present invention relates to a method for preparing (R) -o-chloromandelic acid using the above reaction system of the present invention.
  • a method for preparing (R) -o-chloromandelic acid using the circulating biological enzyme catalytic reaction system of the present invention characterized in that it includes at least the following steps:
  • reaction liquid reaches the bottom layer of the reaction tank (1), is transported to the pre-stirring device (2) under the action of the stirring blade (21), and then continues to be sprayed into the reaction tank (1); preferably, the above (The steps a) to (c) are performed in order 1 to 20 times; preferably 5 to 20 times, more preferably 6 to 18 times, and more preferably 8 to 15 times;
  • step (e) extracting (R) -o-chloromandelic acid in the supernatant to obtain a flow-through liquid, optionally the flow-through liquid is recycled as the reaction buffer at least once in step (b), It is preferably 5 to 20 times, more preferably 6 to 18 times, and even more preferably 8 to 15 times.
  • the reaction liquid is introduced into the temporary storage tank (9) upstream of the resin column, and the ion exchange resin pretreatment is performed in advance in the resin column body (6), and then the upstream of the resin column is temporarily
  • the reaction liquid in the storage tank (9) is sent to the resin column body (6) for adsorption, and flows out of the resin column body (6) into the downstream storage tank (7) of the resin column to become a flow-through liquid; the flow-through The liquid is sent to the pre-stirring device (2) as a buffer solution.
  • the eluent is used to enter the resin column body (6) to elute the resin, the eluent is collected, and then extracted and concentrated to obtain (R ) -O-chloromandelic acid finished product.
  • reaction system of the present invention is used to perform the following steps.
  • the Escherichia coli engineering bacteria containing nitrilase is subjected to an immobilization reaction to form the fixed bacteria required for the reaction, and it is evenly spread on the reaction plates (bed layers) 11 of each layer where the reaction tank 1 is located, according to Figure 1
  • the substrate o-chloromandenitrile
  • the reaction buffer solution are added to the pre-stirring device 2 through the addition funnel 23. After the substrate is dissolved, it enters the reaction tank 1 through the spray device 3, respectively Fully contact and react with the fixed bacteria on the reaction plate 11 of each layer.
  • reaction liquid When the reaction liquid reaches the bottom layer of the reaction tank 1 by permeation, it is sent to the pre-stirring device 2 through the pipeline by the power device under the action of the stirring paddle 21, and then sprayed to the reaction tank 1 at the lower end, and so on. This is a catalytic reaction step.
  • the reaction liquid enters into the temporary storage tank 9 upstream of the resin column through the pipeline under the action of the stirring blade 21, and at the same time, the ion exchange resin is pretreated in the resin column body 6 in advance
  • the reaction liquid in the temporary storage tank 9 upstream of the resin column is sent to the resin column body 6 through a power device for adsorption, and flows out of the resin column body 6 into the temporary storage tank 7 downstream of the resin column at a certain flow rate Becomes a flow-through liquid, and the flow-through liquid can be sent to the pre-stirring device 2 as a buffer solution by a power device.
  • the prepared eluent enters the resin column body 6 through the vacuum tube 81 to elute the resin.
  • the eluted eluent passes through the valve under the resin column body 6 Collected, extracted with an extractant, and concentrated to obtain the finished product of (R) -o-chloromandelic acid. This is the ion exchange step.
  • reaction liquid processed by the resin column body 6 flows out of the resin column body 6, it becomes a flow-through liquid, and then enters into the pre-stirring device 2 through the power device, and can continue to be used as a buffer for the next reaction, and the circulation is continued 10 times the above. This is a cyclic reaction step.
  • reaction solution transfer the reaction solution from 1 to 250ml Erlenmeyer flask, add 1.5g of engineered bacterial cells, 30mM substrate o-chloromandenitrile and co-solvent (methanol, the amount of addition is 1% of the volume of the reaction system), shake at 220rpm Medium reaction, in which the substrate was fed in 6 batches, each time the substrate concentration was 25 mM, and the end point of the reaction was monitored by thin layer chromatography.
  • substrate o-chloromandenitrile and co-solvent methanol, the amount of addition is 1% of the volume of the reaction system
  • the substrate is o-chloromandelonitrile racemate.
  • Example 2 only after measuring the volume of the collected reaction liquid, the lost volume is replenished.
  • the other implementations are the same as in Example 1.
  • the results of the obtained eluent products are shown in Table 2.
  • the substrate is o-chloromandelonitrile racemate.
  • Example 3 only after each measurement of the volume of the collected reaction solution, the lost volume was replenished and continued for 20 times. Other embodiments are the same as in Example 1, and the resulting eluent product results are shown in Table 3.
  • the substrate is o-chloromandelonitrile racemate.
  • the product yield can be significantly improved by adding a new reaction buffer.
  • the yield of the product tends to decrease, so the number of cycles in the present invention is preferably 8 to 15 times.
  • Example 4 Compared with Example 1, in Example 4 in Step 1 and Step 2, the substrate is fed in batches in 5 batches, and other embodiments are the same as in Example 1; the results of the obtained eluent products are shown in Table 4.
  • the substrate is o-chloromandelonitrile racemate.
  • Example 4 It can be seen from the experimental results of Example 4: 5 batches of substrate are added to each circulation flow, and although there are no significant changes in the various quality indicators of the reaction, under the same conditions, the buffer is circulated 6 times, and the catalytic concentration of the substrate in Example 4 is 750 mM , Lower than 900mM in Example 1. The product capacity of the buffer solution is not fully utilized. From the perspective of productivity and economy, the output ratio corresponding to the number of substrate replenishment times selected in Example 4 is not as good as the output ratio corresponding to the substrate replenishment times of Example 1.
  • Comparative Example 1 Compared with Example 1, in Comparative Example 1, in step 1 and step 2, the substrate concentration is 8 mM for each feeding, other embodiments are the same as in Example 1;
  • Comparative Example 2 Compared with Example 1, in Comparative Example 2 in step 1 and step 2, the concentration of the substrate is 55 mM for each feeding, and other embodiments are the same as in Example 1;
  • Comparative Example 3 Compared with Example 1, in Comparative Example 3, in step 1 and step 2, the substrate is fed in batches in 8 batches, and other embodiments are the same as in Example 1.
  • the substrate is fed in batches in 8 batches.
  • the cumulative concentration of substrate in each cycle exceeds the optimal catalytic efficiency of the enzyme catalyst, which will lead to a significant reduction in the reaction rate of the system, and a large amount of impurities such as aldehyde and substrate will accumulate, reducing the reaction. Yield.
  • more impurities in the system under this condition will affect the efficiency of resin purification and increase the cost of resin regeneration.
  • the 8 batches of supplementary times selected in Comparative Example 3 are not suitable for actual production.
  • the pH 7.58, (R) -orthochloromandelic acid content of 143mmol / L aqueous phase fixed bacteria catalytic reaction liquid is the liquid to be treated, and HZ-202 type resin is used as the strong basic anion resin.
  • the pH 4.3, (R) -o-chloromandelic acid content of 140mmol / L aqueous phase fixed bacteria catalytic reaction liquid is the liquid to be treated, and HZ-202 type resin is used as the strong basic anion resin.
  • Pre-treatment at room temperature first infiltrate and wash with 1M NaOH solution, the ratio of the volume of NaOH solution (unit is ml) to the mass of strong basic anion resin (unit is g) is 3: 1, then use 1M dilute hydrochloric acid Wetting and washing, the ratio of the volume of dilute hydrochloric acid (in ml) to the mass of strongly basic anion resin (in g) is 3: 1, both are soaked for 15 minutes and washed at a rate of 2 ml / min;
  • the pH 7.2, (R) -o-chloromandelic acid content of 156mmol / L aqueous phase free bacteria catalytic reaction liquid is the liquid to be treated, and HZ202 type resin is used as the strong basic anion resin.
  • the resin In the exchange column at room temperature, the resin is used for adsorption once after recycling, and the product o-mandelic acid is extracted by dynamic adsorption. After 15 minutes of ion exchange, the liquid is separated from the resin at a rate of 2.5ml / min;
  • the pH 3.9, (R) -orthochloromandelic acid content of 205mmol / L aqueous phase fixed bacteria catalytic reaction liquid is the liquid to be treated, and HZ202 type resin is used as the strong basic anion resin.
  • the pH 5.4, (R) -o-chloromandelic acid content of 143mmol / L aqueous phase free bacteria catalytic reaction liquid is the liquid to be treated, and HZ-202 type resin is used as the strong basic anion resin.
  • the resin In the exchange column at room temperature, the resin is used after three cycles of adsorption, and the product is extracted by dynamic adsorption. After 15 minutes of ion exchange, the liquid is separated from the resin at a rate of 2.5ml / min. ;
  • the pH 3.1, (R) -o-chloromandelic acid content of 143mmol / L aqueous phase free bacteria catalytic reaction liquid is to be treated, and HZ-202 type resin is used as the strong basic anion resin.
  • the pH 6.39, (R) -orthochloromandelic acid content of 425 mmol / L aqueous phase fixed bacteria catalytic reaction liquid is the liquid to be treated, and HZ-202 type resin is used as the strong basic anion resin.
  • the resin after 4 cycles of recycling is used for adsorption, and the product o-chloromandelic acid is extracted by dynamic adsorption. After 15 minutes of ion exchange, the liquid is separated from the resin at a rate of 2.5ml / min ;
  • the pH 5.2, (R) -orthochloromandelic acid content is 143mmol / L, the aqueous phase fixed bacteria catalytic reaction liquid is the liquid to be treated.
  • the resin is regenerated: a 2mol / L NaOH solution and a 10% NaCl mixed alkaline solution are used First soak for 30min, the ratio of the volume of the mixed alkaline solution (in ml) to the mass of the strong basic anion resin (in g) is 3: 1; then wash at a rate of 2.0ml / min; then use the concentration of 2mol / The mixed acid solution of L HCl and ethanol with a concentration of 30% is soaked in the resin for 30 minutes, and the ratio of the volume of the mixed acid solution (in ml) to the mass of the strongly basic anion resin (in g) is 3: 1; Wash again at a rate of 2.0 ml / min. The regenerated resin is used to extract (R) -o-chloromandelic acid to be extracted in accordance with the adsorption operation.
  • Example 6 Separation and extraction were carried out according to Example 6, the only difference was that the pretreatment step was first infiltration washing with 5M NaOH solution, then infiltration washing with 5M dilute hydrochloric acid, both soaked for 10 minutes, and then washed at a rate of 1 ml / min.
  • the experimental results are shown in Table 6.
  • Example 6 Separation and extraction were carried out according to Example 6, the only difference was that the pretreatment step was to first infiltrate and wash with 0.5M NaOH solution, and then infiltrate and wash with 0.5M dilute hydrochloric acid, both soaked for 60 minutes, and then washed at a rate of 5 ml / min.
  • the experimental results are shown in Table 6.
  • Example 6 Separation and extraction were carried out according to Example 6, the only difference was that the ratio of NaOH solution (unit: ml) to the mass (unit: g) of strongly basic anion resin in the pretreatment step was 6: 1, and HCl (unit: ml) was The ratio of the mass (in g) of the strong basic anion resin is 6: 1.
  • Table 6 The experimental results are shown in Table 6.
  • Example 6 The separation and extraction were carried out according to Example 6, the only difference was that: the washing speed of the NaOH solution in the pretreatment step was 0.5 ml / min, and the washing speed of the HCl solution was 0.5 ml / min.
  • the experimental results are shown in Table 6.
  • Example 6 The separation and extraction were carried out according to Example 6, the only difference was that the washing speed of the NaOH solution in the pretreatment step was 5.5 ml / min, and the washing speed of the HCl solution was 5.5 ml / min.
  • the experimental results are shown in Table 6.
  • the engineered bacteria after fermentation are immobilized, and 90 g of the obtained immobilized bacteria are evenly spread on the sand core reaction plate 11 of the reaction tank 1.
  • the reaction plate 11 is composed of three layers, each layer is covered with 30 g of immobilized bacteria, and the reaction tank 1 is installed according to the experimental device diagram (FIG. 1).
