CN110699343A - Enzyme for synthesizing gamma-D glutamyl peptide and synthesis method of gamma-D glutamyl peptide - Google Patents
Enzyme for synthesizing gamma-D glutamyl peptide and synthesis method of gamma-D glutamyl peptide Download PDFInfo
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Abstract
The invention relates to the technical field of preparation of small molecule peptides, and particularly discloses an enzyme for synthesizing gamma-D glutamyl peptide and a synthesis method of the gamma-D glutamyl peptide. The enzyme for synthesizing the gamma-D glutamyl peptide is characterized by being prepared by the following method, (1) transcribing a glutaminase gene fragment in bacillus subtilis into escherichia coli, then inoculating the escherichia coli into a fermentation culture medium for culture, centrifuging and filtering fermentation liquor, and taking supernate to obtain crude enzyme liquid; (2) adding glutamine into water, then adjusting the pH value to be alkaline, then adding crude enzyme liquid, and reacting to obtain the enzyme for synthesizing the gamma-D glutamyl peptide. When the enzyme for synthesizing the gamma-D glutamyl peptide is used in the synthesis process of the gamma-D glutamyl peptide, the generation of byproducts can be avoided, the synthesis yield of the gamma-D glutamyl peptide can be improved, and a novel method is provided for large-scale industrial production of the gamma-D glutamyl peptide.
Description
Technical Field
The invention relates to the technical field of preparation of small molecule peptides, in particular to an enzyme for synthesizing gamma-D glutamyl peptide and a method for synthesizing gamma-D glutamyl peptide.
Background
The gamma-D-glutamyl-L-tryptophan (SCV-07) is used as an immunoregulation peptide, has various physiological functions, such as being used as a clinical treatment medicament, can treat pulmonary tuberculosis and relieve oral mucositis of head and neck cancer patients in the process of radioactive chemotherapy; inhibiting the growth of various tumor cell lines such as leukemia, lymphoma, herpes simplex virus type 2, head and neck tumors, and the like; can stimulate T-lymphocyte division and specific immune response, and increase the production stimulation of interleukin 2 and interferon of mice.
Gamma-glutamyl peptides are widely present in a variety of animals, plants and microorganisms, and are involved in the metabolic activity and physiological function of the organism. Most of the gamma-glutamyl peptide has special physiological functions of calming, reducing pressure, relieving fatigue, resisting cancer, detoxifying, complexing heavy metal ions, maintaining the balance of metal ions in cells and the like. For example, gamma-Glu-Cys is used as a precursor substance of glutathione, and has various physiological activities similar to glutathione. For example, the pharmaceutical composition has an anti-inflammatory effect in acute pancreatitis, and the gamma-Glu-Cys and the gamma-Glu-D-Cys are injected intravenously to prevent reperfusion injury in the cerebral ischemic stroke medication process and stabilize cerebral vessels so as to prevent injury in the thrombolytic process. Gamma-Glu-Cys and gamma-Glu-Val are useful in the treatment of chronic inflammatory diseases such as Inflammatory Bowel Disease (IBD). It is known that gamma-glutamylation of drugs increases the affinity as well as the potency of the drug, e.g. the antioxidant capacity of Vasoactive Intestinal Peptide (VIP) is increased after gamma-glutamylation. Liraglutide is a glucagon peptide analogue, can inhibit DPP-4 enzyme activity, is used for treating type II diabetes, and is researched to find that the drug effect duration of Liraglutide is increased after gamma-glutamylation. The gamma-glutamyl compound can be used as a precursor of a medicament, and can release a corresponding medicament in vivo after the catalysis of GGT enzyme, for example, gamma-Glu-enkephalin can release enkephalin in vivo after entering the body under the catalysis of GGT enzyme. Amino acids with low solubility have increased solubility after gamma-glutamylation, for example, tyrosine can be replaced by gamma-Glu-Tyr generated after gamma-glutamylation of tyrosine to be used as Total Parenteral Nutrition (TPN) supplement.
At present, the preparation methods of gamma-glutamyl peptide mainly include extraction methods, chemical synthesis methods, synthesis methods of gamma-transglutaminase, and the like. However, the existing synthesis method has the problems of more byproducts and low reaction yield; therefore, large-scale industrial production of γ -glutamyl peptide is limited.
Disclosure of Invention
The primary technical problem to be solved by the present invention is to provide an enzyme for the synthesis of gamma-D glutamyl peptide; the enzyme is prepared by a brand new method, and can reduce the generation of byproducts in the synthesis of gamma-D glutamyl peptide.
