Aminopeptidase APs8-A297V, aminopeptidase APs8-A599V and application thereof

Abstract

The invention discloses an aminopeptidase APs8-A297V, an aminopeptidase APs8-A599V, and an application of the aminopeptidase APs8-A297V and the aminopeptidase APs8-A599V in degradation of bitter peptides. The amino acid sequence of the aminopeptidase APs8-A297V is as shown in SEQ ID NO.5, and the amino acid sequence of the aminopeptidase APs8-A599V is as shown in SEQ ID NO.6. The invention further discloses a preparation method of the aminopeptidase. The aminopeptidase APs8-A297V and the aminopeptidase APs8-A599V are obtained through site-specific mutagenesis of the aminopeptidase APs8, optimization of substrate specificity and remarkable improvement of catalytic activity are achieved, good temperature stability, pH stability and excellent high-salt tolerance are shown, accurate hydrolysis can be achieved according to bitter peptide characteristics of different food source proteins, and the application prospect is wide. The bitter taste of the food is reduced to the maximum extent, the quality of the food is improved, and theoretical and practical support is provided for diversified application of the aminopeptidase in the food industry.

Description

Technical Field

[0001] This invention relates to aminopeptidase APs8-A297V, aminopeptidase APs8-A599V and their applications, and belongs to the field of functional enzyme technology. Background Technology

[0002] Aminopeptidases (APs, EC 3.4.11) are a class of exopeptidases that catalyze the cleavage of N-terminal amino acids in peptide chains. They are widely used in the food, pharmaceutical, and biotechnology fields, with one of their core applications being the degradation of bitter peptides produced during protein hydrolysis to improve food palatability. The amino acid composition of proteins from different food sources varies significantly. After treatment with endopeptidases, the types and quantities of hydrophobic amino acids (leucine, proline, alanine, phenylalanine, isoleucine, valine, etc.) exposed differ. Therefore, selecting the appropriate aminopeptidase for a specific protein hydrolysis process is crucial for improving food quality. Currently reported aminopeptidases mostly exhibit high hydrolytic activity only on leucine or proline substrates; this single substrate selectivity limits their widespread application in different protein degradation processes.

[0003] Semi-rational design is a method for targeted enzyme modification based on fundamental knowledge of enzyme sequences, structures, and catalytic mechanisms, combined with computer-aided design. It integrates the advantages of both rational and irrational design, achieving precise regulation of enzyme activity and substrate specificity through an iterative optimization process combining experiments and computation. This makes it a highly efficient method for functional enzyme modification. FireProt, a commonly used computer-aided tool in semi-rational design, can integrate multiple computational methods to predict potential mutation sites, providing scientific guidance for targeted enzyme modification. Summary of the Invention

[0004] In response to the above-mentioned prior art, the present invention provides two mutants of aminopeptidase APs8—aminopeptidase APs8-A297V and aminopeptidase APs8-A599V—and their applications in the degradation of bitter peptides.

[0005] This invention is achieved through the following technical solution: The aminopeptidase APs8-A297V has the amino acid sequence shown in SEQ ID NO.5.

[0006] The aminopeptidase APs8-A599V has the amino acid sequence shown in SEQ ID NO.6.

[0007] The application of aminopeptidase APs8-A297V and aminopeptidase APs8-A599V in the degradation of bitter peptides, and in the preparation of formulations that degrade bitter peptides.

[0008] Further, the hydrophobic amino acid exposed by the bitter peptide is any one or more of alanine, phenylalanine, leucine, methionine, proline, arginine, isoleucine, and valine. More specifically, for aminopeptidase APs8-A297V, the hydrophobic amino acid exposed by the bitter peptide is preferably alanine, leucine, arginine, or valine; for aminopeptidase APs8-A599V, the hydrophobic amino acid exposed by the bitter peptide is preferably alanine, leucine, or methionine.

[0009] This invention utilizes FireProt-assisted semi-rational design to perform site-directed mutagenesis on key sites of aminopeptidase APs8, resulting in two core mutants—APs8-A297V and APs8-A599V. Both mutants achieve substrate-specific optimization and significantly enhanced catalytic activity, exhibiting good temperature stability, pH stability, and excellent high-salt tolerance, making them particularly suitable for the production processes of high-salt fermented foods such as fish sauce and soy sauce.