  • the configured 1M dilute hydrochloric acid eluent is used to elute the adsorbed resin at a rate of 2.5 ml / min, and the eluent after elution is collected to obtain 3 L of eluent and use 0.3 times
  • a volume of 1 L of ethyl acetate was used to extract it, a small amount of times, and the extracted organic phase was concentrated under reduced pressure (0.09 MPa, 45 ° C.) to obtain (R) -o-chloromandelic acid.
  • the engineered bacteria after fermentation are immobilized, and 90 g of the obtained immobilized bacteria are evenly spread on the sand core reaction plate device 11 of the reaction tank 1.
  • the reaction plate 11 is composed of three layers, each layer is covered with 30 g of immobilized bacteria, and the reaction tank 1 is installed according to the experimental device diagram (FIG. 1).
  • the liquid passes through the three-layer bed reaction plate 11 in turn, and under the action of the stirring blade 21, the liquid flows out of the reaction tank 1 and is delivered to the top pre-stirring device 2 through the power device. Repeat the above process. After 2 hours of reaction, continue feeding through the addition funnel 23, feeding 6 times, and react for 12 hours. That is, the flow-through liquid is used once as a reaction buffer.
  • the configured 1M dilute hydrochloric acid eluent is used to elute the adsorbed resin at a rate of 2.5 ml / min, and the eluent after elution is collected to obtain 3 L of eluent, which is used 0.3 times
  • a volume of 1 L of ethyl acetate was used to extract it, a small amount of times, and the extracted organic phase was concentrated under reduced pressure (0.09 MPa, 45 ° C.) to obtain (R) -o-chloromandelic acid.
  • Example 16 The procedure of Example 16 is repeated, except that in step (4) of Example 16, that is, when the flow-through solution is recycled as a reaction buffer, the step (3) of Example 16 is repeated to obtain the flow-through solution again , And then perform step (4) of Example 16 again. That is, the flow-through solution was used twice as the reaction buffer. Then, step (5) of Example 16 was carried out to obtain (R) -o-chloromandelic acid.
  • Table 7 The experimental results are shown in Table 7.
  • Example 16 The steps of Example 16 were repeated except that the steps of Example 16 were followed as follows: "Step (1)-Step (2)-Step (3)-Step (4)-Step (3)-Step (4)-Step (3) -Step (4) -Step (5) ", that is, the flow-through solution is used as a reaction buffer for three times. The finished product of (R) -o-chloromandelic acid was obtained. The experimental results are shown in Table 7.
  • Example 16 The procedure of Example 16 was repeated, except that the flow-through liquid was used as the reaction buffer for 4 to 20 times.
  • the finished product of (R) -o-chloromandelic acid was obtained.
  • the experimental results are shown in Table 7.
  • the method for preparing (R) -o-chloromandelic acid and the circulating biological enzyme catalytic reaction system provided according to the embodiments of the present invention can be applied to the fields of medicinal chemistry and biological enzyme catalytic equipment.

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Abstract

本发明涉及一种(R)-邻氯扁桃酸的制备方法，通过该制备方法，本发明提高了反应缓冲液的利用率，从而降低了(R)-邻氯扁桃酸的生产成本，显著减少了废液的排放，实现了(R)-邻氯扁桃酸的绿色环保生产。同时，本发明的制备方法分离效果好，收率高，并且使用的强碱性阴离子树脂使用寿命长久，分离的水相体系可继续为下一次的生物催化提供反应体系，降低了生产成本并减少了废水的排放。本发明还涉及一种用于所述(R)-邻氯扁桃酸的制备方法的循环式生物酶催化反应系统，通过该催化反应系统，简化了生产操作，提高了工作效率。本发明还涉及一种使用所述循环式生物酶催化反应系统制备(R)-邻氯扁桃酸的方法。

Description

一种(R)-邻氯扁桃酸的制备方法和用于该制备方法的一种循环式生物酶催化反应系统 技术领域
本发明属于药物化学领域，具体讲，涉及一种(R)-邻氯扁桃酸的制备方法。本发明还涉及生物酶催化设备领域，具体地说，涉及一种用于该制备方法的循环式生物酶催化反应系统。
背景技术
手性药物的合成开发技术已经成为当今社会制药领域的研究热点和制高点，手性药物的开发与相关技术的提升也极大促进了相关药物的制备方法和提取方法的探索。
(R)-邻氯扁桃酸为白色结晶性粉末，化学式为C 8H 7ClO 3，分子量为186.59，熔点为86～92℃，是一种重要的医药中间体和精细化工品，是用于合成新型安全有效的抗血小板聚集药物氯吡格雷的原料。氯吡格雷作为全球重磅级药物，是目前市场最畅销的药物。随着氯吡格雷专利到期，全球掀起了一股仿制热潮，因此作为其重要的中间体的(R)-邻氯扁桃酸在国内外的需求不断增长。因此如何高效廉价的制备大量高纯度的(R)-邻氯扁桃酸将具有重要的市场价值。
国内外通过腈水解酶法制备(R)-邻氯扁桃酸已有许多，如下式所示：
Figure PCTCN2019114309-appb-000001
但无一不需要根据反应条件使用合适的反应缓冲液。由于上述所涉及到的腈水解只能在一定酸碱度下进行特异性催化，且底物浓度过高会使得对该催化剂的酶活性下降，甚至不能催化。所以，只能配制适合该催化环境的缓冲液以及底物浓度的反应体系。因此，在工业级水平生产(R)-邻氯扁桃酸， 将产生大量的废液排放，对环境造成一定的危害。
鉴于此，特提出本发明的一种(R)-邻氯扁桃酸的制备方法。
离子交换法是利用特定的有机高分子树脂直接分离有机物，具有设备简单，操作容易，工艺流程短，选择性高，树脂容易再生的优点，利用离子交换树脂对邻氯扁桃酸进行吸附分离，可以从催化反应体系中直接提取邻氯扁桃酸，其主要依据是离子交换树脂对扁桃酸和杂质的亲和力的不同。离子交换树脂对邻氯扁桃酸的亲和力取决于扁桃酸的理化性质，树脂的功能团特点以及溶液中的其他杂质和离子的影响，由于邻氯扁桃酸为弱酸性有机酸，根据离子交换理论，应该使用碱性树脂对其分离提取。
鉴于此，本发明的制备方法中采用了利用离子交换法分离提取(R)-邻氯扁桃酸的步骤。
再一方面，本发明提出一种用于所述制备方法的循环式生物酶催化反应系统。
进一步地，本发明提出一种使用所述循环式生物酶催化反应系统制备(R)-邻氯扁桃酸的方法。
总的来说，本发明涉及如下总的发明构思：在循环使用反应缓冲液制备(R)-邻氯扁桃酸后，以利用离子交换树脂的方法将(R)-邻氯扁桃酸提取出来。所述制备和提取这两个过程都在本发明的所述循环式生物酶催化反应系统中进行。
发明内容
技术问题
有鉴于此，本发明要解决的技术问题是显著提高反应缓冲液的利用率，从而降低了(R)-邻氯扁桃酸的生产成本，进而降低氯吡格雷的生产成本。另一方面，本发明要解决的技术问题是提高邻氯扁桃酸的分离效果，提高产品收率，提高强碱性阴离子树脂使用寿命，降低生产成本以及减少废水的排放。再一方面，本发明的目的是提供一种循环式生物酶催化反应系统，以解决现有技术中的问题，简化操作，提高工作效率。
解决方案
为了解决上述技术问题，根据本发明的一实施例，提供了一种(R)-邻氯 扁桃酸的制备方法，至少包括以下步骤：
(1)培养表达腈水解酶的大肠杆菌工程菌；
(2)采用培养获得的工程菌作为催化剂、邻氯扁桃腈作为底物、反应缓冲液作为反应介质进行反应；
(3)反应结束后，分离收集上清液；
(4)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，任选地所述流穿液在步骤(2)中作为所述反应缓冲液循环使用至少一次，优选为5～20次，进一步优选为6～18次，更优选为8～15次。
可选的，当所述流穿液在步骤(2)中作为所述反应缓冲液循环使用造成反应体积减少时，补充新的反应缓冲液。
可选的，所述反应缓冲液的pH为7.0～9.0；
优选的，所述反应缓冲液选自KH 2PO 4/K 2HPO 4缓冲液、NaH 2PO 4/Na 2HPO 4缓冲液或三羟甲基氨基甲烷-HCl缓冲液中的至少一种；
更优选地，所述步骤(2)反应体系的pH为7.5～8.5。
可选的，在步骤(2)中，所述转化反应的反应温度为25～50℃，优选为25～45℃，更优选为30℃。
