One-pot method for preparing gamma-glutamy small peptides compounds

Abstract

The invention discloses a one-pot method for preparing gamma-glutamy small peptides compounds. The method comprises the following steps of carrying out water phase peptide reaction on 5-carboxyl ethyl hydantoin and amino acid so as to form 5-carboxyl ethyl hydantoin small peptide compounds; after reaction, directly adding thallus containing both hydantoinase and carbamoyl enzyme without extraction and separation, and carrying out enzymatic hydrolysis ring opening reaction by taking the thallus as a catalyst so as to obtain the gamma-glutamy small peptides compounds. According to the method, a hydantoin ring is adopted for protecting both the alpha-amino and the alpha-carboxyl of glutamic acid, and only the gamma-carboxyl is exposed, so that the position specificity of peptide reaction is guaranteed; by adopting water phase peptides, the medium product does not need to be extracted and separated and can be directly used for enzyme reaction of the next step, in the 'one-pot method' reaction process, the reaction total recovery can be effectively improved, and the water phase reaction avoids environment contamination caused by an organic solvent; and the enzymatic hydrolysis ring opening reaction avoids repeated protection and deprotection steps in most of the chemical synthesis reactions and reduces side reactions.

Description

technical field [0001] The invention relates to a method for preparing gamma-glutamyl small peptide compounds by adopting a one-pot method. Background technique [0002] [0003] γ-glutamyl small peptide compound (1) is a small peptide compound (generally not more than two amino acids). Because of the special γ-peptide bond structure, γ-glutamyl small peptide compounds are not easy to be hydrolyzed by general proteases in organisms, so these compounds are widely involved in some important metabolic processes in organisms. As an important class of amino acid derivative compounds, γ-glutamyl small peptide compounds have been widely used in food, medicine, cosmetics, feed additives and other fields. Recent studies have shown that the synthetic precursor of glutathione, γ-L-glutamyl-L-cysteine, has similar physiological functions to glutathione, and can effectively achieve transmembrane transport. In terms of the level of intracellular glutathione, its function is better t...

AI Technical Summary

Problems solved by technology

The extraction method is a classic production method, which mainly uses animal and plant tissues and yeasts containing γ-glutamyl small peptide compounds as raw materials, and then separates and refines them by adding appropriate solvents or combining amylase and protease treatment, but Due to the low content of γ-glutamyl small peptide compounds in raw materials, it needs to consume a lot of raw materials and solvents, so it is rarely used

Fermentation production is to select (construct) microorganisms with strong synthesis ability of γ-glutamyl small peptides and high content of intracellular products, screen and optimize culture formula, establish and optimize fermentation control strategy, improve and enhance downstream process technology etc., finally improve the yield and quality of the product. The main problems of the fermentation method are low conversion efficiency, long fermentation cycle, too low product content requires complex separation process, large equipment investment, high production cost, and product purity and yield Relatively low, thus limiting the further promotion of the law

The chemical synthesis method usually needs to protect the α-amino group and α-carboxyl group of glutamic acid step by step, and only the γ-carboxyl group is freed for peptide grafting reaction. The protective group of the α-carboxyl group is removed, so that in the process of repeated protection and deprotection, it is easy to cause peptide bond breakage, racemization, etc., resulting in reduced yield and reduced optical purity

Suzuki et al. used γ-glutamyl transpeptidase to conduct a more detailed study on the enzymatic synthesis of γ-glutamyl small peptide compounds, and prepared γ-L-glutamyl-L-histidine, γ -L-glutamyl-L-tyrosine methyl ester, γ-L-glutamyl-L-phenylalanine and γ-D-glutamyl-L-tryptophan and a series of γ-glutamine Amino Acids (Suzuki H, Yamada C, Kato K.γ-Glutamyl Compounds and Their Enzymatic Production Using Bacterialγ-Glutamyltranspeptidase. Amino Acids, 2007, 32:333~340.), but due to γ-glutamyl transpeptidase The enzyme has both transpeptide and hydrolysis activities. During the reaction process, it is often accompanied by side reactions such as autorotation peptide and product hydrolysis, which makes the reaction process difficult to control and seriously affects the reaction efficiency.

Our previous research (Cao Fei, Zhou Jiadong, Wang Yuexia, Zhang Furong, Huang Rongxing, Wei Ping. A method for enzymatically preparing glutathione from hydantoin. CN: ZL200910213531.8, 2009-11-05.; Cao Fei , Wei Ping, Zhou Jiadong, Wang Yuexia, Wu Hongli, Huang Rongxing, Zhang Furong. A method for enzymatically preparing optically active theanine. CN: ZL200910183579.9, 2009-09-23.), the hydantoin enzymatic method was applied to The synthesis of γ-glutamyl compounds such as glutathione and theanine also has the following two problems: on the one hand, when carrying out the γ-glutamyl peptide reaction, the organic phase peptide reagent is used. It is carried out in an organic solvent, and the organic solvent is not friendly to the environment and is harmful to the human body. At the same time, the organic solvent generally has a high boiling point, and requires a higher price when the product is separated; on the other hand, the product of the peptide reaction needs to be separated from the organic solvent. Separation and extraction from the medium, and then added to the water phase for enzyme-catalyzed hydrolysis ring-opening reaction, the extraction process will cause part of the product loss, resulting in a decrease in the overall yield

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