Aminopeptidases for Protein Hydrolysis

Abstract

To provide a single component peptidase enzyme which has activity useful for improving the properties and degree of hydrolysis of protein hydrolysates used in food and or feed products.SOLUTION: The present disclosure provides an isolated polypeptide having aminopeptidase activity, where the isolated polypeptide has a specific amino acid sequence and can hydrolyze a polypeptide having a proline residue in position 2, as numbered from the N-terminus. The present disclosure further provides methods of obtaining protein hydrolysates useful as flavor improving agents.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 185,503, filed June 26, 2015. and the priority and benefit of application Ser. No. 62 / 235937 filed on October 1, 2015. The provisional applications each claim an invention entitled "Novel Aminopeptide for Hydrolyzing Proteins." The name is "Chidase".

[0002] Incorporation by reference of sequence listing 192,505 by 2016-06-10, filed herewith. "20160610_NB40989-PCT sequence The sequence listing shown in the file named "listing prj_ST25.txt" is No. 6,399,433, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Various food and feed products contain protein hydrolysates. This hydrolysis: Traditionally, this has been accomplished by chemical hydrolysis. However, such chemical hydrolysis , decomposes a large amount of amino acids obtained during hydrolysis, and also produces harmful by-products during the chemical reaction. Concerns about using protein hydrolysates obtained by chemical hydrolysis The increasing demand for enzyme-based hydrolysis has led to the development of enzyme-based hydrolysis processes. Summary of the Invention [Problem to be solved by the invention]

[0004] The enzymatic hydrolysis process of proteinaceous substances aims at obtaining a high degree of hydrolysis. Polypeptides having aminopeptidase activity include peptides, polypeptides and and catalyzes the removal of one or more amino acid residues from the N-terminus of a protein. To produce a protein hydrolysate having a high degree of hydrolysis, a peptidyl ester is generally used. It is necessary to use a mixture of enzyme activities, either alone or in combination with other enzymes, to It is useful for improving the properties and degree of hydrolysis of protein hydrolysates used in food and feed products. It would be desirable to provide a single component peptidase enzyme with useful activity. [Means for solving the problem]

[0005] The present disclosure relates to novel polypeptides having high aminopeptidase activity, as well as desirable The present invention provides a method for obtaining protein hydrolysates having high quality and a high degree of hydrolysis.

[0006] Further features and advantages of the present invention will become apparent from the following detailed description which illustrates certain illustrative embodiments in which the principles of the invention are utilized. A better understanding may be obtained by reference to the accompanying drawings, in which: [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows the overall design of the synthetic gene encoding the pepN_2 enzyme at the top, with a close-up of the leader sequence used in place of the endogenous secretion signal sequence at the bottom. [Diagram 2] Figure 2 shows the pH dependence of glutamate release of PepN2 from Neosartorya fischeri during gluten hydrolysis (without pH adjustment during hydrolysis). [Diagram 3] Figure 3 shows the temperature dependence of glutamic acid release of PepN2 from Neosartorya fischeri during gluten hydrolysis (without pH adjustment during hydrolysis). [Figure 4] FIG. 4 shows glutamate release of different PepN2s. [Diagram 5] FIG. 5 shows the hydrolysis of WHWLQLKPGQPMY (SEQ ID NO:26). [Figure 6] FIG. 6 shows the hydrolysis of KPGQPMY (SEQ ID NO:27). [Figure 7] FIG. 7 shows the hydrolysis of QPMY. (SEQ ID NO:28). [Figure 8] FIG. 8 shows that the peptide sequences of the substrate and cleavage products were entered into Skyline and the intensities of each sample were calculated. [Figure 9] FIG. 9 shows the spatial arrangement of six panels, FIGS. 9A-9F, which list sequence alignments of several PepN2 enzymes. [Figure 10] FIG. 10. Hydrolysis of TPAAR (SEQ ID NO: 29) by TRI032, TRI035, TRI063 (Aspergillus oryzae (A. oryzae)) and COROLASE® LAP (fungal exopeptidase; DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The present disclosure provides polypeptides having aminopeptidase activity. Nucleic acid sequences encoding the polypeptides of the present invention, nucleic acid constructs and vectors containing the nucleic acid sequences The disclosure also relates to polypeptides and host cells, as well as methods for producing the polypeptides. A method for obtaining a hydrolysate from a proteinaceous substrate, comprising the steps of: It has aminopeptidase activity in combination with a protease, e.g., an endopeptidase. The present disclosure also relates to a method for the production of a polypeptide from a protein substrate, comprising exposing the polypeptide to a polypeptide that: Hydrolysates rich in free glutamic acid and / or peptides with bound glutamic acid residues The method for obtaining said compound comprises exposing a substrate to a polypeptide having aminopeptidase activity. The present disclosure further relates to methods comprising: The composition may further comprise an additional enzymatic activity.

[0009] In another aspect, the methods described herein can be used to enhance flavor in food-related applications, such as baking. Alternatively, flavor improvement in foods can be achieved by the method of the present invention. In some embodiments, this can be achieved by the addition of a hydrolysate obtained by The hydrolysates produced using the aminopeptidases described herein also contain untreated fermented Compared to the hydrolysate, the bitterness can be reduced.

[0010] In some embodiments, the present invention provides a novel fungal aminopeptidase (PepN). , a production host (e.g., Trichoderma reesei )) in high yields, as well as for example for debittering and for the production of glutamine. Their use for producing protein hydrolysates for acid production is provided. As shown, the type 2 aminopeptidase of the present invention has a greater activity than the type 1 aminopeptidase. Initial results surprisingly showed that these PepNs were able to increase glutamate release from other It was shown that it has a higher tolerance to proline in P1 than the known aminopeptidases. Thus, the proline-tolerant amino acids taught for use in the present invention Peptidases can act on a wide range of peptide and / or protein substrates, such as Due to its broad substrate specificity, it is possible to selectively bind certain amino acids (e.g., proline and / or or lysine and / or arginine and / or glycine)-rich substrates. is not hindered by

[0011] Surprisingly, the inventors have found that type 2 aminopeptidases are more potent than type 1 aminopeptidases. Furthermore, the inventors have surprisingly found that the good We found a group of fungal type 2 aminopeptidases that have the following structure: Some tidases have long N-termini in their mature amino acid sequences. 1 shows an alignment of amino acid sequences of type 2 aminopeptidases from

[0012] The term "aminopeptidase activity" as used herein refers to a peptide, oligopeptide, or It is defined as the peptidase activity that catalyzes the cleavage of amino acids from the N-terminus of a peptide or protein. In a general definition, aminopeptidase activity is the activity of a peptide, oligopeptide or or amino acid X from the N-terminus of a protein (where X is Ala, Arg, Asn, or As p, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Ph e, Pro, Ser, Thr, Trp, Tyr, and Val Amino acid residues, at least Leu, Glu, Gly, Ala, and / or Pro residues The aminopeptidase activity of the present invention can cleave the aminopeptidase group. The peptide is non-specific with respect to the amino acid that is cleaved from the N-terminus of the peptide or polypeptide substrate. It will be appreciated that this may be objective.

[0013] In some embodiments, the present invention provides a method for the preparation of a 67 The amino acid sequence of the predicted mature sequence includes more than 13 residues N-terminal to the conserved residues I / V at positions An isolated polypeptide, or fragment thereof, having peptidase activity, provided that Fragments of have aminopeptidase activity.

[0014] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least one amino acid sequence similar to that of SEQ ID NO:1. at least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Preferably, the amino acid sequence has at least about 95% identity, and most preferably at least about 97% identity. an isolated polypeptide having an amino acid sequence and having aminopeptidase activity; In some embodiments, the homologous polypeptide The peptide may have the amino acid sequence of SEQ ID NO:1 and a sequence of 5 amino acids, preferably 4 amino acids. , more preferably 3 amino acids, even more preferably 2 amino acids, and most preferably In some embodiments, the amino acid sequences of the present invention differ by one amino acid. The polypeptide may have the amino acid sequence of SEQ ID NO:1, the mature amino acid sequence or an allelic variant. and fragments thereof, which fragments have aminopeptidase activity In some embodiments, the polypeptide of the invention comprises SEQ ID NO: 1. In one embodiment, the polypeptide of the present invention has the amino acid sequence of SEQ ID NO: 1, or a fragment thereof. The fragment of SEQ ID NO:1 has aminopeptidase activity. The fragment may be one or more amino and / or carboxy termini of the amino acid sequence. In some embodiments, the fragment is a polypeptide in which at least one amino acid has been deleted. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 18. 1 amino acid sequence.

