阅读说明：本技术 一种用于γ-D谷氨酰肽合成的酶及γ-D谷氨酰肽的合成方法 (Enzyme for synthesizing gamma-D glutamyl peptide and synthesis method of gamma-D glutamyl peptide ) 是由 杨娟 周威佃 曾晓房 白卫东 于 2019-10-12 设计创作，主要内容包括：本发明涉及小分子肽制备技术领域,具体公开了一种用于γ-D谷氨酰肽合成的酶及γ-D谷氨酰肽的合成方法。所述的用于γ-D谷氨酰肽合成的酶,其特征在于,通过如下方法制备得到,(1)将枯草芽孢杆菌中的谷氨酰胺酶基因片段转录到大肠杆菌中,然后将大肠杆菌接种到发酵培养基中培养,取发酵液经离心、过滤后取上清液得粗酶液；(2)在水中加入谷氨酰胺,然后调节pH值至碱性,再加入粗酶液,进行反应即得所述的用于γ-D谷氨酰肽合成的酶。本发明所述的用于γ-D谷氨酰肽合成的酶用于γ-D谷氨酰肽的合成过程中可以杜绝副产物的生成以及提高γ-D谷氨酰肽的合成产率,为γ-D谷氨酰肽的大规模工业化生产提供了一种新的方法。(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.)

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.

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.