阅读说明：本技术 一种化学酶法制备d-谷氨酰胺的方法 (Method for preparing D-glutamine by chemical enzyme method ) 是由 陈诚 吴聪 程文莹 甘飞 *** 刘均忠 焦庆才 于 2019-11-14 设计创作，主要内容包括：本发明提供了一种化学酶法制备D-谷氨酰胺的方法。所述方法按以下步骤进行：1、制备N-邻苯二甲酰-DL-谷氨酸；2、制备N-邻苯二甲酰-DL-谷氨酸酐；3、制备N-邻苯二甲酰-DL-谷氨酰胺；4、制备DL-谷氨酰胺；5、培养制备氨肽酶；6、氨肽酶拆分DL-谷氨酰胺。本发明利用成本低廉的DL-谷氨酸为原料,经四步简单的化学反应制备DL-谷氨酰胺,并对工艺参数进行优化,DL-谷氨酰胺收率由57%提高至67%；利用氨肽酶拆分DL-谷氨酰胺,得到L-谷氨酸和D-谷氨酰胺,D-谷氨酰胺达到理论收率的95%。本发明原料成本低、工艺路线简单、绿色环保,有利于工业化实施。(The invention provides a method for preparing D-glutamine by a chemical enzyme method. The method comprises the following steps: 1. preparing N-phthaloyl-DL-glutamic acid; 2. preparing N-phthaloyl-DL-glutamic anhydride; 3. preparing N-phthaloyl-DL-glutamine; 4. preparing DL-glutamine; 5. culturing to prepare aminopeptidase; 6. aminopeptidase cleaves DL-glutamine. The method utilizes low-cost DL-glutamic acid as a raw material, prepares DL-glutamine through four simple chemical reactions, optimizes process parameters, and improves the yield of the DL-glutamine from 57 percent to 67 percent; the DL-glutamine is resolved by aminopeptidase to obtain L-glutamic acid and D-glutamine, wherein the yield of the D-glutamine reaches 95 percent of the theoretical yield. The invention has low cost of raw materials, simple process route, environmental protection and contribution to industrial implementation.)

1. A method for preparing D-glutamine by a chemoenzymatic method is characterized by comprising the following steps: the method comprises the following steps:

(1) preparing N-phthaloyl-DL-glutamic acid, namely adding DL-glutamic acid and phthalic anhydride with the molar ratio of 1:1 ~ 1:1.2 into a round-bottom flask, carrying out oil bath for 10 ~ 40 minutes at the temperature of 150 ~ 200 ℃, naturally cooling, precipitating crystals, and drying to obtain the N-phthaloyl-DL-glutamic acid;

(2) preparing N-phthaloyl-DL-glutamic anhydride, dissolving the N-phthaloyl-DL-glutamic acid generated in the step (1) in acetic anhydride, refluxing for 20 ~ 60 minutes, naturally cooling, separating out crystals, filtering, washing the crystals with ethyl acetate, and drying to obtain the N-phthaloyl-DL-glutamic anhydride;

(3) dissolving the N-phthaloyl-DL-glutamine generated in the step (2) into an ammonia water solution with the concentration of 2 ~ 4 mol/L, stirring and reacting for 20 ~ 60 minutes at room temperature, then adding hydrochloric acid with the concentration of 6mol/L to acidify until the pH value is 3, separating out crystals, filtering and drying to obtain the N-phthaloyl-DL-glutamine;

(4) preparing DL-glutamine, namely dissolving the N-phthaloyl-DL-glutamine generated in the step (3) in hydrazine hydrate solution with the concentration of 0.5 ~ 2mol/L, standing at room temperature for 10 ~ 40 hours, acidifying reaction liquid by hydrochloric acid with the concentration of 6mol/L until the pH value is 3, filtering to remove separated phthalhydrazide, concentrating filtrate under reduced pressure to separate DL-glutamine, and finally refining and drying to obtain DL-glutamine;

(5) culturing the strain with aminopeptidase activity in culture medium to produce high-activity aminopeptidase, wherein the mass concentration of the total carbon source in the culture medium is 1 ~ 20 g/L, and the mass concentration of the total nitrogen source in the culture medium is 1 ~ 20 g/L;

(6) and (2) resolving DL-glutamine by aminopeptidase, namely preparing the DL-glutamine generated in the step (4) into a solution with the concentration of 10 ~ 100 g/L, adding aminopeptidase bacterial cells or crude enzyme solution with the final concentration of 5 ~ 40 g/L and 0.05 ~ 0.1 g/L Tween 80 or Triton X-100, controlling the pH value to be 7 ~ 10 by using a sodium carbonate buffer solution with the concentration of 2 ~ 3mol/L at the temperature of 20 ~ 45 ℃, performing enzymatic conversion to obtain L-glutamic acid and D-glutamine, and separating by using a method combining isoelectric point crystallization and ion exchange resin to obtain the high-purity D-glutamine.

