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CN-122012473-A - Self-cutting resistant high specific activity AspN enzyme mutant and encoding gene and application thereof

CN122012473ACN 122012473 ACN122012473 ACN 122012473ACN-122012473-A

Abstract

The invention relates to an AspN enzyme mutant with self-cleavage resistance and high specific activity, and a coding gene and application thereof. Aims at solving the problems of easy self-cutting inactivation and poor stability of wild type AspN enzyme. The mutant is obtained by mutating one or more aspartic acid sites in positions 94, 108, 132, 141, 182 and 184 on the basis of the amino acid sequence shown in SEQ ID NO. 1. Preferred mutations include D182N, D182A, D182V, D N, D132Q, D184E, D G, D108T, D141G and combinations of D182N and D132N. Compared with the wild type, the mutant has obviously improved specific activity and/or enzyme activity retention rate. The invention effectively improves the stability and the catalytic efficiency of the AspN enzyme, and has good application prospects in protein sequencing, polypeptide preparation and mass spectrometry analysis.

Inventors

  • WANG HONGBIN
  • LIN XUE
  • WANG YUYING
  • LU WANBO
  • LIU SHASHA
  • LU FUPING

Assignees

  • 天津科技大学

Dates

Publication Date
20260512
Application Date
20260202

Claims (10)

  1. 1. A self-cutting resistant AspN enzyme mutant with specific activity improvement is characterized in that the mutant is obtained by carrying out mutation at one or more aspartic acid sites selected from 94 th, 108 th, 132 th, 141 th, 182 th and 184 th sites on the basis of an amino acid sequence shown in SEQ ID NO. 1.
  2. 2. The AspN enzyme mutant according to claim 1, wherein the mutation is any one or more of D182N, D182A, D182V, D N, D132Q, D184E, D94G, D108T, D141G and a combination of D182N and D132N, the mutant having increased specific activity and/or retention of enzyme activity compared to the wild type.
  3. 3. Use of an AspN enzyme mutant according to any one of claims 1-2 in protein sequencing, polypeptide preparation or mass spectrometry.
  4. 4. A gene encoding the AspN enzyme mutant according to any one of claims 1-3.
  5. 5. The gene according to claim 4, wherein the nucleotide sequence of the gene is shown in any one of SEQ ID NO. 3 to SEQ ID NO. 12, wherein SEQ ID NO. 3 to SEQ ID NO. 11 correspond to the genes encoding single-point mutants D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D T, D G, respectively, and SEQ ID NO. 12 corresponds to the genes encoding combined mutants D182N and D132N.
  6. 6. A recombinant expression vector comprising the gene according to claim 4 or 5.
  7. 7. The recombinant expression vector of claim 6, wherein the vector is selected from the pET series, or wherein the vector is an additional expression vector suitable for use in prokaryotic expression systems that is replaceable with the pET series vector.
  8. 8. A recombinant strain comprising the recombinant expression vector of claim 6 or 7.
  9. 9. The recombinant strain according to claim 8, wherein the host cell is E.coli BL21 (DE 3) or the host cell is another prokaryotic host cell which is suitable for recombinant protein expression and which is exchangeable with E.coli BL21 (DE 3).
  10. 10. A method of preparing a mutant AspN enzyme according to any one of claims 1 to 3, comprising the steps of: ⑴ Constructing a gene for encoding the mutant into an expression vector, transforming the expression vector into a host cell to obtain a recombinant strain, fermenting and culturing the recombinant strain ⑵ to induce and express the AspN enzyme, collecting thalli by ⑶ and cracking the thalli to obtain a protein component containing the AspN enzyme mutant, and purifying and/or renaturating the protein component by ⑷ to obtain the AspN enzyme mutant.

