<i>Methylopila </i>sp. and use thereof in selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate

Abstract

Methylopila sp. and use thereof in the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate. Methylopila sp. that produces enzymes is subjected to cell immobilization, and is then applied to the biological resolution of a racemate (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester to prepare high optically pure (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester, which is further subjected to a hydrolysis reaction to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetate. The present invention achieves a high conversion yield up to 50.0% or more, a good stereoselectivity, and an enantiomeric excess value e.e..sub.s (%) of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester not less than 99.5; the catalytic efficiency is high; the concentration of the racemic substrate in the resolution reaction is up to 500 g/L, the reaction time does not exceed 15 h, the number of reuse times of the immobilized cells is not lower than 35.

Claims

1. A method for a selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate, comprising the following three steps: (1) immobilizing bacterial cells of Methylopila sp. in a cell immobilization method using a bacterial solution of Methylopila sp. to obtain an immobilized bacterial agent containing an immobilized resolution enzyme, wherein the Methylopila sp. is a Methylopila sp. cxzy-L013 strain deposited in China Center for Type Culture Collection on Sep. 18, 2016 under the conservation number CCTCC M2016494; (2) reacting (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester as a substrate with a predetermined amount of water and the immobilized bacterial agent to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester; and (3) hydrolysing (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester using immobilized ester hydrolase or alkaline to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetate.

2. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein the bacterial solution of Methylopila sp. is an enzyme-containing bacterial suspension containing not less than 50 wt % of wet cells, which is obtained by subjecting the Methylopila sp. to slant culture, seed liquid culture, inoculation fermentation and concentration steps.

3. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein the cell immobilization method comprises dissolving the bacterial solution of Methylopila sp. in a buffer solution, adding at least one adsorbent and/or cross-linking agent, and stirring and suction-filtrating the same to obtain the immobilized bacterial agent; the adsorbent is selected from any one of diatomaceous earth or activated carbon; and the cross-linking agent is selected from any one of glutaraldehyde, toluene diisocyanate or bis-diazotized benzidine.

4. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 3, wherein the immobilized resolution enzyme achieved in the immobilized bacterial agent using the cell immobilization method has an enzyme activity recovery of not less than 90% and an adsorption rate of not lower than 95%, and the number of reuse times of the immobilized bacterial agent is not lower than 35.

5. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 4, wherein the cell immobilization method comprises first adding the adsorbent, then stirring and mixing uniformly, and adding the crosslinking agent.

6. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 5, further comprising adding polyethyleneimine during the addition of the crosslinking agent.

7. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein the temperature of the resolution reaction is 25-37° C., and the method further comprises adding a 15-25 v/v % aqueous sodium carbonate solution dropwise to control the pH of the reaction process at 6.5-8.5, and carrying out the reaction for 2-15 h.

8. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 7, wherein, in the resolution reaction, the mass concentration of (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester in the reaction system is 160-500 g/L.

9. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 8, wherein the immobilized bacterial agent has a dosage concentration of 5-25 g/L on a wet weight basis in the reaction system.

10. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 9, wherein the enantiomeric excess value e.e..sub.s (%) is not less than 99.5 and the conversion rate is not lower than 49%, as detected by HPLC after the completion of the resolution reaction.

11. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein after said step (2), (R)-α-ethyl-2-oxo-1-pyrrolidineacetic acid in the aqueous phase is recovered, and is subjected to racemization and esterification to obtain a starting substrate material.

12. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein, in said step (3), (S)-α-ethyl-2-oxo-1-pyrrolidineacetate is obtained by the immobilized ester hydrolase hydrolysis, and is then concentrated and crystallized to obtain a crude product thereof.

13. The method for the selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate according to claim 1, wherein, in said step (3), the alkaline solution used for the alkaline hydrolysis is 200-400 g/L of an ionic membrane alkaline solution, allowing the pH value of the reaction to be not less than 13, the reaction temperature is 10-20° C. until the substrate hydrolysis reaction is completed, and then the crude product is obtained by concentration and crystallization.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a peak appearance chromatogram of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid;

(2) FIG. 2 is a peak appearance chromatogram of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid;

(3) FIG. 3 is an HPLC chromatogram of an organic phase after the catalytic resolution of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester for 3 h;

(4) FIG. 4 is an HPLC chromatogram of an aqueous phase after the catalytic resolution of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester for 3 h;

(5) FIG. 5 is an HPLC chromatogram of an organic phase at the end of the catalytic resolution of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester;

(6) FIG. 6 is an HPLC chromatogram of an aqueous phase at the end of the catalytic resolution of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester; and

(7) FIG. 7 shows a chart of changing trend of the reaction time as a function of the number of times of use of the enzyme in test example 3.

