Self-cutting-resistant and high-specific-activity AspN enzyme mutant as well as coding gene and application of self-cutting-resistant and high-specific-activity AspN enzyme mutant

Abstract

The invention relates to an anti-autotomy and high-specific-activity AspN enzyme mutant as well as a coding gene and application thereof. The invention aims to solve the problems that the wild type AspN enzyme is easy to self-dissect and inactivate and is poor in stability. The mutant is obtained by mutating one or more aspartic acid sites in the 94th site, the 108th site, the 132 site, the 141 site, the 182 site and the 184 site on the basis of an amino acid sequence shown in SEQ ID NO: 1. The preferred mutations include D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D108T, D141G, and a combination of D182N and D132N. Compared with a wild type, the specific activity and / or the enzyme activity retention rate of the mutant are / is remarkably improved. The stability and catalytic efficiency of the AspN enzyme are effectively improved, and the method has good application prospects in protein sequencing, polypeptide preparation and mass spectrometry.

Description

Technical Field

[0001] This invention relates to obtaining protein endopeptidase AspN mutants and their encoding genes, recombinant expression systems, and applications through molecular modification, specifically to an AspN enzyme mutant with anti-autolysis ability and / or increased specific activity, belonging to the fields of enzyme engineering and genetic engineering technology. Background Technology

[0002] AspN (EC 3.4.24.33) is a zinc-dependent metalloproteinase that specifically cleaves the N-terminal peptide bond of aspartic acid (Asp) residues, making it valuable in proteomics analysis, mass spectrometry sequencing, and peptide preparation. AspN can not only serve as an effective supplement to trypsin but can also be used in combination with various proteases such as Glu-C, Lys-C, and chymotrypsin to significantly improve the coverage of protein sequence identification.

[0003] Currently, commercially available AspN enzymes are mainly derived from Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia The natural secretion of ) has problems such as long cultivation cycle, low yield, difficult purification and high cost, which seriously limit its further application in scientific research and industrial fields.

[0004] Furthermore, while performing its catalytic function, AspN enzymes can recognize and cleave exposed aspartic acid sites in their own molecules, resulting in autocleavage. This autocleavage leads to structural damage, decreased activity, or even complete inactivation of the enzyme. This autocleavage problem is particularly prominent during enzyme storage, transportation, and use, and is a significant technical bottleneck restricting the practical application of AspN enzymes.

[0005] Therefore, it is urgent to rationally modify AspN enzymes through molecular engineering to obtain novel AspN enzyme molecules that possess both anti-autocleavage ability and / or improved specific activity, in order to meet the pressing need for high-performance AspN enzymes in the fields of protein analysis and peptide preparation. Summary of the Invention

[0006] The purpose of this invention is to provide an AspN enzyme mutant with significantly improved resistance to autolysis and / or specific activity, as well as the gene encoding the mutant, recombinant expression vector, recombinant strain, preparation method and application thereof, in order to solve the problems of easy autolysis inactivation and poor stability of existing AspN enzymes.

[0007] To achieve the above objectives, this invention focuses on AspN enzyme derived from Stenotrophomonas maltophilia, the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:2. By analyzing the amino acid composition and potential autocleavage-related sites of the AspN enzyme, the inventors selected aspartic acid residues at positions 94, 108, 132, 141, 182, and 184 as mutation targets. One or more of these sites were replaced using site-directed mutagenesis, thereby obtaining AspN enzyme mutants with increased anti-autocleavage ability and / or specific activity.

[0008] In some embodiments, the AspN enzyme mutant is obtained by a single-point mutation at 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.

[0009] In a preferred embodiment, the mutations include, but are not limited to: D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D108T, and D141G.

[0010] Experimental results show that, compared with wild-type AspN enzyme, the above mutants have varying degrees of improved initial specific activity and / or higher enzyme activity retention, significantly improving the stability of AspN enzyme during use and storage.

[0011] Given the positive effects exhibited by single-point mutants, those skilled in the art can anticipate that combining the aforementioned effective mutation sites (e.g., a combination of D182N and D132N) may also yield mutants with improved performance, as demonstrated by a preferred embodiment of the present invention, MT10 (D182N / D132N).

[0012] The present invention also provides a gene encoding the above-mentioned AspN enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO:3 to SEQ ID NO:12, corresponding to mutants MT1 to MT10 respectively.