[0010] The aminopeptidases APs8-A297V and APs8-A599V of this invention achieve substrate-specific directional regulation and significantly enhance catalytic activity. They can precisely hydrolyze the bitter peptides of proteins from different food sources, minimizing bitterness and improving food quality. This provides theoretical and practical support for the diversified application of aminopeptidases in the food industry.

[0011] This invention combines the exposure characteristics of different hydrophobic amino acids in food proteins under actual enzymatic hydrolysis conditions, and through semi-rational design, it has carried out targeted modification of aminopeptidase, optimized its substrate selectivity, and developed an aminopeptidase mutant with high hydrolytic activity for a variety of hydrophobic amino acids. This mutant can minimize the bitterness of food, improve product quality, and meet the diverse application needs of the food industry.

[0012] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0013] Figure 1 The results of the determination of the relative enzyme activity of each mutant are shown in the figure. APs(Sm)-8 represents aminopeptidase APs8.

[0014] Figure 2 The results of pH stability determination are as follows: APs8 represents aminopeptidase APs8, A297V represents aminopeptidase APs8-A297V, and A599V represents aminopeptidase APs8-A599V.

[0015] Figure 3 Results of temperature stability measurements.

[0016] Figure 4Results of NaCl stability determination. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0018] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0019] Example 1: Heterologous expression of aminopeptidase APs8 This invention has extracted a natural aminopeptidase, aminopeptidase APs8, from Stenotrophomonas maltophilia. Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0020] Specific primers APs8-F (nucleotide sequence shown in SEQ ID NO.3) and APs1-R (nucleotide sequence shown in SEQ ID NO.4) were designed to amplify the DNA fragment of aminopeptidase APs8 from the genomic DNA of Stenotrophomonas maltophilia. The fragment was then transformed into Escherichia coli BL21(DE3) using standard methods to construct the recombinant engineered strain. Transformants were inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 h for activation; then, 3% of the transformants were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 200 rpm until OD (open-circuit retrieval). 600 When the pH value reaches 0.6–0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a concentration of 0.4 mM, and induce culture at 16°C and 200 rpm for 16 h.

[0021] The bacterial cells were collected by centrifugation, resuspended in an appropriate amount of buffer, and the cells were disrupted by low-temperature sonication. Cell debris was removed by centrifugation, and the supernatant was the crude enzyme solution. The crude enzyme solution was loaded onto Ni-NTA resin, and impurities were washed with elution buffer containing 20 mM imidazole. The target protein was then eluted with lysis buffer (80 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl). The eluent was collected and concentrated by ultrafiltration through a 30 kDa ultrafiltration tube to obtain the pure enzyme solution.

[0022] Example 2: Determination of the specific activity of aminopeptidase APs8 The method for determining aminopeptidase activity was as follows: the enzyme solution was mixed with 50 mM Tris-HCl buffer at pH 8.0 and incubated at 37°C for 5 min; then, nine different chromogenic substrates (aminoacyl-p-nitroanilines) (Leu-pNA, Arg-pNA, Met-pNA, Ala-pNA, Ile-pNA, Val-pNA, Pro-pNA, Phe-pNA, and Glu-pNA, all at a concentration of 2 mM) were added, and the reaction was carried out at 37°C for 5 min; the reaction was terminated by adding glacial acetic acid; and the absorbance at 405 nm was measured using a microplate reader.

[0023] Enzyme activity is defined as the amount of enzyme required to catalyze the formation of 1 μmol of 4-nitroaniline per minute from a substrate at 37°C.

[0024] The specific activity of aminopeptidase APs8 is shown in Table 1. In the table, WT represents aminopeptidase APs8, A297 represents aminopeptidase APs8-A297V, and A599V represents aminopeptidase APs8-A599V.

[0025] Table 1 Results of specific activity determination

[0026] As shown in Table 1, aminopeptidase APs8 is effective for Pro- p NA exhibited the highest catalytic activity (0.56 U / mg, relative enzyme activity 100%), followed by Leu-pNA (0.36 U / mg, relative enzyme activity 64.29%). Besides hydrolyzing Pro and Leu, aminopeptidase APs8 also hydrolyzed Ala-pNA (0.28 U / mg, relative enzyme activity 50%) and Arg-pNA (0.11 U / mg, relative enzyme activity 19.64%). Furthermore, it also showed some catalytic activity towards Ile-pNA (0.06 U / mg, relative enzyme activity 10.71%), Met-pNA (0.04 U / mg, relative enzyme activity 7.14%), and Val-pNA (0.03 U / mg, relative enzyme activity 5.36%).

