CN-121343952-B - Glutamyl transpeptidase mutant and application thereof

Abstract

The invention provides a glutamyl transpeptidase mutant and application thereof. Wherein the glutamyl transpeptidase mutant comprises (a) a protein mutated based on the wild-type glutamyl transpeptidase shown in SEQ ID NO:1, the mutation comprising a mutation at any one or more of H87, Q89, D190, T193, E197, M323, T412, Q430, E548 or S552, or (b) a protein having 70% or more homology with the amino acid sequence defined in (a) and having glutamyl transpeptidase activity. Can solve the problem of low activity of glutamyl transpeptidase under the acidic condition in the prior art, and is suitable for the technical field of microorganisms.

Inventors

• XIONG TAO
• HUANG QIANQIAN
• ZHANG YAN
• YU HUASHUN
• ZHENG XIANLIANG
• YU CHEN
• LI NA

Assignees

• 安琪酵母股份有限公司
• 安琪酶制剂(宜昌)有限公司

Dates

Publication Date

20260508

Application Date

20251222

Claims (11)

1. 1. A glutamyl transpeptidase mutant, characterized in that the glutamyl transpeptidase mutant is: A protein mutated on the basis of the wild-type glutamyl transpeptidase shown in SEQ ID No. 1, said mutation being any one of the amino acid mutations: h87Y, H87y+e548T, H y+t193k+e548T, H y+q89r+t193k+e548T, H y+q89r+t193k+q430e+e548T or h87y+q89r+t193k+q430e+e548t+s552R, wherein the pre-numerical letter represents the original amino acid and the post-numerical letter represents the mutated amino acid.
2. 2. A DNA molecule, characterized in that, the DNA molecule encodes the glutamyl transpeptidase mutant of claim 1.
3. 3. A recombinant plasmid is characterized in that, the recombinant plasmid contains the DNA molecule of claim 2.
4. 4. The recombinant plasmid of claim 3, wherein the recombinant plasmid comprises a pBE-SDNA plasmid.
5. 5. The recombinant plasmid according to claim 3 or 4, wherein the recombinant plasmid comprises apre promoter.
6. 6. A host cell comprising the DNA molecule of claim 2 or the recombinant plasmid of any one of claims 3 to 5.
7. 7. The host cell of claim 6, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell.
8. 8. The host cell of claim 7, wherein the prokaryotic cell comprises e.coli or bacillus subtilis.
9. 9. A method for producing a glutamyl transpeptidase mutant, comprising culturing the host cell of any one of claims 6 to 8, and obtaining the glutamyl transpeptidase mutant.
10. 10. A method for producing a gamma-glutamyl peptide, comprising catalyzing the reaction of gamma-glutamine with an amino acid substrate comprising a polypeptide or an amino acid using the glutamyl transpeptidase mutant of claim 1.
11. 11. The method according to claim 10, wherein the pH of the reaction system in the method is 4.5 to 10.

Description

Glutamyl transpeptidase mutant and application thereof Technical Field The invention relates to the technical field of microorganisms, in particular to a glutamyl transpeptidase mutant and application thereof. Background Glutamyl transpeptidase is a bifunctional enzyme widely existing in mammals and bacteria, and is capable of catalyzing the transfer of gamma-glutamyl molecules of gamma-glutamyl compounds to receptor molecules such as other amino acids, short peptides, etc. (transpeptidation reaction) or water molecules (hydrolysis reaction). The enzyme has wide application prospect in the field of food processing, can be used for catalyzing and synthesizing amino acid derivatives and prodrugs, such as theanine, glutathione and the like, and can also be used for improving the flavor of certain bitter amino acids. However, the use of glutamyl transpeptidase in the food industry still faces a number of challenges. Since most of the food processing is carried out under acidic conditions, the wild enzymes have low activity under acidic conditions, resulting in limited effectiveness. In addition, the existing transpeptidase can not meet the requirements of various food processing substrates and processing procedures in terms of substrate specificity, thermal stability and the like. These problems severely limit the range of applications of glutamyl transpeptidase in the food processing field, affecting its potential value in improving food quality and flavor. Disclosure of Invention The invention mainly aims to provide a glutamyl transpeptidase mutant, which aims to solve the problem of low activity of glutamyl transpeptidase under acidic conditions in the prior art. In order to achieve the above object, according to a first aspect of the present invention, there is provided a glutamyl transpeptidase mutant comprising (a) a protein mutated based on a wild-type glutamyl transpeptidase as shown in SEQ ID NO:1, said mutation comprising a mutation occurring at any one or more of the following positions H87, Q89, D190, T193, E197, M323, T412, Q430, E548 or S552, or (b) a protein having 70% or more homology to the amino acid sequence defined in (a) and having glutamyl transpeptidase activity. Further, in the above (a), the mutation is selected from any one or more of the following mutations, H87Y, Q89, R, D, S, T193K, E, 197R, M323I, T, Y, Q, 430, E, E T or S552R, wherein the pre-numeral letter represents an original amino acid and the post-numeral letter represents a mutated amino acid. Further, the above mutation includes any one of the following amino acid mutations ：H87Y、Q89R、D190S、T193K、E197R、M323I、T412Y、Q430E、E548T、S552R、H87Y+E548T、Q89R+T193K、Q430E+S552R、H87Y+T193K+E548T、Q89R+T193K+Q430E、H87Y+Q89R+T193K+E548T、H87Y+Q89R+T193K+Q430E+E548T、H87Y+Q89R+T193K+Q430E+E548T+S552R. In order to achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the above glutamyl transpeptidase mutant. In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid containing the above DNA molecule. Further, the recombinant plasmids include pBE-SDNA plasmids. Further, the sequence of the pBE-SDNA plasmid comprises the nucleic acid sequence shown in SEQ ID NO. 2. Further, the recombinant plasmid includes apre promoter. Further, apre includes the nucleic acid sequence shown in SEQ ID NO. 2. In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell containing the above DNA molecule or the above recombinant plasmid. Further, the host cell includes a prokaryotic cell or a eukaryotic cell. Further, the prokaryotic cell includes Escherichia coli or Bacillus subtilis. In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a method for producing a glutamyl transpeptidase mutant, the method comprising culturing the above-described host cell and obtaining the above-described glutamyl transpeptidase mutant. In order to achieve the above object, according to a sixth aspect of the present invention, there is provided a method for producing a gamma-glutamyl peptide, the method comprising catalyzing a reaction of gamma-glutamine with an amino acid substrate comprising a polypeptide or an amino acid using the glutamyl transpeptidase mutant described above to obtain the gamma-glutamyl peptide. Further, the pH of the reaction system in the above preparation method is 4.5-10. By applying the technical scheme of the invention, the activity of the wild type glutamyl transpeptidase can be improved by using the glutamyl transpeptidase mutant, and the gamma-glutamyl peptide can be prepared and obtained. Compared with wild enzyme, the glutamyl transpeptidase mutant has better activity under the acidic condition, higher efficiency for preparing the gamma-glutamyl peptide, is suitable for the acidic condition in