[0013] The present invention further provides a recombinant expression vector containing the above-mentioned coding gene. The vector is selected from the pET series; or, the vector is another expression vector suitable for prokaryotic expression systems and interchangeable with the pET series vectors.

[0014] The present invention also provides a recombinant strain comprising the above-described recombinant expression vector. The host cell is selected from *Escherichia coli* BL21(DE3); or, the host cell is another prokaryotic host cell suitable for recombinant protein expression and replaceable with *Escherichia coli* BL21(DE3).

[0015] The present invention also provides a method for preparing the above-mentioned AspN enzyme mutant, comprising the following steps: (1) constructing the gene encoding the AspN enzyme mutant into an expression vector and transforming it into a host cell to obtain a recombinant strain; (2) culturing the recombinant strain and inducing expression of the AspN enzyme mutant; (3) collecting the bacterial cells and lysing them to obtain a protein component containing the AspN enzyme mutant; (4) purifying and / or refolding the protein component to obtain the AspN enzyme mutant.

[0016] This invention also provides the application of the above-mentioned AspN enzyme mutant in protein sequencing, peptide preparation and mass spectrometry analysis.

[0017] Beneficial effects of the present invention Compared with existing technologies, the AspN enzyme mutants provided by this invention significantly improve autolysis resistance and / or specific activity without affecting substrate specificity. Specifically, the specific activity of some mutants can be increased by up to 1.76 times that of wild-type AspN enzymes, and the autolysis resistance can be increased by up to 1.81 times that of wild-type AspN enzymes. This effectively alleviates the problem of easy autolysis inactivation of AspN enzymes during storage and use, and has good prospects for industrial application. Attached Figure Description

[0018] Figure 1 The initial specific activities of wild-type AspN enzyme and its single-point mutants MT1-MT10 were compared.

[0019] Figure 2 Comparison of enzyme activity retention rates of wild-type AspN enzyme and its single-point mutant MT1-MT10 after incubation at 37°C for 4 h. Detailed Implementation

[0020] To make the technical solution, technical effects, and implementation path of the present invention clearer, the present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only for explaining the present invention and should not be construed as limiting the scope of protection of the present invention. Without departing from the technical concept of the present invention, those skilled in the art can make various modifications or combinations to the above embodiments, and all such modifications or combinations should fall within the scope of protection of the present invention.

[0021] In this invention, the "specific activity" of AspN enzyme refers to the catalytic ability of a unit mass of enzyme protein on a specific substrate under standard reaction conditions; the "enzyme activity retention rate" refers to the ratio of residual enzyme activity to initial enzyme activity after incubation for a certain period of time under specific conditions, which is used to characterize the anti-autolysis performance of AspN enzyme.

[0022] The AspN enzyme mutant described in this invention can be recombinantly expressed using various prokaryotic or eukaryotic expression systems known in the art. The examples use a commonly used *E. coli* expression system as an example, with the pET-28a vector and BL21(DE3) host strain being merely exemplary choices. Those skilled in the art will understand that, based on similar expression principles, other commonly used expression vectors suitable for *E. coli* (such as other variants of the pET series, pBAD series, pCold series, etc.) and other host strains suitable for recombinant protein expression (such as other strains of the BL21 series, C41(DE3), C43(DE3), Rosetta series, Origami / Shuffle series, etc.) can also be used to implement this invention. Such substitutions of conventional expression elements fall within the scope of ordinary technical knowledge for those skilled in the art and are expected to achieve the objectives of this invention.

[0023] Example 1: Obtaining the AspN prozymogen gene and constructing wild-type engineered bacteria Based on the amino acid sequence of the AspN enzyme from Stenotrophomonas maltophilia in the Uniprot database (Entry number: B2FQP3, SEQ ID NO:1), codon optimization was performed on *E. coli* and a gene was synthesized (SEQ ID NO:2). This gene was inserted into plasmid pET-28a to obtain the recombinant plasmid pET-28a-AspN (wild-type). This plasmid was transformed into *E. coli* BL21(DE3) competent cells to obtain a strain expressing the wild-type AspN enzyme, which was then stored at -80°C.