DETAILED DESCRIPTION OF EMBODIMENTS

(8) The present invention will be further described with reference to particular embodiments. It should be noted that unless otherwise specified, the percentage concentrations described below are mass percentage concentrations.

Example 1

(9) Methylopila sp. cxzy-L013, which strain is deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan 430072, P. R. China, on Sep. 18, 2016, under the conservation number CCTCC M2016494.

(10) The Methylopila sp. cxzy-L013 is obtained from the soil in the factory area of Huahai Pharmaceutical Co., Ltd., Duqiao Town, Linhai City, Zhejiang Province, by preliminary screening of colony characteristics on plates, using primary fermentation and shake-flask culture one by one, detecting the enzymatic activity, and comparing the enzymatic activity of the stereoselective ester hydrolases.

(11) The characteristics of the colonies are as follows: colonies are regularly rounded, and have neat edges, diameters of 0.5-1 mm and raised surfaces, and are moist, shiny and milky white; the cells are in short round rod shapes, and are singly and dispersively arranged with a size of (0.3-0.4) μm×(1.0-1.2) μm; Gram-negative bacteria; especially, they grow slowly on a medium when using glucose, glycerol and ethanol as carbon sources, and grow faster when using methanol, methylamine hydrochloride and ammonium formate as carbon sources.

Example 2

(12) The Methylopila sp. cxzy-L013 obtained in example 1 should be further activated through culturing for fermentation to obtain a bacterial liquid of Methylopila sp. cxzy-L013. The specific obtaining steps are as follows:

(13) Slant Culture

(14) The Methylopila sp. cxzy-L013 strain in a glycerol tube is streaked on an LB slant test tube and is cultured at 30° C. for 2-3 days.

(15) Seed Liquid Culture

(16) The slant cells are inoculated into a seed culture medium, and cultured at 30° C. for 2-3 days, so as to obtain a seed liquid; and the concentrations and compositions of the seed medium are: MgSO.sub.4.7H.sub.2O 1.0 g/L, K.sub.2HPO.sub.4 1.8 g/L, (NH.sub.4).sub.2SO.sub.4 1.0 g/L, yeast leaching powder 5.0 g/L, a methanol solution with a volume fraction of 75% 5.0 mL/L (added before inoculation), and ammonia water is used to adjust the pH to 7.0.

(17) Inoculation Fermentation

(18) The seed liquid is inoculated into a 7 L fermentation tank for fermentation: inoculation volume: 100 mL, initial volume of the fermentation broth: 5 L, fermentation temperature: 30° C., pH controlled by ammonia water: 6.5-7.0, aeration rate: 0.5-1 vvm, gradually increasing the mechanical stirring speed from 100 r/min to 900 r/min, so that DO≥30%, concentration of 75% methanol intermittently supplemented during fermentation: 5.0 mL/L, and fermentation time: 3-4 days; when the pH does not fall but rise, the cells are released from the tank and collected; and at this time OD600≥40, the wet weight of the cells can be up to 70-90 g/L.

(19) The concentrations and compositions of the fermentation medium are: NaCl 0.5 g/L, MgSO.sub.4.7H.sub.2O 3.6 g/L, K.sub.2HPO.sub.4 1.0 g/L, (NH.sub.4).sub.2SO.sub.4 1.0 g/L, yeast extract powder 6.0 g, a methanol solution with a volume fraction of 75% 5.0 mL/L (added before inoculation).

(20) As shown in Table 1, when the methanol used in the fermentation process is changed to use glucose, glycerol and ethanol as a carbon source, the cell growth is very slow, and it can be seen that methanol as the carbon source is significantly superior to the other three carbon sources. The medium formulation used is the same as the medium formulation of the seed liquid medium except for the carbon source, and the culture method is also the same as that for the seed liquid culture.