可选的，在步骤(2)的反应体系中，所述工程菌细胞的浓度为10g/L～40g/L；
优选的，步骤(2)中的反应体系中还添加助溶剂；助溶剂优选碳原子数为1～8的醇、碳原子数为6～12的烷烃中的至少一种；优选的，助溶剂添加的量为反应体系总体积的1％～10％。
可选的，在步骤(2)中，所述底物通过5～7次分批流加，优选为6次；每次流加底物的浓度为10～50mM，优选为20～45mM，更优选为25～30mM。
可选的，在步骤(3)中，所述分离的方法选自离心、抽滤、压滤中的至少一种；
优选的，所述离心采用6000～10000rpm，优选为6000～8000rpm，更优选为7000rpm。
可选的，在步骤(4)中，所述提取的方法为采用阴离子树脂进行吸附，优选采用强碱性阴离子树脂。
可选的，所述制备方法还包括以下步骤：
(5)使用洗脱剂对步骤(4)中吸附后的阴离子树脂进行洗脱，得到洗脱液；所述洗脱剂优选为1～2M的盐酸；
(6)采用有机溶剂萃取所述洗脱液水相中的(R)-邻氯扁桃酸，分离有机溶剂，得到所述(R)-邻氯扁桃酸的晶体；
所述有机溶剂优选乙酸乙酯或氯仿；所述分离的方法优选为旋转蒸发。
为了解决上述技术问题，根据本发明的另一实施例，在本发明的(R)-邻氯扁桃酸的制备方法中，所述提取(R)-邻氯扁桃酸的步骤至少包括以下步骤：
(i)将强碱性阴离子树脂进行预处理，所述预处理包括依次进行碱洗和酸洗的步骤；所述强碱性阴离子树脂优选季胺盐强碱性阴离子树脂；
(ii)采用步骤(i)处理后的强碱性阴离子树脂对(R)-邻氯扁桃酸的待处理液进行吸附；
(iii)采用洗脱剂对步骤(ii)的强碱性阴离子树脂进行洗脱，得到洗脱液，所述洗脱液经萃取、浓缩后得到所述(R)-邻氯扁桃酸。
可选的，在步骤(i)中，所述碱洗的步骤包括：先用浓度为0.5～5mol/L的碱液浸泡强碱性阴离子树脂10～60min，然后用所述碱液以1.0～5.0ml/min的速度进行洗涤；
优选的，浸泡时所述碱液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述碱液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述碱液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
更优选的，所述碱选自一元碱。
可选的，在步骤(i)中，所述酸洗的步骤包括：先用浓度为0.5～5mol/L的酸液浸泡强碱性阴离子树脂15～30min，然后用所述酸液以1.0～5.0ml/min的速度进行洗涤；
优选的，浸泡时所述酸液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述酸液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述酸液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
更优选的，所述酸选自一元酸。
可选的，在步骤(i)中，所述酸洗后还包括水洗至中性的步骤；优选的，所述碱洗前还包括水洗去机械杂质的步骤；
更优选的，所述水选自纯化水。
可选的，在步骤(ii)中，所述吸附前还包括浸润的步骤，优选的，所述浸润的步骤包括：向所述强碱性阴离子树脂中加入缓冲液浸润10～30min，所述缓冲液的体积为强碱性阴离子树脂质量的2～5倍；所述缓冲液的pH值优选为7～9。优选的，所述缓冲液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述缓冲液的体积以mL计时，所述强碱性阴离子树脂的质量以g计。
可选的，将洗脱后的强碱性阴离子树脂水洗至中性后进入步骤(ii)循环使用，当循环使用15～20次后，对所述强碱性阴离子树脂进行再生处理；优选的，所述再生处理包括以下方式中的至少一种：
方式1：用混合碱液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤；所述混合碱液中含有浓度为2～5mol/L的一元碱和质量百分比浓度为10％～30％的氯化钠；优选的，所述混合碱液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述混合碱液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述混合碱液的体积以mL计时，所述强碱性阴离子树脂的质量以g计。
方式2：用混合酸液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤；所述混合酸液中含有2～5mol/L的一元酸和体积百分比浓度为10％～50％的低级醇；优选的，所述低级醇选自碳原子为1～6的醇；更优选的，所述混合酸液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述混合酸液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述混合酸液的体积以mL计时，所述强碱性阴离子树脂的质量以g计。
进一步优选的，所述再生处理还包括水洗至中性的步骤，所述水优选为纯化水。
可选的，在步骤(ii)中，所述吸附的温度为10～60℃；
在步骤(iii)中，所述洗脱的温度为10～60℃；优选的，所述萃取采用的有机溶剂选自酯类有机溶剂、醚类有机溶剂、醇类有机溶剂中的至少一种； 更优选碳原子数为4～8的酯类有机溶剂、碳原子数为2～6的醚类有机溶剂、碳原子数为1～8的醇类有机溶剂；更优选乙酸乙酯、乙醚、乙醇中的至少一种。
可选的，在步骤(ii)中，所述吸附的流速为0.1～10ml/min；在步骤(iii)中，所述洗脱的流速为0.1～10ml/min。
可选的，所述待处理液为水相反应液或包含与水互溶的有机相的水相反应液；
优选的，所述待处理液包括化学合成、生物催化合成、拆分合成所得的反应液；
更优选的，所述待处理液的pH值为2～8。
可选的，当所述待处理液为生物催化合成反应液时，步骤(ii)吸附后的反应液作为生物催化反应体系的反应液循环使用。
为了解决上述技术问题，根据本发明的再一实施例，提供了一种用于本发明的制备方法的循环式生物酶催化反应系统，其包括顺次连接的循环式固定化反应床、树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐；
所述循环式固定化反应床包括：
反应罐；
设置在反应罐上方的预搅拌装置，树脂柱下游暂存罐还与预搅拌装置连接；
设置在预搅拌装置与反应罐之间的喷淋装置，所述喷淋装置伸至反应罐顶部的内部，用于向反应罐喷射反应液；
间隔的设置在反应罐内部的至少一个反应板，将反应罐内部分隔为多个反应空间，所述反应板内部填充有催化剂；
设置在反应罐底部的底部搅拌装置，且反应罐的底部与树脂柱上游暂存罐连接，反应罐的底部还与预搅拌装置连接。
作为优选，所述预搅拌装置包括：
与喷淋装置连接的搅拌罐；
设置在搅拌罐内部的搅拌桨与设置在搅拌罐外部的搅拌电机，搅拌电机与搅拌桨连接；
设置在搅拌罐上的加料漏斗。
作为优选，所述底部搅拌装置包括设置在反应罐内部的搅拌桨和设置在反应罐外部的搅拌电机，所述搅拌电机与搅拌桨连接。
作为优选，所述循环式生物酶催化反应系统还包括废气收集装置、动力泵和负压抽气管道，每个反应空间处的反应罐上均设置有抽气孔，废气收集装置依次通过动力泵、负压抽气管道与每个反应空间的抽气孔连接。
作为优选，所述反应板内部设置有用于容纳催化剂的填充腔，填充腔上下表面均设置有可拆卸的砂芯筛网。
作为优选，所述反应板与搅拌罐的侧壁可拆卸连接。
作为优选，所述树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐均连接有真空管、压缩空气管及温度探头。
作为优选，每个反应空间处的反应罐上均设置有观察窗。
作为优选，所述搅拌罐、树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐外部均设置有控温夹套。
为了解决上述技术问题，根据本发明的再一实施例，提供了一种使用本发明所述的循环式生物酶催化反应系统制备(R)-邻氯扁桃酸的方法，其特征在于，至少包括以下步骤：
(a)培养表达腈水解酶的大肠杆菌工程菌；
(b)将上述大肠杆菌工程菌进行固定化反应形成固定菌，将其均匀平铺在反应罐(1)中各层的反应板(11)上，通过加料漏斗(23)将邻氯扁桃腈以及反应的缓冲溶液加入至预搅拌装置(2)中，通过喷淋装置(3)进入到反应罐(1)中，分别与各层的反应板(11)上的固定菌反应；
(c)反应液到达反应罐(1)最底层，在搅拌桨(21)的作用下输送至预搅拌装置(2)中，再继续喷淋至反应罐(1)中；优选地，上述(a)～(c)的步骤按顺序进行1～20次；优选为5～20次，进一步优选为6～18次，更优选为8～15次；
(d)反应结束后，分离收集上清液；
(e)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，任选地所述流穿液在步骤(b)中作为所述反应缓冲液循环使用至少一次，优选为5～20 次，进一步优选为6～18次，更优选为8～15次。
作为优选，在步骤(d)之后，使反应液进入至树脂柱上游暂存罐(9)中，同时在树脂柱本体(6)中提前进行离子交换树脂的预处理，然后将树脂柱上游暂存罐(9)中的反应液输送至树脂柱本体(6)中进行吸附，流出树脂柱本体(6)进入至树脂柱下游暂存罐(7)中，成为流穿液；所述流穿液输送至预搅拌装置(2)中作为缓冲液。
作为优选，在反应液全部流出树脂柱本体(6)后，使用洗脱剂进入到树脂柱本体(6)中对树脂进行洗脱，洗脱液被收集，再经过萃取、浓缩后得到(R)-邻氯扁桃酸成品。
有益效果
本发明采用分离上清液中的产物，从而获得含极少量产物的流穿液，本发明通过将所得流穿液直接用于转化反应作为缓冲液，从而显著提高了反应缓冲液的利用率，从而降低了(R)-邻氯扁桃酸的生产成本，进而降低氯吡格雷的生产成本，将会产生良好的经济和社会效益。本发明的方法显著减少了废液的排放，实现(R)-邻氯扁桃酸的绿色环保生产，对实现(R)-邻氯扁桃酸工业化生产具有重要意义，还可以提高我国的生物、化工、医药等领域的手性药物的生产水平，有利于促进我国绿色生产、工业生物技术的发展，对我国经济的可持续发展具有重要意义。
另一方面，本发明的制备方法中分离效果好，设备简单，操作简便，得到的产品收率较高；并且使用的强碱性阴离子树脂使用寿命长久(再生处理后可继续使用)，分离的水相体系可继续为下一次的生物催化提供反应体系，大大降低了生产成本以及减少了废水的排放。
再一方面，本发明提供的循环式生物酶催化反应系统具备以下有益效果：
(1)本发明实施例的反应板内部填充有催化剂，使得可以提前准备好填充了酶催化剂的反应板，而且，循环式固定化反应床的反应板与反应罐间采用可拆卸连接，使得催化剂更换简便，抽出前批次反应板迅速更换本批次反应板，极大节约时间与简化操作；
(2)循环式固定化反应床、树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐可设置多组，可以在更换催化剂或离子交换的树脂的时候，迅速 将反应液管道切换至新循环式固定化反应床或树脂柱，催化反应不会被中断；
(3)本发明采用“双反应液体系”，可以保证反应液由循环式固定化反应床进入树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐组成的产物分离纯化装置后，有“替补反应液”进入循环式固定化反应床，避免设备闲置，有效提高生产效率；同理，反应液由产物分离纯化装置进入循环式固定化反应床后，也有“替补反应液”进入产物分离纯化装置，避免设备闲置，提高了时空产率；
(4)本发明设有挥发性物质的废气收集装置，收集后按相关技术标准处理，满足企业对清洁生产及绿色催化的需求；
(5)本发明操作简便，且相关设备结构简洁无死角，便于生产后清洗，极大减轻了操作者的劳动强度，利于提高生产效率。
根据下面参考附图对示例性实施例的详细说明，本发明的其它特征及方面将变得清楚。
附图说明
包含在说明书中并且构成说明书的一部分的附图与说明书一起示出了本发明的示例性实施例、特征和方面，并且用于解释本发明的原理。
图1为本发明实施例提供的循环式生物酶催化反应系统的正视图。
附图标记说明：