[0015] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least the amino acid sequence of SEQ ID NO:2. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95% and most preferably at least about 97% homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO:2 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:2, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:2. In another embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:2, The sequence includes a fragment thereof, which fragment has aminopeptidase activity. The fragment numbered 2 is the amino- and / or carboxy-terminal end of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from In some embodiments, the fragment comprises the sequence of SEQ ID NO: 19. This has the amino acid sequence of SEQ ID NO:2.

[0016] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least the amino acid sequence of SEQ ID NO:3. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95% and most preferably at least about 97% homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO:3 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:3, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:3. In another embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:3, The sequence includes a fragment thereof, which fragment has aminopeptidase activity. The fragment numbered 3 corresponds to the amino and / or carboxy termini of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from In some embodiments, the fragment comprises the sequence of SEQ ID NO: 20. The domain has the amino acid sequence of SEQ ID NO:3.

[0017] In some embodiments, the present invention provides a method for the preparation of a nucleic acid sequence having at least one amino acid sequence similar to that of the mature amino acid sequence of SEQ ID NO:4. at least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Preferably, the amino acid sequence has at least about 95% homology, and most preferably at least about 97% homology. an isolated polypeptide having an amino acid sequence and having aminopeptidase activity; In some embodiments, the homologous polypeptide The peptide may have the amino acid sequence of SEQ ID NO: 4 and 5 amino acids, preferably 4 amino acids. , more preferably 3 amino acids, even more preferably 2 amino acids, and most preferably In some embodiments, the amino acid sequences of the present invention differ by one amino acid. The polypeptide may have the amino acid sequence of SEQ ID NO:4, the mature amino acid sequence or an allelic variant. and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:4. In another embodiment, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:4: or a fragment thereof, which fragment has aminopeptidase activity. A fragment of SEQ ID NO:4 may be any amino- and / or carboxy-terminal fragment of this amino acid sequence. A polypeptide having one or more amino acids deleted from its terminus. In some embodiments, the fragment comprises the sequence of SEQ ID NO: 21. The peptide has the amino acid sequence of SEQ ID NO:4.

[0018] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least the amino acid sequence of SEQ ID NO:5. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95% and most preferably at least about 97% homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO:5 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:5, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity In some embodiments, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:5. In another embodiment, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:5. or a fragment thereof, which fragment has aminopeptidase activity. The fragment in column number 5 corresponds to the amino and / or carboxy termini of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from the end. In some embodiments, the fragment comprises the sequence of SEQ ID NO: 22. The tide has the amino acid sequence of SEQ ID NO:5.

[0019] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least the amino acid sequence of SEQ ID NO:6. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95% and most preferably at least about 97% homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO:6 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:6, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:6. In another embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:6, The sequence includes a fragment thereof, which fragment has aminopeptidase activity. Fragment number 6 is the amino- and / or carboxy-terminal end of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from In some embodiments, the fragment comprises the sequence of SEQ ID NO: 23. The domain has the amino acid sequence of SEQ ID NO:6.

[0020] In some embodiments, the present invention provides a method for the preparation of a polypeptide having at least the amino acid sequence of SEQ ID NO:7. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95%, and most preferably at least about 97%, homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO: 7 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:7, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:7. In another embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO: 7, The sequence includes a fragment thereof, which fragment has aminopeptidase activity. Fragment number 7 is the amino- and / or carboxy-terminal end of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from In some embodiments, the fragment comprises the sequence of SEQ ID NO: 24. The domain has the amino acid sequence of SEQ ID NO:7.

[0021] In some embodiments, the present invention provides a polypeptide having at least the amino acid sequence of SEQ ID NO:8. At least about 50%, preferably at least about 60%, more preferably at least about 70%, Preferably at least about 80%, even more preferably at least about 90%, and most preferably Amino acids having at least about 95% and most preferably at least about 97% homology thereto. an isolated polypeptide having an aminopeptidase activity, In some embodiments, the homologous polypeptide The peptide has the amino acid sequence of SEQ ID NO:8 and 5 amino acids, preferably 4 amino acids, More preferably, 3 amino acids, even more preferably, 2 amino acids, and most preferably, 1 In some embodiments, the polypeptides of the present invention have different amino acid sequences. The polypeptide has the amino acid sequence of SEQ ID NO:8, the mature amino acid sequence or an allelic variant; and fragments thereof, which fragments have aminopeptidase activity. In some embodiments, the polypeptide of the invention comprises the amino acid sequence of SEQ ID NO:8. In another embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO:8, The sequence includes a fragment thereof, which fragment has aminopeptidase activity. Fragment number 8 is the amino- and / or carboxy-terminal end of this amino acid sequence. In some embodiments, the polypeptide is a polypeptide having one or more amino acids deleted from In some embodiments, the fragment comprises the sequence of SEQ ID NO: 25. The domain has the amino acid sequence of SEQ ID NO:8.

[0022] Preferably, the fragment is at least 330 amino acid residues, more preferably It contains at least 380 amino acid residues, most preferably 430 amino acid residues.

[0023] An allelic variant is any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally by mutation and leads to phenotypic polymorphism within a population. obtain.

[0024] As used herein, a "parent enzyme" refers to a gene having the structure set forth in SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the sequence It is an aminopeptidase having all of the amino acid residues of the polypeptide shown in number 8. In this regard, for example, the parent may have a modification of the mutant polypeptide (which may vary from 0 to 1 depending on the type of mutation). The criterion for determining whether the criterion is true will be 1 or 2.

[0025] In some embodiments, the present invention is more preferably carried out under conditions of low stringency. The enzyme is preferably cleaved under moderate stringency conditions, and most preferably under high stringency conditions. By a nucleic acid sequence that hybridizes with an oligonucleotide probe under the conditions An isolated polypeptide having aminopeptidase activity, Allelic variants and fragments of tide (which have aminopeptidase activity) The oligonucleotide probe is selected from SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:11, or SEQ ID NO:12, or SEQ ID NO:13, SEQ ID NO:14, or is a polypeptide code for the nucleic acid sequence of SEQ ID NO: 15, or SEQ ID NO: 16, or a complementary strand thereof Hybridization is carried out under the same conditions as for the ligated portion (J. Sambrook, EF Fritsch, and T. Maniatus, 1989, Molecular Cl oning,A Laboratory Manual,2d edition,Col. d Spring Harbor, New York).

[0026] Hybridization refers to the nucleic acid sequence being identical to SEQ ID NO:9, or SEQ ID NO:10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: SEQ ID NO:15, or an oligonucleotide corresponding to the polypeptide-encoding portion of the nucleic acid sequence shown in SEQ ID NO:16. The oligonucleotide probe was subjected to low to high stringency conditions (i.e., 5× SS PE, 0.3% SDS, 200 pg / ml fragmented denatured salmon sperm DNA, and , 25, 35 or 50% formamide for low, medium and high stringency, respectively. Prehybridization and hybridization at 42°C in 100°C. The results show that the hybridization occurs according to the tandem blot method.

[0027] SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or is the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:8; or a subsequence thereof may be used to design an oligonucleotide probe, For example, SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or No. 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or the sequence A nucleic acid sequence encoding a polypeptide of the present invention, such as the polypeptide-encoding portion of the nucleic acid sequence of SEQ ID NO:16. The nucleic acid sequence, or a subsequence thereof, can be isolated by amplification according to methods well known in the art. A strain of the genus or species encoding a polypeptide having aminopeptidase activity In particular, such probes can be used to identify or clone NAs. To identify and isolate the corresponding gene in Used to hybridize with the genomic or cDNA of a genus or species of interest Such probes may be considerably shorter than the entire sequence, but may be of any length. At least 15, preferably at least 25, more preferably at least 40 nucleic acids. Longer probes can also be used. Both probes and RNA probes can be used. These probes usually correspond to To detect genes (e.g., using 32P, 3H, 35S, biotin or avidin) The markers are labeled accordingly.

[0028] Thus, genomes, cDNA or recombinant DNA sequences prepared from such other organisms may be used. The chemical library was used to select the peptides that hybridized with the probe and had aminopeptidase activity. It is possible to screen for DNA encoding a polypeptide having such properties. Genomic or other DNA from other organisms such as The proteins may be separated by gel electrophoresis or other separation techniques. The DNA or isolated DNA is transferred to and immobilized on nitrocellulose or other suitable carrier material. SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or Column number 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: To identify clones or DNA that are homologous to the polypeptide-encoding portions of the 16 nucleic acid sequences, To achieve this, a carrier material was used in a Southern blot, which was then incubated for 30 minutes at 4°C for 10 min. Three separate incubations were performed in 2×SSC, 0.2% SDS, preferably for at least 50 min. °C, more preferably at least 55 °C, more preferably at least 60 °C, more preferably is at least 65°C, more preferably at least 70°C, and most preferably at least The oligonucleotide probe hybridizes under these conditions and is then washed at 75°C. The molecules that bind are detected using X-ray film.