The technical problem to be solved by the invention is realized by the following technical scheme:
an enzyme for gamma-D glutamyl peptide synthesis, which is prepared by the following method,
(1) transcribing a glutaminase gene fragment in bacillus subtilis into escherichia coli, inoculating the escherichia coli into a fermentation medium for culture, centrifuging and filtering fermentation liquor, and taking supernate to obtain crude enzyme liquid;
(2) adding glutamine into water, then adjusting the pH value to be alkaline, then adding crude enzyme liquid, and reacting to obtain the enzyme for synthesizing the gamma-D glutamyl peptide.
Preferably, the fermentation medium in the step (1) comprises the following components in percentage by weight: 3-5% of soybean meal, 1-3% of bran and 1-3% of corn flour.
Most preferably, the fermentation medium in step (1) comprises the following components in percentage by weight: 4% of soybean meal, 2% of bran and 2% of corn flour.
Preferably, the amount of the Escherichia coli inoculated in the step (1) is 0.5-2% of the total weight of the fermentation medium.
Most preferably, the amount of Escherichia coli inoculated in step (1) is 1% by weight based on the total weight of the fermentation medium.
Preferably, the culture in the step (1) is fermentation culture at 37-39 ℃ for 24-36 h.
Preferably, the dosage ratio of the glutamine, the crude enzyme liquid and the water in the step (2) is 80-120 mM: 3-5 g: 100 g.
Most preferably, the dosage ratio of the glutamine, the crude enzyme solution and the water in the step (2) is 100 mM: 4 g: 100 g.
Preferably, the reaction in the step (2) is carried out at 38-42 ℃ for 3-5 h.
The invention also provides a method for synthesizing gamma-D glutamyl peptide, which comprises the following steps:
mixing D-glutamine and L-amino acid, dissolving in water, adjusting pH value to alkalinity, adding enzyme for synthesizing gamma-D glutamyl peptide, and reacting to obtain the gamma-D glutamyl peptide.
Preferably, the ratio of the amount of D-glutamine, L-amino acid and water is 100 mM: 50-1000 mM: 100 g.
Most preferably, the ratio of the amount of D-glutamine, L-amino acid and water is 100 mM: 100 to 200 mM: 100 g.
Preferably, the weight ratio of the addition amount of the enzyme for synthesizing the gamma-D glutamyl peptide to the weight amount of water is 0.03-0.1: 100.
most preferably, the weight ratio of the addition amount of the enzyme for gamma-D glutamyl peptide synthesis to the amount of water is 0.05: 100.
preferably, the reaction is carried out at 30-55 ℃ for 1-12 hours.
Most preferably, the pH value is adjusted to be alkaline, namely the pH value is adjusted to be 8.0-11.0.
Preferably, the L-amino acid is selected from one or more of L-tryptophan, L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-arginine, L-glutamic acid, L-glycine, L-alanine, L-cysteine, L-methionine, L-lysine, L-proline, L-threonine, L-histidine, L-aspartic acid, L-asparagine and L-tyrosine.
Has the advantages that: the invention synthesizes a brand-new enzyme for synthesizing gamma-D glutamyl peptide by a brand-new method. When the enzyme is used in the synthesis process of the gamma-D glutamyl peptide, the generation of byproducts can be avoided, the synthesis yield of the gamma-D glutamyl peptide can be improved, and a new method is provided for large-scale industrial production of the gamma-D glutamyl peptide.
Drawings
FIG. 1 is a primary mass spectrum of gamma-D-glutamyl-L-tryptophan.
FIG. 2 is a secondary mass spectrum of gamma-D-glutamyl-L-tryptophan.
FIG. 3 is a NMR chart of γ -D-glutamyl-L-tryptophan.
FIG. 4 is a primary mass spectrum of the by-product γ -D-glutamyl- γ -D-glutamyl-L-tryptophan.
FIG. 5 is a secondary mass spectrum of gamma-D-Glu-L-Ile.
FIG. 6 is a secondary mass spectrum of gamma-D-Glu-L-Val.
FIG. 7 is a secondary mass spectrum of γ -D-Glu-L-Tyr.
FIG. 8 is the second-order mass spectrum of gamma-D-Glu-L-Leu.
FIG. 9 is a secondary mass spectrum of γ -D-Glu-L-Phe.
