Method for preparing D-glutamine through whole-cell catalysis

Abstract

The invention belongs to the technical field of biological catalysis, provides a method for preparing D-glutamine through whole-cell catalysis, and discloses a novel method for preparing D-glutamine from myxococcus glutamine deaminase (also known as glutaminase), L-glutamine is used as a substrate, and the D-glutamine is obtained through racemization of alanine racemase. According to the present invention, the method comprises the following steps: 1) preparing the DL-glutamine from the DL-glutamine, 2) carrying out deamination on the DL-glutamine through the glutaminase to obtain the D-glutamine, the ee value of the obtained D-glutamine exceeds 99%, and the liquid phase purity of the obtained D-glutamine reaches 99.9%, and the preparation method has characteristics of high efficiency, low cost, environmental protection and no pollution, and has broad industrial application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing D-glutamine by whole-cell catalysis. Background Art

[0002] D-glutamine has been used to study its role in protecting the monolayer of Caco-2 cells against acetaldehyde-induced disruption of barrier function;

[0003] The lack of effect of D-glutamine in protecting against acetaldehyde indicates that L-glutamine-mediated protection is stereospecific.

[0004] It is a medicinal amino acid used to treat gastric and duodenal ulcers and mental retardation in infants, etc., and is also an intermediate for leukemia drugs.

[0005] However, the existing preparation processes of D-glutamine usually use chemical enzymatic methods and biological enzymatic methods for preparation. The chemical enzymatic method mainly uses the Chinese patent with the publication number CN 1 10643670 A. Through N-phthaloyl-DL-glutamic acid, it is chemically converted into N-phthaloyl-DL-glutamine, and D-glutamine is obtained by splitting through the action of aminopeptidase. However, its process is relatively cumbersome and the chemical pollution is serious, which is not suitable for industrial production. The biological enzymatic method mainly uses DL-glutamine as a substrate by Qian Shaosong et al. (Modern Chemical Industry, 2005, (25)9:44-46). Using L-glutamine decarboxylase to selectively cleave L-glutamine to generate 4-aminobutyramide, retaining D-glutamine, and the yield of D-glutamine reaches 92% of the theoretical yield.

[0006] Therefore, those skilled in the art have proposed a method for preparing D-glutamine by whole-cell catalysis, aiming to obtain optically pure D-glutamine with L-glutamine as a substrate through biocatalysts alanine racemase and L-glutamine deaminase, so as to prepare D-glutamine efficiently, at low cost, and with green and pollution-free. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides a method for preparing D-glutamine by whole-cell catalysis to solve the problems raised in the background art.

[0008] According to a first aspect of the present disclosure, a method for preparing D-glutamine by whole-cell catalysis is proposed, including the following steps:

[0009] S1. Use alanine racemase and perform a racemization reaction with L-glutamine as the initial substrate. After racemization, obtain the mother liquor of DL-glutamine by filtration and sterilization;

[0010] S2. Use the obtained DL-glutamine mother liquor for a resolution reaction with glutaminase. After the resolution is completed, sterilize by ultrafiltration and concentrate and crystallize to obtain the target product D-glutamine.

[0011] Preferably, the alanine racemase is constructed using pET-20b as the vector and Escherichia coli BL21(DE3) as the expression host.

[0012] Preferably, the glutaminase is constructed using pET-28a as the vector and Escherichia coli BL21(DE3) as the expression host.

[0013] Preferably, the D-glutamine is obtained through a whole-cell catalytic reaction using a recombinant bacterium constructed with alanine racemase and glutaminase.

[0014] Preferably, the catalytic reaction process is as follows:

[0015] Add 4 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, and make up the volume to 100 ml.

[0016] Add 0.6 g of alanine racemase, stir and react at 37 °C and 200 rpm for 8 - 10 h, and detect that the specific rotation is 0.

[0017] Centrifuge to collect the supernatant, remove proteins from the supernatant by ultrafiltration, adjust the pH of the filtrate to 8.0, make up the volume to 100 ml with pure water, add 0.6 g of L-glutaminase, and react at 30 °C for 12 - 24 h.

[0018] Preferably, the chiral purity of the target product D-glutamine detected by liquid chromatography is 99.9%.

