CN106480009B - Immobilized amino acid ester acyl transferase and application thereof in preparation of glutamine dipeptide - Google Patents
Immobilized amino acid ester acyl transferase and application thereof in preparation of glutamine dipeptide Download PDFInfo
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- CN106480009B CN106480009B CN201611211842.7A CN201611211842A CN106480009B CN 106480009 B CN106480009 B CN 106480009B CN 201611211842 A CN201611211842 A CN 201611211842A CN 106480009 B CN106480009 B CN 106480009B
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Abstract
The invention relates to an immobilized amino acid ester acyltransferase, a preparation method and an application thereof, wherein the immobilized amino acid ester acyltransferase is prepared by immobilizing amino acid ester acyltransferase in an immobilized enzyme carrier in an adsorption, covalent bonding, embedding, microencapsulation or crosslinking immobilization manner, wherein the mass percentage of the amino acid ester acyltransferase in the immobilized enzyme carrier is 0.01-5%. Compared with liquid enzyme, the immobilized enzyme of the invention reduces the production cost and labor intensity, improves the utilization efficiency and product quality of the enzyme, reduces the discharge of three wastes, and is green and environment-friendly. In addition, the immobilized enzyme is easy to separate from the reaction liquid mixture and can be repeatedly used; the reactor design is flexible; suitable for a plurality of different reaction media and reaction systems; process development and downstream processing are facilitated, and process control is particularly facilitated.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemicals, and particularly relates to immobilized amino acid ester acyl transferase, a preparation method thereof, and a method for preparing glutamine dipeptide by adopting the immobilized amino acid ester acyl transferase.
Background
The glutamine dipeptide (N- (2) -L-alanyl-L-glutamine) has higher stability and solubility in water solution than glutamine, and is widely applied to injection and serum-free culture medium. Glutamine has very important physiological functions and pharmacological actions, so that the application of glutamine in parenteral nutrition is generally regarded by people. The research proves that the glutamine dipeptide can be quickly decomposed into alanine and glutamine in vivo, the half-life period is very short, only a small amount of dipeptide can be detected in blood, and only a small amount of dipeptide is discharged from urine, so that the glutamine dipeptide can be effectively utilized and cannot be accumulated in the blood, and the possible pharmacological and physiological damage is avoided.
The synthesis of glutamine dipeptide by chemical method has the disadvantages of long complex synthetic route, great environmental pollution and high cost. The enzymatic method for producing glutamine dipeptide is simple in process, green, environment-friendly and low in cost. At present, liquid enzyme is mostly used for reaction, compared with immobilized enzyme, each batch of reaction needs to be replaced by new enzyme liquid, the separation is difficult, the operation is complicated, the utilization efficiency of the enzyme is reduced, waste liquid cannot be recycled, a large amount of three-waste pollutants are generated, and meanwhile, the product possibly contains protein and other thallus impurities.
The development of immobilized enzyme technology has been over 100 years old, and by the end of the last 70 th century, the technology has been well developed, and suitable immobilization methods such as embedding, microencapsulation, covalent bonding, adsorption or combination methods, suitable carriers such as inorganic or organic carriers, natural or synthetic carriers, porous or non-porous carriers, membranes, granules, foams, capsules, etc., and suitable immobilization conditions such as aqueous phase, organic solvents, pH, temperature, etc., have been selected. With the development of scientific technology, the technology of immobilized enzymes is continuously improved, and the performances of a plurality of enzymes without industrial application are improved through the continuous improvement of immobilized carriers, immobilization methods and enzymes, so that the immobilized enzymes have higher selectivity, activity and durability. The use of immobilized enzyme facilitates the separation of reaction mixture, the reactor design is flexible, and the reactor is suitable for various different reaction media and reaction systems, facilitates the process development and downstream treatment, and is particularly convenient for process control. However, because the application time is short, many industrial reactions, especially some pharmaceutical intermediate preparation processes, do not adopt immobilized enzyme technology.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the prior art and provides an immobilized amino acid ester acyl transferase.