FIG. 10 is a secondary mass spectrum of γ -D-Glu-L-Met.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
In the examples, the reaction products were qualitatively analyzed by UPLC-MS/MS: the method comprises the following steps: agilent1290series UPLC system (Agilent Technologies) was used to separate the individual gamma-D-glutamyl-L-peptides, and qualitative analysis was carried out using a mass spectrometry system with ESI (Q-TOF MS/MS, Bruker Daltonics). The chromatographic column is as follows: a Agilent ZORBAX RRHD SB-C18(2.1X 50mm,1.8 μm), the sample size was 5 μ L. The liquid phase conditions were: solution A: 0.1% formic acid-water, and solution B: 0.1% formic acid-acetonitrile, gradient elution procedure: 0-10% of B,0-5.0 min; 10-15% of B,5.0-10.0 min; then 100% B,10.0-12.0 min. MS conditions: ionization: a positive ion mode; drying gas: 10L/min at 350 ℃; nebulizer (Nebulizer pressure): 25 psig; fragment (capillary voltage): 30V.
The reaction products were quantitatively analyzed by HPLC in the examples: and (3) analyzing the column: XSelect HST 35 μm4.6x 250 mm; mobile phase: solution A: 0.1% (V/V) formic acid-water solution; and B, liquid B: 0.1% (V/V) formic acid-acetonitrile solution; column temperature: 40 ℃, flow rate: 1 mL/min. The amount of the sample was 10. mu.L. The mobile phase was subjected to gradient change as shown in table 1.
TABLE 1 gradient conditions for mobile phase
|
Time/ |
0 | 5 | 10 | 15 | 20 |
| A/％ | 90 | 85 | 20 | 90 | 90 |
EXAMPLE 1 preparation of enzyme for Gamma-D glutamyl peptide Synthesis
(1) Transcribing a glutaminase gene fragment in bacillus subtilis into escherichia coli, inoculating the escherichia coli into a fermentation medium, fermenting and culturing for 32 hours at 37-39 ℃, centrifuging a fermentation liquid at a high speed for 10 minutes under the conditions of 4 ℃ and 10000r/min, and filtering to obtain a supernatant to obtain a crude enzyme liquid;
the fermentation medium comprises the following components in percentage by weight: 4% of soybean meal, 2% of bran, 2% of corn flour and 92% of water; fermenting and culturing for 30 hours at 37-39 ℃. Centrifuging the fermentation liquor at 4 deg.C and 10000r/min for 10min, and filtering to obtain supernatant, i.e. crude glutaminase solution. The amount of the Escherichia coli inoculated was 1% of the total weight of the fermentation medium.
(2) 100mM of glutamine is added into 100g of water, then the pH value is adjusted to 10.0, 4g of crude enzyme liquid is added, and the reaction is carried out for 4 hours at 40 ℃ to obtain the enzyme for synthesizing the gamma-D glutamyl peptide.
Example 2 Synthesis of gamma-D-glutamyl-L-tryptophan
Mixing D-glutamine (100 mM) and L-amino acid (100 mM), dissolving in 100g of water, adjusting pH to 10.0, adding 0.05g of the enzyme for synthesizing gamma-D-glutamyl peptide prepared in example 1, and reacting at 37 ℃ for 3 hours to obtain the gamma-D-glutamyl-L-tryptophan. As can be seen from the primary mass spectrum of the product in FIG. 1, the secondary mass spectrum of the product in FIG. 2, and the NMR chart of the product in FIG. 3, γ -D-glutamyl-L-tryptophan was successfully synthesized in this example. As a result, all the products prepared in this example were γ -D-glutamyl-L-tryptophan, and no by-product was produced, the yield was 88.76mM, and the yield was 88.76% relative to L-tryptophan.
Therefore, the gamma-D glutamyl peptide synthesized enzyme prepared by the invention has no by-product and high yield; the reaction condition is mild, and the reaction can be finished after 3 hours at 37 ℃; therefore, the method can be used for large-scale industrial production of the gamma-D glutamyl peptide.
