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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- 102000057234 Acyl transferases Human genes 0.000 title claims abstract description 42
- 108700016155 Acyl transferases Proteins 0.000 title claims abstract description 42
- -1 amino acid ester Chemical class 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 title claims description 39
- 108010016626 Dipeptides Proteins 0.000 title claims description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 42
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 38
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 229940024606 amino acid Drugs 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 17
- 239000000758 substrate Substances 0.000 claims description 16
- IYUKFAFDFHZKPI-DFWYDOINSA-N methyl (2s)-2-aminopropanoate;hydrochloride Chemical compound Cl.COC(=O)[C@H](C)N IYUKFAFDFHZKPI-DFWYDOINSA-N 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 6
- 239000008055 phosphate buffer solution Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 5
- 238000006555 catalytic reaction Methods 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 239000008213 purified water Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 239000011942 biocatalyst Substances 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 125000003700 epoxy group Chemical group 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 8
- 239000002699 waste material Substances 0.000 abstract description 4
- 238000013461 design Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004886 process control Methods 0.000 abstract description 3
- 238000011165 process development Methods 0.000 abstract description 3
- 239000012429 reaction media Substances 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 2
- 238000004132 cross linking Methods 0.000 abstract description 2
- 238000011143 downstream manufacturing Methods 0.000 abstract description 2
- 230000003100 immobilizing effect Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 235000001014 amino acid Nutrition 0.000 description 27
- 239000007853 buffer solution Substances 0.000 description 7
- 239000000969 carrier Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- DWKPPFQULDPWHX-VKHMYHEASA-N l-alanyl ester Chemical compound COC(=O)[C@H](C)N DWKPPFQULDPWHX-VKHMYHEASA-N 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 3
- 239000008346 aqueous phase Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000016236 parenteral nutrition Nutrition 0.000 description 1
- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/104—Aminoacyltransferases (2.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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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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| 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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