[0019] According to the second aspect of the present disclosure, there is provided the use of the recombinant engineering bacterium of the first aspect or a bacterial agent containing the recombinant engineering bacterium in the preparation of D-glutamine.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] By using alanine racemase such as SEQ ID NO:1 and glutaminase such as SEQ ID NO:2 in the present invention, with 200 mM of L-glutamine as the substrate, through a two-step enzymatic catalytic reaction, the ee value of the product D-glutamine exceeds 99.9%, and the product purity exceeds 99%, showing good prospects for industrial development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the synthetic transformation route diagram of the present invention;

[0023] Figure 2 It is the alanine racemase provided by the embodiment of the present invention;

[0024] Figure 3 This is the protein electrophoresis map of the glutaminase of the present invention;

[0025] Figure 4 This is the determination of the liquid-phase purity map of D-glutamine provided by the embodiment of the present invention;

[0026] Figure 5 This is the determination of the chiral purity map of D-glutamine provided by the embodiment of the present invention;

[0027] Figure 6 This is the schematic diagram of the plasmid map of alanine racemase provided by the embodiment of the present invention;

[0028] Figure 7 This is the schematic diagram of the plasmid map of glutaminase provided by the embodiment of the present invention. Detailed implementation manners

[0029] The following further describes in detail the implementation manners of the present invention in conjunction with the drawings and embodiments. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0030] The total gene synthesis, primer synthesis and sequencing in the embodiments were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0031] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (Third Edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0032] Example 1: The present invention provides an alanine racemase biocatalyst derived from Geobacillus stearothermophilus and an L-glutaminase deaminase biocatalyst derived from Myxococcales bacterium. This catalyst can be used for the racemization and resolution of L-glutamine to obtain D-glutamine with an ee value > 99%. Its synthetic transformation route is as Figure 1 shown.

[0033] A method for enzymatic resolution of DL-glutamine, which has two steps: one is using L-glutamine as a substrate and catalyzing the racemization of L-glutamine to DL-glutamine with alanine racemase, and the other is using DL-glutamine as a substrate and resolving DL-glutamine through glutaminase to obtain D-glutamine.

[0034] In the above method, only by adding the substrates and enzymes corresponding to the reaction steps to the reaction system can the reaction proceed continuously.

[0035] In this application, a first target gene and a second target gene are provided. Among them, the first target gene includes the gene sequence of alanine dehydrogenase, and the second target gene includes the gene sequence of glutaminase.

[0036] In one embodiment, the alanine racemase used in the above method is the wild-type alanine racemase SEQ ID NO:1 derived from Geobacillus stearothermophilus.

[0037] In one embodiment, the glutaminase used in the above method is the glutaminase SEQ ID NO:2 derived from Myxococcales bacterium.

[0038] The expression vector is selected from pET series vectors, pASK series vectors or pBad series vectors. Preferably, it is a pET series vector.

[0039] In the examples of the present invention, the selected pET series vector is pET-28a(+) or pET-20b(+). Those skilled in the art can select a plasmid containing a strong promoter as the expression vector plasmid.

[0040] In the examples of the present invention, the target gene alanine racemase was inserted into pET-20b(+) by restriction endonucleases NcoI and HindIII through total gene synthesis, and the target gene glutaminase was inserted into pET-28a(+) by restriction endonucleases NdeII and HindIII to obtain the expression plasmid. It was transformed into Escherichia coli BL21(DE3) for expression, and the correct recombinant engineering bacteria were preliminarily screened by colony PCR and sequenced. The results showed that the target gene sequence was the sequence shown in SEQ ID NO:1-2, and its encoded amino acid sequence was the sequence shown in SEQ ID NO:3-4.

[0041] A preparation method for preparing D-glutamine includes the following steps:

[0042] (1) Seed activation: Pick the recombinant engineering bacteria described in the present invention and inoculate them into a solid medium;

[0043] (2) Liquid seed activation: Pick the bacterial lawn from the medium in step (1) into a liquid seed medium for activation culture;

[0044] (3) Fermentation culture: Inoculate the liquid seeds in step (2) into a liquid fermentation medium for culture;

[0045] (4) Collect the bacterial cells and freeze them at -20°C to -60°C;

[0046] (5) Racemization: Dissolve L-glutamine and add alanine racemase for racemization;

[0047] (6) Recover DL-glutamine: After sterilizing and ultrafiltering the racemized DL-glutamine reaction solution (5), obtain the DL-glutamine reaction solution;

[0048] (7) Prepare D-glutamine: Add the feed liquid in step (6) to glutaminase for reaction. After the reaction is completed, sterilize, filter, decolorize, and crystallize to obtain D-glutamine.

