An aminopeptidase s derived from streptococcus thermophilus and its preparation method and application

Abstract

The application discloses an aminopeptidase S derived from Streptococcus thermophilus and a preparation method and application thereof, relates to the technical field of genetically engineered recombinant protease, and has the technical points that the aminopeptidase is an aminopeptidase S (PepS) derived from Streptococcus thermophilus, is a proteolytic enzyme, and the amino acid sequence of the PepS is shown in SEQ ID NO:1. The application provides a new PepS coding gene and realizes secretory expression of the PepS in Pichia pastoris for the first time, and provides a separation and preparation method of the PepS. The application also provides biological characteristics of the PepS and potential application value of the PepS.

Description

Technical Field

[0001] This invention relates to the field of recombinant protease technology, specifically to an aminopeptidase S derived from Streptococcus thermophilus, its preparation method, and its application. Background Technology

[0002] Aminopeptidases are a class of exopeptidases that selectively cleave amino acid residues from the N-terminus of proteins or peptide chains. They generally have a broad range of applications and were among the earliest discovered enzymes. Based on their specificity in hydrolyzing N-terminal amino acid residues, aminopeptidases are divided into two main categories: one type exhibits strict specificity for N-terminal amino acid residues, specifically hydrolyzing only one or a few amino acid residues, such as aspartic acid and glutamate, which can only be hydrolyzed by PepA; this type includes PepP (hydrolyzing terminal proline residues), PepX (hydrolyzing peptides with a proline residue at the second terminal position), and PepI (hydrolyzing terminal proline imino groups). The other type exhibits weak specificity for N-terminal amino acid residues, hydrolyzing almost all amino acid residues, such as lysine aminopeptidase (PepN), leucine aminopeptidase (LAP), and phenylalanine aminopeptidase (PepM). The enzymatic hydrolysis products of aminopeptidase are small peptides and free amino acids. Since small peptides and free amino acids are important nutrients and flavorings in food, aminopeptidase has received widespread attention in the food seasoning industry.

[0003] Lactic acid bacteria (LAB) is a general term for a class of heterologous Gram-positive bacteria, all characterized by the fermentation of carbohydrates to form lactic acid as the final product. Most LABs are auxotrophic amino acids, but they can grow well on complex protein media, indicating a robust proteolytic system. Therefore, LABs are a rich resource of proteolytic enzymes. *Streptococcus thermophilus* is an important member of the LAB family, widely used in the dairy industry, such as in the production of yogurt and hard cheese. Compared to other LAB members, *Streptococcus thermophilus* has a more abundant proteolytic system, and its aminopeptidase S (Pep S) is unique to *Streptococcus thermophilus*, suggesting unique proteolytic characteristics and application value. Currently, there are no reports on the preparation of this enzyme using recombinant DNA technology, the study of the biological characteristics of the recombinant enzyme, or any guidance for its application.

[0004] Therefore, the present invention aims to provide a PepS derived from Streptococcus thermophilus, its preparation method, biological characteristics, and potential applications to solve the above-mentioned problems. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide an aminopeptidase S derived from Streptococcus thermophilus, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: The present invention provides a PepS, wherein the PepS is a protease, and the amino acid sequence of the PepS is shown in SEQ ID NO: 1.

[0007] The present invention also provides a nucleotide fragment for encoding the PepS.

[0008] Furthermore, the sequence of the nucleotide fragment is shown in SEQ ID NO: 2.

[0009] The present invention also provides a recombinant expression vector, wherein the expression vector is an expression vector carrying the nucleotide fragment described above. It is constructed by inserting the nucleotide fragment encoding the above-mentioned PepS into the vector.

[0010] Furthermore, the recombinant expression vector is the Pichia pastoris expression vector pHB905M-PepS.

[0011] This invention also provides a method for expressing PepS, which involves transforming the aforementioned recombinant expression vector into a host cell, performing high-density fermentation, and inducing PepS expression with an inducer. Further, the host cell is Pichia pastoris GS115.

