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CN-120966803-B - Glutaminase mutant and application thereof

CN120966803BCN 120966803 BCN120966803 BCN 120966803BCN-120966803-B

Abstract

The application provides a glutaminase mutant and application thereof. Wherein the glutaminase mutant comprises (a) a protein which is subjected to amino acid mutation and has glutaminase activity function at least one site of S467, Y334, K400, N521, R337, E402 or D447 in the amino acid sequence shown in SEQ ID NO. 2, and (b) a protein which has more than 80% homology with the amino acid sequence defined in (a) and has glutaminase function. The glutaminase mutant with the amino acid sequence has higher specific enzyme activity when being subjected to enzyme catalytic reaction, and has low cost and strong market competitiveness when being used for commercial food processing.

Inventors

  • ZHANG YAN
  • Mao Zejing
  • XIAO MINGHUA
  • ZHENG XIANLIANG
  • He Yunlou
  • HU YUE
  • YU HUASHUN
  • WEI XUETUAN

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
20251020

Claims (6)

  1. 1. A glutaminase mutant, characterized in that the glutaminase mutant has a mutation in the amino acid sequence shown in SEQ ID No. 2, wherein the mutation comprises the following mutation: S467H+Y334A, S467H+R337A or s467h+d447A.
  2. 2. A DNA molecule, characterized in that, the DNA molecule encodes the glutaminase mutant of claim 1.
  3. 3. A recombinant plasmid, wherein said recombinant plasmid is linked to the DNA molecule of claim 2.
  4. 4. The recombinant plasmid of claim 3, wherein the recombinant plasmid comprises a bacillus expression vector pHY300PLK.
  5. 5. A host cell transformed with the recombinant plasmid of claim 3 or 4.
  6. 6. Use of the glutaminase mutant of claim 1 in food processing.

Description

Glutaminase mutant and application thereof Technical Field The invention relates to the technical field of fermentation, in particular to a glutaminase mutant and application thereof. Background Glutaminase is an enzyme capable of catalyzing the hydrolysis of L-glutamine to produce L-glutamic acid and ammonia. Glutamic acid plays an important role in the food industry and can increase the delicate flavor and nutrition of food. In recent years, glutaminase has been rapidly developed for use in foods, for example, in the production of soy sauce, by hydrolyzing glutamine in soy sauce to glutamic acid while forming more flavor peptides by transpeptidation, increasing the flavor of soy sauce, and reducing the formation of pyroglutamic acid from glutamine in soy sauce. At present, commercial glutaminase has realized large-scale production and application, but the production cost is still higher. In the prior art, the protein rational design and site-directed mutagenesis of glutaminase from different strains are also studied to improve the stability or salt tolerance of the glutaminase, but the specific enzyme rational design of glutaminase from bacillus amyloliquefaciens is not reported yet. In view of the excellent application effect of the glutaminase derived from the bacillus amyloliquefaciens, the development of the glutaminase with high specific activity and low production cost has important significance. Disclosure of Invention The invention mainly aims to provide a glutaminase mutant and application thereof, so as to solve the problem of lower enzyme activity of glutaminase in the prior art. In order to achieve the above object, according to a first aspect of the present invention, there is provided a glutaminase mutant comprising (a) a protein having an amino acid mutation at least one site of S467, Y334, K400, N521, R337, E402 or D447 in the amino acid sequence shown in SEQ ID NO. 2 and having a glutaminase activity function, and (b) a protein having 80% or more homology with the amino acid sequence defined in (a) and having a glutaminase function. Further, the amino acid mutations of (a) are each independently selected from S467H, Y334A, K400A, N521A, R337A, E402A, D447F or D447A, wherein the pre-numerical letter represents the original amino acid and the post-numerical letter represents the mutated amino acid. Further, in (c), a protein having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 99% or more homology to the amino acid sequence defined in (a) or (b) and having a glutaminase function. Further, the mutation of the glutaminase mutant includes any one of the amino acid mutations S467H, S467 H+Y330A, S467H+R337A, S467H+K400A, S467H+E402A, S467H+D447A, S467H+N521A, and D447F. In order to achieve the above object, according to a second aspect of the present invention, there is provided a DNA molecule encoding the glutaminase mutant described above. Further, the DNA molecule is selected from 1) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO.1, and 2) a polynucleotide having 80% or more, more preferably 90% or more, still more preferably 95% or more homology with the nucleotide sequence shown in SEQ ID NO. 1. In order to achieve the above object, according to a third aspect of the present invention, there is provided a recombinant plasmid having the above DNA molecule attached thereto. Further, the recombinant plasmid includes bacillus expression vector pHY300PLK. In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a host cell into which the above recombinant plasmid is transformed. In order to achieve the above object, according to a fifth aspect of the present invention, there is provided the use of the glutaminase mutant described above in food processing. The glutaminase mutant with the amino acid sequence has higher specific enzyme activity when being subjected to enzyme catalytic reaction, and has low cost and strong market competitiveness when being used for commercial food processing. Drawings The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings: FIG. 1 shows a schematic diagram of the structure of the Bacillus expression vector pHY300PLK in experimental materials according to examples in the description of the present application. Detailed Description It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples. As mentioned in the background, wild-type glutaminase has a relatively high cost for commercial use due to its relatively low catalytic efficiency. Therefore,