1-反应罐，11-反应板，12-观察窗，13-循环管道，2-预搅拌装置，21-搅拌桨，22-搅拌电机，23-加料漏斗，3-喷淋装置，4-控温夹套，5-负压抽气管道，51-动力泵，6-树脂柱本体，7-树脂柱下游暂存罐，81-真空管，82-压缩空气管，83-温度探头，9-树脂柱上游暂存罐。
具体实施方式
以下将参考附图详细说明本发明的各种示例性实施例、特征和方面。附图中相同的附图标记表示功能相同或相似的元件。尽管在附图中示出了实施例的各种方面，但是除非特别指出，不必按比例绘制附图。
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段和元件未作详细描述，以便于凸显本发明的主旨。
本发明的一个实施例涉及一种(R)-邻氯扁桃酸的制备方法，通过培养获得的工程菌作为催化剂、邻氯扁桃腈作为底物、反应缓冲液作为反应介质进行反应；反应结束后，分离收集上清液；提取上清液中的(R)-邻氯扁桃酸后获得流穿液，流穿液中含有极少量的产物，本发明实施例通过循环使用反应缓冲液，反应循环次数可达10次以上，因此本发明实施例与等量的反应缓冲液相比，缓冲液利用率提高了10倍以上。
具体的，本发明实施例的制备方法至少包括以下步骤：
(1)培养表达腈水解酶的大肠杆菌工程菌；
(2)采用培养获得的工程菌作为催化剂、邻氯扁桃腈作为底物、反应缓冲液作为反应介质进行反应；
(3)反应结束后，分离收集上清液；
(4)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，流穿液在步骤(2)中作为所述反应缓冲液循环使用至少一次。
具体的，在步骤(1)中所使用的表达腈水解酶的大肠杆菌工程菌，可采用市售菌株，并优选采用申请号CN 201510978973.7公开的方法中构建的工程菌。
可选的，步骤(1)中的培养包括通过摇瓶培养或者发酵罐培养，离心收集工程菌细胞。
可选的，步骤(2)中的反应体系中还可添加助溶剂；具体的，助溶剂可选自碳原子数为1～8的醇、碳原子数为6～12的烷烃中的至少一种；具体可选自乙醇、甲醇、正己烷、正丙醇、异丙醇、正丁醇、乙腈、正己醇、正戊醇、正辛醇等有机溶剂中的一种或者几种，助溶剂添加的量为反应体系总体积的1％～10％。
可选的，流穿液在步骤(2)中作为反应缓冲液循环使用5～20次，如果循环次数过高，则缓冲液中离子强度等参数变化显著，不利于反应正常进行；如果循环次数过少，反应缓冲液重复利用率低，单位体积缓冲液的产品 产出比低，减少废水排放的优势不明显。
优选的，流穿液循环使用6～18次，更优选的，流穿液循环使用8～15次。
可选的，当流穿液作为反应缓冲液循环使用造成反应体积减少时，补充新的反应缓冲液。本发明实施例中的步骤(3)和(4)会造成反应体系体积的减少，添加新的反应缓冲液以弥补损失的体积，从而可进一步提高产物的收率。
可选的，在步骤(2)中，反应缓冲液的pH为7.0～9.0。
优选的，反应缓冲液可选自KH 2PO 4/K 2HPO 4缓冲液、NaH 2PO 4/Na 2HPO 4缓冲液或三羟甲基氨基甲烷-HCl缓冲液中的至少一种，并不限于此。
优选的，步骤(2)中反应体系的pH为7.5～8.5。
优选的，在反应过程中可采用1～2M HCl和1～2M NaOH调节反应体系的pH值。
可选的，在步骤(2)中，转化反应的反应温度为25～50℃，转化反应的反应温度的上限可为50℃、48℃、45℃、42℃、40℃、38℃，转化反应的反应温度的下限可为25℃、28℃、30℃、35℃，温度范围可由上限、下限中的任意数值组成。转化反应的反应温度优选为25～45℃，更优选为30℃。
可选的，在步骤(2)的反应体系中，工程菌细胞的浓度为10g/L～40g/L。工程菌细胞的浓度的上限可为40g/L、35g/L、30g/L、25g/L、22g/L，工程菌细胞的浓度的下限可为10g/L、12g/L、15g/L、18g/L、20g/L；工程菌细胞的浓度的范围可由上限、下限中的任意数值组成。
可选的，在步骤(2)中，底物通过5～7次分批流加，优选为6次。如果底物补加次数过多，则反应后程速率明显降低，而且会积累大量醛、底物等杂质，降低反应收率；如果底物补加次数过低，则体系积累的产物浓度低，体系内酶催化剂的催化潜力没有得到充分发挥，造成经济上的浪费。
每次流加底物浓度为10～50mM。如果底物浓度过高则会引起酶催化剂的不可逆失活；如果底物浓度过低则产物对映体过量会偏低，而且体系产物浓度积累速率过慢，造成后处理成本的浪费。
每次流加底物浓度的上限可为50mM、48mM、45mM、40mM、35mM、30mM，每次流加底物浓度的下限可为10mM、12mM、15mM、20mM、22mM、 25mM。每次流加底物浓度的范围可由上限、下限中的任意数值组成。优选的，每次流加底物浓度为20～45mM，更优选为25～30mM。
当本发明实施例中每次流加底物浓度为25mM，当每次常规反应流加6次底物时，则底物催化浓度可达到1500mM，从而可显著提高缓冲液的产物产出比。
可选的，在步骤(2)中，催化剂、底物、反应缓冲液以及助溶剂通过磁力搅拌混合均匀。
可选的，在步骤(3)中，分离的方法选自离心、抽滤、压滤中的至少一种；并优选离心。
可选的，离心采用6000～10000rpm，优选为6000～8000rpm，更优选为7000rpm。
可选的，在步骤(4)中，提取的方法为采用阴离子树脂进行吸附，优选采用强碱性阴离子树脂。具体可选择HZ-202或711型树脂。本发明实施例中的树脂使用前参照所购买树脂产商的使用说明书进行相应的活化处理。
可选的，本发明实施例的制备方法还包括以下步骤：
(5)使用洗脱剂对步骤(4)中吸附后的阴离子树脂进行洗脱，得到洗脱液；
优选的，洗脱剂为1～2M的盐酸；
(6)采用有机溶剂萃取洗脱液水相中的(R)-邻氯扁桃酸，分离有机溶剂，得到(R)-邻氯扁桃酸的晶体；
优选的，有机溶剂选自乙酸乙酯或氯仿；
优选的，分离的方法为旋转蒸发。
本发明的另一个实施例提出一种在本发明的(R)-邻氯扁桃酸制备方法中分离提取(R)-邻氯扁桃酸的方法，本发明实施例选择对(R)-邻氯扁桃酸亲和力较强、吸附能力较强的强碱性阴离子树脂对(R)-邻氯扁桃酸进行吸附，离子交换吸附后使得缓冲液中的蛋白质分子和其他的无机离子(缓冲液体系)随着液体流出，强碱性阴离子树脂中只存在(R)-邻氯扁桃酸，再使用洗脱剂对强碱性阴离子树脂进行解吸操作，使得产物(R)-邻氯扁桃酸随着洗脱液一同被洗脱下来，最后进行有机相萃取、减压浓缩，干燥即得到(R)-邻氯扁桃 酸。本发明实施例的方法具有分离效果好、产品收率较高的技术优势，并且使用的强碱性阴离子以及体系的反应液均可实现循环利用，大大降低了生产成本并减少了废水的排放。
具体的，本发明实施例的分离提取方法包括以下步骤：
(i)将强碱性阴离子树脂进行预处理，优选的，预处理包括水洗去机械杂质、碱洗、酸洗和水洗至中性的步骤，优选的：
碱洗的步骤为：先用浓度为0.5～5mol/L的碱液浸泡强碱性阴离子树脂10～60min，然后用碱液以1.0～5.0ml/min的速度进行洗涤至液体全部流下；优选的，碱选自一元碱，更优选的，碱液选自NaOH溶液、KOH溶液；
如果所用碱液浓度过低，则树脂未能全部转化为OH型，若过高，则会使碱液的量增加，造成成本的增加；同样地，若处理时间过短，则也会造成树脂未能全部转化，时间过久，则会增加工艺的时间，不适合工业生产。
优选的，酸洗的步骤包括：先用浓度为0.5～5mol/L的酸液浸泡强碱性阴离子树脂15～30min，然后用酸液以1.0～5.0ml/min的速度进行洗涤至液体全部流下；优选的，酸选自一元酸，更优选的，酸液选自稀盐酸；
如果所用酸液浓度过低，则树脂未能全部转化为Cl型，若过高，则会使酸液的量增加，造成成本的增加；同样地，若处理时间过短，则也会造成树脂未能全部转化，时间过久，则会增加工艺的时间，不适合工业生产。
(ii)采用步骤(i)处理后的强碱性阴离子树脂采用动态吸附的方式对(R)-邻氯扁桃酸的待处理液进行吸附；其中，所述(R)-邻氯扁桃酸的待处理液即为包含(R)-邻氯扁桃酸的上清液；
(3)采用洗脱剂对步骤(2)的强碱性阴离子树脂进行洗脱，得到洗脱液，洗脱液经萃取、浓缩后得到(R)-邻氯扁桃酸的粗品，最后对粗品精制后得到(R)-邻氯扁桃酸。
进一步可选的，强碱性阴离子树脂选自季胺盐强碱性阴离子树脂，并优选HZ-202型树脂。
可选的，浸泡时碱液的体积与强碱性阴离子树脂的质量之比为2～5：1，浸泡时酸液的体积与强碱性阴离子树脂的质量之比为2～5：1。碱液或酸液体积的单位为L时，强碱性阴离子树脂质量的单位为Kg；或者碱液或酸液体积的单位为mL时，强碱性阴离子树脂质量的单位为g。如果所用碱液体 积过小，则树脂未能全部转化为OH型，若过大，则碱液用量增加，会造成成本的增加。如果所用酸液体积过小，则树脂未能全部转化为Cl型，若过大，则酸液用量增加，会造成成本的增加。
可选的，水洗的步骤选用纯化水进行洗涤。
可选的，在步骤(ii)中，吸附前还包括浸润的步骤，优选的，浸润的步骤包括：向强碱性阴离子树脂中加入缓冲液浸润10～30min，缓冲液的体积为强碱性阴离子树脂质量的2～5倍。优选的，所述缓冲液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述缓冲液的体积以mL计时，所述强碱性阴离子树脂的质量以g计。
进一步可选的，缓冲液的pH值为7～9。
进一步可选的，缓冲液与待处理液中缓冲液的pH值一致。当待处理液为生物催化合成反应液时，缓冲液可采用生物催化合成的缓冲液，例如KH 2PO 4/K 2HPO 4缓冲液、NaH 2PO 4/Na 2HPO 4缓冲液。
可选的，将洗脱后的强碱性阴离子树脂水洗至中性后进入步骤(ii)循环使用，从而可大大降低了生产成本。当循环使用15～20次后，强碱性离子交换树脂的吸附效率发生显著下降时，则应对树脂进行再生处理，优选的，再生处理包括以下方式中的至少一种：
方式1：用混合碱液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤至液体全部流下；混合碱液中含有浓度为2～5mol/L的一元碱和质量百分比浓度为10％～30％的氯化钠；优选的，混合碱液的体积与强碱性阴离子树脂的质量之比为2～5：1；混合碱液体积的单位为L时，树脂质量的单位为Kg，或者混合碱液体积的单位为mL时，树脂质量的单位为g。
方式2：用混合酸液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤至液体全部流下；混合酸液中含有2～5mol/L的一元酸和体积百分比浓度为10％～50％的低级醇；优选的，低级醇选自碳原子为1～6的醇；更优选的，混合酸液的体积与强碱性阴离子树脂的质量之比为2～5：1；混合酸液体积的单位为L时，树脂质量的单位为Kg；或者混合酸液体积的单位为mL时，树脂质量的单位为g。
进一步优选的，再生处理还包括水洗至中性的步骤，水优选为纯化水。
在再生处理中，可使用方式1和方式2中的任意一种，也可先采用方式 1处理，再采用方式2处理。处理两次相当于再生了两次，洗去了树脂中残留的有机物杂质，更有利于提高树脂的性能。
可选的，在步骤(2)中，吸附的温度为10～60℃，优选15～50℃。
可选的，在步骤(3)中，洗脱的温度为10～60℃，优选15～50℃。
可选的，萃取采用的有机溶剂选自酯类有机溶剂、醚类有机溶剂、醇类有机溶剂中的至少一种；优选碳原子数为4～8的酯类有机溶剂、碳原子数为2～6的醚类有机溶剂、碳原子数为1～8的醇类有机溶剂；更优选乙酸乙酯、乙醚、乙醇中的至少一种。
可选的，在步骤(2)中，吸附的流速为0.1～10ml/min，优选为1～9ml/min，更优选2～8ml/min。
可选的，在步骤(3)中，洗脱的流速为0.1～10ml/min，优选为1～9ml/min，更优选2～8ml/min。
可选的，待处理液为水相反应液或包含与水互溶的有机相的水相反应液；具体的，待处理液可选自化学合成、生物催化合成、拆分合成所得的反应液，并不限于此。
优选的，待处理液可选自生物催化合成所得的反应液。当待处理液为生物催化合成反应液时，步骤(ii)吸附后的反应液可作为生物催化反应体系的反应液循环使用，从而大大减少了废水的排放。
可选的，强碱性阴离子树脂的pH应与所使用树脂吸附(R)-邻氯扁桃酸最适宜的pH一致，强碱性阴离子树脂吸附(R)-邻氯扁桃酸最适宜的pH为2～8。因此，将待吸附液配制成pH为2～8的溶液再对其吸附可进一步提高吸附效率。
可选的，在步骤(iii)中，浓缩的方式可为减压浓缩，具体条件为：45～55℃、0.09MPa；优选50℃、0.09MPa。