2. The chemoenzymatic method for producing D-glutamine according to claim 1, characterized in that: the carbon source in the culture medium in the step (5) of the method is one or more of glucose, maltose, sucrose and/or lactose.

3. The chemoenzymatic method for producing D-glutamine according to claim 1, characterized in that: the nitrogen source in the culture medium in the step (5) of the method is one or more of beef extract, yeast extract, corn steep liquor, peptone and/or bean cake hydrolysate.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for preparing D-glutamine by a chemical enzyme method.

Background

The content of D-amino acid in nature is far lower than that of L-amino acid, and protein polypeptide consisting of D-amino acid is generally not hydrolyzed or slowly hydrolyzed by polypeptide enzyme, so that D-amino acid has been used for synthesizing beta-lactam antibiotics and physiologically active peptides, and is widely applied to the fields of medicine, chemical industry, food and the like.

The molecular formula of the D-glutamine is C10H20N4O6, the relative molecular mass is 146.14, the D-glutamine is white needle-shaped crystal, and the D-glutamine has no odor, has slightly sweet fragrance, and has the melting point of 184-185 ℃ (decomposition). Dissolving in methanol, ethanol, ether, benzene, acetone, chloroform and ethyl acetate. Stable in neutral solution, and easily decomposed into glutamic acid in acid, alkali or hot water, or lactonized into pyrrole carboxylic acid. D-glutamine is a medicinal amino acid, is used for treating gastric ulcer, duodenal ulcer, infantile mental retardation and the like, is an intermediate of medicaments for treating leukemia, and has very important application prospect.

The main method for industrially producing D-amino acid reported at present is to prepare D-amino acid by taking L-amino acid as a raw material. Firstly racemizing the L-amino acid into DL-amino acid by a chemical method or a biological method, and then splitting the DL-amino acid by the chemical method or the biological method to obtain the D-amino acid. The common chemical resolution methods include a derivative resolution method and a cyclic polyether resolution method, and the common biological resolution methods include an acylase method, a carboxypeptidase method and an enzyme asymmetric degradation method.

(1) Derivative resolution method: the method comprises the steps of generating double salt by racemic amino acid and pure chiral acid or alkali, separating two enantiomers by physical methods such as recrystallization or chromatography, and obtaining pure D-type or L-type amino acid again by a hydrolysis method. The commonly used chiral bases are strychnine and its salts, and the commonly used chiral acids are tartaric acid, camphor, sulfonic acid (CA) S and its salts. The racemate resolution by the method is carried out, amino acid derivatization is carried out before resolution, hydrolysis is carried out after resolution to remove derivatization, the non-target enantiomer needs to be subjected to repeated cyclic racemization, the product needs to be subjected to repeated recrystallization, the process is complex, and the yield is low. In particular, the chiral resolving agent is expensive, which greatly increases the production cost.

(2) A cyclic polyether splitting method: the optically active cyclic polyether has unique optical isomer distinguishing property, and may form stable compound with the identified isomer in the enantiomer, while the other isomer with opposite optical activity has poor combination stability. The crown ether is used for resolving the alpha-amino acid, amino protection is not needed, the operation is convenient, but the crown ether is expensive to synthesize at present, so that the method is only limited to the research and application in a laboratory.

(3) A biological resolution method: the enzyme is a chiral molecule and has catalytic specificity and stereoselectivity. After α -amino acetylation (or carboxyamidation) of a racemic amino acid, hydrolysis is carried out using a hydrolase such as an acylase, carboxypeptidase or aminopeptidase, and since the hydrolase recognizes and hydrolyzes only an amide bond formed by an L-amino acid, the L-amino acid can be liberated; the amido bond formed by D-amino acid which can not be hydrolyzed by enzyme still exists in the form of acetylamino acid or carboxyl acylated amino acid, thus achieving the purpose of separation. Or directly carrying out enzymatic degradation on DL-amino acid to eliminate L-enantiomer in the DL-amino acid so as to obtain the D-amino acid.

The Qiashaoxing and the like (modern chemical engineering, 2005, (25)9: 44-46) use DL-glutamine as a substrate, utilize L-glutamine decarboxylase to selectively crack L-glutamine to generate 4-aminobutanamide, and keep D-glutamine, wherein the yield of the D-glutamine reaches 92% of the theoretical yield.

At present, the commercially available glutamine is mainly L-glutamine, and the gamma amido bond of the glutamine causes the glutamine to be unstable to acid, alkali or high temperature and is easy to form pyroglutamic acid or decompose into glutamic acid, so that the DL-glutamine is difficult to prepare by utilizing chemical racemic L-glutamine. The Qianshogsong et al (organic chemistry, 2006, (26)4: 514-517) chemically synthesize DL-glutamine by four-step reaction with a yield of 57% by taking DL-glutamic acid as a raw material.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing D-glutamine with high efficiency and low cost.

