Method for asymmetric synthesis of high-optical-purity ethyl (R)-3-hydroxybutyrate through enzyme catalysis

Abstract

The invention provides a method for asymmetrically synthesizing high-optical-purity (R)-3-hydroxybutyric acid ethyl ester through enzyme catalysis, which comprises the following steps: taking ethyl acetoacetate as a substrate, introducing a reducing equivalent supply component into a water-phase medium, and adding an enzyme catalyst to start an asymmetric reduction reaction; in the reaction process, the pH value of a reaction solution is collected on line, continuous or intermittent dropwise adding of an alkali solution is carried out according to a collection result, the pH value is maintained in a preset interval, and the pH value stability judgment of a set continuous time window is used as an end point control criterion to terminate the reaction. Through online collection and closed-loop regulation of the pH of the reaction liquid, the pH in the reaction process is continuously maintained in a preset interval, and the influence of pH fluctuation caused by the condition change of a multi-phase system on an enzyme catalysis microenvironment is avoided, so that the reaction stereoselectivity is stabilized, the side reaction occurrence probability is reduced, and the reaction yield is improved. The (R)-3-hydroxybutyric acid ethyl ester can continuously obtain high optical purity, and the difference between batches is reduced.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of enzyme catalysis, in particular to a method for enzyme-catalyzed asymmetric synthesis of high-optical-purity (R)-3-hydroxybutyric acid ethyl ester. BACKGROUND

[0002] (R)-3-hydroxybutyric acid ethyl ester is an important chiral hydroxyl ester compound, which contains both hydroxyl and ester functional groups in the molecule, and is often used as a chiral synthesis block for the preparation of various drugs, fine chemicals and functional materials; because the downstream application is sensitive to the configuration, the optical purity and impurity level of (R)-3-hydroxybutyric acid ethyl ester are usually regarded as key quality indicators, therefore, it is of practical significance to develop a preparation process which can stably obtain high-optical-purity products and is suitable for scale-up production.

[0003] In the prior art, such enzyme-catalyzed asymmetric reduction system usually belongs to a multi-phase reaction system coexisting with a solvent / water phase, and the substrate addition mode, reduction equivalent supply state, enzyme activity change and dynamic change of the solvent / water phase ratio during the reaction process can all cause the fluctuation of the pH of the reaction solution, the pH fluctuation can change the microenvironment of the enzyme catalytic reaction, and then affect the stereoselectivity and stability of the reaction path, and under the scale-up production conditions, the conversion rate fluctuation, optical purity deviation and impurity spectrum instability are easily caused, so that the product is difficult to continuously and stably meet the requirements of high-optical-purity and impurity limit.

[0004] Therefore, a method for enzyme-catalyzed asymmetric synthesis of high-optical-purity (R)-3-hydroxybutyric acid ethyl ester is provided. SUMMARY

[0005] Therefore, the application provides a method for enzyme-catalyzed asymmetric synthesis of high-optical-purity (R)-3-hydroxybutyric acid ethyl ester to solve or alleviate the technical problems in the prior art, and at least provides a beneficial choice.

[0006] The technical scheme of the application is as follows: a method for enzyme-catalyzed asymmetric synthesis of high-optical-purity (R)-3-hydroxybutyric acid ethyl ester, comprising the following steps:

[0007] S1, acetyl acetic acid ethyl ester, glucose and an aqueous medium are added to a reaction kettle, stirring is started to make the system uniform, the temperature is controlled to be 30-35 DEG C, and pre-equilibrium is carried out for 10-60 minutes; wherein the volume fraction of acetyl acetic acid ethyl ester in the reaction system is 20%-55%, and the addition amount of glucose is 0.5%-15%;

[0008] The volume fraction of acetyl acetic acid ethyl ester is 30%-50%, the stirring speed is 200-600 rpm, and the pre-equilibrium time is 20-40 minutes.