[0012] This invention also provides a one-step method for separating and purifying PepS, namely, immobilized Ni 2+ Affinity chromatography yielded PepS purity exceeding 95%.

[0013] This invention also provides the aforementioned biological characteristics of PepS. Specifically, this enzyme is a weakly specific aminopeptidase, hydrolyzing peptides with N-terminal basic amino acids (Arg), aromatic amino acids (Trp, Tyr, Phe), and hydrophobic amino acids (Leu), but not acidic amino acids (Asp, Glu). This enzyme can be inhibited by most divalent cation and metalloproteinase inhibitors. Disulfide reducing agents and metal ion chelating agents such as EDTA also have strong inhibitory effects on this enzyme, but serine protease inhibitors and carboxypeptidase inhibitors have no inhibitory effect. These findings are of great significance for guiding the application of this enzyme.

[0014] This invention also provides potential applications of the aforementioned PepS in the food condiment industry. Specifically, this enzyme can be used in combination with other endopeptidases and exopeptidases, such as glutamate aminopeptidase PepA (which hydrolyzes peptide chains with acidic amino acids Asp or Glu at their amino terminals), to effectively degrade bitter peptides in food while increasing the content of aromatic amino acids (Trp, Tyr, Phe) and acidic amino acids (Glu, Asp) in food, thus enhancing its aroma and flavor.

[0015] The following are the sequences involved in the present invention:

[0016] SEQ ID NO: 1 (PepS amino acid sequence):

[0017] MVLPNFKENLEKYAKLLVTNGINVQPGHTVALSIDVEQAELAHLLVKEAYALGAAEVIVQ

[0018] WSDDTINRERFLHAEMNRIEEVPAYKKAEMEYLLEKKASRLGVRSSDPDAFNGVAPERLS

[0019] AHAKAIGAAFKPMQVATQSNKVSWTVAAAAGKEWAKKVFPNASSDEEAVDLLWNQIFKTC

[0020] RVYEKDPVRAWKEHADRLDAKARILNEAQFSALHYTAPGTDLTLGLPKNHVWESAGAINA

[0021] QGESFLPNMPTEEVFTAPDFRRAYGYVRSTKPLSYGGNIIEGIKVTFKDGEAVDITADQG

[0022] EKVLKNLVFNNNGARALGECALVPDSSPISQSNITFFNTLFDENASNHLAIGAAYAFSVE

[0023] GGADMTEEELKAAGLNRSDVHVDFIIGSNQMNIDGIHHDGSRVPIFRNGDWVI

[0024]

[0025] Compared with existing technologies, the beneficial effects of this solution are as follows: This invention provides a novel PepS encoding gene and achieves secretory expression of PepS in Pichia pastoris for the first time, and provides a method for its isolation and preparation. This invention also provides the biological characteristics of PepS and its potential application value. Attached Figure Description

[0026] Figure 1 This is a comparison between the PepS encoding gene obtained in the embodiments of the present invention and the PepS encoding gene of the reported Streptococcus thermophilus strain S.thermophilus CNRZ302.

[0027] Figure 2 This is the construction procedure for the PepS expression vector pHB905M-PepS in the embodiments of the present invention;

[0028] Figure 3 This invention demonstrates the efficient secretory expression of PepS in Pichia pastoris GS115 in this embodiment (A, SDS-PAGE analysis of the supernatant of Pichia pastoris GS115 induced by pHB905M-PepS vector, arrows indicate the target protein PepS, M is the protein molecular weight marker (kDa). B, PepS immobilized in Ni 2+ SDS-PAGE analysis of protein purified by affinity chromatography in one step, where M is the protein molecular weight marker (kDa) and 1 is the PepS value.

[0029] Figure 4 This invention relates to the substrate specificity and activity of PepS in the embodiments of the present invention (A, determination principle, specific hydrolysis of L-amino acid-pNA to form amide bonds. B, quantitative curve of enzyme activity. C, substrate specificity and enzyme activity of PepS);

[0030] Figure 5 These are the optimal temperature, optimal pH, thermal stability, and acid-base stability of PepS in the embodiments of the present invention (A, optimal temperature and thermal stability; B, optimal pH and acid-base stability).