Description

Self-cutting resistant high specific activity AspN enzyme mutant and encoding gene and application thereof Technical Field The invention relates to an AspN mutant of endopeptidase obtained by molecular transformation, a coding gene, a recombinant expression system and application thereof, in particular to an AspN mutant with self-cutting resistance and/or specific activity improvement, belonging to the technical fields of enzyme engineering and genetic engineering. Background The endopeptidase AspN (EC 3.4.24.33) is a zinc-dependent endoprotease, can specifically cut an N-terminal peptide bond of an aspartic acid (Asp) residue, and has important application value in the fields of proteomics analysis, mass spectrometry, polypeptide preparation and the like. The AspN enzyme not only can be used as effective supplement of trypsin, but also can be used in combination with Glu-C, lys-C, chymotrypsin and other proteases, so that the coverage rate of protein sequence identification is obviously improved. The commercial AspN enzyme is mainly derived from natural secretion of the stenotrophomonas maltophilia (Stenotrophomonas maltophilia), and the production process has the problems of long culture period, low yield, difficult purification, high cost and the like, so that the further application of the AspN enzyme in the fields of scientific research and industry is severely limited. In addition, aspN enzyme can recognize and cleave an exposed aspartic acid site in self molecules while playing a catalytic function, so that a self-cleavage reaction occurs, and the molecular structure of the enzyme is damaged, the activity is reduced and even the enzyme is completely inactivated. The self-cutting problem is particularly prominent in the processes of storing, transporting and using the enzyme, and is an important technical bottleneck for restricting the practical application of the AspN enzyme. Therefore, there is a need to rationally modify AspN enzyme by molecular engineering means to obtain novel AspN enzyme molecules with self-cleavage resistance and/or specific activity improvement so as to meet urgent requirements of high-performance AspN enzymes in the fields of protein analysis and polypeptide preparation. Disclosure of Invention The invention aims to provide an AspN enzyme mutant with obviously improved self-cutting resistance and/or specific activity, and a gene, a recombinant expression vector, a recombinant strain and a preparation method and application thereof for encoding the mutant, so as to solve the problems of easy self-cutting inactivation and poor use stability of the existing AspN enzyme. In order to achieve the purpose, the AspN enzyme from the stenotrophomonas maltophilia is taken as a research object, the amino acid sequence of the AspN enzyme is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The inventor selects aspartic acid residues at 94 th, 108 th, 132 th, 141 th, 182 th and 184 th positions as mutation targets by analyzing the amino acid composition and potential self-cutting related sites of the AspN enzyme, and replaces one or more of the above sites by a site-directed mutagenesis mode, so that the AspN enzyme mutant with improved self-cutting resistance and/or specific activity is obtained. In some embodiments, the AspN enzyme mutant is obtained by single point mutation of one of the aspartic acid residues at positions 94, 108, 132, 141, 182 and 184 based on the amino acid sequence shown in SEQ ID NO. 1. In a preferred embodiment, the mutations include, but are not limited to, D182N, D182A, D V, D132Q, D184E, D4994G, D108T and D141G. Experimental results show that compared with the wild type AspN enzyme, the mutant has improved initial specific activity and/or higher enzyme activity retention rate to different degrees, and the stability of the AspN enzyme in the use and storage processes is remarkably improved. In view of the positive effects exhibited by single point mutants, one skilled in the art would expect that combining the above effective mutation sites (e.g., a combination of D182N and D132N) would also result in mutants with improved performance, as demonstrated by a preferred embodiment MT10 (D182N/D132N) of the present invention. The invention also provides a gene for encoding the AspN enzyme mutant, the nucleotide sequences of which are shown in SEQ ID NO. 3-SEQ ID NO. 12, and the mutants MT 1-MT 10 are respectively corresponding. The invention further provides a recombinant expression vector containing the coding gene. The vector is selected from pET series, or other expression vector which is suitable for prokaryotic expression system and has replaceability with pET series vector. The invention also provides a recombinant strain containing the recombinant expression vector. The host cell is selected from Escherichia coli BL21 (DE 3), or the host cell is other prokaryotic host cell which is suitable for recombinant protein