[0049] Example 2: I. Preparation of culture medium and reagents

[0050] (1) Preparation of LB medium: Peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L (for solid medium), natural pH, sterilize at 121°C for 20 min.

[0051] (2) 50 mg / mL ampicillin solution (Amp): Dissolve 1 g of ampicillin in 20 mL of sterilized deionized water, filter and sterilize with a 0.22 μm filter membrane, aliquot into sterilized EP tubes, 1 mL per tube, and store at -20°C for later use. Before use, add 1‰ of the amount, that is, the final concentration is 50 μg·mL -1 .

[0052] (3) 50 mg·mL -1 Kanamycin solution (Kan): Dissolve 1 g of kanamycin in 20 mL of sterilized deionized water, filter and sterilize with a 0.22 μm filter membrane, aliquot into sterilized EP tubes, 1 mL per tube, and store at -20°C for later use. Before use, add 1‰ of the amount, that is, the final concentration is 50 μg·mL -1 .

[0053] (4) 1 M isopropyl-β-D-thiogalactoside (IPTG): Dissolve 2.38 g of IPTG in sterilized deionized water, filter and sterilize with a 0.22 μm filter membrane, aliquot into sterilized EP tubes, 1 mL per tube, and store at -20°C for later use.

[0054] (5) Protein electrophoresis electrode buffer: 3 g of tris(hydroxymethyl)aminomethane (Tris), 14.4 g of glycine, 1.0 g of SDS, pH = 8.3, make up to 1 L with deionized water, and store at room temperature.

[0055] (6) Protein electrophoresis staining buffer: 2.5 g of Coomassie Brilliant Blue R250, 454 mL of absolute ethanol, 46 mL of glacial acetic acid, 454 mL of deionized water, stir to dissolve. Store in the dark at room temperature.

[0056] (7) The protein electrophoresis kit uses the SDS-PAGE denaturing acrylamide gel rapid preparation kit from Sangon Biotech (Shanghai).

[0057] II. Construction of alanine racemase-expressing strains:

[0058] For the wild-type alanine racemase SEQ ID NO:1 from Geobacillus stearothermophilus, codon optimization was performed, and the coding gene sequence SEQ ID NO:5 was synthesized by gene synthesis. Restriction enzyme sites NcoI and HindIII were designed at both ends of the gene and cloned into the pET-20b plasmid vector to obtain the recombinant plasmid pET20b-ALR. The recombinant plasmid pET20b-ALR was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli ALR expressing the wild enzyme.

[0059] III. Construction of glutaminase-expressing strains:

[0060] For the wild-type glutaminase SEQ ID NO:2 from Myxococcales bacterium, codon optimization was performed, and the coding gene sequence SEQ ID NO:6 was synthesized by gene synthesis. It was cloned into the pET-28a plasmid vector at both ends with NdeI and HindIII restriction enzyme sites to obtain the recombinant plasmid pET28a-glnA. The recombinant plasmid pET28a-glnA was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli glnA expressing the wild enzyme.

[0061] IV. Inductive expression of strains:

[0062] Inductive expression of alanine racemase:

[0063] Pick the alanine racemase transformed into BL21(DE3), perform overnight culture and colony PCR verification. Those with successful verification were sent for sequencing. According to an inoculation amount of 1‰, ampicillin was inoculated, and according to an inoculation amount of 1%, it was inoculated into the secondary medium. When OD reached 0.6 - 0.8, inductive culture was carried out, 0.2 mM IPTG was added, the temperature was lowered to 30 °C, and inductive culture was carried out for 8 h.

[0064] Inductive expression of glutaminase:

[0065] Pick the glutaminase transformed into BL21(DE3), conduct overnight culture and verify by colony PCR. Those with successful verification are sent for sequencing. Inoculate kanamycin at an inoculation amount of 1‰, and inoculate into the secondary medium at an inoculation amount of 1%. When the OD reaches 0.6 - 0.8, perform induction culture, add 0.2 mM IPTG, cool down to 28°C, and conduct induction culture for 8 h.