The design idea of the invention is to use low-cost DL-glutamic acid as a raw material, use low-cost phthaloyl as a protective group, prepare DL-glutamine through four simple chemical reactions, and then use aminopeptidase to split DL-glutamine to obtain D-glutamine. The reaction product of each step of chemical synthesis of DL-glutamine is easy to crystallize and separate, the DL-glutamine is resolved by an enzyme method without modifying a substrate, the catalysis efficiency is high, the stereoselectivity is strong, the reaction condition is mild, the product separation method is simple, and the industrial implementation is facilitated. The process route adopted by the invention is as follows:

the invention optimizes the process route of chemically synthesizing DL-glutamine, improves the DL-glutamine yield to 67 percent, and obtains L-glutamic acid and D-glutamine by splitting the DL-glutamine by aminopeptidase, wherein the D-glutamine reaches 95 percent of the theoretical yield. And the other product L-glutamic acid can be racemized to obtain DL-glutamic acid, so that the whole process route for preparing D-glutamine by the chemoenzymatic method is circulated.

In order to achieve the purpose, the invention is achieved by the following technical scheme:

a method for preparing D-glutamine by a chemoenzymatic method, which comprises the following steps:

1. preparing N-phthaloyl-DL-glutamic acid, namely adding DL-glutamic acid and phthalic anhydride with the molar ratio of 1:1 ~ 1:1.2 into a round-bottom flask, carrying out oil bath for 10 ~ 40 minutes at the temperature of 150 ~ 200 ℃, naturally cooling, precipitating crystals, and drying to obtain the N-phthaloyl-DL-glutamic acid;

2. preparing N-phthaloyl-DL-glutamic anhydride, dissolving the N-phthaloyl-DL-glutamic acid generated in the step 1 in acetic anhydride, refluxing for 20 ~ 60 minutes, naturally cooling, separating out crystals, filtering, washing the crystals with ethyl acetate, and drying to obtain the N-phthaloyl-DL-glutamic anhydride;

3. dissolving the N-phthaloyl-DL-glutamine generated in the step 2 in ammonia water solution with the concentration of 2 ~ 4 mol/L, stirring and reacting for 20 ~ 60 minutes at room temperature, then adding hydrochloric acid with the concentration of 6mol/L to acidify until the pH value is 3, separating out crystals, filtering and drying to obtain the N-phthaloyl-DL-glutamine;

4. preparing DL-glutamine by dissolving the N-phthaloyl-DL-glutamine generated in the step 3 in hydrazine hydrate solution with the concentration of 0.5-2 mol/L, standing at room temperature for 12 ~ 40 hours, acidifying the reaction liquid to the pH value of 3 by hydrochloric acid with the concentration of 6mol/L, filtering to remove the separated phthalhydrazide, concentrating the filtrate under reduced pressure to separate out the DL-glutamine, and finally refining and drying to obtain the DL-glutamine;

5. culturing aminopeptidase in culture medium to produce high activity aminopeptidase, wherein the total carbon source concentration in the culture medium is 1 ~ 20 g/L and the total nitrogen source concentration in the culture medium is 1 ~ 20 g/L;

6. and (2) resolving DL-glutamine by using aminopeptidase, namely preparing the DL-glutamine obtained in the step (4) into a solution with the concentration of 10 ~ 100 g/L, adding aminopeptidase strains or crude enzyme solution with the final concentration of 5 ~ 40 g/L and 0.05 ~ 0.1 g/L Tween 80 or Triton X-100, controlling the pH value to be 7 ~ 10 by using sodium carbonate buffer solution with the concentration of 2 ~ 3mol/L at the temperature of 20 ~ 45 ℃, performing enzymatic conversion to obtain L-glutamic acid and D-glutamine, and separating by using a method combining isoelectric point crystallization and ion exchange resin to obtain high-purity D-glutamine.

The carbon source in the culture medium in the step 5 of the method is one or more of glucose, maltose, sucrose and/or lactose.

In the method, the nitrogen source in the culture medium in the step 5 is one or more of beef extract, yeast extract, corn steep liquor, peptone and/or bean cake hydrolysate.

Compared with the prior art, the invention has the following advantages:

1. the method utilizes low-cost DL-glutamic acid as a raw material, prepares DL-glutamine through four simple chemical reactions, optimizes process parameters, and improves the yield of the DL-glutamine from 57 percent to 67 percent;

2. the method utilizes aminopeptidase to split DL-glutamine, has the advantages of high substrate concentration, high catalytic efficiency, strong stereoselectivity, mild reaction conditions and the like, and the yield of the product D-glutamine can reach 95 percent of the theoretical yield;

3. the invention utilizes L-glutamic acid which is one of products of splitting DL-glutamine by an enzyme method, can obtain DL-glutamic acid through racemization, and can also be used as a raw material for preparing DL-glutamine, so that the whole process for splitting DL-glutamine by a chemical enzyme method is circulated;

4. the aminopeptidase adopted by the invention can directly catalyze and split the substrate DL-glutamine without modifying the substrate, and the method has the advantages of simple process route, environmental protection and contribution to industrial implementation.

Detailed Description

The technology of the present invention will be further described in detail and fully with reference to the following examples.