[0029] As used herein, the terms "modifying" and "modification" refer to the closest homology. The comparison refers to a substitution as compared to the wild-type aminopeptidase polypeptide sequence of the A variant aminopeptidase polypeptide and SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or Two of the wild-type aminopeptidases have reference sequences shown as sequence number 8. This is done by aligning the

[0030] Similarly, as used herein, "equivalent modifications" refer to modifications at equivalent positions in other aminopeptidases. It refers to making the same modification (usually a substitution) to the amino acids in

[0031] In one embodiment, the aminopeptidase sequences of the present invention are in isolated form. The term "aminopeptidase" refers to an aminopeptidase sequence that is naturally associated with and found in nature. It means that the composition is at least substantially free of at least one other component that is present in the composition. The distinct aminopeptidase sequences effectively bind one or more contaminants with which the substance may otherwise associate. Thus, for example, it may be provided in a form that is substantially free of one or more potentially contaminating The nucleic acid may be substantially free of contaminating polypeptides and / or nucleic acid molecules.

[0032] In one embodiment, the aminopeptidase sequences of the present invention are in purified form. The term "constituted" means that a given component is present at a high level. The component is preferably the major component present in the composition. At least about 90%, or at least about 95%, or at least about 98% Preferably, said levels are present in the total composition under consideration in a dry weight ratio. / Measured on a dry weight basis.

[0033] As used herein, the singular forms "a," "an," and "the" are used where the context requires. Unless otherwise indicated, nucleic acids are represented in a 5' to 3' orientation. The amino acid sequence is written from left to right in the amino to carboxy direction. Unless otherwise indicated, the present disclosure is directed to the specific methods, protocols, and tests described herein. Note that this is not limited to drugs.

[0034] Terms and abbreviations not defined are defined according to their normal usage in the art. Unless otherwise specifically defined herein, the terms used herein shall have the following meaning: All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the practice of this disclosure requires prior art knowledge in molecular biology, protein engineering and microbiology. This includes prior art commonly used in the art. Although various methods and materials can be used in the practice of this disclosure, some preferred methods and materials are described herein. The terms defined immediately below are more fully understood by reference to the entire specification. Well defined.

[0035] Nucleotide sequence The scope of the present invention is directed to aminopeptidases encoding the specific properties defined herein. The present invention includes a nucleotide sequence which encodes the

[0036] In some embodiments, the nucleic acid sequence is selected from the group consisting of Aspergillus For example, Aspergillus clavatus In some embodiments, the nucleic acid sequence encodes a polypeptide obtained from Neosartorya, e.g. Aspergillus fischeri The polypeptide encoded by the compound is obtained from artorya fischeri.

[0037] In some embodiments, the present invention provides a method for the preparation of a 67 The amino acid sequence of the predicted mature sequence includes more than 13 residues N-terminal to the conserved residues I / V at positions Nucleic acids encoding polypeptides or fragments thereof having peptidase activity However, the fragment has aminopeptidase activity.

[0038] In some embodiments, the polynucleotides of the invention are similar to the exemplified polynucleotides. In some embodiments, the polynucleotide has specific nucleic acid homology to The nucleotide is SEQ ID NO:9, or SEQ ID NO:10, or SEQ ID NO:11, or No. 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16 At least 50, 60, 65, 70, 7 5, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or In some embodiments, the polynucleotide comprises a nucleic acid sequence having 100% identity. SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or is selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. In another embodiment, the polynucleotide of the present invention also comprises a nucleic acid sequence selected from the sequence Sequence number 9, or sequence number 10, or sequence number 11, or sequence number 12, or sequence Selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 In some embodiments, the polynucleotide may have a nucleic acid sequence complementary to a nucleic acid sequence to be cloned. The tide is SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or Selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:8 At least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 51 Recombinant polypeptides or polypeptides comprising an amino acid sequence having 8, 99 or 100% identity thereto. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an active fragment thereof. The nucleotide is SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. A recombinant polypeptide or an active fragment thereof comprising the amino acid sequence of Homology can be determined by amino acid sequence alignment, e.g., as described herein. Determined using programs such as BLAST, ALIGN, or CLUSTAL as described It is possible.

[0039] The present invention also relates to a method for the preparation of a nucleic acid sequence similar to that of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: No. 3, or SEQ ID NO: 4, or SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, which is different from SEQ ID NO: 8, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: The present invention encompasses nucleic acid sequences encoding a polypeptide having the amino acid sequence of SEQ ID NO:8. Also, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or the sequence SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: No. 11, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or a partial sequence of SEQ ID NO: 16. SEQ ID NO: 9, or SEQ ID NO: 1 0, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or or SEQ ID NO: 15, or a partial sequence of SEQ ID NO: 16, SEQ ID NO: 9, except for one or more nucleotide deletions, or SEQ ID NO: 10, or SEQ ID NO:11, or SEQ ID NO:12, or SEQ ID NO:13, SEQ ID NO:14, or a nucleic acid sequence encompassed by SEQ ID NO: 15, or SEQ ID NO: 16. Preferably, the number of the nucleotides is at least 990, more preferably at least 1140, and most preferably at least 1000. Most contain at least 1290 nucleotides.

[0040] As used herein, the term "nucleotide sequence" refers to an oligonucleotide The sequences or polynucleotide sequences, as well as variants, homologues, fragments and The nucleotide sequence may be genomic or synthetic. or of recombinant origin, whether it represents the sense or antisense strand. It may be double-stranded or single-stranded regardless.

[0041] The term "nucleotide sequence" in the context of the present invention includes genomic DNA, cDNA, synthetic It includes DNA and RNA. Preferably, it means DNA encoding the present invention. And more preferably, it means a cDNA sequence.

[0042] In a preferred embodiment, the present invention relates to a method for producing a nucleophilic compound which is encompassed by the scope of the present invention itself. A nucleotide sequence is understood to be a nucleic acid sequence that is expressed as it occurs in its natural environment and as a sequence with which it is naturally associated. When bound to a string (which also exists in its natural environment), the native nucleoside according to the invention For ease of reference, applicants refer to this preferred embodiment as a "non- In this regard, the term "native nucleotide sequence" is used. The promoter is present in its natural environment and is naturally associated with it (the promoter is also present in its natural environment). When the entire sequence is operably linked to the entire nucleic acid sequence (present in However, the amino acid sequences encompassed within the scope of the present invention may be identical to the nucleotide sequences in their natural form. The sequence may be isolated and / or purified after expression. However, preferably, The amino acid sequences encompassed within the scope of the present invention are generated by nucleotide sequences in their natural organisms. can be expressed in a manner similar to that with which it is naturally associated in the organism. Not under the control of the promoter.

[0043] Typically, nucleotide sequences encompassed by the present invention can be prepared using recombinant DNA techniques (i.e. However, in an alternative embodiment of the invention, The nucleotide sequence may be determined in its entirety using chemical methods well known in the art. Alternatively, some of the peptides can be synthesized (Caruthers MH et al., (19 80)Nuc Acids Res Symp Ser 215-23, and Horn T et al.,(1980)Nuc Acids Res Symp Ser 2 (see 25-232).

[0044] The present invention also relates to the nucleic acid sequence of SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: No. 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16 At least about 50%, preferably about 60%, preferably about 70%, At least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably For purposes of the present invention, the term "nucleotide sequence" refers to a nucleic acid sequence having approximately 97% homology between two nucleic acid sequences. The degree of homology was determined by the CLUSTAL method (Higgins, 1989, see above). , same table, gap penalty 10, and gap length penalty 10. The decision is made based on the above.

[0045] Modifications of a nucleic acid sequence encoding a polypeptide of the present invention can be used to produce a polypeptide that is substantially analogous to the polypeptide. "Substantially similar" to a polypeptide may be necessary for the synthesis of a similar polypeptide. The term refers to non-naturally occurring forms of polypeptides. These polypeptides may be any They may differ from polypeptides isolated from natural sources by engineering techniques. For example, specific activity, thermostability, pH optimum, etc. may be used to identify variants of different polypeptides, e.g. It may be interesting to synthesize similar sequences by site-directed mutagenesis. No. 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: The polypeptide of the nucleic acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 and / or The enzyme does not give rise to any other amino acid sequence of the polypeptide encoded by the nucleic acid sequence. by the introduction of nucleotide substitutions that correspond to the codon usage of the host organism for the production of or by introducing nucleotide substitutions that can result in different amino acid sequences. For a summary of nucleotide substitutions, see, for example, Ford et al., 199 1.Protein Expression and Purification 2: See 95-107.