(21) TABLE-US-00001 TABLE 1 Effects of different carbon sources in the fermentation of the bacterial solution Carbon Cell Carbon source source Fermentation concentration type concentration method OD.sub.600 methanol with a 5.0 mL/L shake-flask 5 ± 0.5 volume fraction of 75% fermentation glucose 5 g/L shake-flask 2 ± 0.5 fermentation glycerol 5 g/L shake-flask 1 ± 0.5 fermentation ethanol with a 5.0 mL/L shake-flask 2 ± 0.5 volume fraction of 75% fermentation

(22) As shown in Table 2, if the yeast extract powder in the fermentation medium is changed to use corn steep liquor powder, tryptone, beef extract or the like as a carbon source, the growth rate of the cell is common, and the yeast extract powder is superior to the other three carbon sources. The medium formulation used is the same as the medium formulation of the seed liquid medium except for the nitrogen source, and the culture method is also the same as that for the seed liquid culture.

(23) TABLE-US-00002 TABLE 2 Effects of different carbon sources as the carbon source of the fermentation medium for the bacterial solution Carbon Cell Carbon source source Fermentation concentration type concentration method OD.sub.600 yeast extract 6 g/L shake-flask   5 ± 0.5 powder fermentation corn steep liquor 6 g/L shake-flask 3.5 ± 0.5 powder fermentation beef extract 6 g/L shake-flask   2 ± 0.5 fermentation tryptone 6 g/L shake-flask 2.5 ± 0.5 fermentation

(24) In order to obtain an enzyme-containing bacterial suspension of not less than 50 wt %, enzyme-containing wet cells are obtained after centrifugal separation of the fermentation broth with a high-speed centrifuge; according to an equal mass ratio, the wet cells are diluted with water, stirred uniformly, and refrigerated for use; or the fermented broth is directly filtered and concentrated through a microfiltration membrane to obtain a bacterial suspension containing wet cells in a mass fraction of about 50 wt %, which is refrigerated for use.

Example 3

(25) (S)-α-ethyl-2-oxo-1-pyrrolidineacetate is prepared by stereoselective resolution using Methylopila sp. cxzy-L013 of example 1, comprising the following steps:

(26) (1) treating a bacterial solution of Methylopila sp. cxzy-L013 by a cell immobilization method to obtain an immobilized bacterial agent containing an immobilized resolution enzyme, wherein the method for obtaining the bacterial solution of Methylopila sp. cxzy-L013 has been set forth in example 2;

(27) (2) with (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester as a substrate, adding a certain amount of water and the immobilized bacterial agent for a resolution reaction to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester; and

(28) (3) using immobilized ester hydrolase hydrolysis or alkaline hydrolysis to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetate.

(29) (4) after step (2), recovering the (R)-α-ethyl-2-oxo-1-pyrrolidineacetic acid in the aqueous phase, and performing racemization and esterification to obtain the starting substrate material.

(30) The cell immobilization method in step (1) consists of first adding the adsorbent, and after stirring and mixing uniformly, then adding the crosslinking agent. The mechanical strength can be significantly increased by first using the adsorption method to form embedded balls, and then using the cross-linking method in combination. The immobilized resolution enzyme achieved by the cell immobilization method has an enzyme activity recovery of not less than 90% and an adsorption rate of not lower than 95%, and the number of reuse times of the immobilized bacterial agent is not lower than 35.

(31) In step (2), the (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester is 500-900 g/L in a toluene solution, preferably at a concentration of 700-800 g/L. The mass concentration of (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester in the reaction system is 160-500 g/L. The immobilized bacterial agent has a dosage concentration of 5-25 g/L on a wet weight basis in the reaction system.

(32) The conditions of the resolution reaction are as follows: the reaction temperature is 25-37° C., an aqueous solution of sodium carbonate with a volume fraction of 15-25% (V/V) is dropwise added to control the pH of the reaction process at 6.5-8.5, the reaction is carried out for 2-15 h, the reaction conversion result is detected by HPLC, the enantiomeric excess value e.e..sub.s (%) is not less than 99.5, and the conversion rate is up to 50%.

(33) After the resolution reaction, a separation and purification method for the reaction mixture is as follows: the reaction solution is directly centrifuged by a high-speed centrifuge or filtered through a plate filter. After the liquid is partially layered, the organic phases are retained, and 1:0.3-1.5 of toluene is added to the aqueous layer for extraction 3-5 times. The organic phases are combined and the toluene is distilled off at 35-60° C., so as to obtain a concentrate of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester.