[0066] Preservation of the induced product:

[0067] Centrifuge the induced culture at 6000 rpm and 4°C for 1 h to collect the wet bacterial cells, and store the collected wet bacterial cells frozen at -20°C.

[0068] V. Verification of enzyme activity in small-scale test

[0069] Determination of alanine racemase activity:

[0070] Dissolve 4 g of L-glutamine in 100 ml of water, add 0.2 g / L of PLP, adjust the pH to between 8.0 and 9.0, adjust the temperature to 37°C. When the temperature of the reaction substrate solution rises to 35°C, add 1 g of alanine racemase for racemization reaction, and monitor the pH every 1 h and adjust it to between 8.0 and 9.0. The racemization reaction is completed after 6 h of conversion. Sample and centrifuge to collect the supernatant, and measure the specific rotation of the supernatant. A specific rotation of 0 indicates the completion of the reaction.

[0071] Determination of glutaminase activity;

[0072] Take the above reaction supernatant, adjust the pH to 8.0, adjust the temperature to 30°C, add wet bacterial cells of glutaminase according to the ratio of substrate to bacterial cell mass of 6:1, stop the reaction after catalyzing the reaction for 12 h - 24 h. After the reaction is completed, send the sample for liquid phase detection. When the chiral purity of D-glutamine reaches 99.9%, the reaction is completed.

[0073] Determination of D-glutamine purity

[0074] HPLC detection method:

[0075] Chromatographic column: CAPCELL PAK C18 250*4.6*50.00

[0076] Mobile phase: water (0.05% TFA) CAN (0.05% TFA)

[0077] Column temperature: 30°C

[0078] Injection volume: 2 μl

[0079] Flow rate: 1.0 ml / min

[0080] Method:

[0081]

[0082] Sample preparation:

[0083] 1. Preparation of FMOC-CL solution: Accurately weigh 52 mg of FMOC into a 20-ml volumetric flask, and mix with acetonitrile to the calibration line.

[0084] 2. Borate buffer: Prepare 0.01 mol of sodium tetraborate hydrate buffer in water, and adjust the pH to 10.2 with diluted sodium hydroxide.

[0085] 3. Weigh 50 mg of the conversion solution sample. Add 1.0 ml of sodium tetraborate to dissolve it, and react with 1.0 ml of FMOC-CL for 5 min.

[0086] 4. Weigh 2 mg of the GLn standard. Add 1.0 ml of sodium tetraborate to dissolve it, and react with 1.0 ml of FMOC-CL for 5 min.

[0087] HPLC chiral detection method

[0088] Chromatographic column: CR-I(-) 150*3*5

[0089] Detection wavelength: 210 nm

[0090] Sample solvent: Mobile phase

[0091] Sample concentration: 0.5 mg / ml

[0092] Injection volume: 5 μl

[0093] Column temperature: 17 °C

[0094] Mobile phase: H2O adjusted to pH 1.5 with perchloric acid

[0095] Flow rate: 0.4 ml / min

[0096] Running time: 25 min

[0097] VI. Whole-cell catalysis of L-glutamine to produce DL-glutamine and then resolution into D-glutamine

[0098] In a 250 ml three-necked flask, add 4 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, make up the volume to 100 ml, add 0.6 g of alanine racemase obtained from the construction of the alanine racemase-expressing strain, stir and react at 37 °C and 200 rpm for 8 - 10 h, and the specific rotation is detected to be 0. Centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, adjust the pH of the filtrate to 8.0, add pure water to make up the volume to 100 ml, add 0.4 g of L-glutaminase, and react at 37 °C for 12 - 24 h. The chiral purity is detected by liquid phase to be 99.2%. After the conversion is completed, centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, and concentrate and crystallize (the concentration temperature is not higher than 45 °C) to obtain 1.4 g of the product, and the yield is about 60%.

[0099] VII. Whole-cell catalysis of L-glutamine to produce DL-glutamine and then resolution into D-glutamine

[0100] In a 250 ml three-necked flask, add 5 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, make up the volume to 100 ml, add 0.6 g of alanine racemase obtained from the construction of the alanine racemase-expressing strain, stir and react at 37 °C and 200 rpm for 8 - 10 h, and the specific rotation is detected to be 0. Centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, adjust the pH of the filtrate to 8.0, add pure water to make up the volume to 100 ml, add 0.4 g of L-glutaminase, and react at 37 °C for 12 - 24 h. The chiral purity is detected by liquid phase to be 99.3%. After the conversion is completed, centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, and concentrate and crystallize (the concentration temperature is not higher than 45 °C) to obtain 1.4 g of the product, reaching 56% of the theoretical yield.