[0046] Such substitutions may be made outside regions essential to the function of the molecule and still produce an active polypeptide. It will be apparent to one skilled in the art that the isolated nucleic acid sequences of the present invention may be used to Amino acids essential for the activity of the polypeptide are preferably not substituted. The amino acid residues can be selected by methods known in the art, such as site-directed mutagenesis or alanine substitution induction. can be identified by procedures that are used to lls, 1989, Science 244:1081-1085). In this study, we introduced mutations into all the positively charged residues in a molecule and synthesized an aminopeptide derivative of the resulting molecule. The enzyme activity of the molecule is tested to identify the amino acid residues that are essential for the activity of the molecule. Interaction sites may also be identified by techniques such as nuclear magnetic resonance spectroscopy, crystallography, or photoaffinity labeling. The three-dimensional structure is determined by the de Vos method (e.g., t al.,1992,Science 255:306-312;Smith et al. al.,1992,Journal of Molecular Biology 22 4:899-904; Wlodaver et al., 1992, FEBS Lett. (See Matthew 309:59-64).

[0047] composition In one aspect, the present invention also relates to the aminopeptidases described herein, as well as aminopeptidases. The present invention relates to compositions comprising the amino acid and / or nucleotide sequences.

[0048] In some embodiments, the present invention provides a method for the preparation of a ribosome-specific ribosome-specific nucleotide sequence comprising the steps of: Aminopeptides with predicted mature sequences containing more than 13 residues N-terminal to the conserved residues I / V The present invention provides a polypeptide, or a fragment thereof, having tidylserinase activity, wherein the fragment In some embodiments, the composition comprises a fragment having aminopeptidase activity. As shown in the sequence alignment of Fig. 1, there are more than 13 residues N-terminal to the conserved residue I / V at position 67. For a polypeptide having aminopeptidase activity, the predicted mature sequence includes the following amino acid sequence: at least about 50%, preferably at least about 60%, preferably at least about 70%, More preferably, it is at least about 80%, even more preferably, it is at least about 90%, and most preferably, it is at least about 90%. or at least about 95%, and most preferably at least about 97% homology thereto. Contains a polypeptide.

[0049] In some embodiments, the present invention provides a method for the preparation of a nucleic acid sequence comprising at least one amino acid sequence selected from the group consisting of: At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:1 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence or allelic variant of SEQ ID NO:1. variants; and fragments thereof, provided that the fragments are aminopeptidase In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the composition has the amino acid sequence of SEQ ID NO:1 or a fragment thereof. aminopeptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:1. Includes do.

[0050] In some embodiments, the present invention provides a method for the preparation of a nucleic acid sequence comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:2 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence or allelic variant of SEQ ID NO:2. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:2. In one embodiment, the composition comprises an amino acid having the amino acid sequence of SEQ ID NO:2 or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:2. nothing.

[0051] In some embodiments, the present invention provides a method for the preparation of a nucleic acid sequence comprising at least one amino acid sequence selected from the group consisting of: At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:3 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:3 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:3. In one embodiment, the composition comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:3, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:3. nothing.

[0052] In some embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:4. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:4 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:4 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:4. In one embodiment, the composition comprises an amino acid having the amino acid sequence of SEQ ID NO:4, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:4. nothing.

[0053] In some embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:5. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:5 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:5 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:5. In one embodiment, the composition comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:5, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:5. nothing.

[0054] In some embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:6. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:6 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:6 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:6. In one embodiment, the composition comprises an amino acid having the amino acid sequence of SEQ ID NO:6, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:6. nothing.

[0055] In some embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:7. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO: 7 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:7 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the composition comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:7, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:7. nothing.

[0056] In some embodiments, the present invention provides a method for the preparation of a nucleic acid molecule comprising at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:8. At least about 50%, preferably at least about 60%, more preferably at least about 70%, or at least about 80%, even more preferably at least about 90%, and most preferably at least Amino acids with at least about 95% identity, and most preferably at least about 97% identity The present invention provides a general composition comprising at least one aminopeptidase having a sequence. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:8 and five amino acids, preferably At most 4 amino acids, more preferably 3 amino acids, even more preferably 2 amino acids. and most preferably homologous polypeptides having amino acid sequences differing by one amino acid. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:8 or an allelic variant. variants; and fragments thereof, which fragments do not exhibit aminopeptidase activity. In some embodiments, the composition comprises the amino acid sequence of SEQ ID NO:8. In one embodiment, the composition comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:8, or a fragment thereof. peptidase, provided that fragments thereof have aminopeptidase activity. In some embodiments, the composition comprises a polypeptide having the amino acid sequence of SEQ ID NO:8. nothing.

[0057] In some embodiments, the compositions contain specific nucleic acid homologs to the exemplified polynucleotides. In some embodiments, the composition comprises a polynucleotide having the sequence of SEQ ID NO:9, or SEQ ID NO:10, or SEQ ID NO:11, or SEQ ID NO:12, or SEQ ID NO:13, The polypeptide code of the nucleic acid sequence of SEQ ID NO:14, or SEQ ID NO:15, or SEQ ID NO:16. At least 50, 60, 65, 70, 75, 80, 85, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity In some embodiments, the polynucleotide comprises a nucleic acid sequence having the The tide is SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16 In other embodiments, the composition comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, or the sequence Column number 10, or SEQ ID NO: 11, or SEQ ID NO: 12, or SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, the composition comprises a polynucleotide having a complementary nucleic acid sequence. SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO: 5, SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:8 At least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or a recombinant polypeptide having an amino acid sequence having 100% identity thereto, or an activity thereof. In some embodiments, the polynucleotide comprises a nucleic acid sequence that encodes a specific fragment. In one embodiment, the composition comprises SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO: 4, or the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 The recombinant polypeptide or an active fragment thereof comprising an amino acid sequence selected from the group consisting of The present invention includes a polynucleotide comprising a nucleic acid sequence that encodes the nucleic acid sequence.

[0058] In some embodiments, the composition comprises an aminopeptidase, an amylase, a carboxylase, a glycerol ... Dolanase, carboxypeptidase, catalase, cellulase, chitinase, cutinase , cyclodextrin glycosyltransferase, deoxyribonuclease, enzyme Sterase, alpha-galactosidase, beta-galactosidase, glucoamylase alpha-glucosidase, beta-glucosidase, haloperoxidase, inbeta Lutase, laccase, lipase, mannosidase, oxidase, pectin decomposition enzyme, Peptide glutaminase, peroxidase, phytase, polyphenol oxidase , proteolytic enzymes, ribonucleases, transglutaminases or xylanases Further enzymes may be included in the Aspergillus sp. Aspergillus, preferably Aspergillus aculeatus uleatus, Aspergillus awamori i) Aspergillus niger or Aspergillus Aspergillus oryzae, or Trichoderma spp. Trichoderma, or Humicola, preferably Humicola Humicola insolens, or Fusarium spp. Fusarium, preferably Fusarium bactridioides bactridioides, Fusarium cere alis), Fusarium crookwellense ense), Fusarium culmorum, Fusari Fusarium graminearum, Fusarium graminearum raminum (Fusarium graminum), Fusarium heterosporum (Fu sarium heterosporum, Fusarium negunji negundi), Fusarium oxysporum um), Fusarium reticulatum, Fusarium Fusarium roseum, Fusarium sambusinum ( Fusarium sambucinum, Fusarium sarcochromium ium sarcochroum, Fusarium sporotrichioides um sporotrichioides, Fusarium sulphureum um sulphureum, Fusarium torurosum losum, Fusarium trichothecioides ecioides) or Fusarium venenatum It can be produced by microorganisms belonging to the Bacillus subtilis (Diptera).

[0059] In some embodiments, the invention provides a polypeptide having aminopeptidase activity, and and a suitable carrier. Any suitable carrier known in the art may be used. In another embodiment, the composition further comprises an endopeptide. In some embodiments, the composition further comprises one or more non-specifically acting enzymes. In some embodiments, the enzymes include endopeptidase and / or exopeptidase. In some embodiments, the composition further comprises one or more specifically acting endopeptidases and / or contains an exopeptidase enzyme.

[0060] In some embodiments, the specific acting proteolytic enzyme is glutamyl endo Peptidase (EC 3.4.21.19); Lysyl endopeptidase (EC 3.4 .21.50); ​​leucyl endopeptidase (EC 3.4.21.57); glycyl Endopeptidase (EC 3.4.22.25); Prolyl endopeptidase (EC 3.4.21.26); trypsin (EC 3.4.21.4) or trypsin-like (Lysine / Arginine specific) endopeptidase, or peptidyl-Asp metalloenzyme and endopeptidases such as endopeptidase (EC 3.4.24.33).

[0061] In some embodiments, the exopeptidase enzyme is a tripeptidyl aminopeptidase. aminopeptidases, dipeptidyl aminopeptidases, carboxypeptinases and other aminopeptidases The enzyme is selected from the group consisting of tidases.