[0009] S2, under stirring, the pH of the reaction solution is adjusted to 7.0±0.1 by adding alkali solution and maintained for 1-20 minutes, and then an enzyme catalyst is added to start the asymmetric reduction reaction;

[0010] S3, the temperature is controlled at 33-37℃ during the reaction, the pH of the reaction solution is collected on-line, and intermittent or continuous dropping of alkali solution is performed according to the collected value to maintain the pH of the reaction solution at 6.5-7.0; the reaction is terminated when the end point determination condition is met; wherein the end point determination condition is that within a determination time window of 10-60 minutes, the pH of the reaction solution is always at 6.5-7.0 and the maximum pH fluctuation amplitude is ≤0.10;

[0011] In S2 and S3, the alkali solution is a 20%-35% sodium hydroxide aqueous solution by mass fraction; the dropping is performed by pulse dropping rule, i.e. when the on-line collected pH is <6.5, the dropping is started, and when the pH reaches 6.9-7.0, the dropping is stopped, and the next sampling cycle is entered to continue to determine whether dropping is needed;

[0012] The on-line collection is performed according to a fixed sampling period, and the sampling period is 5-60 seconds; each sampling period takes the deviation ΔpH of the current pH from the lower limit 6.5 of the target interval as the trigger, determines the dropping time according to ΔpH classification, and performs dropping once, wherein:

[0013] When ΔpH≥0.30, the dropping time is 5-10 seconds;

[0014] When 0.10≤ΔpH<0.30, the dropping time is 2-5 seconds;

[0015] When 0<ΔpH<0.10, the dropping time is 0.5-2 seconds.

[0016] The end point determination condition further includes any one or both of the following:

[0017] (1) within the determination time window, the cumulative dropping amount of alkali solution is ≤0.05% of the total mass of the reaction system;

[0018] (2) within the determination time window, the pH fluctuation amplitude is ≤0.05, and no dropping operation is performed continuously for not less than 30 minutes.

[0019] S4, after the reaction is terminated, a filter cake is removed by plate and frame filtration to obtain a filtrate, the filtrate is left to stratify for 30-240 minutes, and an organic phase is separated;

[0020] The plate and frame filtration is completed within 0-30 minutes after the reaction is terminated; the stratification temperature is 20-35℃; when the organic phase is separated, the volume Vorg of the organic phase is recorded, and Vorg is taken as a measurement reference for water washing in S5.

[0021] S5, after the organic phase is washed with water for 1-3 times, activated carbon is added for decolorization and filtration to obtain a crude organic phase;

[0022] The activated carbon is added in an amount of 0.1%-2.0% of the mass of the crude organic phase, the decolorization temperature is 20-40 DEG C, and the decolorization time is 10-60 minutes, and after decolorization, the crude organic phase is filtered to obtain a crude organic phase;

[0023] S6, the crude organic phase is subjected to vacuum distillation, the front fraction is removed first, and then the target fraction is collected to obtain (R)-3-hydroxybutyric acid ethyl ester product;

[0024] The operation pressure of the vacuum distillation is 5-50 kPa, and the target fraction is collected after the front fraction is removed until the water content of the distillate is less than or equal to 0.2%.

[0025] Further preferably, the enzyme catalyst is a ketoreductase or a dehydrogenase, and is added in the form of wet bacteria, crude enzyme liquid, enzyme liquid or immobilized enzyme, and the amount of the enzyme catalyst added is 1%-20% of the mass of the substrate ethyl acetoacetate based on the wet bacteria, or 2%-20% of the volume of the reaction system based on the enzyme liquid.

[0026] Further preferably, the glucose is added in one time or in stages;

[0027] When added in stages, 50%-80% of the total amount is added first into S1, and the remaining 20%-50% is added in 2-6 times in S3, the interval is 10-60 minutes, and the pH is maintained at 6.7-7.0 within 1-10 minutes after each addition.

[0028] Further preferably, the number of water washing in S5 is 2 times, the water volume added in each time is 10%-30% of Vorg, the stirring time is 5-20 minutes, the standing and layering time is 10-90 minutes, and the organic phase is separated into step S6.

[0029] The embodiment of the application has the following advantages due to the use of the above technical solutions:

[0030] First, the application continuously maintains the pH in the preset interval during the reaction process through online collection and closed-loop adjustment of the pH of the reaction liquid, avoids the influence of pH fluctuation caused by the change of the multi-phase system condition on the enzyme catalytic microenvironment, stabilizes the reaction stereoselectivity, reduces the probability of side reactions, enables (R)-3-hydroxybutyric acid ethyl ester to continuously obtain high optical purity, and reduces the batch-to-batch difference.