[0031] Figure 6 This describes the effect of metal ions (A) and inhibitors (B) on PepS activity in this embodiment of the invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0034] Example:

[0035] The implementation process of the present invention is as follows:

[0036] 1. Cloning of the PepS encoding gene

[0037] Primers were designed based on the PepS nucleotide sequence (Accession number: AF102860) of *S. thermophilus* CNRZ 302 from the GeneBank database. Using a homologous cloning strategy, the coding sequence of PepS was amplified from the laboratory-isolated *S. thermophilus* strain *LMY005*. The primers were P1: 5′-ATGGTTTTACCAAATTTTAAAGA-3′ and P2: 5′-TTAAATCACCCAATCACCATTT-3′. Single colonies of *S. thermophilus* LMY005 were picked from M17 agar plates and transferred to 1.5 mL EP tubes. The cells were resuspended in 100 μL PBS, incubated in a boiling water bath for 10 min, and the EP tubes were quickly placed on ice to cool. The cells were centrifuged, and the supernatant was collected. Using the prepared supernatant as a template, the PepS coding gene was amplified by PCR. The reaction conditions are as follows: 5 μL template, 1 μL each of primers P1 and P2, and 2 μL of 10× buffer (containing Mg). 2+ Add 1 μL of Taq enzyme and sterile water to a final volume of 20 μL. The thermal cycling is as follows: 95℃ for 5 min, 95℃ for 1 min, 55℃ for 1 min 30 s, 72℃ for 2 min, for 30 cycles, followed by 72℃ for 5 min. The amplified product is recovered and ligated into the pMD-18T vector using AT cloning for sequence analysis.

[0038] like Figure 1As shown, the nucleotide fragment encoding Pep S was successfully amplified from S. thermophilus LMY005. Sequence analysis showed that the homology with the Pep S encoding nucleotide sequence of the reported strain S. thermophilus CNRZ 302 was 98.71%. In the 1239bp reading frame encoding 413 amino acids, the following changes were made: T at position 802 changed to C, C at position 803 changed to G, A at position 804 changed to T, A at position 826 changed to G, A at position 827 changed to G, A at position 874 changed to G, T at position 875 changed to C, A at position 910 changed to C, G at position 997 changed to A, G at position 998 changed to A, G at position 999 changed to C, A at position 1069 changed to T, and C at position 1070 changed to T. This resulted in the following changes: Ser at position 268 was changed to Arg, Asn at position 276 to Gly, Ile at position 292 to Ala, Met at position 304 to Leu, Gly at position 333 to Asn, and Thr at position 357 to Phe, achieving an amino acid homology of 98.55%.

[0039] 2. Construction of PepS expression vector

[0040] The PepS coding gene obtained above was optimized to use a Pichia pastoris-preferred codon, and a histidine tag coding sequence was added to the C-terminus. The PepS coding gene was synthesized using a gene synthesis method, with CpoI and NotI restriction sites introduced at the 5′ and 3′ ends, respectively. The synthesized gene was inserted into the vector pMD-18T, forming the cloning vector pMD-18T-PepS. The plasmid pMD-18T-PepS and pHB905M were double-digested with CpoI / NotI. The PepS gene fragment and the linearized plasmid vector pHB905M were recovered. T4 DNA ligase was used to ligate the PepS gene fragment and the linearized plasmid vector pHB905M. The PepS coding gene was then inserted into the expression cassette of the vector pHB905M, forming the PepS expression vector pHB905M-PepS. The construction process of the expression vector is as follows: Figure 2 As shown, the PepS encoding gene is inserted into the expression vector pHB905M into an expression cassette consisting of the promoter (5′AOX) and terminator (3′AOXTT) of Pichia pastoris alcohol dehydrogenase, with a secretion signal peptide of Pichia pastoris α-factor upstream of it. Induction with methanol can efficiently express PepS and secrete it into the culture medium.