(34) Any of the immobilized ester hydrolases capable of hydrolyzing the (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester ester bond can be used in step (3). 1 part of industrial water and 1-2 parts of a concentrate of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester are added to a reactor, 1/20-1/5 parts of the immobilized ester hydrolase are added and stirred, the temperature and pH are controlled until the ester hydrolysis reaction is finished, the ester hydrolase is filtered off to obtain a salt solution of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid, which is then subjected to concentration and crystallization to obtain a solid crude product of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate.

(35) In step (3), for the obtained (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester, it is also possible to obtain the salt of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid by an alkaline hydrolysis method: adding 2-3 parts of deionized water and 2-3 parts of the concentrate of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester to a reactor, stirring, and then adding 1-2 parts of 300 g/L of an ionic membrane alkaline solution so that the pH reaches 14, hydrolyzing at 10-20° C. for 1-5 h until the hydrolysis reaction of the substrate is finished, so as to obtain a salt solution of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid, followed by concentration and crystallization to obtain a solid crude product of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate.

Example 4

(36) Based on example 3, this example further optimizes the cell immobilization method in step (1): with 20 g of a bacterial suspension containing wet cells dissolved in a mass fraction of 50 wt % in 100 mL of an ammonium formate buffer solution (pH 7.0) for reaction as a preferred example, stirring and mixing the same uniformly, adding 0.4-0.8 g of diatomaceous earth or activated carbon, adding 3-4.5 mL of 5% polyethyleneimine for cross-linking for 1 h, then adding 1-1.5 mL of 25% glutaraldehyde for cross-linking for 1 h, and finally filtering under vacuum to obtain an immobilized bacterial agent containing the immobilized resolution enzyme, washing same with tap water 2 times, suction-filtrating and placing same at 4° C. for refrigerated preservation. The immobilized resolution enzyme has an enzyme activity recovery≥90% and an adsorption rate≥95%.

Example 5

(37) Based on the method of example 3, this example selects Protin AP Conc. lipase from Amano Enzyme Inc., Janpan as an immobilized ester hydrolase to obtain (S)-α-ethyl-2-oxo-1-pyrrolidineacetate.

(38) 2 g of a Protin AP Conc. lipase powder is dissolved in 100 mL of purified water, 2 g of sodium alginate is added and slowly stirred until completely dissolved, and the mixed solution is uniformly pumped with a needle or sprayer into a 100 mM calcium chloride solution, and is slowly stirred until the bead-like gel is hardened, and then is repeatedly washed with distilled water 2-3 times to obtain an ester hydrolase immobilized by calcium alginate, which is preserved at 4° C. for use.

Example 6

(39) A cell immobilized bacterial agent can be used to isolate (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester from its racemate solution. The immobilized resolution enzyme has an activity recovery of not less than 90% and an adsorption rate of not lower than 95%, and the number of reuse times of the immobilized bacterial agent is not lower than 35. The bacterial agent is obtained by the following method:

(40) The bacterial suspension of Methylopila sp. cxzy-L013 as described in example 2 is dissolved in a buffer solution, diatomaceous earth is at least added as an adsorbent, then polyethyleneimine is added, then glutaraldehyde is added as a cross-linking agent, and an immobilized cell solution is obtained after stirring. The immobilized bacterial agent is obtained by vacuum filtration. The specific method is described with reference to example 4.

Example 7

(41) Based on example 3, this example further describes the step of resolution of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester in step (2):

(42) taking 300 mL of a catalytic reaction system as an example: 100 mL of a toluene solution with (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester at a concentration of about 500 g/L is used to prepare a cell immobilized bacterial agent according to the method of example 4 and example 6.

(43) Specifically: 200 mL of water is added to a 500 mL conversion bottle, 100 mL of a toluene solution containing about 50 g of (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester is added to allow the initial concentration of (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester in the reaction system to be about 166 g/L, stirring is initiated, a Na.sub.2CO.sub.3 solution (20%, V/V) is used to adjust the pH to 7.0, 2 g of the cell immobilized bacterial agent prepared in example 6 is added, the immobilization bacterial agent has a dosage concentration of 6.6 g/L, at 37° C., the Na.sub.2CO.sub.3 solution (20%, V/V) is used to maintain the pH at 7.0-7.5, and the resolution reaction is carried out for 2-3 h. The reaction conversion result is detected by HPLC, the enantiomeric excess value e.e..sub.s (%) is not less than 99.8 and the conversion rate is 50.0%.