[0031] Second, the application matches the reaction termination point with the actual process of the system entering a steady state based on the pH stability end point determination of a continuous time window, and avoids the problem of over-reaction or under-reaction caused by relying on a fixed reaction time.

[0032] The above summary is intended to illustrate only and is not intended to be limiting in any way. Further aspects, embodiments and features of the present application will be apparent from the detailed description that follows, taken in conjunction with the accompanying drawings and the description of the state of the art. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0034] Figure 1 The step flow chart of the present application. DETAILED DESCRIPTION

[0035] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature rather than limiting.

[0036] The embodiments of the present application will be described in detail below with reference to the drawings.

[0037] As Figure 1 shown, the embodiments of the present application provide a method for enzyme-catalyzed asymmetric synthesis of high-optical-purity (R)-3-hydroxybutyric acid ethyl ester, and the specific steps are as follows:

[0038] I. Equipment and detection configuration

[0039] Reaction equipment: 5L glass-lined or stainless steel reaction kettle with mechanical stirring, jacketed temperature control, dropping interface and online detection interface; Kettle body is configured with temperature probe (PT100 or equivalent), pH electrode (solvent-resistant composite electrode), metering pump or peristaltic pump dropping system.

[0040] Online pH calibration: Before reaction, two-point calibration of online pH electrode is carried out using pH 6.86 and pH 7.00 (or pH 7.00 and pH 9.18) standard buffer solution to ensure stable reading in the range of 6.5-7.0 for subsequent closed-loop dropping control.

[0041] End point determination data recording: Online signal acquisition is connected to data acquisition module to record sampling time, pH value, dropping action (start / stop), single dropping time and cumulative dropping amount for end point determination window and "no need to drop" determination of the embodiments.

[0042] S1, add the following into the reactor in sequence:

[0043] Deionized water 2.70 L (as the aqueous medium); ethyl acetoacetate 2.00 L, so that its volume fraction in the reaction system is 40%; glucose 150 g, which is about 3.0% according to the total mass of the system in this embodiment.

[0044] Start stirring, set the stirring speed to 400 rpm, and raise the temperature in the reactor to 32°C through the jacket, maintain pre-equilibrium for 30 minutes, observe the system state during the pre-equilibrium process, ensure that the ethyl acetoacetate forms a uniform dispersed phase under the action of stirring, there is no obvious dead corner deposition in the reactor, and the temperature and pH probe readings tend to be stable, providing uniform initial conditions for subsequent accurate pH adjustment and enzyme reaction.

[0045] S2, under the condition of stirring at 400 rpm, start the alkali solution dropping system, the alkali solution is a 30% by mass sodium hydroxide aqueous solution, and the pH of the reaction solution is adjusted to 7.0±0.1 by dropping;

[0046] When the online pH reading enters 6.9-7.1 and is stable, continue to maintain for 10 minutes to eliminate short-term fluctuations of the system mixing and electrode response, so that the pH and temperature are in a controllable steady state.

[0047] Then add the enzyme catalyst to start the asymmetric reduction reaction:

[0048] The enzyme catalyst "ketone reductase crude enzyme solution" is added in the form of a solution, and the amount is 400 mL, which accounts for about 8% of the volume of the reaction system.

[0049] After adding the enzyme, continue to stir at 400 rpm, and switch the temperature control setting to step S3 of the reaction temperature control.

[0050] S3, control the temperature at 35°C during the reaction, and start the online pH acquisition and closed-loop dropping logic:

[0051] The online sampling period is set to 10 seconds (within the range of 5-60 seconds). The "current pH" is recorded in each sampling period and the deviation ΔpH is calculated.

[0052] ΔpH = 6.5 - current pH, i.e. the deviation of the "current pH" from the lower limit 6.5 of the target interval, as a trigger, to determine whether to drop and the dropping duration.