[0041] 3. Transformation of yeast cells and induction of PepS expression

[0042] Take GS115 competent cells, add 5 μL (100 ng) of linearized vector pHB905M-PepS (SalI digested), mix well, and transfer the liquid into a 0.2 ml electroporation cuvette. Transformation is performed at 1.5 kV, 25 μF, 400 Ω, and 4.2 s. The electroporated GS115 cells are then plated on MD medium and incubated upside down at 28°C until single colonies appear. Single colonies are picked and inoculated into 250 mL shake flasks containing 25 ml of BMGY, and incubated at 28°C and 250-300 rpm until OD500. 600 =2-6 (approximately 16-18 hours). Inoculate 25 ml of culture medium into a 3-4 L shake flask containing 1 L BMGY, and shake vigorously at 28°C (250-300 rpm) until the logarithmic growth phase (OD200). 600 =2-6). Collect cells by centrifuging at 1500-3000g for 5 min in sterile centrifuge tubes at room temperature. For induction of expression, remove the supernatant and resuspend the cells in BMMY until OD200. 600 =1.0 (2-6 L). Aliquot the culture into several 3-4 L split-plate shake flasks, cover with two layers of sterile gauze or cheesecloth, and incubate on a shaker at 28 °C. Every 24 h, add methanol to a final concentration of 0.5% until the optimal induction time is reached. Centrifuge at 1500-3000 g for 5 min at room temperature, retain the supernatant, pre-cool at 4 °C, and concentrate if necessary. Observe protein expression using SDS / PAGE.

[0043] like Figure 3 As shown in Figure A, PepS was successfully secreted into the culture medium after 120 h of methanol-induced expression, with a molecular weight of approximately 45 kDa, consistent with the theoretical value.

[0044] 4. Isolation and purification of PepS

[0045] Add an equal volume of 100% saturated ammonium sulfate to 1 L of the above expression supernatant, and incubate overnight at 4°C with gentle shaking to ensure complete protein precipitation. Centrifuge at 12000g for 30 min to collect the precipitate, resuspend the precipitate in 100 mL of dialysis buffer (20 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl), place it in a dialysis bag, and place it in 1000 mL of dialysis buffer. Dialyze at 4°C with stirring for 24 h, changing the dialysis buffer every 8 h to thoroughly remove ammonium sulfate and other ions from the protein. Subsequently, the protein is passed through immobilized Ni 2+ Purification was performed using affinity chromatography. Briefly, the dialyzed protein was loaded onto NiO2 buffer equilibrated with 10 volumes of dialysis buffer. 2+Affinity chromatography column (Ni-NTA) was used at a flow rate of 0.5 mL / min. After sample flow-through, the sample was washed to baseline with buffer (20 mmol / L Tris-HCl (pH 8.0), 500 mmol / L NaCl, 20 mmol / L imidazole) at a flow rate of 1 mL / min to thoroughly remove unbound proteins. Finally, the protein was eluted with buffer (20 mmol / L Tris-HCl (pH 8.0), 150 mmol / L NaCl, 500 mmol / L imidazole) at a flow rate of 1 mL / min. The protein peak was collected, analyzed by SDS-PAGE, and dialyzed against 100 volumes of 1×PBS buffer for 24 h, with the buffer changed every 8 h to thoroughly remove imidazole and other inorganic ions from the protein. The dialyzed protein was centrifuged at 12000 g for 20 min, filtered through a 0.22 μm filter to remove small amounts of protein precipitate and potential microorganisms, quantified by Bradford chromatography, and lyophilized for later use.

[0046] like Figure 3 As shown in B, the immobilized Ni 2+ Affinity chromatography can be used for one-step purification, achieving a purity of over 95% for PepS. 50-60 mg of PepS can be routinely obtained from 1 L of expression supernatant.