Comparative example 1 Synthesis of gamma-D-glutamyl-L-tryptophan
D-glutamine (100 mM) and L-amino acid (100 mM) were mixed and dissolved in 100g of water, the pH was adjusted to 10.0, and 0.05g of the crude enzyme solution (prepared in step (1) of example 1) was added and reacted at 37 ℃ for 3 hours to obtain a product. The product contains a large amount of byproduct gamma-D-glutamyl-L-tryptophan besides gamma-D-glutamyl-L-tryptophan by analysis; the primary mass spectrum of the byproduct gamma-D-glutamyl-L-tryptophan is shown in FIG. 4. The yield of gamma-D-glutamyl-L-tryptophan obtained by this comparative preparation was 33.75mM and the yield thereof was 33.75% relative to L-tryptophan by analysis; the yield of the by-product, γ -D-glutamyl- γ -D-glutamyl-L-tryptophan, was 19.57mM, and the yield thereof was 19.57% relative to L-tryptophan.
As can be seen from comparative example 1, comparative example 1 used the crude enzyme solution of step (1) of example 1 in the synthesis of γ -D-glutamyl-L-tryptophan; as a result, it was confirmed that the yield was greatly reduced from that of gamma-D-glutamyl-L-tryptophan prepared in example 1, and a large amount of by-product gamma-D-glutamyl-L-tryptophan was produced. This indicates that the crude enzyme solution obtained in step (1) of example 1 must be subjected to step (2) of reaction with glutamine to obtain an enzyme for synthesizing γ -D-glutamyl-L-tryptophan with high efficiency and no by-product.
Comparative example 2 Synthesis of gamma-D-glutamyl-L-tryptophan
Mixing D-glutamine (100 mM) and L-amino acid (100 mM), dissolving in 100g of water, adjusting pH to 10.0, adding glutaminase (100 GTU/g activity, manufactured by Amano of Japan) and reacting at 37 deg.C for 3 hr to obtain the product.
The product contains a large amount of by-product gamma-D-glutamyl-L-tryptophan with the structure shown in the primary mass spectrum of FIG. 4 besides gamma-D-glutamyl-L-tryptophan. The yield of gamma-D-glutamyl-L-tryptophan obtained by this comparative preparation was 58.54mM according to analysis, and the yield relative to L-tryptophan was 58.54%; the yield of the by-product, γ -D-glutamyl- γ -D-glutamyl-L-tryptophan, was 11.32mM, and the yield thereof was 11.32% relative to L-tryptophan.
As can be seen from comparative example 2, the use of a conventional glutaminase for the synthesis of gamma-D-glutamyl-L-tryptophan also results in the presence of the by-product gamma-D-glutamyl-L-tryptophan; and the yield is only 58.54%; much less than the yield of the enzyme used in example 1 for the synthesis of gamma-D glutamyl peptide. Therefore, compared with the prior art, the enzyme for synthesizing the gamma-D glutamyl peptide can greatly improve the yield, eliminate the generation of byproducts and reduce the problem of the post-treatment of the byproducts.
EXAMPLE 3 Synthesis of other gamma-D glutamyl peptides
100mM of D-glutamine and 100mM of different L-amino acids (L-Leu, L-Ile, L-Val, L-Met, L-Phe and L-Tyr) are mixed and dissolved in 100g of water, then the pH value is adjusted to 10.0, 0.05g of the enzyme for synthesizing gamma-D glutamyl peptide prepared in the embodiment 1 is added to react for 3 hours at 37 ℃, and then the gamma-D-Glu-L-Ile, the gamma-D-Glu-L-Val, the gamma-D-Glu-L-Tyr, the gamma-D-Glu-L-Leu, the gamma-D-Glu-L-Phe and the gamma-D-Glu-L-Met are obtained. Secondary mass spectrograms of gamma-D-Glu-L-Ile, gamma-D-Glu-L-Val, gamma-D-Glu-L-Tyr, gamma-D-Glu-L-Leu, gamma-D-Glu-L-Phe and gamma-D-Glu-L-Met are respectively shown in figures 5-10.
Example 3 experiments show that the enzyme for gamma-D glutamyl peptide synthesis prepared in example 1 can synthesize other gamma-D glutamyl peptides in addition to gamma-D-glutamyl-L-tryptophan efficiently.
Claims (10)
1. An enzyme for gamma-D glutamyl peptide synthesis, which is prepared by the following method,
(1) transcribing a glutaminase gene fragment in bacillus subtilis into escherichia coli, inoculating the escherichia coli into a fermentation medium for culture, centrifuging and filtering fermentation liquor, and taking supernate to obtain crude enzyme liquid;
(2) adding glutamine into water, then adjusting the pH value to be alkaline, then adding crude enzyme liquid, and reacting to obtain the enzyme for synthesizing the gamma-D glutamyl peptide.