[0024] Example 2: Site-directed mutagenesis of AspN enzyme Using the pET-28a-AspN plasmid constructed in Example 1 as a template, site-directed mutations were introduced at aspartic acid residues at positions 94, 108, 132, 141, 182, and 184 via reverse PCR. The PCR amplification products were digested with Dpn I enzyme, then verified by agarose gel electrophoresis and purified. The purified products were circularized using a seamless cloning enzyme to obtain single-point mutant plasmids, named pET-28a-MT1 to pET-28a-MT9, respectively. The ligation products were transformed into *E. coli* DH5α competent cells, and clones were selected for sequencing verification. The correctly sequenced mutant plasmids were extracted and transformed again into *E. coli* BL21(DE3) competent cells to obtain recombinant expression strains of each mutant, which were stored at -80°C. Based on the performance evaluation of the single-point mutants, multi-point mutants were further introduced. The amino acid mutation sites and their corresponding relationships for each mutant are shown in Table 1 below.

[0025] Table 1. Correspondence between AspN enzyme mutants and their mutation sites

[0026] Example 3: Expression and preparation method of AspN enzyme mutant (inclusion body implementation method) Escherichia coli BL21(DE3) containing the recombinant plasmid was inoculated into LB medium and cultured at 37°C with shaking until the OD600 reached 0.6–0.8. IPTG was then added for induction, with a final IPTG concentration of 0.1–1.0 mM, and the culture was continued for 2–8 h. After induction, the bacterial cells were collected by centrifugation, and the cells were lysed using sonication. Inclusion bodies containing the AspN enzyme were obtained by centrifugation.

[0027] The obtained inclusion bodies were added to 8 M urea solution or an equivalent denaturing agent (such as guanidine hydrochloride) at a mass-to-volume ratio of 1:10 for complete denaturation and dissolution. Then, a serial dilution method was used for renaturation, such as diluting the urea concentration stepwise from 8 M to 6 M to 4 M to 2 M to 1 M, with an interval of 30-120 min between each dilution, to finally obtain the renatured AspN enzyme mutant solution.

[0028] Example 4: Method for determining the specific activity of AspN enzyme Using the short peptide EEMQRRDPF as a substrate, hydrolysis was performed with AspN enzyme under standard reaction conditions. Enzyme activity was characterized by detecting the peak area of ​​the resulting DPF tripeptide using LC-MS. One unit of enzyme activity (U) was defined as the amount of reaction with a DPF peak area of ​​10000. Specific activity (U / mg) was defined as the enzyme activity corresponding to a unit mass of protein. This definition is only used for comparisons between different mutants in this invention.

[0029] Example 5: Method for determining the autoclast resistance of AspN enzyme AspN enzyme solution was treated with a solution containing 1 mM Ca 2+ The enzyme was diluted 10-fold with 50 mM ammonium bicarbonate buffer and incubated at 37°C for 4 h. After incubation, the residual enzyme activity was measured and compared with the initial enzyme activity to calculate the enzyme activity retention rate, which was used as an evaluation index of the autoclast resistance of AspN enzyme.

[0030] Example 6: Performance Comparison of AspN Enzyme Mutants Using the wild-type and mutant AspN enzymes prepared in Example 3 as samples, their initial specific activity and autoclast resistance were determined using the above method. The results are as follows: Figure 1 and Figure 2 As shown, compared with wild-type AspN enzymes, mutants MT1 to MT10 showed varying degrees of improvement in specific activity and / or anti-autolysis properties.

[0031] Initial specific vitality: such as Figure 1As shown, all mutants exhibited varying degrees of increased specific activity, with MT1 (D182N) and MT4 (D132N) showing the most significant increases, reaching 1.56-fold and 1.51-fold compared to the wild-type, respectively. Anti-autolysis ability (enzyme activity retention): After incubation at 37°C for 4 hours, the wild-type AspN enzyme retained 40.79% of its activity. Figure 2 As shown, most mutants exhibited improved enzyme activity retention compared to the wild type, indicating enhanced resistance to autolysis. MT4 (D132N), in particular, showed the most significant improvement in autolysis resistance, with an enzyme activity retention rate 1.55 times that of the wild type.

[0032] Among the single-point mutants, MT1 (D182N) and MT4 (D132N) showed the best results. Combining these two mutants to create the MT10 mutant showed even better performance improvement, with specific activity increased to 1.76 times that of wild type and enzyme activity retention rate increased to 1.81 times that of wild type.