[0053] Graded dropping rules:

[0054] When ΔpH≥0.30 (corresponding to current pH≤6.20), drop 8 seconds of 30% NaOH solution;

[0055] When 0.10≤ΔpH<0.30 (corresponding to 6.20<current pH≤6.40), drop for 4 seconds;

[0056] When 0<ΔpH<0.10 (corresponding to 6.40<current pH<6.50), drop for 1 second;

[0057] When the current pH≥6.5, do not drop, and enter the next sampling period to continue to judge.

[0058] The above drop action is a single execution in a sampling period, and after execution, continue to stir and mix and wait for the next sampling period, so that the pH is maintained in the interval of 6.5-7.0 during the reaction.

[0059] When the pH reaches 6.9-7.0, stop dropping and enter the next sampling period to judge, which is used to avoid the pH exceeding the upper limit of the target interval and reduce the excessive addition of lye.

[0060] When the reaction proceeds to about 7 hours, the system enters the end point judgment state, and whether to terminate the reaction is judged according to the following conditions:

[0061] Select 45 consecutive minutes as the judgment time window, and the pH interval in the window meets the condition: the online pH record is always in the interval of 6.5-7.0 within the 45 minutes.

[0062] The maximum fluctuation amplitude of the pH in the time window is 0.04, which meets the requirement of “≤0.10” and the more stringent condition of “≤0.05”.

[0063] Optionally, in this embodiment, the drop amount is restricted, and the cumulative drop amount of the lye in the time window is less than 0.03% of the total mass of the reaction system, which meets the requirement of “≤0.05%”.

[0064] Optionally, in this embodiment, without drop restriction, there is a continuous 30 minutes without performing drop action in the time window, which indicates that the system has reached a stable state.

[0065] After meeting the above end point judgment conditions, stop dropping and stop the reaction and enter the post-processing, terminate the reaction, and enter the post-processing step S4.

[0066] S4, complete plate and frame filtration to remove slag within 15 minutes after terminating the reaction to obtain a clear filtrate; place the filtrate at 25°C and stand for 90 minutes to separate the two phases after the oil-water two-phase interface is clear.

[0067] Separate the organic phase and measure and record the volume of the organic phase as Vorg=1.95L, which is used as a measurement reference for the water washing amount in step S5.

[0068] S5, measure twice according to Vorg

[0069] First water washing: add 0.39 L of deionized water, stir for 10 minutes, stand for 30 minutes, separate the organic phase;

[0070] Second water washing: repeat the above operation, and also add 0.39 L of deionized water, stir for 10 minutes, stand for 30 minutes, separate the organic phase into the decolorization step.

[0071] Decolorization treatment: add 12 g of activated carbon to the water-washed organic phase, and the amount of activated carbon added is about 0.6% of the mass of the crude organic phase, and decolorize at 30°C for 30 minutes. After decolorization, remove the activated carbon by filtration to obtain the crude organic phase.

[0072] S6, distill the crude organic phase under reduced pressure, and control the distillation operation pressure at 10 kPa. During the distillation process, first remove the front fraction, and then start collecting the target fraction after confirming that the moisture content of the distillate is ≤0.2% by online or spot checking. (R)-3-hydroxybutyric acid ethyl ester product is obtained.

[0073] The obtained product is detected, and the results are as follows:

[0074] Appearance: colorless transparent liquid;

[0075] Identification: the retention time of the main peak in the test sample chromatogram is consistent with that of the reference substance;

[0076] Moisture: 0.08% (≤0.1%); Maximum impurity: 0.12% (≤0.2%); Purity: 99.3% (≥99.0%); ee value: 99.1% (≥99.0%).

[0077] Example 2

[0078] This example is based on Example 1, and the volume fraction of ethyl acetoacetate and the glucose addition method in the reaction system are adjusted. The remaining steps, device configuration and control logic are consistent with Example 1, and the only difference is as follows:

[0079] In step S1, add 2.50 L of ethyl acetoacetate and 2.50 L of aqueous medium to the reaction kettle, so that the volume fraction of the substrate is 50%; The total amount of glucose added is 5.0% of the total mass of the reaction system, of which 70% is added at one time in S1 stage, and the remaining 30% is supplemented in three times in S3 stage, with an interval of 30 minutes each time.

[0080] After each glucose addition, the pH of the reaction solution is adjusted and maintained at 6.7-7.0 within 1-10 minutes according to the online pH collection and closed-loop dropping rules, and then the reaction continues.