[0062] In some embodiments, one or more endopeptidases and / or exopeptidases The enzymes are fungal acid endopeptidase, neutral metalloendopeptidase, and alkaline endopeptidase. Serine endopeptidase, subtilisin, bromelain, heat-stable bacterial neutral enzyme and alkaline serine endopeptidase (overlapping). can be.

[0063] In some embodiments, the endopeptidase and / or The exopeptidase enzyme is one or more proteases from one or more of the following commercially available products: That's fine.

[0064] [Table 1]

[0065] Additionally or alternatively, endopeptidase and / or exopeptidase enzymes. The ingredient may be contained in one or more of the following commercially available products: KANNA SE(trademark), NOVOCARNETM Tender , and Novozym 370 20, NOVO-PRO™ D (both available from Novozymes); Bio Sorb-ACDP (Noor Creations, India); ANGEL (Registered Trademark)Acid Protease(Angel Yeast Co, Ltd.,Chi na) or COROLASE® LAP (from AB Enzymes).

[0066] In some embodiments, the invention also provides methods for producing at least one of the aminopeptidases described herein. The present invention provides a feed additive composition and / or a food additive composition comprising at least one of the above-mentioned compounds.

[0067] In other embodiments, compositions and / or feed additive compositions comprising the hydrolysates of the present invention are and / or a food additive composition are provided. Preferably, such a food additive composition and and / or the feed additive composition further comprises an aminopeptidase (optionally an endoprotease In some embodiments, the glycerol may be present in combination with a glycerol-containing enzyme.

[0068] The enzyme-containing liquid may contain additives that improve the properties of the liquid composition. Non-limiting examples of additives include salts (e.g., alkali salts, earth metal salts, additional chloride salts, Sulfates, nitrates, carbonates (wherein examples of counterions are calcium, potassium and sodium ions), inorganic minerals or clays (e.g., zeolites, kaolin, ventona talc and / or silicates), carbohydrates (e.g., sucrose and / or starch), color pigments (e.g., titanium dioxide), biocides (e.g., RODALON ( (registered trademark), PROXEL (registered trademark) dispersant, defoamer, reducing agent, acidic agent, alkaline agent agents, enzyme stabilizers (polyols such as glycerol, propylene glycol, sorbitol, etc. , inorganic salts, sugars, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, and combinations thereof), enzyme inhibitors, preservatives (e.g., methylparaben, propylparaben, (preservatives such as benzoates, sorbates, or other food approved preservatives), and Excipients that may be used in the present preparations / compositions include maltitol, ... glucose syrup or dried glucose syrup starch, modified starch, gelatinized starch, L-lactic acid, ascorbyl palmitate, Tocopherol, lecithin, citric acid, citrate, phosphoric acid, phosphate, sodium alginate sodium, carrageenan, locust bean gum, guar gum, xanthan gum, pectin, Sodium carboxymethylcellulose, mono- and diglycerides, monoglycerides Citrate esters of glycerides and diglycerides, sucrose esters, carbon dioxide, alkanes Gon, helium, nitrogen, nitrous oxide, oxygen, hydrogen, and starch octenyl succinate As shown in Example 7 herein, the aminopeptide of the present invention can be used as a medicament for the treatment of osteoporosis. Tidase can retain enzymatic activity in compositions containing sodium chloride.

[0069] form The products and / or compositions of the present invention, whether used alone or included in a composition, Similarly, the aminopeptidases of the present invention can be used in any suitable form, e.g. In the food industry, when combined with exo- and / or endo-proteases food processing aids or food additives (i.e. food ingredients, functional food ingredients or pharmaceutical ingredients) The composition may be used in any form suitable for use as a component (such as a component of a pharmaceutical composition).

[0070] Suitable examples of forms include immediate release, delayed release, controlled release, sustained release, pulsed release, or the like. or tablets, pills, capsules, ovoids, solutions, or suspensions for controlled release applications. These may contain flavoring or coloring agents.

[0071] As an example, when the product and / or composition is used in the form of a tablet (functional component), When used as a tablet, the tablets also contain microcrystalline cellulose, lactose, citric acid, Excipients such as sodium carbonate, calcium carbonate, dibasic calcium phosphate, and glycine starch (preferably corn, potato, or tapioca starch); Sodium starch glycolate, croscarmellose sodium, and certain complexes Disintegrants such as silicates; polyvinylpyrrolidone, hydroxypropyl methylcellulose ( HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and Granulation binders such as gum acacia; magnesium stearate, stearic acid, behenic acid One or more lubricating agents, such as glyceryl and talc, may be included.

[0072] Examples of nutritionally acceptable carriers that may be used in preparing these dosage forms include, for example: , water, saline solution, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene Licorice, propylene glycol, liposomes, sugar, gelatin, lactose, amylose , magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fat Fatty acid monoglycerides and diglycerides, petroleum fatty acid esters, hydroxymethyl Examples of the copolymer include ethyl cellulose and polyvinylpyrrolidone.

[0073] Preferred excipients for the dosage form include lactose, starch, cellulose, milk sugar, or High molecular weight polyethylene glycols are included.

[0074] In the case of aqueous suspensions and / or elixirs, the aminopeptidase and / or The compositions may also contain various sweetening or flavoring agents, coloring substances or dyes, as well as emulsifying agents and and / or suspending agents, and also water, ethanol, propylene glycol, and glycerin. It may be used with any diluent, as well as combinations thereof.

[0075] Dosage forms include gelatin capsules, fiber capsules, and fiber tablets. Can be obtained.

[0076] method In some embodiments, the polypeptides of the present invention are useful in the production of protein hydrolysates. For example, to increase the degree of hydrolysis, it is necessary to remove the bitterness of protein hydrolysates in general. for enhancing flavor development, for the production of glutamate, and / or for the It can be used for other purposes such as nitrile production or FAN production during brewing.

[0077] The present invention further provides a method for the preparation of a protein-rich material comprising the steps of: The present invention relates to methods of using tides in combination with proteases (eg, endopeptidases). The method comprises treating a proteinaceous substrate with the polypeptide and an endopeptidase. The substrate may be treated with the enzymes simultaneously or sequentially.

[0078] The polypeptides of the present invention may be used in effective amounts conventionally used in protein hydrolysis processes. In some embodiments, the polypeptides of the invention are added to a proteinaceous substrate. Protein substrates contain approximately 0.1 to 100,000 aminopeptides per 100 g of protein. The range is from about 1 to about 10,000 aminopeptidase units, or about 100 g of protein. As defined herein, the amount of each aminopeptidase unit is in the range of 1 aminopeptidase unit. The APU was calculated by the synthesis of Ala-p-nitroanilide (Sigma Chemical 1 micromole of p-nitride per minute from the Ionic Co., St. Louis MO. The amount of enzyme required to release the peptide is called the aminopeptidase assay. Hydrolysis of the peptide substrate H-Ala-nitroanilide to release p-nitroanilide (pNA) The absorbance of pNA at a wavelength of 405 nm is measured using an ELISA reader. The reaction mixture was 180 μl of 20 mM CPB buffer, 15 μl of diluted enzyme and 20 μl of substrate. The reaction is carried out at 30°C. The CPB buffer is 20 mM citrate, 20 mM phosphate, The substrate was prepared by dissolving the buffer in 1 ml of DMSO (SI BACHEM in dimethyl sulfoxide (GMA, Catalog No. D2650) H-Ala-pNA (L-1070) 20 mg.

[0079] The endopeptidase may be derived from a Bacillus strain, preferably Bacillus rickettsiae. Bacillus licheniformis or Bacillus subtilis Bacillus subtilis strains, Staphylococcus lococcus strains, preferably Staphylococcus aureus Coccus aureus strains, Streptomyces, Preferably, Streptomyces thermovaleris vularis or Streptomyces griseus griseus strains, Aspergillus strains, preferably Aspergillus Aspergillus aculeatus, Aspergillus Aspergillus awamori, Aspergillus fetish Aspergillus foetidus, Aspergillus nidulans Aspergillus nidulans, Aspergillus niger illus niger) or Aspergillus oryzae oryzae strain, or Trichoderma strain, preferably Trichoderma Trichoderma reesei strain, Fusarium arium strains, preferably Fusarium venenatum In some embodiments, the endopeptidase can be obtained from a strain of A. LPHALASE® AFP, FOODPRO® PAL, FOODP RO® PNL, FOODPRO® Alkaline Proteases e, FOODPRO® PXT, FOODPRO® PBR, FOOD FOODPRO® PHT, FOODPRO® 30L, and FOODPRO® (registered trademark) 51FP.