[0101] VIII. Whole-cell catalysis of L-glutamine to produce DL-glutamine and then resolution into D-glutamine

[0102] In a 250 ml three-necked flask, add 5 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, make up the volume to 100 ml, add 0.6 g of alanine racemase obtained from the construction of the alanine racemase-expressing strain, stir and react at 37 °C and 200 rpm for 8 - 10 h, and the specific rotation is detected to be 0. Centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, adjust the pH of the filtrate to 8.0, add pure water to make up the volume to 100 ml, add 0.4 g of L-glutaminase, and react at 30 °C for 12 - 24 h. The chiral purity is detected by liquid phase to be 99.5%. After the conversion is completed, centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, and concentrate and crystallize (the concentration temperature is not higher than 45 °C) to obtain 1.5 g of the product, reaching 60% of the theoretical yield.

[0103] IX. In a 250-ml three-necked flask, add 4 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, make up the volume to 100 ml, add 0.6 g of alanine racemase obtained from the construction of an alanine racemase-expressing strain, and stir and react at 37°C and 200 rpm for 8 - 10 h. The specific rotation is detected to be 0. Centrifuge to collect the supernatant, and ultrafilter the supernatant to remove proteins. Adjust the pH of the filtrate to 8.0, add pure water to make up the volume to 100 ml, add 0.6 g of L-glutaminase, and react at 30°C for 12 - 24 h. The chiral purity is detected by liquid phase to be 99.9%. After the conversion is completed, centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, and concentrate and crystallize (the concentration temperature is not higher than 45°C) to obtain 1.7 g of the product, reaching 85% of the theoretical yield.

[0104] In summary: The alanine racemase SEQ ID NO: 1 and glutaminase SEQ ID NO: 2 constructed in the present invention can catalyze the synthesis of D-glutamine using L-glutamine as a substrate. For example, at a substrate concentration of 40 g / L, complete conversion can be achieved within 2 reactions, a total of 30 h. Due to losses during the reaction and extraction process, the highest yield calculated according to the theoretical yield can reach 85%. However, both the product purity and chiral purity exceed 99%, showing potential for industrial development and application.

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Claims

1. A method for preparing D-glutamine by whole-cell catalysis, characterized in that, It includes the following steps: S1. Use alanine racemase and perform a racemization reaction with L-glutamine as the initial substrate. After racemization, obtain the mother liquor of DL-glutamine by filtration and sterilization. S2. Use the obtained mother liquor of DL-glutamine for a resolution reaction with glutaminase. After the resolution is completed, obtain the target product D-glutamine by ultrafiltration and sterilization, concentration and crystallization.

2. The method for preparing D-glutamine by whole-cell catalysis according to claim 1, wherein: The alanine racemase is constructed with pET-20b as the vector and Escherichia coli BL21(DE3) as the expression host.

3. The method for preparing D-glutamine by whole-cell catalysis according to claim 1, wherein: The glutaminase is constructed with pET-28a as the vector and Escherichia coli BL21(DE3) as the expression host.

4. The method for preparing D-glutamine by whole-cell catalysis according to claim 1, wherein: The D-glutamine is obtained through a whole-cell catalytic reaction by a recombinant bacterium constructed with alanine racemase and glutaminase.

5. The method for preparing D-glutamine by whole-cell catalysis according to claim 4, characterized in that: The process of the catalytic reaction is as follows: Add 4 g of L-glutamine, adjust the pH to 8.0 with 6 M hydrochloric acid, and make up the volume to 100 ml. Add 0.6 g of alanine racemase, stir and react at 37 °C and 200 rpm for 8 - 10 h, and detect that the specific rotation is 0. Centrifuge to collect the supernatant, ultrafilter the supernatant to remove proteins, adjust the pH of the filtrate to 8.0, make up the volume to 100 ml with pure water, add 0.6 g of glutaminase, and react at 30 °C for 12 - 24 h.

6. The method for preparing D-glutamine by whole-cell catalysis according to claim 1, characterized in that: The chiral purity of the target product D-glutamine detected by liquid phase is 99.9%.

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