[0081] The remaining steps S2-S6 are completely consistent with Example 1.

[0082] Results:

[0083] The final product (R)-ethyl 3-hydroxybutyrate was detected as follows:

[0084] Purity: 99.2%; ee value: 99.0%; maximum impurity: 0.15%;

[0085] The results show that under the condition of high substrate volume fraction, high optical purity product can still be stably obtained by adding reducing equivalent in sections and cooperating with pH closed-loop control, verifying the adaptability of the method of the application to high load reaction system.

[0086] Example 3

[0087] This example is used to verify the universality of the pH control strategy of the application under different enzyme carrier forms.

[0088] The difference from Example 1 is that the enzyme catalyst adopts the form of immobilized enzyme, and the remaining steps remain unchanged.

[0089] In step S2, the immobilized ketone reductase particles are added, and the amount is equivalent to 10% of the mass of ethyl acetoacetate based on the effective enzyme activity in the immobilized enzyme. The immobilized enzyme is suspended in the form of a mesh bag in the stirring area of the reaction kettle to ensure good mass transfer.

[0090] During the reaction, the pH is still collected and added dropwise on line to maintain the pH at 6.5-7.0.

[0091] After the reaction is completed, the immobilized enzyme mesh bag is removed before S4, and the remaining post-processing steps S4-S6 are consistent with Example 1.

[0092] Results:

[0093] The detection results of the product obtained are as follows:

[0094] Purity: 99.1%; ee value: 99.2%; moisture: 0.09%.

[0095] The results show that the method of the application does not depend on a specific enzyme form, and the pH control and end point determination strategy is also effective in the immobilized system.

[0096] Example 4

[0097] This example is used to verify the technical effect of the end point determination condition.

[0098] Compared with Example 1, the main difference of this example is:

[0099] The end point determination time window is adjusted to 20 minutes;

[0100] The end point determination meets both claims 5(1) and 5(2) at the same time.

[0101] In the later stage of step S3, when the reaction enters the stable stage, 20 minutes of continuous time is selected as the end point determination time window. In this time window:

[0102] The pH is always maintained at 6.5-7.0;

[0103] The maximum fluctuation amplitude of pH is 0.03;

[0104] No base solution dropping operation is performed in 20 minutes of continuous time;

[0105] The cumulative dropping amount of base solution is 0.02% of the total mass of the reaction system.

[0106] The reaction is terminated after meeting the above conditions, and the remaining steps S4-S6 are consistent with Example 1.

[0107] Results:

[0108] The ee value of the obtained product is 99.0%, and the purity is 99.1%;

[0109] The batch repeatability is good, and the end point determination window can be reasonably shortened according to the actual working condition without affecting the product quality.

[0110] Comparative Example 1 (without pH online closed-loop control)

[0111] Except that the pH online collection and closed-loop dropping of step S3 is not performed, the remaining steps are the same as Example 1, and only the pH is adjusted to 7.0 at the beginning of the reaction and is not dynamically adjusted thereafter;

[0112] The results show that the pH gradually decreases to 6.1-6.3 during the reaction, and the pH fluctuates obviously in the later stage of the reaction; the ee value of the obtained (R)-3-hydroxybutyric acid ethyl ester is 95.8%, and the batch difference is large.

[0113] Comparative Example 2 (without end point determination condition)

[0114] Except that the pH determination condition of the continuous time window is not set, the remaining steps are the same as Example 1, and only the fixed reaction time of 8 hours is used as the reaction termination standard;

[0115] The results show that slight pH fluctuation still occurs in the later stage of the reaction in some batches, and the cumulative dropping amount of base solution increases significantly; the ee value of the obtained product fluctuates between 97.2% and 98.4%.

[0116] Test Example

[0117] 1. Detection method and determination criterion

[0118] (1) Purity and maximum impurity, gas chromatography or liquid chromatography is used for content determination, the area percentage of target peak in the chromatogram of test sample is recorded as the normalized purity, and the area percentage of the largest single impurity peak is calculated as the maximum impurity index.