[0080] The endopeptidase of the present invention is a useful enzyme that is conventionally used in protein hydrolysis processes. The effective amount is preferably in the range of about 0.05 to about 15 AU / 100 g protein, and more preferably The range of about 0.1 to about 8 AU / 100 g protein is added to the proteinaceous substrate. 1 AU (Anson unit) is the unit of measurement under standard conditions (i.e., 25°C, pH 7.5, and reaction time (10 min) per minute, the amount of phenol that produces the same color as 1 milligram equivalent of tyrosine It is defined as the amount of enzyme that digests hemoglobin at an initial rate that releases 100 TCA-soluble products. In some embodiments, the endoprotease is present in an amount of about 1000 to about 10000 mg / kg of protein substrate. About 10 to about 3000 mg of enzyme, e.g., per metric ton (MT) of protein substrate Amounts of between 0.01 and 3 g of enzyme may be administered.

[0081] Treatment with enzymes, i.e. incubation of the substrate with an enzyme preparation, results in the production of enzyme- The reaction is carried out at a convenient temperature at which the agent is not inactivated, preferably at a temperature in the range of about 20° C. to about 70° C. According to normal procedures, the enzyme preparation can be used to adjust the temperature of the incubation mixture to the temperature of the enzyme. By raising the temperature to a temperature at which the dye is inactivated, for example above about 70° C., or by The pH of the incubation mixture is lowered to a level at which the enzyme is inactivated, e.g., below about 4.0. By allowing the enzyme to react with the protein, it is possible to appropriately inactivate the protein.

[0082] Additionally, the methods of the present invention increase the degree of hydrolysis of proteinaceous substrates. When used in a protein synthesis, the degree of hydrolysis (DH) is the amount of protein hydrolyzed by a proteolytic enzyme. In one embodiment, the enzyme according to the present invention is The DH of the protein substrate is at least about 5, 7, 10, 12, 15, 17, 20, 22, 2 5, 27, 30, 35, 40, 45, or 50% increase or enhancement. This increase is relative to type 1 aminopeptidase, although any suitable comparison is possible. In a preferred embodiment, the protein hydrolysate is The content of Er, Asp, Asn, Pro, Cys, Ala and / or Gln, e.g. If the number of samples is 1.1, the number of samples increases.

[0083] In some embodiments, the protein hydrolysate has an increased content of Glu. In some embodiments, the protein hydrolysate is enriched in Leu. In an embodiment, the protein hydrolysate has an increased content of Gly. In some embodiments, the protein hydrolysate is enriched in Ser. In some embodiments, the protein hydrolysate is enriched in Asp. In some embodiments, the protein hydrolysate is enriched in Asn. The hydrolysate is enriched in Pro. In some embodiments, the protein hydrolysate is The hydrolysate is enriched in Cys. In some embodiments, the protein hydrolysate is In another more preferred embodiment, the protein hydrolysate has an increased Ala content. , the Gln content increases.

[0084] The aminopeptidase enzymes of the present invention can reduce inhibition by enzyme reaction products. As shown in Example 8 of the specification, the two PepN2 enzymes of the present invention are Leucine amine from Aspergillus oryzae RIB40 Nopeptidase 2 enzyme (NCBI reference sequence: XP_001819545.1) The results showed less product inhibition than the 5'-terminal ... [ka]

[0085] Thus, in one embodiment, the invention relates to SEQ ID NO: 17 (herein referred to as TRI063). (see, e.g., Examples) In particular, the present invention provides an isolated polypeptide having aminopeptidase activity. The isolated polypeptide having minopeptidase activity has a product concentration of greater than 2, 3, 4 or 5 mM. In a preferred embodiment, the compound has an aminopeptidase activity. The isolated polypeptide is a type 2 aminopeptidase enzyme. Thus, the type 2 aminopeptidase enzyme is any one of SEQ ID NOs: 1-8 as defined herein. , in particular as shown in SEQ ID NO: 1 or 5.

[0086] The aminopeptidase enzyme of the present invention has a 2-position numbered from the N-terminus. It may be capable of hydrolyzing polypeptides having proline residues. Aminopeptidase enzymes contain a proline residue at position 2 from the N-terminus. A polypeptide having the same activity was added to a concentration of more than half the starting concentration for a 2-hour incubation. The degree of hydrolysis that can be achieved by hydrolysis can be determined by any suitable protocol known in the art. For example, it can be measured by the method shown in Example 10. As shown in Example 10, The two PepN2 enzymes of the present invention (TRI032 and TRI035) are TPAAR was slowly hydrolyzed to less than half its concentration after 2 hours of incubation. However, TRI063 (Aspergillus oryzae (A. oryzae)) was not able to inhibit the oxidative stress. e)) and COROLASE® LAP are capable of activating TPAAR within 12 hours. No hydrolysis is observed.

[0087] The present invention also relates to free glutamic acid and / or peptides having glutamic acid residues attached thereto. A method for obtaining a hydrolysate enriched in aminopeptidase, comprising: The present invention also relates to a method comprising exposing free glutamine or free glutamine to the action of a peptide. Free glutamic acid and / or oligopeptides containing glutamine or glutamic acid residues A method for obtaining a peptide-rich hydrolysate, comprising: The present invention relates to a method comprising exposing the subject to the action of a polypeptide.

[0088] The present invention also provides a method for removing the bitterness of a protein hydrolysate, comprising: The method further comprises exposing the subject to the action of a polypeptide having peptidase activity.

[0089] In some embodiments, the method further comprises subjecting the substrate to a deamidation process. The deamidation process involves converting a substrate into a polypeptide having aminopeptidase activity. This can be done simultaneously with, before or after exposure to the action of the tide.

[0090] In some embodiments, the methods of the invention provide a method for the production of ribozymes, whether free or bound. Whether it is an oligopeptide or not, glutamic acid (Glu) is responsible for the flavor and taste of protein hydrolysates. Because it plays an important role in flavor, protein hydrolysates with enhanced flavors are produced. In some embodiments, the method provides improved functionality, particularly improved solubility and emulsification. , a protein hydrolysate with increased degree of hydrolysis and improved foaming properties is also produced. .

[0091] The release of ammonia converts the amide (glutamine or asparagine) to a charged acid (glutamine). The conversion of the carboxyl group to a non-carboxyl group (such as carboxylic acid or aspartic acid) is known as deamidation. It may be carried out as an enzymatic deamidation process or as an enzymatic deamidation process.

[0092] In some embodiments, deamidation can be achieved by an enzymatic deamidation process, e.g., by deamidating a substrate to Exposure to glutaminase, transglutaminase and / or peptidoglutaminase This is done by:

[0093] In some embodiments, the glutaminase is glutaminase SD-C100STM ( Amano, Japan).

[0094] In some embodiments, glutaminase is present at about 1 mg to 2 mg per gram of substrate protein. In some embodiments, glutaminase can be administered in an amount of 0 mg. It can be administered in an amount of about 5 mg to 15 mg per gram of protein. In the form, glutaminase should be administered in an amount of approximately 10 mg per gram of substrate protein. can be done.

[0095] The present invention also provides a method for producing glutamate, comprising: The method further comprises exposing the subject to the action of a polypeptide having putidase activity.

[0096] The transglutaminase may be from any convenient source, such as a mammalian source (e.g., J. P1050382 and JP5023182), activated factor XIII (See, for example, WO 93 / 15234) (see, e.g., EP 555,649), or derived from a microorganism (e.g. , EP379,606, WO 96 / 06931, WO 96 / In some embodiments, the transformer may be a Glutaminase can be produced from oomycetes, e.g., phytophthora sp., preferably or Phytophthora cactorum strains, Alternatively, the genus Pythium, preferably Pythium irregulare um irregulare strains, Pythium spinosum strains , Pythium intermedium strain, Pythium Pythium ultimum strain, or Pythium periirum (Pythium periilum) (or Pythium periclocum) In some embodiments, the trans The glutaminase is of bacterial origin, preferably from the genus Bacillus. Bacillus subtilis strain, Streptoverte Streptoverticillium, preferably Streptoverticillium Streptoverticillium mobaensis raensis strain, Streptoverticillium griseoverticillatum (Str eptoverticillium griseoverticillatum strain, or Streptoverticillium cinnamoneum ium cinnamoneum strains, and Streptomyces sp. ces, preferably Streptomyces lydicus dicus strain.

[0097] Peptidoglutaminase is a type of enzyme that is involved in the synthesis of peptidoglutaminase I (peptidyl-glutaminase EC.3.5.1.43), or peptidoglutaminase II (protein-glutaminase EC.3.5.1.44) or a mixture of these. Peptide glutaminase may be used in the preparation of Aspergillus sp. ), preferably Aspergillus japonicus us strain, Bacillus spp., preferably Bacillus circans Cryptococcus circulans strains, s), preferably Cryptococcus albidus dus, or Debaryomyces, preferably Debaryomyces Obtained from Debaryomyces kloecheri strain can be done.