[0027] Example 3 Semi-rational design of aminopeptidase APs8 The results from Example 2 show that aminopeptidase APs8 exhibits catalytic activity against Leu, Ala, and Arg substrates, and can also hydrolyze hydrophobic amino acid substrates such as Ile and Val. However, the overall catalytic activity is still not ideal, and there is room for improvement in substrate selectivity. Therefore, this invention attempts to modify it through semi-rational design to obtain a mutant enzyme with better catalytic activity and broader substrate selectivity.

[0028] The specific process of the semi-rational design is as follows: A three-dimensional structural model of aminopeptidase APs8 was constructed using the SWISS-MODEL server. An aminopeptidase derived from *Vibrio refractoryta* was selected as the template, which showed 47.14% amino acid sequence identity with APs8, a GMQE score of 0.80, and a QMEAN Z-Score of -2.48, indicating reliable model quality. This structural model was submitted to the FireProt 2.0 server for mutation site prediction, and potential mutation sites were screened and identified. Thirteen potential mutation sites were obtained through screening: G544P, A620M, D199P, D467Y, T135M, A473G, E231Y, A297V, Q561L, N453W, S405M, V504L, and A599V.

[0029] For the 13 potential mutation sites identified through screening, corresponding overlapping primers were designed, and site-directed mutants were constructed by whole-plasmid PCR. Crude enzyme solutions of each mutant were obtained by expression using conventional methods. Using Ala-pNA as the detection substrate, the enzyme activities of aminopeptidase APs8 and the crude enzyme solutions of each mutant were measured (method as in Example 2). The relative enzyme activity of each mutant was calculated with the enzyme activity of the parent enzyme as 100%.

[0030] The relative enzyme activities of each mutant were measured as follows: Figure 1 As shown, the relative enzyme activities of mutants A297V and A599V are significantly increased compared to the parental enzyme, reaching up to 2.1 times. Therefore, mutants A297V and A599V will be used as the subjects for subsequent experimental verification.

[0031] The crude enzyme solutions of mutants A297V and A599V were purified (using the same method as in Example 1) to obtain pure enzyme solutions of mutants A297V and A599V. The specific activity of the pure enzyme solutions of mutants A297V and A599V was determined using the same method as in Example 2. The results of the specific activity determination are shown in Table 1.

[0032] As shown in Table 1, the specific enzyme activities of mutant A297V for Arg and Val substrates were significantly improved compared with WT. Specifically, the catalytic activities for Arg substrate (from 0.11 U / mg to 0.23 U / mg, which is 2.1 times that before the mutation) and Val substrate (from 0.03 U / mg to 0.07 U / mg, which is 2.33 times that before the mutation) were both significantly improved. The specific enzyme activities for Ala, Leu, Met and Ile substrates were all improved compared with WT, while the specific enzyme activity for Pro substrate was decreased compared with WT.

[0033] As shown in Table 1, the specific enzyme activities of mutant A599V for Met and Ala substrates were significantly improved compared to WT. Specifically, the catalytic activities for Met substrates (increased from 0.04 U / mg to 0.08 U / mg, which is twice that before the mutation) and Ala substrates (increased from 0.28 U / mg to 0.49 U / mg, which is 1.75 times that before the mutation) were both significantly improved. The specific enzyme activities for Ile substrates were also improved compared to WT, while the specific enzyme activities for Pro substrates were decreased compared to WT.

[0034] The above results indicate that mutants A297V and A599V exhibit superior catalytic activity and significantly improved substrate selectivity. Mutant A297V is formally named aminopeptidase APs8-A297V, and its amino acid sequence is shown in SEQ ID NO. 5. Mutant A599V is formally named aminopeptidase APs8-A599V, and its amino acid sequence is shown in SEQ ID NO. 6.

[0035] When heterologously expressing aminopeptidases APs8-A297V and APs8-A599V, the gene was cloned under the T7 promoter of the pET-28a(+) expression vector using a 6×histidine tag as the homologous sequence at both ends. The DNA fragment of the mutant APs8-A297V was amplified using overlapping primers APs8-A297V-F (nucleotide sequence as shown in SEQ ID NO.7) and APs8-A297V-R (nucleotide sequence as shown in SEQ ID NO.8), and the DNA fragment of the mutant APs8-A599V was amplified using overlapping primers APs8-A599V-F (nucleotide sequence as shown in SEQ ID NO.9) and APs8-A599V-R (nucleotide sequence as shown in SEQ ID NO.10).