可选的，待提取液中(R)-邻氯扁桃酸的最大量应不大于强碱性阴离子树脂的最小交换容量，即所需的树脂应为过量。
可选的，待提取液中含有(R)-邻氯扁桃酸、(S)-邻氯扁桃酸、(R,S)-邻氯扁桃酸中的至少一种。
优选的，本发明实施例的使用强碱性阴离子树脂分离提取(R)-邻氯扁桃酸方法的具体的操作步骤如下：
1、强碱性阴离子树脂进行预处理：取一定量的强碱性阴离子树脂(含水)于交换柱中，用纯化水洗去机械杂质，加入0.5～5M NaOH溶液浸泡15min，NaOH溶液的体积与强碱性阴离子树脂的质量之比为2～5：1，用0.5～5M NaOH溶液以1.0～5.0ml/min洗涤，再加入0.5～5M稀盐酸浸泡，稀盐酸液的体积与强碱性阴离子树脂的质量之比为2～5：1，用0.5～5M稀盐酸以1.0～5.0ml/min洗涤，最后用纯化水洗至中性待用。
2、吸附：取2～5倍强碱性阴离子树脂体积的缓冲液(pH＝7～9)加入强碱性阴离子树脂中浸泡至树脂的pH为7～8后将缓冲液流下，再将待处理液加入至强碱性阴离子树脂中，采用静态吸附或动态吸附的方式对产物(R)-邻氯扁桃酸进行提取，吸附的温度为10～60℃，静态吸附的时间为2～6小时，动态吸附的时间为10～20min；其中，静态吸附可以得到树脂的最大吸附量，测量最为准确，但是操作难度大，不能反复对树脂进行操作，不适合工业化生产；动态吸附增加了实验的可操作性，可重复操作，但是吸附的量会比静态吸附少。
吸附完全后，将吸附后的反应液以0.1～10ml/min从交换柱中流下；吸附后的反应液作为生物催化反应体系的反应液循环使用；
3、树脂解吸洗脱：用1M稀盐酸做洗脱剂，对树脂进行洗脱，洗脱的温度为10～60℃，用5倍于强碱性阴离子树脂质量的体积的洗脱剂以0.1～10ml/min进行洗脱解吸，使产物(R)-邻氯扁桃酸被洗脱剂解吸后进入洗脱液中；
4、对洗脱液用0.3～1倍洗脱液体积的乙酸乙酯萃取，萃取后的有机相减压浓缩(50～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。
采用HPLC检测，即对单一构型(光学纯度)进行液相检测，具体检测条件为：Ultimate Cellu-J HPLC色谱柱，手性柱(4.6nm×250nm,5μm)(Welch Materials,Inc,USA)，流动相：正己烷：乙醇：甲醇：三氟乙酸＝90：8：2：0.1，检测波长：254nm，流速：0.8ml/min，并可通过对映体过量值(e.e.)来表示其提取的(R)-邻氯扁桃酸的光学纯度(e.e＝([R]-[S])/([R]+[S])×100％)。
如图1所示，本发明实施例提供一种用于本发明制备方法的循环式生物酶催化反应系统，包括顺次连接的循环式固定化反应床、树脂柱上游暂存罐 9、树脂柱本体6、树脂柱下游暂存罐7；
所述循环式固定化反应床包括：
反应罐1；
设置在反应罐1上方的预搅拌装置2，树脂柱下游暂存罐7还与预搅拌装置2连接；
设置在预搅拌装置2与反应罐1之间的喷淋装置3，所述喷淋装置3伸至反应罐1顶部的内部，用于向反应罐1喷射反应液；
间隔的设置在反应罐1内部的至少一个反应板11，将反应罐1内部分隔为多个反应空间，所述反应板11内部填充有催化剂；
设置在反应罐1底部的底部搅拌装置，且反应罐1的底部与树脂柱上游暂存罐9连接，反应罐1的底部还与预搅拌装置2连接。
作为优选，所述预搅拌装置2包括：
与喷淋装置3连接的搅拌罐；
设置在搅拌罐内部的搅拌桨21与设置在搅拌罐外部的搅拌电机22，搅拌电机22与搅拌桨21连接；
设置在搅拌罐上的加料漏斗23。
作为优选，所述底部搅拌装置包括设置在反应罐1内部的搅拌桨21和设置在反应罐1外部的搅拌电机22，所述搅拌电机22与搅拌桨21连接。
作为优选，所述循环式生物酶催化反应系统还包括废气收集装置、动力泵51和负压抽气管道5，每个反应空间处的反应罐1上均设置有抽气孔，废气收集装置依次通过动力泵51、负压抽气管道5与每个反应空间的抽气孔连接。
作为优选，所述反应板11内部设置有用于容纳催化剂的填充腔，填充腔上下表面均设置有可拆卸的砂芯筛网。
作为优选，所述反应板11与搅拌罐的侧壁可拆卸连接。优选地，可采用抽屉式的抽拉式可拆卸连接。
作为优选，所述树脂柱上游暂存罐9、树脂柱本体6、树脂柱下游暂存罐7均连接有真空管81、压缩空气管82及温度探头83。
作为优选，每个反应空间处的反应罐1上均设置有观察窗12。
作为优选，所述搅拌罐、树脂柱上游暂存罐9、树脂柱本体6、树脂柱下游暂存罐7外部均设置有控温夹套4。
优选地，反应罐1为圆柱体罐状结构，罐顶与罐底为顺滑曲面半球状结构。反应罐1顶层外部有料液预搅拌装置2，预搅拌装置2顶部侧面有加料漏斗23与搅拌电机22，反应罐1顶部有反应液进样口及喷淋装置3，反应罐1外部设有控温夹套4，反应罐1外部设有负压抽气管道5及抽气孔，并由动力泵51提供管道负压，反应罐1为多级“蒸笼”状结构，并由罐体间连接机构串联在一起，所述反应罐1侧面有观察窗12，所述反应罐1内部水平设置有填充了固定化酶的反应板11，所述反应罐1最底层中心设有搅拌桨21，所述搅拌桨21由置于反应罐1外部的搅拌电机22提供动力，所述反应罐1底部设有物料取样管，反应罐1与料液预搅拌装置2之间由反应罐1料液外部循环管道13及动力泵51连通形成循环通路，反应罐1底部与树脂柱上游暂存罐9之间由物料管道连通，所述树脂柱上游暂存罐9上方设有真空管81、压缩空气管82、温度探头83，侧面设有控温夹套4，底部设有取样管，底部与树脂柱本体6间由物料管道连通，树脂柱本体6上方同样设置真空管81、压缩空气管82、温度探头83，侧面设有控温夹套4，底部设有取样管，所述树脂柱本体6与树脂柱下游暂存罐7之间由物料管道连通，所述树脂柱下游暂存罐7上方设有真空管81、压缩空气管82、温度探头83，侧面设有控温夹套4，底部设有取样管，所述树脂柱下游暂存罐7与反应罐1顶层外部的料液预搅拌装置2之间设有物料连通管道。整套管路体系均由阀门控制物料走向，可以通过阀门的开关配合，使物料在催化反应釜内循环反应，或是进入纯化系统进行产物提纯。
反应罐1可采用可拆卸式多级串联结构，本实施例中利用罐体间连接机构将各个单元串联在一起，形成多级“蒸笼”结构。
具体地，本发明实施例中，循环式固定化反应床的外壳，即反应罐1为圆柱体，罐顶与罐底为顺滑曲面半球状无死角结构，便于反应后清洗，反应罐1为多级“蒸笼”状结构，并由连接机构串联在一起，便于设备快速拆装检修等工作。
反应罐1顶层外部有反应液预搅拌装置2，能将物料混合均匀，混匀的物料通过反应罐1顶层内部的喷淋装置3，均匀分布于反应板11上，能使催化剂均匀接触物料。反应板11上下底面为砂芯筛网结构，反应板11内可以填充固定化的大颗粒酶催化剂(下称固定化酶)，反应板11上下底面为可拆卸结构，便于酶的更换、清洗等操作，反应板11侧面为不透水耐腐蚀软性密封机构， 可以与反应罐1贴合，反应板11由反应罐1卡槽部位的锁定装置固定。
反应罐1侧面有负压抽气管道5，通过负压动力泵51，能将催化反应产生的挥发性物质带离反应罐1，有利于推动催化反应进行；抽离的挥发性物质进入专门废气收集装置集中处理，符合清洁生产的理念。
反应罐1底部有底部搅拌装置，可以将催化反应后的液体混合均匀，并导入顶部的预搅拌装置2，参与下一个循环反应。反应罐1底部有反应液的循环管道13及循环管道13上的循环泵，将反应液经由循环管道13上行进入预搅拌装置2，反应液即可顺流再次通过喷淋装置3进入下一个循环反应；反应液循环式反应的好处在于，首先避免传统的搅拌式反应过程中搅拌装置对酶催化剂的机械损伤；其次便于酶催化剂与反应液的分离；另外，对产生挥发性物质的反应，便于收集挥发性物质集中处理。
本发明还设置了连续性的产物分离装置，在本实施例中，采用树脂分离系统，即树脂分离系统包括树脂柱上游暂存罐9、树脂柱本体6、树脂柱下游暂存罐7。反应进行特定时间后，经TLC、HPLC等检测方法确保反应液产物浓度达到既定的分离标准。可关闭反应罐1底部循环管道13的上行阀门，开启连通树脂分离系统的管道阀门，利用树脂柱上游暂存罐9的真空管81，从而可将反应液抽入树脂柱上游暂存罐9内。接着关闭连通树脂分离系统的管道阀门，利用压缩空气管82将树脂柱上游暂存罐9内的反应液压入树脂柱本体6。经过一段时间的树脂吸附后，反应液匀速滴出树脂柱本体6，进入树脂柱下游暂存罐7。树脂柱下游暂存罐7中分离了产物的反应液可通过管道再次进入反应罐1顶层外部的反应液预搅拌装置2。加入反应底物，搅匀之后即可参与下一批次的循环反应。
为了实现本循环催化系统的连续催化与连续纯化的双重特性。首先，可以配置数组离子交换的树脂柱。在实际操作过程中，设置两份全量缓冲液依次反应：第一份全量缓冲液经反应液预搅拌装置2，加入底物搅拌形成底物分散液，进入反应罐1(反应板11含预置的固定化酶)，利用反应罐1底部侧面反应液循环管道13实现封闭式循环反应，待底物投加量达一定比例，且反应液产物浓度达到既定的分离标准后，缓冲液进入树脂柱上游暂存罐9。紧接着，第二份全量缓冲液进入反应罐1参与催化反应，此时反应板11中的固定化酶不更换。而进入树脂柱上游暂存罐9的第一份缓冲液经过树脂柱本体6 纯化后，泵入树脂柱下游暂存罐7。待第二份缓冲液反应结束进入空出来的树脂柱上游暂存罐9后，第一份纯化后的缓冲液进入反应罐1接力反应，依次循环，从而实现催化与纯化两步过程的无缝衔接。换言之，实现了反应缓冲液的循环利用。
另外，为了实现反应罐1的无缝衔接，可以配置数组反应罐1与数组反应板11，在第一组反应罐1中的酶活性降低至不能有效推动反应进程时，有两种方案可供选择：①可以将前次反应罐1上的管道快速切换至当前反应罐1上，实现反应罐1催化反应快速衔接；②还可以迅速更换新准备的含固定化酶的反应板11，实现催化反应快速衔接。
本发明工作原理：在实际操作过程中，设置两份全量缓冲液依次反应：第一份全量缓冲液经反应液预搅拌装置2，加入底物搅拌形成底物分散液，进入反应罐1(反应板11含预置的固定化酶)，物料通过循环管道13，开启动力泵51实现封闭式循环反应，反应全程开启负压抽气管道5上的负压动力泵51，能将催化反应产生的挥发性物质带离反应罐1，有利于推动催化反应进行；抽离的挥发性物质进入专门废气收集装置集中处理，符合清洁生产的理念。待反应液产物浓度达到既定的分离标准后，缓冲液进入树脂柱上游暂存罐9。紧接着，第二份全量缓冲液进入反应罐1参与催化反应。而进入树脂柱上游暂存罐9的第一份缓冲液经过树脂柱本体6纯化后，泵入树脂柱下游暂存罐7。待第二份缓冲液反应结束进入空出来的树脂柱上游暂存罐9后，第一份纯化后的缓冲液进入反应罐1接力反应，依次循环，从而实现催化与纯化两步过程的无缝衔接，即实现了反应缓冲液的循环利用。另外，为了实现反应罐1的无缝衔接，同样配置了数组反应罐1与数组反应板11，在第一组反应罐1中的酶活性降低至不能有效推动反应进程时，有两种方案可供选择：①可以将前次反应罐1上的管道快速切换至当此反应罐1上，实现反应罐1催化反应快速衔接；②还可以迅速更换新准备的含固定化酶的反应板11，实现催化反应快速衔接。
综上所述，对于本发明的反应罐1，通过反应罐1顶层外部有反应液预搅拌装置2，可以形成均一反应体系，保证体系中底物浓度均一。通过反应罐1顶部的喷淋装置3，可以将反应缓冲液均匀喷洒于催化剂上，使催化剂充分发挥催化效果。通过反应罐1可拆卸式多级串联“蒸笼”式结构，可以降低 操作人员清洗反应罐1及更换酶催化剂过程的劳动强度。通过反应罐1侧面的负压抽气管道5及抽气孔，由动力泵51提供管道负压，能将催化反应产生的挥发性物质带离反应罐1，即有利于推动催化反应进行，又可以将抽离的挥发性物质进行集中处理，符合清洁生产的理念。
反应罐1每一个串联单元均具备独立控温夹套4，便于反应体系精准控温。通过反应罐1的独立观察窗12，方便观察反应液面高度等体系状态。通过将固定化酶填充于反应板11中，可以有效防止固定化酶受液流冲击而杂乱分布于反应液中。通过反应罐1的反应液外部循环管道13及反应罐1的反应液外部循环管道13的动力泵51，形成反应液循环通路，反应液循环式反应的好处在于：避免传统的搅拌式反应过程中，搅拌装置对酶催化剂的机械损伤；其次便于酶催化剂与反应液的分离。通过纯化系统树脂柱本体6的上游与下游设立的暂存罐，可以实现缓冲液循环与纯化之间的无缝链接。本发明的各个管道上均设置有阀门，通过管路体系的阀门开关配合，可以控制物料走向，使物料或在催化反应罐1内循环反应，或是进入纯化系统进行产物提纯。
根据本发明的另一实施例，本发明涉及一种使用本发明的上述反应系统制备(R)-邻氯扁桃酸的方法。