[0098] Transglutaminase is traditionally used for protein substrates in the deamidation process. The effective amount is preferably in the range of about 0.01 to about 5% by weight of the enzyme preparation relative to the amount of the substrate, and more preferably in the range of about 0.01 to about 5% by weight of the enzyme preparation relative to the amount of the substrate. Preferably, it is added in the range of about 0.1 to about 1% by weight.

[0099] Peptidoglutaminase is traditionally used for protein substrates in the deamidation process. The effective amount is preferably about 0.01 to about 100,000 peptide groups per 100 g of substrate. More preferably, about 0.1 to about 10,000 peptide glutaminase units. The amount of amine added is in the range of amine units.

[0100] Peptidoglutaminase activity was determined according to the method of Cedrangoro et al. (1965, E It can be measured according to the procedure in the Journal of Nutrition and Nutrition Vol. 29, p. 143. In this procedure, 0.5 ml of enzyme sample adjusted to pH 6.5 with 1N NaOH is Put the mixture into a small container. Then add 1 ml of a pH 10.8 borate buffer to the container. The released ammonia is absorbed with 5N sulfuric acid and the mixture is then cooled using Nessler's reagent. The color is developed and measured at 420 nm. One peptidoglutaminase unit is This is the amount of enzyme that can produce 1 micromole of ammonia per minute.

[0101] The present invention also provides a method for producing free amino nitrogen (FAN) during malting and / or brewing. The method comprises the steps of: converting a substrate into an aminopeptide during the malting and / or brewing process; The method relates to a method comprising exposing the subject to the action of a polypeptide having enzyme activity.

[0102] In some embodiments of the methods of the invention, the protein substrate is exposed to a polypeptide of the invention. The peptidoglutaminase of the present invention is capable of hydrolyzing proteins on a protein substrate. An effective amount conventionally used in the solution process, preferably about 0.001 to about 0.5 AU / l 00 g substrate, more preferably in the range of about 0.01 to about 0.1 AU / 100 g substrate. is added.

[0103] In another embodiment, the free glutamic acid and / or glutamic acid residues of the present invention The method for producing hydrolysates enriched in conjugated peptides involves treating the substrate with one or more non-specifically engineered peptides. This involves exposing the enzyme to endopeptidases and / or exopeptidases. This step may be carried out simultaneously with the step of exposing the protein substrate to the polypeptide of the present invention, and then You may go to.

[0104] In a more preferred embodiment, non-specifically acting endopeptidases and / or endopeptidases are The xopeptidase enzyme is derived from Aspergillus strains, or from Bacillus subtilis. It is obtained from a Bacillus strain.

[0105] Non-specifically acting endopeptidase and / or exopeptidase enzymes An effective amount conventionally used in protein hydrolysis processes, preferably in the range of about , is added to the substrate.

[0106] In some embodiments, the endopeptidase and / or exopeptidase is About 50 to about 3000 mg of enzyme per kg of protein substrate, e.g. It can be administered in amounts of 0.05 to 3 g of enzyme per metric ton (MT).

[0107] Suitably, the endopeptidase and / or exopeptidase is a protein substrate. It may be administered in amounts of less than about 4.0 g of enzyme per MT.

[0108] In another embodiment, the endopeptidase and / or exopeptidase is The enzyme can be administered at about 0.5 g to about 5.0 g per 1 MT of protein substrate. endopeptidases and / or exopeptidases act on the protein substrate 1MT The enzyme can be administered at a dose of about 0.5 g to about 3.0 g per unit time. Protease is dosed at about 1.0 g to about 2.0 g of enzyme per MT of protein substrate. It can be given.

[0109] In some embodiments, the polypeptides of the invention are The enzyme is administered in an amount of about 0.5 mg to about 2 g per kg of the food and / or feed additive composition. Suitably, the polypeptide of the present invention can be administered to a protein substrate and / or or about 1 mg to about 2 g of enzyme per kg of food and / or feed additive composition. More preferably, the protein substrate and / or the food and / or The amount of enzyme is about 5 mg to about 1.5 g per kg of the feed additive composition.

[0110] In the preparation of the hydrolysate, the polypeptide of the invention is present at about 1000 g / kg of protein substrate. The amount of the enzyme can be administered in the range of 0.5 mg to about 2 g. The enzyme can be administered in an amount of about 1 mg to about 2 g per kg of protein substrate. More preferably, the amount of enzyme is about 5 mg to about 1.5 g per kg of protein substrate.

[0111] In one embodiment, the polypeptide of the invention is present at about 5 mg / kg of protein substrate. The polypeptide of the present invention can be administered in an amount of about 500 mg of enzyme. The enzyme can be administered in amounts of about 50 mg to about 500 mg per kg of protein substrate. Preferably, the polypeptide of the present invention is present in an amount of about 100 mg to about 4 mg per kg of protein substrate. A dose of 50 mg of enzyme can be administered.

[0112] Each treatment with an enzyme is carried out at a temperature at which the enzyme preparation is not inactivated, preferably at about 20° C. to about 70° C. The enzyme preparation can then be heated to a temperature in the range of about 70° C. or by lowering the pH, for example, to less than about 4.0. It can be inactivated by

[0113] The proteinaceous substrates used in the method of the present invention may be intact proteins, prehydrolyzed proteins, It may be composed of proteins (i.e., peptides), or mixtures thereof. The porous substrate may be of vegetable or animal origin. In the form, the proteinaceous substrate may be of vegetable origin, e.g., soy protein, cereal protein, Proteins, such as wheat gluten, corn gluten, barley, rye, oats, rice, Zein, lupin, cottonseed protein, peanut, alfalfa protein, ene Bean protein, fabaceous bean protein Proteinaceous substrates of animal origin include hominid proteins, sesame proteins, and sunflower proteins. A protein, casein, meat protein, fish protein, red blood cells, egg whites, gelatin, It may be lactalbumin, hair protein or feather protein.

[0114] The invention also relates to the protein hydrolysates produced by these processes.

[0115] Preparation of nucleotide sequences Proteins having particular properties as defined herein or suitable for modification The nucleotide sequence encoding the protein may be obtained from any cell or organism that produces said protein. It can be identified and / or isolated and / or purified. Various methods are available for the identification and / or isolation and / or purification of nucleotide sequences. It is well known. By way of example, once suitable sequences have been identified and / or isolated and / or purified, PCR amplification techniques can be used to prepare larger numbers of sequences.

[0116] As a further example, chromosomal DNA or messenger RNA from the organism that produces the enzyme. A genomic DNA and / or cDNA library can be constructed using Once the amino acid sequence of the enzyme is known, a labeled oligonucleotide probe can be synthesized and used to to identify enzyme-encoding clones from a genomic library prepared from the organism. It is possible. Alternatively, a labeled oligonucleotide probe containing a sequence homologous to another known enzyme gene may be used. The probe can be used to identify clones that encode enzymes. Low stringency hybridization and washing conditions are used.

[0117] Alternatively, the enzyme-encoding clone may be incorporated into an expression vector such as a plasmid carrying the genomic DNA. The fragment of A was inserted, and the resulting genomic DNA library was transformed into enzyme-negative bacteria. The transformed bacteria are then cultured in a broth containing a substrate for the enzyme (i.e., maltose). The transformed bacteria are plated on agar plates, which identifies the clones expressing the enzyme. This can be done by enabling the identification of the loan.

[0118] In yet a further alternative, the nucleotide sequence encoding the enzyme is selected from established standards. Semi-standard methods, e.g., Beucage SLet al., (1981) Tetrah Phosphoramida described in Edron Letters 22, p 1859-1869 or Matthes et al., (1984) EMBO J.3, p80 The phosphoramidite can be prepared by synthesis according to the method described in US Pat. In the method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, and , annealed, ligated and cloned into an appropriate vector.

[0119] Nucleotide sequences may be derived by standard techniques, from synthetic, genomic or cDNA sources (as appropriate). Mixed genomic and synthetic origins, prepared by ligating pieces of mixed synthetic and c The ligated fragments may be of DNA origin, or of mixed genomic and cDNA origin. Each fragment corresponds to a different portion of the overall nucleotide sequence. No. 4,683,202 or Saiki RK et al., (S As described in the 1988 239, pp 487-491 It can be prepared by polymerase chain reaction (PCR) using primers.

[0120] Amino acid sequence The scope of the present invention also includes the amino acid sequences of enzymes having the specific properties defined herein. It is included.

[0121] As used herein, the term "amino acid sequence" is intended to be used interchangeably with the terms "polypeptide" and / or "amino acid sequence." or the term "protein." In some instances, the term "amino acid sequence," the term " In some instances, the term "amino acid sequence" is synonymous with the term "enzyme." It is.