[0036] Example 4 Enzymatic Characterization (1) Determination of pH stability Aminopeptidase APs8, Aminopeptidase APs8-A297V, and Aminopeptidase APs8-A599V were incubated in phosphate buffer at pH 7.0 at 35°C for different times (0 h, 2 h, 4 h, 6 h, 8 h, 10 h). The enzyme activity was measured using Ala-pNA as a substrate (method as in Example 2). The enzyme activity before incubation was taken as 100%, and the relative enzyme activity after different treatments was calculated.

[0037] The results of the pH stability test are as follows: Figure 2As shown in the figure. The results showed that aminopeptidase APs8 and aminopeptidase APs8-A297V could retain more than 80% of their enzyme activity after being incubated at pH 7.0 for 10 h. Aminopeptidase APs8-A297V exhibited extremely high pH stability (relative enzyme activity of 88.39%), and was adapted to synergistic effects with neutral endopeptidases in food processing.

[0038] (2) Determination of temperature stability Aminopeptidase APs8, Aminopeptidase APs8-A297V, and Aminopeptidase APs8-A599V were incubated at 40°C and pH 8.0 in Tris-HCl buffer for different times (0 h, 2 h, 4 h, 6 h, 8 h, 10 h). The enzyme activity was measured using Ala-pNA as a substrate (method as in Example 2). The enzyme activity before incubation was taken as 100%, and the relative enzyme activity after different treatments was calculated.

[0039] The results of the temperature stability test are as follows: Figure 3 As shown in the figure. The results showed that after 10 h of heat treatment, the residual enzyme activities of aminopeptidase APs8, aminopeptidase APs8-A297V, and aminopeptidase APs8-A599V were 82.90%, 89.80%, and 64.71%, respectively. Aminopeptidase APs8-A297V exhibited excellent temperature tolerance and was suitable for the conventional temperature conditions of food processing.

[0040] Example 5: Effect of NaCl concentration on enzyme activity Salt-tolerant aminopeptidases have great application potential in the production of high-salt fermented foods such as fish sauce and soy sauce. Therefore, the NaCl stability of aminopeptidase APs8, aminopeptidase APs8-A297V, and aminopeptidase APs8-A599V was determined.

[0041] Aminopeptidase APs8, Aminopeptidase APs8-A297V, and Aminopeptidase APs8-A599V were incubated at 37°C for 10 h in NaCl solutions of different concentrations (0%, 1%, 3%, 5%, 10%, g / ml). The enzyme activity was measured using Ala-pNA as a substrate (method as in Example 2). The enzyme activity before treatment was taken as 100%, and the relative enzyme activity under different conditions was calculated.

[0042] The results of the NaCl stability test are as follows: Figure 4As shown in the figure, aminopeptidases APs8, APs8-A297V, and APs8-A599V all exhibited good salt tolerance. As the NaCl concentration increased from 0% to 10%, enzyme activity was not significantly inhibited, and even increased at some concentrations. Therefore, aminopeptidases APs8-A297V and APs8-A599V can be directly applied to the degradation of bitter peptides in high-salt fermented foods without additional adjustment of the salt concentration in the processing system, demonstrating significant practical industrial application value.

[0043] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An aminopeptidase APs8-A297V, characterized by: The amino acid sequence is shown in SEQ ID NO.

5.

2. The use of the aminopeptidase APs8-A297V according to claim 1 in the degradation of bitter peptides, or in the preparation of formulations that degrade bitter peptides.

3. Use according to claim 2, characterized in that: The hydrophobic amino acids exposed by the bitter peptide are any one or more of alanine, phenylalanine, leucine, methionine, proline, arginine, isoleucine, and valine.

4. The application according to claim 3, characterized in that: The hydrophobic amino acids exposed by the bitter peptides are alanine, leucine, arginine, or valine.

5. An aminopeptidase APs8-A599V, characterized in that: The amino acid sequence is shown in SEQ ID NO.

6.

6. The use of the aminopeptidase APs8-A599V according to claim 5 in the degradation of bitter peptides, or in the preparation of formulations for degrading bitter peptides.

7. The application according to claim 6, characterized in that: The hydrophobic amino acids exposed by the bitter peptide are any one or more of alanine, phenylalanine, leucine, methionine, proline, arginine, isoleucine, and valine.

8. The application according to claim 7, characterized in that: The hydrophobic amino acids exposed by the bitter peptides are alanine, leucine, or methionine.

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