In order to solve the problems, the invention adopts the following technical scheme:
the immobilized amino acid ester acyltransferase is prepared by immobilizing amino acid ester acyltransferase in an immobilized enzyme carrier in an adsorption, covalent bonding, embedding, microencapsulation or crosslinking immobilization manner, wherein the mass percent of the amino acid ester acyltransferase in the immobilized enzyme carrier is 0.01-5%.
Preferably, the immobilized amino acid ester acyltransferase of the present invention is 0.1-2.5% by mass of the immobilized enzyme vector.
The immobilized amino acid ester acyltransferase of the present invention is a wild-type or mutant amino acid ester acyltransferase.
Further, the immobilized amino acid ester acyltransferase of the present invention is one in which the immobilization is covalent bonding and the immobilized enzyme carrier is selected from an epoxy immobilized enzyme carrier and an amino immobilized enzyme carrier.
Further, the immobilized amino acid ester acyl transferase of the present invention is produced by a method comprising: respectively adding amino acid ester acyl transferase and an immobilized enzyme carrier into a buffer solution, wherein the pH value of the buffer solution is 5-10, the immobilization reaction temperature is controlled to be 5-40 ℃, stirring and reacting for 5-72 hours, collecting solids, and drying to obtain the immobilized enzyme carrier.
Preferably, the pH value of the buffer solution is 7-9, more preferably 8, the immobilization reaction temperature is 15-25 ℃, more preferably 20 ℃, and the buffer solution is selected from a phosphate buffer solution, a Tris-HCl buffer solution or a triethanolamine buffer solution.
The invention also aims to provide a biological preparation method for producing glutamine dipeptide by using the immobilized enzyme.
The technical scheme provided by the invention is as follows:
a method for preparing glutamine dipeptide by using the immobilized amino acid ester acyltransferase of the invention as a biocatalyst, which comprises the steps of taking glutamine and L-alanine methyl ester or L-alanine methyl ester hydrochloride as substrates, stirring the substrates in an aqueous solution with the pH value of 5.0-9.5 at the reaction temperature of 5-30 ℃, and reacting the two substrates under the catalysis of the immobilized amino acid ester acyltransferase of any one of claims 1-4 to generate the glutamine dipeptide.
Preferably, in the method for preparing glutamine dipeptide, the concentration of glutamine in a reaction system at the beginning of the reaction is 1% -25% in unit g/mL, the concentration of L-alanine methyl ester or L-alanine methyl ester hydrochloride is 1% -25% in unit g/mL, and the concentration of immobilized amino acid ester acyl transferase is 1% -25% in unit g/mL.
Further preferably, in the preparation method of glutamine dipeptide, the concentration of L-alanine methyl ester or L-alanine methyl ester hydrochloride in a reaction system at the beginning of the reaction is 5% -25% in unit g/mL, and the concentration of glutamine is 5% -15% in unit g/mL.
Preferably, in the preparation method of glutamine dipeptide, the concentration of the immobilized amino acid ester acyl transferase in the reaction initiation system is 5% -15%, and the unit g/mL.
Preferably, in the preparation method of glutamine dipeptide, the pH value of the aqueous phase solution is 7.0-9.5 in the reaction system at the beginning of the reaction.
Preferably, in the preparation method of glutamine dipeptide, the reaction temperature in the reaction system at the beginning of the reaction is 5-20 ℃.
Preferably, the method for preparing glutamine dipeptide according to the present invention is a buffered solution.
The substrate (L-alanine methyl ester or L-alanine methyl ester hydrochloride, glutamine), amino acid ester acyltransferase, immobilized enzyme vector, etc. used in the present invention are commercially available.
The preparation method of the invention is implemented as follows: adding a buffer solution into a reaction container, sequentially adding substrates L-alanine methyl ester or L-alanine methyl ester hydrochloride, glutamine and immobilized amino acid ester acyl transferase, stirring and reacting at the temperature of 5-30 ℃, monitoring a reaction product by using a high performance liquid chromatography, and stopping the reaction until the conversion rate reaches more than 80%.