Examples 8-13

(44) The resolution method in examples 8-13 is basically the same as that in example 7, except that the dosage of the cell immobilized bacterial agent, the concentration of the substrate used in the toluene solution, and the concentration of the substrate in the reaction system are different. The differences in resolution effect of different examples can be seen from Table 3.

(45) TABLE-US-00003 TABLE 3 Results for biocatalytic resolution of the substrate (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester Initial Dosage concentration concentration Concentration of the of the of the substrate immobilized substrate in the Enantiomeric bacterial in reaction Resolution Main excess Conversion agent toluene system time product value rate Examples (g/L) (g/L) (g/L) (h) configuration e.e..sub.s (%) C (%) Example 8 6.6 500 166 2-3 S 99.8 50.0 Example 9 16.6 500 250 2-3 S 99.9 49.1 Example 16.6 500 333 10-12 S 99.6 49.5 10 Example 16.6 600 200 5-6 S 99.9 49.8 11 Example 25 800 400 8-9 S 99.8 49.8 12 Example 25 800 533 12-14 S 99.9 50.0 13

(46) Based on examples 8-13, it can be seen that: when the dosage of the toluene solution of the substrate is larger, the reaction time is longer, and the conversion rate is decreased; if the concentration of the substrate in the toluene solution is higher, and the dosage in the reaction system is larger, the reaction will be prolonged; and the conversion rate can still achieve the desired effect by a method of appropriately increasing the dosage of the immobilized bacterial agent.

Test Example 1

Resolution Reaction Liquid Monitoring

(47) Instrument: high performance liquid chromatograph equipped with a UV detector

(48) Chromatographic column: CHIRALPAK AS-H 250×4.6 mm 5 μm

(49) Mobile phase: n-hexane:isopropanol:trifluoroacetic acid=80:20:0.2 (% V/V/V)

(50) Flow rate: 0.8 mL/min, wavelength: 210 nm, column temperature: 30° C., running time: 30 min, injection volume: 20 μL

(51) Diluent: mobile phase, blank solution:diluent

(52) Test sample solution: 20 mg of the test sample is weighed into a 10 mL volumetric flask, and dissolved with a diluent to a fixed volume. The test sample comprises: racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester and its monomers, and racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid and its monomers.

(53) Sample treatment: based on the method steps of example 4, the reaction solution is diluted by 100 folds, mixed uniformly and then filtered through a 0.45 μm microporous filter membrane, and is ready for injection.

(54) The enantiomeric excess value e.e. and substrate conversion rate C of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester are calculated according to the following equations:

(55) $\begin{array}{cc}e.e{.}_s\left(\%\right)=.Math.\frac{{\left[C\right]}_S-{\left[C\right]}_R}{{\left[C\right]}_S+{\left[C\right]}_R}.Math.\times 100\%& \mathrm{Equation}1\\ C\left(\%\right)=\frac{C_P}{C_P+C_S}\times 100\%& \mathrm{Equation}2\end{array}$

(56) in the equations, [C].sub.S and [C].sub.R are respectively the contents of S and R types of substrates in the sample measured by chromatography, e.e..sub.s (%) is the enantiomeric excess value of (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester in the resolution reaction, C.sub.P is the molar concentration of the product, C.sub.S is the molar concentration of the remaining substrate, and C (%) is the conversion rate.

(57) As can be seen from FIG. 1 to FIG. 6, (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester has a peak appearance time of 9.4 min, (R)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester has a peak appearance time of 15.9 min, (S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid has a peak appearance time of 11.5 min, and (R)-α-ethyl-2-oxo-1-pyrrolidineacetic acid has a peak appearance time of 10.4 min.

(58) FIG. 5 is an HPLC chromatogram of an organic phase at the end of the catalytic resolution of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester, and it can be seen that (R)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester has been completely hydrolyzed.

Test Example 2

Comparison of Immobilization Methods

(59) In the present invention, the adsorbent and the cross-linking agent are simultaneously used in the immobilization method, polyethyleneimine in combination with glutaraldehyde improves the cross-linking effect, the immobilization effect of the cells is obviously improved, and then when they participate in an enzyme catalytic reaction with a high-concentration organic substrate, the purpose of industrial production is easily achieved.

(60) Immobilization object: bacterial cells obtained after the fermentation of the Methylopila sp. cxzy-L013 strain of example 2, and an enzyme solution obtained by the ultrasonic disruption of the bacterial cells.