[0047] 5. Substrate specificity and enzyme activity of PepS

[0048] Principle: Aminopeptidase specifically hydrolyzes polypeptides with different amino acids at their N-terminus, cleaving amino acid residues from the N-terminus to form free amino acids. In this example, the substrate specificity and activity of PepS were determined using the classical method for aminopeptidase activity assay. A series of amide compounds (AA-pNA) formed by L-amino acids (L-AA) and p-nitroaniline (p-NA) were synthesized. Aminopeptidases with different specificities can selectively hydrolyze the amide bond formed between the carboxyl group of different amino acids and the amino group of p-NA, releasing free amino acids and p-NA. p-NA is a yellow substance that absorbs at 405 nm. Quantification of p-NA can be achieved through optical analysis, thereby analyzing the substrate specificity and enzyme activity of aminopeptidase. Figure 4 As shown in Figure A.

[0049] Standard reaction system: 200 ml Tris-HCl (50 mM), 100 mM NaCl.

[0050] Standard reaction conditions: Add 1 mM substrate (AA-pNA) and 1 μg PepS to the standard reaction system, react in a water bath at 50°C for 10 min, and terminate the reaction with an equal volume of acetic acid.

[0051] Quantitative method: Prepare standard p-NA solutions of 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM, and measure the absorbance at 405 nm. Plot a standard curve with concentration on the x-axis and absorbance on the y-axis. Figure 4 B). Take the above reaction solution and measure the absorbance at 405 nm. Substitute the absorbance value into the standard curve and calculate the concentration of p-NA generated after the reaction. The enzyme activity unit can then be calculated. Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate into the product p-NA per minute, which is one unit (IU). Enzyme activity is expressed as IU / mg.

[0052] like Figure 4 As shown in Figure C, PepS exhibits strong hydrolytic activity towards peptide chains with N-terminal aromatic amino acids (Trp, Tyr, Phe) and basic amino acids (Arg, Lys). It also shows some hydrolytic activity towards peptide chains with N-terminal hydrophobic amino acids (Leu, Ala, Pro), but this activity is weak. Furthermore, it has almost no hydrolytic activity towards peptide chains with N-terminal acidic amino acids (Glu, Asp). Based on these findings regarding substrate specificity and enzyme activity, potential applications of PepS include: debittering food proteins, as bitter peptides in food are mostly composed of hydrophobic amino acids; and combining it with aminopeptidase A (PepA, which hydrolyzes N-terminal peptide chains of acidic amino acids Glu and Asp) in the food industry or during cooking to increase the content of aromatic amino acids (Trp, Tyr, Phe) and acidic amino acids (Glu, Asp) in food, resulting in a more aromatic and flavorful dish.

[0053] 6. Optimal reaction temperature, thermal stability, optimal pH, and acid-base stability of PepS

[0054] Add 0.5 mmol / L Larg-pNA and 1 μg PepS to 200 μL of the above standard reaction system, and react at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 10 min. Terminate the reaction by adding an equal volume of acetic acid. Determine and calculate the relative enzyme activity (%) at different temperatures using the above standard enzyme activity assay method to determine the optimal reaction temperature. Incubate PepS in a water bath at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 1 h. Use the untreated enzyme activity as a control and determine the residual enzyme activity using the standard assay method to determine the enzyme's thermostability.

[0055] 1 μg PepS, 0.5 mmol / L sodium acetate (pH 5.0-6.0), sodium phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 9.0-11.0) buffer solutions were added to 200 μL of each buffer. The mixture was reacted at the enzyme's optimal temperature for 10 min, and the reaction was terminated by adding an equal volume of acetic acid. The relative enzyme activity (%) at different pH values ​​was determined and calculated using the standard enzyme activity assay method described above to determine the optimal reaction pH. PepS was added to buffer solutions at different pH values ​​(pH 2.0-11.0) to a final concentration of 100 μg / mL, and the mixture was incubated at 4 °C for 1 h. The residual enzyme activity was determined using the standard assay method, with the untreated enzyme activity as a control, to determine the enzyme's pH stability.