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Compared with the prior art, the invention has the following advantages:
1. the method effectively overcomes the defects of low substrate concentration, long enzyme reaction time consumption and the like in the traditional enzyme catalysis production process by adding the auxiliary materials which are cheap and have wide sources, and realizes the efficient and low-cost preparation of the glutamine dipeptide.
2. Compared with liquid enzyme, the immobilized enzyme of the invention reduces the production cost and labor intensity, improves the utilization efficiency and product quality of the enzyme, reduces the discharge of three wastes, and is green and environment-friendly.
3. The immobilized enzyme is easy to separate from the reaction liquid mixture and can be repeatedly used; the reactor design is flexible; suitable for a plurality of different reaction media and reaction systems; process development and downstream processing are facilitated, and process control is particularly facilitated.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples.
Example 1 preparation of immobilized amino acid ester acyl transferase
Taking a 250mL three-neck flask, adding 150mL phosphate buffer solution (100 mM, pH value 8.0), sequentially adding 1g of amino acid ester acyl transferase (or liquid enzyme) and 40g of epoxy group immobilized enzyme carrier (sepabeads EC-EP 113-M), reacting at 20 ℃ and 150rpm for 24 hours, and collecting the immobilized enzyme.
EXAMPLE 2 preparation of immobilized amino acid ester acyltransferase
Taking a 250mL three-mouth flask, adding 150mL phosphate buffer solution (100 mM, pH value 8.0), sequentially adding 1g of amino acid ester acyl transferase (or liquid enzyme) and 40g of activated amino immobilized enzyme carrier (sepabeads EC-HA/M), reacting at 20 ℃ and 150rpm for 24 hours, and collecting the immobilized enzyme.
Example 3 preparation of immobilized amino acid ester acyltransferase
Taking a 250mL three-neck flask, adding 150mL phosphate buffer solution (100 mM, pH value 8.0), sequentially adding 0.04g of amino acid ester acyl transferase (or liquid enzyme) and 40g of activated amino immobilized enzyme carriers (sepabeads EC-HA/M), reacting at the temperature of 20 ℃ and the rotation speed of 150rpm for 36 hours, and collecting the immobilized enzyme.
EXAMPLE 4 enzymatic preparation of Glutamine
A250 mL three-necked flask was charged with 17.25g of L-alanine methyl ester hydrochloride as a substrate, 8g of glutamine, 150mL of purified water, and 10g of the immobilized amino acid ester acyltransferase of example 1, and the reaction was stirred at 20 ℃ and 200rpm, pH 8.5 was controlled with 6moL/L hydrochloric acid, and the conversion of the reaction was monitored by liquid chromatography, and after 30 minutes, the conversion reached 80% or more, and the immobilized enzyme was separated and recovered.
The immobilized enzyme is repeatedly used for 10 times, the reaction time is less than 1 hour, and the conversion rate is more than 80%.
Experimental example 5 enzymatic preparation of glutamine dipeptide
A250 mL three-necked flask was charged with 17.25g of L-alanine methyl ester hydrochloride as a substrate, 8g of glutamine, 150mL of purified water, and 10g of the immobilized amino acid ester acyltransferase of example 2, and the reaction was stirred at 20 ℃ and 200rpm, pH 8.5 was controlled with 6moL/L hydrochloric acid, and the conversion of the reaction was monitored by liquid chromatography, and after 30 minutes, the conversion reached 80% or more, and the immobilized enzyme was separated and recovered.
The immobilized enzyme is repeatedly used for 10 times, the reaction time is less than 1 hour, and the conversion rate is more than 80%.
Claims (2)
1. A method for preparing glutamine dipeptide by using immobilized amino acid ester acyl transferase as a biocatalyst is characterized in that: glutamine and L-alanine methyl ester hydrochloride are taken as substrates, the two substrates are stirred in a water phase solution, and the two substrates react under the catalysis of the immobilized amino acid ester acyl transferase to generate glutamine dipeptide, and the method specifically comprises the following steps: adding 17.25g of substrate L-alanine methyl ester hydrochloride, 8g of glutamine, 150mL of purified water and 10g of immobilized amino acid ester acyl transferase, stirring for reaction at 20 ℃ and 200rpm, controlling the pH value to 8.5 by using 6moL/L hydrochloric acid, monitoring the conversion rate of the reaction by using liquid chromatography, wherein the conversion rate reaches over 80 percent after 30 minutes, and separating and recovering the immobilized enzyme; the preparation method of the immobilized amino acid ester acyl transferase comprises the following steps: 1g of amino acid ester acyl transferase and 40g of epoxy group immobilized enzyme carrier sepabeads EC-EP113-M are sequentially added into 150mL of 100mM phosphate buffer solution with the pH value of 8.0 at the temperature of 20 ℃ and the rotating speed of 150rpm for reaction for 24 hours, and the immobilized enzyme is collected.