[0119] (2) ee value, chiral chromatographic column is used for enantiomer separation of the test sample, and the ee value is calculated according to the peak area of the two enantiomers; ee≥99.0% is used as the high optical purity threshold.

[0120] (3) Moisture, Karl Fischer moisture determination method is used to detect the moisture content of distillate and finished product.

[0121] (4) Appearance and identification, appearance is visually observed; identification is based on the consistency of the retention time of the main peak of the test sample with the reference substance.

[0122] 2. Test design and comparison scheme

[0123] In order to verify the influence of the "pH online closed-loop control and stable time window end point determination" of the application on the optical purity and batch consistency of the product, the following test groups are set:

[0124] Test group: Examples 1-4, all perform pH online collection and closed-loop addition, and use time window end point determination (window 20-45 min) to control pH to maintain at 6.5-7.0.

[0125] Comparison group 1: Comparative Example 1, only initially adjusted to pH≈7.0, and no online closed-loop addition is performed during the reaction.

[0126] Comparison group 2: Comparative Example 2, pH adjustment is performed but no time window end point determination is set, and the reaction is only terminated by fixed reaction time.

[0127] 3. Results and data comparison

[0128] Table 1: Parameter comparison of different examples and comparative examples

[0129] Group Process key difference Moisture (%) Maximum impurity (%) Purity (%) ee (%) Example 1 pH online closed-loop control + 45 min time window end point determination 0.08 0.12 99.3 99.1 Example 2 Substrate volume fraction 50% + glucose segmented addition + pH closed-loop control 0.09 0.15 99.2 99.0 Example 3 Immobilized enzyme system + pH closed-loop control + time window determination 0.09 0.13 99.1 99.2 Example 4 Time window shortened to 20 min + simultaneous satisfaction of dropwise addition amount and no dropwise addition criteria 0.08 0.14 99.1 99.0 Comparative Example 1 No pH online closed-loop control 0.12 0.62 97.9 95.8 Comparative Example 2 No time window end point determination (fixed 8 h, multiple batches) 0.10–0.13 0.28–0.41 98.1–98.7 97.2–98.4

[0130] Results:

[0131] (1) Comparative Example 1 did not perform pH online closed-loop adjustment during the reaction, the pH of the reaction solution decreased and fluctuated, and the ee value of the obtained product was significantly reduced; Examples 1-4 maintained the pH at 6.5-7.0 through online collection and staged addition during the reaction, and the ee value of the obtained product reached more than 99.0%, indicating that stable control of the pH in the enzyme-catalyzed asymmetric reduction system is conducive to obtaining high optical purity product.

[0132] (2) The comparative example 2 adjusts the pH of the reaction solution, but does not set the end point determination condition of the continuous time window, and only terminates the reaction at a fixed reaction time, resulting in fluctuations in the ee value of the product in different batches; the example 4 sets the end point determination condition of the continuous time window, and can obtain a stable high ee value product in a shorter determination time, proving that using the time window as the reaction termination criterion helps to improve the consistency of the reaction termination time point.

[0133] (3) Under the conditions of increasing the substrate volume fraction and using glucose in a segmented manner, and under the conditions of implementing the method of the present application in the immobilized enzyme system, high optical purity products meeting the quality standards can be obtained, indicating that the method of the present application has good adaptability to substrate load and enzyme catalyst form

[0134] In summary, the present application can make the reaction process of the enzyme-catalyzed asymmetric synthesis of (R)-3-hydroxybutyl acetate more stable and controllable by online monitoring and closed-loop regulation of the pH during the reaction, and combining with the end point determination mode of the continuous time window, so as to obtain a product with high optical purity and stable batch.