根据本发明的再一实施例，提供了一种使用本发明所述的循环式生物酶催化反应系统制备(R)-邻氯扁桃酸的方法，其特征在于，至少包括以下步骤：
(a)培养表达腈水解酶的大肠杆菌工程菌；
(b)将上述大肠杆菌工程菌进行固定化反应形成固定菌，将其均匀平铺在反应罐(1)中各层的反应板(11)，通过加料漏斗(23)将邻氯扁桃腈以及反应的缓冲溶液加入至预搅拌装置(2)中，通过喷淋装置(3)进入到反应罐(1)中，分别与各层的反应板(11)上的固定菌反应；
(c)反应液到达反应罐(1)最底层，在搅拌桨(21)的作用下输送至预搅拌装置(2)中，再继续喷淋至反应罐(1)中；优选地，上述(a)～(c)的步骤按顺序进行1～20次；优选为5～20次，进一步优选为6～18次，更优选为8～15次；
(d)反应结束后，分离收集上清液；
(e)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，任选地所述流穿液在步骤(b)中作为所述反应缓冲液循环使用至少一次，优选为5～20 次，进一步优选为6～18次，更优选为8～15次。
作为优选，在步骤(d)之后，使反应液进入至树脂柱上游暂存罐(9)中，同时在树脂柱本体(6)中提前进行离子交换树脂的预处理，然后将树脂柱上游暂存罐(9)中的反应液输送至树脂柱本体(6)中进行吸附，流出树脂柱本体(6)进入至树脂柱下游暂存罐(7)中，成为流穿液；所述流穿液输送至预搅拌装置(2)中作为缓冲液。
作为优选，在反应液全部流出树脂柱本体(6)后，使用洗脱剂进入到树脂柱本体(6)中对树脂进行洗脱，洗脱液被收集，再经过萃取、浓缩后得到(R)-邻氯扁桃酸成品。
更具体地，在本发明的(R)-邻氯扁桃酸的制备方法中，采用本发明的反应系统进行如下工序。
首先，将含腈水解酶的大肠杆菌工程菌，进行固定化反应形成该反应所需的固定菌，并将其均匀平铺在反应罐1所在的各层的反应板(床层)11，按照图1安装后，通过加料漏斗23将底物(邻氯扁桃腈)以及反应的缓冲溶液加入至预搅拌装置2中，待底物溶解后，通过喷淋装置3进入到反应罐1中，分别与各层的反应板11上的固定菌充分接触反应。通过渗透作用，反应液到达反应罐1最底层时，在搅拌桨21的作用下通过管道被动力装置输送至预搅拌装置2中，再继续喷淋至下端的反应罐1中，如此反复进行。此为催化反应步骤。
待反应通过监测达到实验结束后，反应液在搅拌桨21的作用下，通过管道进入至树脂柱上游暂存罐9中，与此同时，在树脂柱本体6中提前进行离子交换树脂的预处理，待处理完毕后，将树脂柱上游暂存罐9中的反应液通过动力装置输送至树脂柱本体6中进行吸附，并按照一定流速流出树脂柱本体6进入至树脂柱下游暂存罐7中，成为流穿液，流穿液可通过动力装置输送至预搅拌装置2中作为缓冲液。在反应液全部流出树脂柱本体6后，再使用配制好的洗脱剂通过真空管81进入到树脂柱本体6中对树脂进行洗脱，洗脱后的洗脱液经过树脂柱本体6下方阀门被收集，再经过萃取剂萃取，浓缩后得到(R)-邻氯扁桃酸成品。此为离子交换步骤。
经过树脂柱本体6处理后的反应液流出树脂柱本体6后，成为流穿液，再经过动力装置进入至预搅拌装置2中，可继续作为下一步反应的缓冲液继续 使用，持续循环10次以上。此为循环反应步骤。
下面结合具体实施例进一步详述本发明。如无特别说明，本发明的实施例中的原料和催化剂均通过商业途径购买。
实施例1
1、在250ml的三角瓶中加入100ml磷酸缓冲液(NaH 2PO 4/Na 2HPO 4缓冲液)，pH在7.5～8.5，加入1.5g的工程菌细胞、30mM底物邻氯扁桃腈以及助溶剂(甲醇，添加量为反应体系体积的1％)，在220rpm的摇床中反应，其中底物通过6次分批流加，每次流加底物浓度为25mM，用薄层层析监测反应的终点。当底物反应完后，离心去除不溶物，将上清液转移到经活化的强碱性阴离子树脂(HZ-202型树脂)，吸附上清液中的产物(R)-邻氯扁桃酸，收集流穿液，记为反应液1；再使用50ml 2M HCl洗脱树脂上的产物，收集洗脱液，记为洗脱液1。
2、转移反应液1至250ml的三角瓶中，加入1.5g的工程菌细胞、30mM底物邻氯扁桃腈以及助溶剂(甲醇，添加量为反应体系体积的1％)，在220rpm的摇床中反应，其中底物通过6次分批流加，每次流加底物浓度为25mM，用薄层层析监测反应的终点。当底物反应完后，离心去除不溶物，将上清液转移到经活化的强碱性阴离子树脂(HZ-202型树脂)，吸附上清液中的产物(R)-邻氯扁桃酸，收集流穿液，记为反应液2；再使用50ml 2M HCl洗脱树脂上的产物，收集洗脱液，记为洗脱液2。
如此循环进行10次，分别收集洗脱液1～11。洗脱液进行HPLC分析，检测结果如下表1。
表1反应液不同循环次数所制备产物(R)-邻氯扁桃酸的检测结果
Figure PCTCN2019114309-appb-000002
注 1：底物为邻氯扁桃腈消旋体。
注 2：反应液体积减少后未补加新的反应缓冲液。
注 3：循环10次，催化的底物浓度相当于1500mM。
注 4：树脂仅在第一次进行预处理，吸附能力逐渐下降。
实施例2
实施例2只在每次测量收集的反应液体积后，补充损失的体积，其他实施同实施例1，所得洗脱液产物结果如表2。
表2
Figure PCTCN2019114309-appb-000003
注 1：底物为邻氯扁桃腈消旋体。
注 2：反应液体积减少后补加新的反应缓冲液。
注 3：循环10次，催化的底物浓度相当于1500mM。
注 4：树脂仅在第一次进行预处理，吸附能力逐渐下降。
由本实施例的实验结果可知，通过补加新的反应缓冲液可进一步显著提高产物的收率。
实施例3
实施例3只在每次测量收集的反应液体积后，补充损失的体积，持续进行20次，其他实施方式同实施例1，所得洗脱液产物结果表3。
表3
Figure PCTCN2019114309-appb-000004
注 1：底物为邻氯扁桃腈消旋体。
注 2：反应液体积减少后补加新的反应缓冲液。
注 3：循环15次，催化的底物浓度相当于2250mM。
注 4：树脂仅在第一次进行预处理，吸附能力逐渐下降。
由本实施例的实验结果可知，通过补加新的反应缓冲液可显著提高产物的收率。随着循环次数的增加，产物的收率出现下降的趋势，因此本发明循环的次数优选为8～15次。
实施例4
和实施例1相比，实施例4在步骤1和步骤2中，底物通过5批分批流加，其他实施方式同实施例1；所得洗脱液产物结果表4。
表4
Figure PCTCN2019114309-appb-000005
注 1：底物为邻氯扁桃腈消旋体。
注 2：反应液体积减少后未补加新的反应缓冲液。
注 3：树脂仅在第一次进行预处理，吸附能力逐渐下降。
由实施例4的实验结果可知：每个循环流加5批底物，虽然反应各种质量指标没有明显变化，但同等条件下，缓冲液循环6次，实施例4中底物催化浓度为750mM，低于实施例1的900mM。缓冲液的产物容量没有得到充 分利用，从产能及经济角度考虑，实施例4选取的底物补加次数对应的产出比不如实施例1的底物补加次数对应的产出比。
对比例1：和实施例1相比，对比例1在步骤1和步骤2中，每次流加底物浓度为8mM，其他实施方式同实施例1；
对比例2：和实施例1相比，对比例2在步骤1和步骤2中，每次流加底物浓度为55mM，其他实施方式同实施例1；
对比例3：和实施例1相比，对比例3在步骤1和步骤2中，底物通过8批分批流加，其他实施方式同实施例1。
所得洗脱液产物结果表5。
表5
Figure PCTCN2019114309-appb-000006
由对比例1的实验结果可知：当补加底物浓度过低时，出现如下缺点：(1)过低的底物浓度，此时酶催化活性大大过剩，这时候由于酶的R型光学选择性不是绝对的，会错误结合少量S型底物并催化生成S型产物(虽然反应能障高，但会发生)。这样会导致反应后的对映体过量值较实施例1相 比偏低3％左右(由于纯化产品的对映体过量值始终偏低，则缓冲液循环使用6次后即停止进行实验)。(2)同等条件下，缓冲液循环6次，对比例1底物催化浓度为288mM，远低于实施例1的900mM。从产能及经济角度考虑，对比例1选取的底物补加浓度对应的催化效果不如实施例1选取的底物补加浓度对应的催化效果好。
由对比例2的实验结果可知：当补加底物浓度过高时，此浓度下的底物浓度会让酶催化剂(蛋白质)发生不可逆失活，在补加第三次底物后，反应不能继续进行，体系产物纯度低，在此基础上纯化收率比正常收率偏低10％左右(第一轮循环未完成，实验终止)。
由对比例3的实验结果可知：
底物通过8批分批流加，每个循环底物累积浓度超出了酶催化剂的最佳催化效能，会导致体系反应后程速率明显降低，而且会积累大量醛、底物等杂质，降低反应收率。此外，此条件下体系中较多的杂质会影响树脂纯化效率，增大树脂再生成本。说明对比例3选择的8批补加次数不适合实际生产。通过对比例与本发明实施例的效果对比，说明本发明相应参数选择对应的技术效果是目前的最优组合，进一步体现出了本发明的技术优势。
实施例5
pH＝7.58，(R)-邻氯扁桃酸含量为143mmol/L的水相固定菌催化反应液为待处理液，采用HZ-202型树脂作为强碱性阴离子树脂。
1、在25℃、220rpm摇床条件下预处理：先用1M的NaOH溶液浸润洗涤，NaOH溶液的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1，再用1M稀盐酸浸润洗涤，稀盐酸的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1，均浸润15min，再以2ml/min的速度洗涤；
2、用pH＝8的缓冲液(NaH 2PO 4/Na 2HPO 4缓冲液)浸泡处理后的树脂，将此反应液采用静态吸附的方式对产物邻氯扁桃酸进行提取，离子交换一定时间(2～6h)后，将液体与树脂分离；
3、用5倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
4、收集到的洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例6
pH＝4.3，(R)-邻氯扁桃酸含量为140mmol/L的水相固定菌催化反应液为待处理液，采用HZ-202型树脂作为强碱性阴离子树脂。
1、常温下预处理：先用1M的NaOH溶液浸润洗涤，NaOH溶液的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1，再用1M稀盐酸浸润洗涤，稀盐酸的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1，均浸润15min，以2ml/min的速度洗涤；
2、用pH＝8的缓冲液(NaH 2PO 4/Na 2HPO 4缓冲液)浸泡处理后的树脂，将此反应液采用动态吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后，以2.5ml/min的速度将液体与树脂分离；
3、用5倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
4、收集到的洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例7
pH＝7.2，(R)-邻氯扁桃酸含量为156mmol/L的水相游离菌催化反应液为待处理液，采用HZ 202型树脂作为强碱性阴离子树脂。
1、常温在交换柱中，采用循环使用1次后的树脂进行吸附，采用动态吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后以2.5ml/min的速度将液体与树脂分离；
2、用5倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
3、收集到的洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50℃～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)- 邻氯扁桃酸粗品。实验结果如表6所示。
实施例8
pH＝3.9，(R)-邻氯扁桃酸含量为205mmol/L的水相固定菌催化反应液为待处理液，采用HZ202型树脂作为强碱性阴离子树脂。
1、常温在交换柱中，采用循环使用2次后的树脂进行吸附，采用动态吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后，以2.5ml/min的速度将液体与树脂分离；