(61) First of all, this test example compares the use of the adsorption method, the embedding method and the adsorption-cross-linking method, and describes the differences in immobilization effect of the enzyme in the cells caused by different treatment methods for the bacterial solution, the results being as shown in table 4:

(62) TABLE-US-00004 TABLE 4 Influence of different immobilization methods and bacterial solution treatment methods on the immobilization effect Test Immobilization Immobilization group method object Specific operation Result A Epoxy disrupted 10 mL enzyme solution + enzyme activity resin enzyme 1 g resin, stirring at 25° C. recovery ≥ 30% adsorption solution for 12 h, and suction-filtrating B Sodium bacterial 10 g bacterial cells + 2% enzyme activity alginate cells sodium alginate, shaking recovery ≥ 90%, but embedding uniformly, dorpwise adding after repeating 5 the mixture to a 0.1 M/L times, enzyme calcium chloride solution, activity decreases to slowly stirring, and 50% of the initial suction-filtrating activity C Adsorption- disrupted 10 mL of bacterial suction-filtration cross-linking enzyme solution + 0.6 g diatomaceous cannot be performed of solution earth, successively adding 3 mL after immobilization, polyethylenimine of 5% polyethyleneimine indicating that the with and 1 mL of 25% enzyme protein and glutaraldehyde glutaraldehyde, stirring for 1 h, the cross-linking and suction-filtrating agent are not fully cross-linked D Adsorption- bacterial 10 g bacterial cells + 0.6 g Enzyme activity cross-linking cells diatomaceous earth, recovery ≥ 90%, of successively adding 3 mL of adsorption rate after polyethylenimine 5% polyethyleneimine and 1 mL cell immobilization ≥ 95%; with of 25% glutaraldehyde, 19 batches are glutaraldehyde stirring for 1 h, and used repeatedly, and suction-filtrating the activity of each batch of enzyme is stable

(63) Secondly, for test group D, an immobilization and optimization experiment is performed on the dosage of the adsorbent (diatomaceous earth or activated carbon) and the cross-linking agent, respectively, and the results are as shown in Table 5:

(64) TABLE-US-00005 TABLE 5 Influence of different immobilization methods and bacterial solution treatment methods on the immobilization effect Cross-linking agent Result 5% 25% enzyme Adsorption Test polyethyl- glutar- activity rate after cell group Adsorbent eneimine aldehyde recovery immobilization D-1 diato- 3 mL 1 mL 83% 82% maceous earth 0.4 g D-2 diato- 3 mL 1 mL 90% 95% maceous earth 0.6 g D-3 diato- 3 mL 1 mL 87% 95% maceous earth 0.8 g D-4 diato- 4.5 mL   1.5 mL   78% 98% maceous earth 0.6 g D-5 activated 3 mL 1 mL 81% 95% carbon 0.6 g D-6 activated 4.5 mL   1.5 mL   73% 96% carbon 0.6 g

Test Example 3

Validation of the Number of Reuse Times after Cell Immobilization

(65) Validation method: 100 mL of racemic (R,S)-α-ethyl-2-oxo-1-pyrrolidineacetic acid ethyl ester is catalyzed by 5 g of the immobilized bacterial agent obtained in example 4, and the content of the racemic substrate in toluene is 500 g/L, the reaction system is 300 mL, and the consumption of 30 mL of a Na.sub.2CO.sub.3 solution (20%, V/V) indicates the end of the reaction; and after the reaction is finished, the immobilized bacterial agent is suction-filtered and the suction-filtered immobilized bacterial agent is put into the same system and subjected to the next reaction under the same reaction conditions.

(66) FIG. 7 shows a chart of changing trend of the time required to finish the reaction as the number of times of use increases. It can be seen from the figure that 1.2 h is required for finishing the 1st reaction, thereafter, the reaction time gradually increases, 2.6 h is required for the 7th reaction, however, the reaction time begins to decrease after the 8th reaction. Due to repeated filtration and weight loss of the enzyme, the overall reaction time after the 12th reaction tends to increase, but the reaction time is substantially maintained within 5 h after the cell immobilized bacterial agent is used 36 times, with an ideal effect as expected.

(67) The above description is merely preferred embodiments of the present invention, and it should be noted that for a person skilled in the art, some improvements and modifications can also be made under the premise of not departing from the principle of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.