[0033] Example 7: Application of AspN enzyme mutants in protein peptide mapping analysis Based on the AspN enzyme mutants (such as MT10) with high specific activity and anti-autocleavage ability provided by this invention, their application efficiency in proteomics analysis can be significantly improved. The following uses bovine serum albumin (BSA) peptide mapping analysis as an example to illustrate its application method and expected advantages: Sample preparation: Take the BSA solution to be tested and dilute it with 50 mM Tris-HCl buffer (pH 8.0) to a concentration of 1 mg / mL. Add CaCl2 to a final concentration of 1 mM.

[0034] Enzymatic hydrolysis: The AspN enzyme mutant of this invention was added to the above antibody solution at a mass ratio of enzyme to substrate of 1:50 (w / w). After mixing, the solution was incubated in a water bath at 37°C to carry out the enzymatic hydrolysis reaction.

[0035] Analysis of Reaction Advantages: Because the specific activity of the mutant strain of this invention is significantly higher than that of the wild type, under the same conditions, it requires less time or less enzyme to complete efficient enzymatic digestion, thereby increasing analytical throughput and reducing costs. Due to its resistance to autolysis, wild-type AspN enzymes are easily inactivated by autolysis during long incubation times or complex sample pretreatment processes, resulting in incomplete digestion, low peptide yield, and poor reproducibility. The mutant strain of this invention maintains high activity even after incubation at 37°C for 4 hours, thus providing more stable, complete, and reproducible digestion results, which is particularly beneficial for the analysis of low-abundance proteins or precious samples.

[0036] Peptide analysis: After the enzymatic digestion reaction is complete, formic acid can be added to terminate the reaction. The resulting peptide mixture can be analyzed directly or after desalting by liquid chromatography-tandem mass spectrometry (LC-MS / MS) for protein identification, sequence verification, or post-translational modification site analysis.

[0037] Conclusion: The AspN enzyme mutant provided by this invention, due to its improved core enzymatic properties, is expected to exhibit advantages over the wild-type enzyme in practical applications such as protein peptide mapping analysis, mass spectrometry sequencing, and peptide preparation, being faster, more stable, and more reliable.

Claims

1. An AspN enzyme mutant resistant to autoclastization and possessing increased specific activity, characterized in that, The mutant was obtained by mutating one or more aspartic acid sites selected from positions 94, 108, 132, 141, 182, and 184 based on the amino acid sequence shown in SEQ ID NO:

1.

2. The AspN enzyme mutant according to claim 1, characterized in that, The mutation is any one or more of the following: D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D108T, D141G, and a combination of D182N and D132N; the mutant has increased specific activity and / or enzyme activity retention compared to the wild type.

3. The application of the AspN enzyme mutant as described in any one of claims 1-2 in protein sequencing, peptide preparation or mass spectrometry analysis.

4. A gene encoding an AspN enzyme mutant as described in any one of claims 1-3.

5. The gene as described in claim 4, characterized in that, 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 coding genes of single-point mutants D182N, D182A, D182V, D132N, D132Q, D184E, D94G, D108T, and D141G, respectively, and SEQ ID NO:12 corresponds to the coding genes of combined mutants D182N and D132N.

6. A recombinant expression vector comprising the gene as described in claim 4 or 5.

7. The recombinant expression vector as described in claim 6, characterized in that, The vector is selected from the pET series; or, the vector is another expression vector that is suitable for prokaryotic expression systems and is replaceable with the pET series vectors.

8. A recombinant strain comprising the recombinant expression vector as described in claim 6 or 7.

9. The recombinant strain according to claim 8, characterized in that, The host cell is Escherichia coli BL21(DE3); or, the host cell is another prokaryotic host cell suitable for recombinant protein expression and replaceable with Escherichia coli BL21(DE3).

10. A method for preparing the AspN enzyme mutant according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Construct the gene encoding the mutant into an expression vector, transform it into a host cell, and obtain a recombinant strain; (2) Ferment and culture the recombinant strain to induce expression of AspN enzyme; (3) Collect the bacterial cells and lyse them to obtain a protein component containing the AspN enzyme mutant; (4) Purify and / or renature the protein component to obtain the AspN enzyme mutant.

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