2、用5倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
3、收集到的洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50℃～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例9
pH＝5.4，(R)-邻氯扁桃酸含量为143mmol/L的水相游离菌催化反应液为待处理液，采用HZ-202型树脂作为强碱性阴离子树脂。
1、常温在交换柱中，采用循环使用3次后的树脂进行吸附，采用动态吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后，以2.5ml/min的速度将液体与树脂分离；
2、用8倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
3、收集到的洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50℃～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例10
pH＝3.1，(R)-邻氯扁桃酸含量为143mmol/L的水相游离菌催化反应液为待处理液，采用HZ-202型树脂作为强碱性阴离子树脂。
1、常温在交换柱中，采用循环使用4次后的树脂进行吸附，采用动态 吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后，将液体与树脂以2.5ml/min的速度将其流下分离；
2、用10倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
3、对洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50℃～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例11
pH＝6.39，(R)-邻氯扁桃酸含量为425mmol/L的水相固定菌催化反应液为待处理液，采用HZ-202型树脂作为强碱性阴离子树脂。
1、常温在交换柱中，采用循环使用4次后的树脂进行吸附，采用动态吸附的方式对产物邻氯扁桃酸进行提取，离子交换15min后，以2.5ml/min的速度将液体与树脂分离；
2、用8倍树脂质量的体积的1M稀盐酸，以2.5ml/min的流速对该吸附后的树脂进行洗脱；
3、对洗脱液用0.3倍洗脱液体积的乙酸乙酯进行萃取，萃取后的有机相减压蒸馏(50℃～60℃，0.09MPa)，得到的固体经过真空干燥得到(R)-邻氯扁桃酸粗品。实验结果如表6所示。
实施例12
pH＝5.2，(R)-邻氯扁桃酸的含量为143mmol/L的水相固定菌催化反应液为待处理液。
使用多次循环后的HZ-202型树脂，其前一次的吸附效率仅为66％，对该树脂进行再生操作：用浓度为2mol/L的NaOH溶液和质量百分比浓度为10％NaCl混合碱液先浸泡30min，混合碱液的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1；再以2.0ml/min的速度洗涤；再用浓度为2mol/L的HCl和浓度为30％的乙醇的混合酸液对该树脂浸润30min，混合酸液的体积(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为3：1；再以2.0ml/min的速度洗涤。再生处理后的树脂则按照吸 附操作对(R)-邻氯扁桃酸待提取液进行提取。
实验结果如表6所示。
实施例13
按照实施例6进行分离提取，区别仅在于：预处理步骤为先用5M的NaOH溶液浸润洗涤，再用5M稀盐酸浸润洗涤，均浸润10min，再以1ml/min的速度洗涤。实验结果如表6所示。
实施例14
按照实施例6进行分离提取，区别仅在于：预处理步骤为先用0.5M的NaOH溶液浸润洗涤，再用0.5M稀盐酸浸润洗涤，均浸润60min，再以5ml/min的速度洗涤。实验结果如表6所示。
对比例4
按照实施例6进行分离提取，区别仅在于：预处理步骤仅采用NaOH溶液浸润洗涤。实验结果如表6所示。
对比例5
按照实施例6进行分离提取，区别仅在于：预处理步骤仅采用稀盐酸溶液浸润洗涤。实验结果如表6所示。
对比例6
按照实施例6进行分离提取，区别仅在于：预处理步骤中依次采用0.4mol/L NaOH溶液、0.4mol/L HCl溶液浸润洗涤。实验结果如表6所示。
对比例7
按照实施例6进行分离提取，区别仅在于：预处理步骤中依次采用6mol/L NaOH溶液、6mol/L HCl溶液浸润洗涤。实验结果如表6所示。
对比例8
按照实施例6进行分离提取，区别仅在于：预处理步骤中NaOH溶液(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为1.5：1，HCl(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为1.5：1。实验结果如表6所示。
对比例9
按照实施例6进行分离提取，区别仅在于：预处理步骤中NaOH溶液(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为6：1，HCl(单位为ml)与强碱性阴离子树脂的质量(单位为g)之比为6：1。实验结果如表6所示。
对比例10
按照实施例6进行分离提取，区别仅在于：预处理步骤中NaOH溶液洗涤的速度为0.5ml/min、HCl溶液洗涤的速度为0.5ml/min。实验结果如表6所示。
对比例11
按照实施例6进行分离提取，区别仅在于：预处理步骤中NaOH溶液洗涤的速度为5.5ml/min、HCl溶液洗涤的速度为5.5ml/min。实验结果如表6所示。
表6
Figure PCTCN2019114309-appb-000007
由对比例4和对比例5的实验结果可知，如果预处理步骤仅采用酸或碱处理，(R)-邻氯扁桃酸粗品的收率显著降低，纯度也较差。由对比例6～对比例11的实验结果可知，如果预处理步骤的具体条件不在本发明实验例的优选范围内，(R)-邻氯扁桃酸粗品的收率和/或纯度均具有下降的趋势。
实施例15
(1)将发酵后的工程菌固定化，得到的固定化菌称取90g均匀平铺在反应罐1的砂芯反应板11上。反应板11为三层，每层平铺30g固定化菌，按照实验装置图(附图1)安装反应罐1。
(2)量取6L的pH＝8的磷酸盐缓冲液(NaH 2PO 4/Na 2HPO 4缓冲液)于预搅拌装置2中，另称取底物原料邻氯扁桃腈24.0g(总共144.0g，分6次称量，每间隔2h投样1次)与其混合，开启搅拌电机22，待固体全部溶解后，通过喷淋装置3进入到反应罐1中。由重力与渗透作用下，液体依次通过三层床层反应板11，在搅拌桨21的作用下，液体流出反应罐1，通过动力装置输送至顶部的预搅拌装置2中。重复如上的过程，反应2h后，继续通过加料漏斗23投料，投料6次，反应12h。
(3)反应过程中，称取HZ-202树脂150g，进行预处理操作。即使用1M的NaOH 200ml泡洗，和使用1M的稀盐酸200ml泡洗，再使用纯化水将树脂洗至中性待用。待反应结束后，将反应液(测得其pH＝7.8)通过动力装置全部装入树脂柱上游暂存罐9，并输送至树脂柱本体6进行吸附，在树脂柱中静置30min，打开树脂柱本体6的底端阀门，使液体以5ml/min的吸附速度流下，再使用压缩空气管82将树脂柱的液体全部排出，流下的液体(即流穿液，可循环作为缓冲液)通过动力系统输送到树脂柱下游暂存罐7。
(4)另外，将配置好的1M稀盐酸洗脱剂以2.5ml/min的速度对吸附后的树脂进行洗脱，洗脱后的洗脱液收集，得到3L洗脱液，并使用0.3倍体积的1L乙酸乙酯对其萃取，少量多次，萃取后的有机相进行减压浓缩(0.09MPa，45℃)，得到(R)-邻氯扁桃酸成品。纯度99.2％，对映体过量ee＝99.5％，收率84.4％。
实施例16
(1)将发酵后的工程菌固定化，得到的固定化菌称取90g均匀平铺在反应罐1的砂芯反应板装置11上。反应板11为三层，每层平铺30g固定化菌，按照实验装置图(附图1)安装反应罐1。
(2)量取6L的pH＝8的磷酸盐缓冲液(NaH 2PO 4/Na 2HPO 4缓冲液)于预搅拌装置2中，另称取底物原料邻氯扁桃腈24.0g(总共144.0g，分6次称量，每间隔2h投样1次)与其混合，开启搅拌电机22，待固体全部溶解后，通过 喷淋装置3进入到反应罐1中。由重力与渗透作用下，液体依次通过三层床层反应板11，在搅拌桨21的作用下，液体流出反应罐1，通过动力装置输送至顶部的预搅拌装置2中。重复如上的过程，反应2h后，继续通过加料漏斗23投料，投料6次，反应12h。
(3)反应过程中，称取HZ-202树脂150g，进行预处理操作。即使用1M的NaOH 200ml泡洗，和使用1M的稀盐酸200ml泡洗，再使用纯化水将树脂洗至中性待用。待反应结束后，将反应液(测得其pH＝7.8)通过动力装置全部装入树脂柱上游暂存罐9，并输送至树脂柱本体6进行吸附，在树脂柱中静置30min，打开树脂柱本体6的底端阀门，使液体以5ml/min的吸附速度流下，再使用压缩空气管82将树脂柱的液体全部排出，流下的液体(即流穿液，可循环作为缓冲液)通过动力系统输送到树脂柱下游暂存罐7。
(4)再将平铺在反应板11上的固定菌收集，回收处理，继续称取新的固定菌90g，按照与实施例15同样的步骤将其铺在三层反应板11上，将树脂柱下游暂存罐7的液体装入至预搅拌装置2。另称取底物原料邻氯扁桃腈24.0g(总共144.0g，分6次称量，每间隔2h投样1次)与其混合，开启搅拌电机22，待固体全部溶解后，通过喷淋装置3进入到反应罐1中。由重力与渗透作用下，液体依次通过三层床层反应板11，在搅拌桨21的作用下，液体流出反应罐1，通过动力装置输送至顶部的预搅拌装置2中。重复如上的过程，反应2h后，继续通过加料漏斗23投料，投料6次，反应12h。即，流穿液作为反应缓冲液循环使用一次。
(5)另外，将配置好的1M稀盐酸洗脱剂以2.5ml/min的速度对吸附后的树脂进行洗脱，洗脱后的洗脱液收集，得到3L洗脱液，并使用0.3倍体积的1L乙酸乙酯对其萃取，少量多次，萃取后的有机相进行减压浓缩(0.09MPa，45℃)，得到(R)-邻氯扁桃酸成品。纯度99.3％，对映体过量ee＝99.1％，收率84.9％。
实施例17
重复实施例16的步骤，不同在于，在实施例16的步骤(4)，即流穿液作为反应缓冲液循环使用一次时，重复进行实施例16的步骤(3)，即再次获得流穿液，然后再次进行实施例16的步骤(4)。即，流穿液作为反应缓冲液循 环使用二次。然后进行实施例16的步骤(5)，得到(R)-邻氯扁桃酸成品。实验结果如表7所示。
实施例18
重复实施例16的步骤，不同在于，使实施例16的步骤按照：“步骤(1)-步骤(2)-步骤(3)-步骤(4)-步骤(3)-步骤(4)-步骤(3)-步骤(4)-步骤(5)”来进行，即流穿液作为反应缓冲液循环使用三次。得到(R)-邻氯扁桃酸成品。实验结果如表7所示。
实施例19～35
重复实施例16的步骤，不同在于，分别使流穿液作为反应缓冲液循环使用4～20次。得到(R)-邻氯扁桃酸成品。实验结果如表7所示。
表7
Figure PCTCN2019114309-appb-000008
注 1：每次循环，树脂均进行预处理使其吸附能力恢复。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限 于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
实用性
根据本发明实施例所提供的(R)-邻氯扁桃酸的制备方法和循环式生物酶催化反应系统可应用于药物化学和生物酶催化设备领域。

Claims (31)

  1. 一种(R)-邻氯扁桃酸的制备方法，其特征在于，至少包括以下步骤：
    (1)培养表达腈水解酶的大肠杆菌工程菌；
    (2)采用培养获得的工程菌作为催化剂、邻氯扁桃腈作为底物、反应缓冲液作为反应介质进行反应；
    (3)反应结束后，分离收集上清液；
    (4)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，任选地所述流穿液在步骤(2)中作为所述反应缓冲液循环使用至少一次，