2. The enzyme for gamma-D glutamyl peptide synthesis according to claim 1, wherein said culturing in step (1) is fermentation culturing at 37-39 ℃ for 24-36 h.
3. The enzyme for γ -D glutamyl peptide synthesis according to claim 1, wherein the dosage ratio of glutamine, crude enzyme solution and water in step (2) is 80-120 mM: 3-5 g: 100g of the total weight of the mixture; most preferably, the dosage ratio of the glutamine, the crude enzyme solution and the water in the step (2) is 100 mM: 4 g: 100 g.
4. The enzyme for gamma-D glutamyl peptide synthesis according to claim 1, wherein said reaction in step (2) is carried out at 38-42 ℃ for 3-5 h.
5. A method for synthesizing gamma-D glutamyl peptide, which is characterized in that the method comprises the following steps:
mixing D-glutamine and L-amino acid, dissolving in water, adjusting the pH value to be alkaline, adding the enzyme for synthesizing the gamma-D glutamyl peptide according to any one of claims 1 to 6, and reacting to obtain the gamma-D glutamyl peptide.
6. The method for synthesizing γ -D glutamyl peptide according to claim 5, wherein the ratio of the amount of D-glutamine, L-amino acid and water is 100 mM: 50-1000 mM: 100g of the total weight of the mixture; most preferably, the ratio of the amount of D-glutamine, L-amino acid and water is 100 mM: 100 to 200 mM: 100 g.
7. The method for synthesizing γ -D glutamyl peptide according to claim 5, wherein the ratio of the amount of the enzyme for γ -D glutamyl peptide synthesis according to any of claims 1 to 4 to the amount of water is 0.03 to 0.1: 100, respectively; most preferably, the weight ratio of the addition amount of the enzyme for gamma-D glutamyl peptide synthesis according to any one of claims 1 to 6 to water is 0.05: 100.
8. the method for synthesizing gamma-D glutamyl peptide according to claim 5, wherein said reaction is carried out at 30-55 ℃ for 1-12 hours.
9. The method for synthesizing gamma-D glutamyl peptide according to claim 5, wherein said adjusting pH to alkaline is adjusting pH to 8.0-11.0.
10. The method for synthesizing gamma-D glutamyl peptide according to claim 5, wherein said L-amino acid is selected from one or more of L-tryptophan, L-valine, L-leucine, L-isoleucine, L-phenylalanine, L-tryptophan, L-arginine, L-glutamic acid, L-glycine, L-alanine, L-cysteine, L-methionine, L-lysine, L-proline, L-threonine, L-histidine, L-aspartic acid, L-asparagine, and L-tyrosine.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1394953A (en) * | 2001-06-21 | 2003-02-05 | 龟甲万株式会社 | Glutaminase, glutaminase gene, new recombination DNA and method for producing glutaminase |
| CN1421527A (en) * | 2001-11-30 | 2003-06-04 | 味之素株式会社 | Noval mutation glutamine synthelase and process for producing amino acid |
| CN105219747A (en) * | 2015-11-11 | 2016-01-06 | 南京师范大学 | A kind of preparation method of gamma glutamyl transpeptidase |
| CN106544381A (en) * | 2016-10-28 | 2017-03-29 | 华南理工大学 | The preparation method and application of γ glutamy dipeptides |
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2019
- 2019-10-12 CN CN201910966725.9A patent/CN110699343A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1394953A (en) * | 2001-06-21 | 2003-02-05 | 龟甲万株式会社 | Glutaminase, glutaminase gene, new recombination DNA and method for producing glutaminase |
| CN1421527A (en) * | 2001-11-30 | 2003-06-04 | 味之素株式会社 | Noval mutation glutamine synthelase and process for producing amino acid |
| CN105219747A (en) * | 2015-11-11 | 2016-01-06 | 南京师范大学 | A kind of preparation method of gamma glutamyl transpeptidase |
| CN106544381A (en) * | 2016-10-28 | 2017-03-29 | 华南理工大学 | The preparation method and application of γ glutamy dipeptides |
Non-Patent Citations (2)
| Title |
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| KATSUNORI KOBAYASHI 等: "Molecular cloning of the transglutaminase gene fromBacillus subtilis and its expression in Escherichia coli.", 《BIOSCI.BIOTECHNOL.BIOCHEM》 * |
| 李秀星 等: "枯草芽孢杆菌谷氨酰胺转胺酶基因的克隆及在大肠杆菌中的表达", 《食品研究与开发》 * |
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