[0135] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for the enzyme catalyzed asymmetric synthesis of high optical purity (R)-3-hydroxybutyric acid ethyl ester, characterized in that, Comprising the following steps: S1, adding ethyl acetoacetate, glucose and aqueous medium into a reaction kettle, starting stirring to make the system uniform, controlling the temperature at 30-35℃ for pre-equilibrium for 10-60 minutes; wherein the volume fraction of ethyl acetoacetate in the reaction system is 20%-55%, and the addition amount of glucose is 0.5%-15%; S2, under stirring, adjusting the pH of the reaction solution to 7.0±0.1 by adding alkali solution and maintaining for 1-20 minutes, then adding enzyme catalyst to start the asymmetric reduction reaction; S3, controlling the temperature at 33-37℃ during the reaction, collecting the pH of the reaction solution on line, and performing intermittent or continuous dropping of alkali solution according to the collected value to maintain the pH of the reaction solution at 6.5-7.0; terminating the reaction when the end point determination condition is met; wherein the end point determination condition is that within a determination time window of 10-60 minutes, the pH of the reaction solution is always at 6.5-7.0 and the maximum pH fluctuation amplitude is ≤0.10; S4, after terminating the reaction, removing the residue by plate and frame filtration to obtain filtrate, standing the filtrate for 30-240 minutes for layer separation and then separating to obtain organic phase; S5, after water washing the organic phase for 1-3 times, adding activated carbon for decolorization and filtering to obtain crude organic phase; S6, performing vacuum distillation on the crude organic phase, cutting off the front fraction first, then collecting the target fraction to obtain (R)-ethyl 3-hydroxybutyrate product.

2. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The volume fraction of ethyl acetoacetate in S1 is 30%-50%; the stirring speed is 200-600 rpm; and the pre-equilibrium time is 20-40 minutes.

3. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The alkali solution in S2 and S3 is 20%-35% sodium hydroxide aqueous solution; the dropping is performed by pulse dropping rule, that is, when the on-line collected pH is <6.5, the dropping is started, when the pH reaches 6.9-7.0, the dropping is stopped, and the next sampling cycle is continued to judge whether dropping is needed.

4. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The on-line collection in S3 is performed according to fixed sampling period, the sampling period is 5-60 seconds, and the deviation ΔpH of "current pH and lower limit 6.5 of target interval" is taken as the trigger amount, the dropping time is determined according to ΔpH grading and performed once, wherein: When ΔpH≥0.30, the dropping time is 5-10 seconds; When 0.10≤ΔpH<0.30, the dropping time is 2-5 seconds; When 0<ΔpH<0.10, the dropping time is 0.5-2 seconds.

5. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The end point determination condition further includes any one or both of the following: (1) within the determination time window, the cumulative dropping amount of alkali solution is ≤0.05% of the total mass of the reaction system; (2) within the determination time window, the pH fluctuation amplitude is ≤0.05, and continuous dropping operation is not needed for at least 30 minutes.

6. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The enzyme catalyst is ketoreductase or dehydrogenase, and is added in the form of wet bacteria, crude enzyme solution, enzyme solution or immobilized enzyme, and the addition amount of the enzyme catalyst is 1%-20% of the mass of substrate ethyl acetoacetate based on wet bacteria, or 2%-20% of the volume of the reaction system based on enzyme solution.

7. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The glucose is added by one-time addition or segmented addition; When using a staged addition method, first add 50% to 80% of the total amount into S1, and then add the remaining 20% ​​to 50% in 2 to 6 times in S3, with an interval of 10 to 60 minutes between additions. After each addition, maintain the pH at 6.7 to 7.0 for 1 to 10 minutes.

8. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The plate and frame filtration in S4 is completed within 0 to 30 minutes after the reaction is terminated; the static stratification temperature is 20 to 35°C; the volume of the organic phase Vorg is recorded when separating the organic phase, and Vorg is used as the measurement standard for the amount of water added for washing in S5.

9. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: The water washing in step S5 is performed twice; the volume of water added for each wash is 10% to 30% of Vorg, the water washing and stirring time is 5 to 20 minutes, the standing and stratification time is 10 to 90 minutes, and the separated organic phase proceeds to step S6.

10. The method of asymmetric synthesis of high optical purity (R)-ethyl 3-hydroxybutanoate catalyzed by the enzyme according to claim 1, characterized by that: In step S5, the amount of activated carbon added is 0.1% to 2.0% of the mass of the crude organic phase, the decolorization temperature is 20 to 40°C, the decolorization time is 10 to 60 minutes, and the crude organic phase is obtained by filtration after decolorization. The operating pressure of the S6 vacuum distillation is 5-50 kPa. After the fore-distillate is removed until the water content of the distillate is ≤0.2%, the target distillate is collected.

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