    优选为5～20次，进一步优选为6～18次，更优选为8～15次。
  2. 根据权利要求1所述的制备方法，其特征在于，当所述流穿液在步骤(2)中作为所述反应缓冲液循环使用造成反应体系的体积减少时，补充新的反应缓冲液。
  3. 根据权利要求1或2所述的制备方法，其特征在于，所述反应缓冲液的pH为7.0～9.0；
    优选的，所述反应缓冲液选自KH 2PO 4/K 2HPO 4缓冲液、NaH 2PO 4/Na 2HPO 4缓冲液或三羟甲基氨基甲烷-HCl缓冲液中的至少一种；
    更优选的，步骤(2)中反应体系的pH为7.5～8.5。
  4. 根据权利要求1～3中任一项所述的制备方法，其特征在于，在步骤(2)中，所述转化反应的反应温度为25～50℃，优选为25～45℃，更优选为30℃。
  5. 根据权利要求1～4中任一项所述的制备方法，其特征在于，在步骤(2)的反应体系中，所述工程菌细胞的浓度为10g/L～40g/L；
    优选的，步骤(2)中的反应体系中还添加助溶剂；助溶剂优选碳原子数为1～8的醇、碳原子数为6～12的烷烃中的至少一种；
    优选的，助溶剂添加的量为反应体系总体积的1％～10％。
  6. 根据权利要求1～5中任一项所述的制备方法，其特征在于，在步骤(2)中，所述底物通过5～7次分批流加，优选为6次；每次流加底物的浓度为10～50mM，优选为20～45mM，更优选为25～30mM。
  7. 根据权利要求1～6中任一项所述的制备方法，其特征在于，在步骤(3)中，所述分离的方法选自离心、抽滤、压滤中的至少一种；
    优选的，所述离心采用6000～10000rpm，优选为6000～8000rpm，更 优选为7000rpm。
  8. 根据权利要求1～7中任一项所述的制备方法，其特征在于，在步骤
    (4)中，所述提取的方法为采用阴离子树脂进行吸附，优选采用强碱性阴离子树脂。
  9. 根据权利要求1～8中任一项所述的制备方法，其特征在于，所述制备方法还包括以下步骤：
    (5)使用洗脱剂对步骤(4)中吸附后的阴离子树脂进行洗脱，得到洗脱液；所述洗脱剂优选为1～2M的盐酸；
    (6)采用有机溶剂萃取所述洗脱液水相中的(R)-邻氯扁桃酸，分离有机溶剂，得到所述(R)-邻氯扁桃酸的晶体；
    所述有机溶剂优选乙酸乙酯或氯仿；所述分离的方法优选为旋转蒸发。
  10. 根据权利要求1～9中任一项所述的制备方法，其特征在于，所述步骤(4)至少包括以下步骤：
    (i)将强碱性阴离子树脂进行预处理，所述预处理包括依次进行碱洗和酸洗的步骤；所述强碱性阴离子树脂优选季胺盐强碱性阴离子树脂；
    (ii)采用步骤(i)处理后的强碱性阴离子树脂对(R)-邻氯扁桃酸的待处理液进行吸附；
    (iii)采用洗脱剂对步骤(ii)的强碱性阴离子树脂进行洗脱，得到洗脱液，所述洗脱液经萃取、浓缩后得到所述(R)-邻氯扁桃酸。
  11. 根据权利要求10所述的制备方法，其特征在于，在步骤(i)中，所述碱洗的步骤包括：先用浓度为0.5～5mol/L的碱液浸泡强碱性阴离子树脂10～60min，然后用所述碱液以1.0～5.0ml/min的速度进行洗涤；
    优选的，浸泡时所述碱液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述碱液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述碱液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
    更优选的，所述碱选自一元碱。
  12. 根据权利要求10或11所述的制备方法，其特征在于，在步骤(i)中，所述酸洗的步骤包括：先用浓度为0.5～5mol/L的酸液浸泡强碱性阴离子树脂15～30min，然后用所述酸液以1.0～5.0ml/min的速度进行洗涤；
    优选的，浸泡时所述酸液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述酸液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述酸液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
    更优选的，所述酸选自一元酸。
  13. 根据权利要求10～12中任一项所述的制备方法，其特征在于，在步骤(i)中，所述酸洗后还包括水洗至中性的步骤；优选的，所述碱洗前还包括水洗去机械杂质的步骤；
    更优选的，所述水选自纯化水。
  14. 根据权利要求10～13中任一项所述的制备方法，其特征在于，在步骤(ii)中，所述吸附前还包括浸润的步骤，优选的，所述浸润的步骤包括：向所述强碱性阴离子树脂中加入缓冲液浸润10～30min，所述缓冲液的体积为强碱性阴离子树脂质量的2～5倍；所述缓冲液的pH值优选为7～9；优选的，所述缓冲液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述缓冲液的体积以mL计时，所述强碱性阴离子树脂的质量以g计。
  15. 根据权利要求10～14中任一项所述的制备方法，其特征在于，将洗脱后的强碱性阴离子树脂水洗至中性后进入步骤(ii)循环使用，当循环使用15～20次后，对所述强碱性阴离子树脂进行再生处理；优选的，所述再生处理包括以下方式中的至少一种：
    方式1：用混合碱液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤；所述混合碱液中含有浓度为2～5mol/L的一元碱和质量百分比浓度为10％～30％的氯化钠；优选的，所述混合碱液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的，所述混合碱液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述混合碱液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
    方式2：用混合酸液先浸泡10～60min，再以1.0～5.0ml/min的速度洗涤；所述混合酸液中含有2～5mol/L的一元酸和体积百分比浓度为10％～50％的低级醇；优选的，所述低级醇选自碳原子为1～6的醇；更优选的，所述混合酸液的体积与强碱性阴离子树脂的质量之比为2～5：1；优选的， 所述混合酸液的体积以L计时，所述强碱性阴离子树脂的质量以Kg计，或者优选的，所述混合酸液的体积以mL计时，所述强碱性阴离子树脂的质量以g计；
    进一步优选的，所述再生处理还包括水洗至中性的步骤，所述水优选为纯化水。
  16. 根据权利要求10～15中任一项所述的制备方法，其特征在于，在步骤(ii)中，所述吸附的温度为10～60℃；
    在步骤(iii)中，所述洗脱的温度为10～60℃；优选的，所述萃取采用的有机溶剂选自酯类有机溶剂、醚类有机溶剂、醇类有机溶剂中的至少一种；更优选碳原子数为4～8的酯类有机溶剂、碳原子数为2～6的醚类有机溶剂、碳原子数为1～8的醇类有机溶剂；更优选乙酸乙酯、乙醚、乙醇中的至少一种。
  17. 根据权利要求10～16中任一项所述的制备方法，其特征在于，在步骤(ii)中，所述吸附的流速为0.1～10ml/min；在步骤(iii)中，所述洗脱的流速为0.1～10ml/min。
  18. 根据权利要求10～17中任一项所述的制备方法，其特征在于，所述待处理液为水相反应液或包含与水互溶的有机相的水相反应液；
    优选的，所述待处理液包括化学合成、生物催化合成、拆分合成所得的反应液；
    更优选的，所述待处理液的pH值为2～8。
  19. 根据权利要求18所述的制备方法，其特征在于，当所述待处理液为生物催化合成反应液时，步骤(ii)吸附后的反应液作为生物催化反应体系的反应液循环使用。
  20. 一种用于权利要求1～19中任一项的制备方法的循环式生物酶催化反应系统，其特征在于，包括顺次连接的循环式固定化反应床、树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐；
    所述循环式固定化反应床包括：
    反应罐；
    设置在反应罐上方的预搅拌装置，树脂柱下游暂存罐还与预搅拌装置连接；
    设置在预搅拌装置与反应罐之间的喷淋装置，所述喷淋装置伸至反应罐顶部的内部，用于向反应罐喷射反应液；
    间隔的设置在反应罐内部的至少一个反应板，将反应罐内部分隔为多个反应空间，所述反应板内部填充有催化剂；
    设置在反应罐底部的底部搅拌装置，且反应罐的底部与树脂柱上游暂存罐连接，反应罐的底部还与预搅拌装置连接。
  21. 根据权利要求20所述的一种循环式生物酶催化反应系统，其特征在于，所述预搅拌装置包括：
    与喷淋装置连接的搅拌罐；
    设置在搅拌罐内部的搅拌桨与设置在搅拌罐外部的搅拌电机，搅拌电机与搅拌桨连接；
    设置在搅拌罐上的加料漏斗。
  22. 根据权利要求20或21所述的一种循环式生物酶催化反应系统，其特征在于，所述底部搅拌装置包括设置在反应罐内部的搅拌桨和设置在反应罐外部的搅拌电机，所述搅拌电机与搅拌桨连接。
  23. 根据权利要求20～22中任一项所述的一种循环式生物酶催化反应系统，其特征在于，所述循环式生物酶催化反应系统还包括废气收集装置、动力泵和负压抽气管道，每个反应空间处的反应罐上均设置有抽气孔，废气收集装置依次通过动力泵、负压抽气管道与每个反应空间的抽气孔连接。
  24. 根据权利要求20～23中任一项所述的一种循环式生物酶催化反应系统，其特征在于，所述反应板内部设置有用于容纳催化剂的填充腔，填充腔上下表面均设置有可拆卸的砂芯筛网。
  25. 根据权利要求21～24中任一项所述的一种循环式生物酶催化反应系统，其特征在于，所述反应板与搅拌罐的侧壁可拆卸连接。
  26. 根据权利要求20～25中任一项所述的一种循环式生物酶催化反应系统，其特征在于，所述树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐均连接有真空管、压缩空气管及温度探头。
  27. 根据权利要求20～26中任一项所述的一种循环式生物酶催化反应系统，其特征在于，每个反应空间处的反应罐上均设置有观察窗。
  28. 根据权利要求21～27中任一项所述的一种循环式生物酶催化反应系 统，其特征在于，所述搅拌罐、树脂柱上游暂存罐、树脂柱本体、树脂柱下游暂存罐外部均设置有控温夹套。
  29. 一种使用权利要求20～28中任一项所述的循环式生物酶催化反应系统制备(R)-邻氯扁桃酸的方法，其特征在于，至少包括以下步骤：
    (a)培养表达腈水解酶的大肠杆菌工程菌；
    (b)将上述大肠杆菌工程菌进行固定化反应形成固定菌，将其均匀平铺在反应罐(1)中各层的反应板(11)，通过加料漏斗(23)将邻氯扁桃腈以及反应的缓冲溶液加入至预搅拌装置(2)中，通过喷淋装置(3)进入到反应罐(1)中，分别与各层的反应板(11)上的固定菌反应；
    (c)反应液到达反应罐(1)最底层，在搅拌桨(21)的作用下输送至预搅拌装置(2)中，再继续喷淋至反应罐(1)中；优选地，上述(a)～(c)的步骤按顺序进行1～20次；优选为5～20次，进一步优选为6～18次，更优选为8～15次；
    (d)反应结束后，分离收集上清液；
    (e)提取所述上清液中的(R)-邻氯扁桃酸后获得流穿液，任选地所述流穿液在步骤(b)中作为所述反应缓冲液循环使用至少一次，优选为5～20次，进一步优选为6～18次，更优选为8～15次。
  30. 根据权利要求29所述的方法，其特征在于，在步骤(d)之后，
    使反应液进入至树脂柱上游暂存罐(9)中，同时在树脂柱本体(6)中提前进行离子交换树脂的预处理，然后将树脂柱上游暂存罐(9)中的反应液输送至树脂柱本体(6)中进行吸附，流出树脂柱本体(6)进入至树脂柱下游暂存罐(7)中，成为流穿液；
    所述流穿液输送至预搅拌装置(2)中作为缓冲液。
  31. 根据权利要求30的方法，其特征在于，在反应液全部流出树脂柱本体(6)后，使用洗脱剂进入到树脂柱本体(6)中对树脂进行洗脱，洗脱液被收集，再经过萃取、浓缩后得到(R)-邻氯扁桃酸成品。
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