[0056] like Figure 5 As shown in Figure A, PepS maintains good catalytic activity (above 40%) in the range of 30-70℃, with an optimal reaction temperature of 60℃. At 70℃, it still retains 65% activity, but the activity decreases rapidly with increasing temperature, becoming almost undetectable at 90℃. PepS is very stable below 50℃, with virtually no change in activity after 1 hour of incubation. At temperatures of 50-70℃, PepS exhibits moderate stability, retaining over 60% residual activity after 1 hour of incubation at 60℃. However, above 60℃, the activity decreases rapidly, with less than 5% residual activity after 1 hour of incubation at 90℃. Figure 5 As shown in Figure B, the optimal reaction pH for PepS is 8.0, and it is relatively stable within the pH range of 6.0-9.0. After incubation for 1 hour, the residual activity remains above 80%. Within the pH range of 1.0-11.0, after incubation for 1 hour, its residual activity remains above 50%, indicating that PepS has a wide range of acid and base stability.

[0057] 7. Effects of divalent metal ions and inhibitors on PepS activity

[0058] Add 0.1 and 1.0 mmol / L of different divalent metal ions, 1 μg PepS, and 0.5 mmol / L Arg-pNA to a 200 μL standard reaction system. React in a 50℃ water bath for 10 min, then terminate the reaction by adding an equal volume of acetic acid. After cooling, measure the absorbance at 405 nm. Use the enzyme activity without added metal ions as a control, and determine and calculate the relative enzyme activity (%) under different divalent metal ions according to standard methods.

[0059] Different organic reagents and protease inhibitors, 1 μg PepS, and 0.5 mmol / L Arg-pNA were added to a 200 μL standard reaction system at final concentrations of 0.1 and 1.0 mmol / L, respectively. The reaction was carried out in a 50 °C water bath for 10 min, and the reaction was terminated by adding an equal volume of acetic acid. After cooling, the absorbance was measured at a wavelength of 405 nm. The enzyme activity without the addition of organic reagents or protease inhibitors was used as a control. The relative enzyme activity (%) at different concentrations of organic reagents or protease inhibitors was determined and calculated according to the above standard determination method.

[0060] like Figure 6 As shown, different divalent metal ions all have a certain inhibitory effect on PepS activity, among which Ca... 2+ and Mg 2 + The inhibitory effect of Co is relatively small. 2+ Mn 2+ Zn 2+ It has a strong inhibitory effect, Ni 2+ and Cu 2+ The inhibitory effect on PepS was the strongest. Cysteine ​​protease inhibitor E64, serine protease inhibitor benzyl sulfonyl fluoride (PMSF), and carboxypeptidase inhibitor Pepstatin A had no significant effect on PepS activity. However, metalloproteinase inhibitors 1,10-phenanthroline, metal chelators EDTA, and disulfide bond reducing agents dithiothreitol (DTT) or β-mercaptoethanol showed more significant inhibitory effects on PepS.

[0061] The above embodiments investigated the biological characteristics of PepS, such as substrate specificity, enzyme activity, optimal reaction temperature, optimal pH, thermal stability, acid-base stability, and the effects of metal ions and inhibitors. This is of great significance for guiding the application of this enzyme. In summary, through the above embodiments of the present invention, a novel PepS encoding gene was cloned, secretory expression of PepS was achieved in Pichia pastoris, and a method for its isolation and purification was provided. The present invention also provides the biological characteristics of PepS and its potential application value.

[0062] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. An aminopeptidase, characterized in that: The aminopeptidase is aminopeptidase S (Pep S) derived from Streptococcus thermophilus, which is a proteolytic enzyme. The amino acid sequence of Pep S is shown in SEQ ID NO:

1.

2. A nucleotide fragment characterized by: The nucleotide fragment is used to encode Pep S as described in claim 1.

3. The nucleotide fragment of claim 2, characterized in that: The sequence of the nucleotide fragment is shown in SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The expression vector is an expression vector carrying the nucleotide fragment as described in claim 2.

5. A method for expressing Pep S as described in claim 1, characterized in that: The expression method involves transferring the recombinant expression vector as described in claim 4 into host cells, performing high-density fermentation, and inducing Pep S expression with an inducer.

6. The expression method as described in claim 5, characterized in that: The host cell was Pichia pastoris GS115.

7. The application of Pep S as described in claim 1 in the food seasoning industry.

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