2. A method for preparing glutamine dipeptide by using immobilized amino acid ester acyl transferase as a biocatalyst is characterized in that: glutamine and L-alanine methyl ester hydrochloride are taken as substrates, the two substrates are stirred in a water phase solution, and the two substrates react under the catalysis of the immobilized amino acid ester acyl transferase to generate glutamine dipeptide, and the method specifically comprises the following steps: adding 17.25g of substrate L-alanine methyl ester hydrochloride, 8g of glutamine, 150mL of purified water and 10g of immobilized amino acid ester acyl transferase, stirring for reaction at 20 ℃ and 200rpm, controlling the pH value to 8.5 by using 6moL/L hydrochloric acid, monitoring the conversion rate of the reaction by using liquid chromatography, wherein the conversion rate reaches over 80 percent after 30 minutes, and separating and recovering the immobilized enzyme; the preparation method of the immobilized amino acid ester acyl transferase comprises the following steps: 1g of amino acid ester acyl transferase and 40g of activated amino immobilized enzyme carrier sepabeads EC-HA/M are sequentially added into 150mL of 100mM phosphate buffer solution with the pH value of 8.0, the temperature is 20 ℃, the rotation speed is 150rpm, the reaction is carried out for 24 hours, and the immobilized enzyme is collected.
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| CN201611211842.7A CN106480009B (en) | 2016-12-25 | 2016-12-25 | Immobilized amino acid ester acyl transferase and application thereof in preparation of glutamine dipeptide |
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| CN201611211842.7A CN106480009B (en) | 2016-12-25 | 2016-12-25 | Immobilized amino acid ester acyl transferase and application thereof in preparation of glutamine dipeptide |
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| CN106480009B true CN106480009B (en) | 2020-12-11 |
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| CN108728424B (en) * | 2018-06-26 | 2021-09-14 | 河北善泉生物科技有限公司 | Method for purifying immobilized alpha-amino acid lipid acyltransferase by one step |
| CN113817790B (en) * | 2021-10-18 | 2023-11-03 | 精晶药业股份有限公司 | Method for preparing alanyl glutamine by immobilized enzyme |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008126783A1 (en) * | 2007-04-06 | 2008-10-23 | Kyowa Hakko Bio Co., Ltd. | Method for producing dipeptide |
| CN105754983A (en) * | 2016-05-19 | 2016-07-13 | 河北周酶生物科技有限公司 | Immobilized enzyme for preparing Ezetimibe midbody and preparation method of immobilized enzyme |
| CN105925558A (en) * | 2016-05-17 | 2016-09-07 | 河北周酶生物科技有限公司 | Compound immobilized enzyme used for preparing statins and preparation method of compound immobilized enzyme |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008126783A1 (en) * | 2007-04-06 | 2008-10-23 | Kyowa Hakko Bio Co., Ltd. | Method for producing dipeptide |
| CN105925558A (en) * | 2016-05-17 | 2016-09-07 | 河北周酶生物科技有限公司 | Compound immobilized enzyme used for preparing statins and preparation method of compound immobilized enzyme |
| CN105754983A (en) * | 2016-05-19 | 2016-07-13 | 河北周酶生物科技有限公司 | Immobilized enzyme for preparing Ezetimibe midbody and preparation method of immobilized enzyme |
Non-Patent Citations (1)
| Title |
|---|
| 产α-氨基酸酯酰基转移酶重组大肠杆菌的构建及发酵优化;何艳春;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20151215;摘要，正文第26-28页 * |
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