[0122] The amino acid sequence can be prepared / isolated from a suitable source or can be produced synthetically. It may be prepared by the use of recombinant DNA techniques.

[0123] Proteins encompassed by the present invention include other proteins, particularly proline endoproteases. , tripeptidyl exopeptidases, and other forms of endoproteases or exopeptidases. Thus, the present invention also relates to a method for the preparation of a protein comprising the steps of: A combination of an aminopeptidase of the invention and another aminopeptidase of the invention. This aspect covers combinations including other enzymes that may be We will consider this issue.

[0124] Preferably, in the context of the present invention, the amino acid sequences encompassed within the scope of the present invention itself are naturally occurring. In this regard, the term "native enzyme" refers to an enzyme that is present in its natural environment and is not an enzyme. It refers to the entire enzyme expressed from the native nucleotide sequence.

[0125] Sequence identity or homology The present invention also provides polypeptides having the amino acid sequences defined herein that have particular properties. or a polypeptide having a degree of sequence identity or homology to This includes the use of any nucleotide sequence encoding Herein, the term "homologue" refers to a homologue of the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" is synonymous with "identity." It can be considered as being.

[0126] Homologous amino acid and / or nucleotide sequences are conferred with respect to the functional activity of aminopeptidases. and / or improve the activity of aminopeptidase. and / or code.

[0127] In the present context, a homologous sequence is at least 75, 85 or 90% identical to the subject sequence, Preferably, it is understood to include an amino acid sequence that may be at least 95 or 98% identical. Typically, a homolog will contain the same active site as the target amino acid sequence. Consider in terms of similarity (i.e., amino acid residues with similar chemical properties / functions) Although it is possible to express homology in terms of sequence identity, in the context of the present invention it is preferred to express homology in terms of sequence identity. I wish.

[0128] In the present context, a homologous sequence is a nucleotide sequence that encodes a polypeptide of the invention. At least 75, 80, 85 or 90% identical to (the subject sequence), preferably at least It is understood to include nucleotide sequences that may be 95 or 98% identical. Homology is the ability to measure similarity between two sequences, including sequences encoding the same active site, etc. It can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions). However, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0129] Homology comparisons can be performed by eye, or, more usually, using readily available sequence comparison programs. These commercially available computer programs can be used to The percentage of homology between the above sequences can be calculated.

[0130] Percent homology can be calculated over a contiguous sequence, i.e., comparing one sequence to another. Align sequences and compare each amino acid in one sequence with the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Such ungapped alignments are performed only over a relatively small number of residues.

[0131] This is a very simple and consistent method, but for example, It is not possible to take into account that the insertion or deletion may cause subsequent amino acid residues to be out of alignment. Therefore, it is possible that the homology rate will be significantly reduced when performing a global alignment. For this reason, most sequence comparison methods unduly penalize the overall homology score. It is designed to produce optimal alignments that take into account possible insertions and deletions without quantification. This is done by inserting "gaps" in the sequence alignment to maximize local homology. This is achieved by attempting

[0132] However, these more complex methods attempt to isolate as many identical amino acids as possible. Sequence alignment with fewer gaps (reflecting a higher degree of relatedness between the two compared sequences) The alignment is then adjusted so that the alignment with the most gaps achieves a higher score than the alignment with the most gaps. A "gap penalty" is assigned to each gap that occurs in a It charges a relatively high cost for the presence of a gap and a higher cost for each subsequent residue in the gap. The "affine gap cost" that imposes the smallest penalty possible is used. A commonly used gap scoring system. High gap penalties are It will, however, produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparisons, the default values ​​should be used. It is preferred to use

[0133] Therefore, the calculation of maximum homology percentage first involves finding the optimal algorithm taking into account gap penalties. It is required to create an alignment. The computer program used was Vector NTI (Invitrogen Corp.). Examples of software capable of performing sequence comparisons include, for example, the BLAST package ( Ausubel et al.1999,Short Protocols in Mo Leucine Biology, 4th Ed-Chapter 18), BLA ST 2(FEMS Microbiol Lett 1999 174(2):247 -50;FEMS Microbiol Lett 1999 177(1):187- 8 and tachiana@ncbi.nlm.nih.gov), FASTA ( Altschul et al, 1990 J.Mol.Biol.403-410), and AlignX. LAST 2 and FASTA are available for offline and online searches ( (see Ausubel et al. 1999, supra, pp. 7-58-7-60).

[0134] The final homology percentage can be measured in terms of identity, but the alignment process itself The body is not usually based on all-or-nothing pairwise comparisons. Instead, it is based on chemical A number that assigns a score to each pairwise comparison based on its functional similarity or evolutionary distance. A scaled similarity score matrix is ​​commonly used. An example of such a matrix commonly used is the BLOSUM62 matrix (BLAST programs). (default matrix for the suite). Vector NTI programs are generally , using the public default values ​​or a custom symbol comparison table if supplied. (See the user manual for further details). For some applications, It is preferable to use the default values ​​for Vector NTI package.

[0135] Alternatively, the homology ratio can be calculated using the CLUSTAL (Higgins DG & Sharp P Based on an algorithm similar to that of M (1988), Gene 73(1), 237-244 Multiple alignment using Vector NTI (Invitrogen Corp.) based on The feature can be calculated using the

[0136] Once the software has produced an optimal alignment, the percentage of homology, preferably sequence identity, is calculated. It is possible to calculate the ratio of homology to homology between two nucleotides; the software typically does this as part of the sequence comparison. The algorithm performs the calculations and produces a numerical result.

[0137] If gap penalties are used when determining sequence identity, pairwise alignment is It is recommended to use the following parameters for the alignment:

[0138] [Table 2]

[0139] In one embodiment, CLUSTAL is a gap penalty set as defined above. It can be used with tees and gap extensions.

[0140] Preferably, the degree of identity for nucleotide sequences is at least 20 consecutive nucleotides. nucleotides, preferably at least 30 contiguous nucleotides, The sequence may span at least 40 contiguous nucleotides, preferably at least 50 contiguous nucleotides. over consecutive nucleotides, preferably over at least 60 consecutive nucleotides, Preferably, it is determined over at least 100 contiguous nucleotides.

[0141] Preferably, the degree of identity for nucleotide sequences is determined over the entire sequence. can.

[0142] Mutants / homologues / derivatives The present invention also relates to any amino acid sequence of a protein, or a polypeptide encoding such a protein. The present invention encompasses the use of variants, homologues and derivatives of any nucleotide sequence which

[0143] As used herein, the term "homolog" refers to a subject amino acid sequence and a subject nucleotide sequence. Here, the term "homology" is synonymous with "identity." It can be considered as being.

[0144] In the present context, a homologous sequence is one that is at least 75, 80, 85 or 90% identical to the subject sequence. Amino acids that may be identical, preferably at least 95, 96, 97, 98 or 99% identical. A homologue is generally understood to include a sequence of amino acids that is identical to the amino acid sequence of interest, such as an active site. Homology would include similarity (i.e., amino acids with similar chemical properties / functions). In the context of the present invention, the term "sequence identity" can be used to refer to the sequence identity of the corresponding amino acid residues. It is preferred to express homology.

[0145] In the present context, a homologous sequence is a nucleotide sequence that encodes an enzyme of the invention (the target sequence 75, 80, 85 or 90% identical, preferably at least 95, 9 It is understood to include nucleotide sequences which may be 6, 97, 98 or 99% identical. A homologue would contain sequences that code for the same active site, etc. as the subject sequence. Consider in terms of similarity (i.e., amino acid residues with similar chemical properties / functions) Although it is possible to express homology in terms of sequence identity, in the context of the present invention it is preferred to express homology in terms of sequence identity. I wish.

[0146] Homology comparisons can be performed by eye, or, more usually, using readily available sequence comparison programs. These commercially available computer programs can be used to The percentage of homology between the above sequences can be calculated. The percentage of homology is calculated over the contiguous sequence. That is, one sequence can be aligned with another sequence and each It directly compares amino acids with corresponding amino acids in other sequences, one residue at a time. Such ungapped alignments are usually called "gapped" alignments. It is performed only over a relatively small number of residues.

[0147] This is a very simple and consistent method, but for example, It is not possible to take into account that the insertion or deletion may cause subsequent amino acid residues to be out of alignment. Therefore, it is possible that the homology rate will be significantly reduced when performing a global alignment. For this reason, most sequence comparison methods unduly penalize the overall homology score. It is designed to produce optimal alignments that take into account possible insertions and deletions without quantification. This is done by inserting "gaps" in the sequence alignment to maximize local homology. This is achieved by attempting

[0148] However, these more complex methods attempt to isolate as many identical amino acids as possible. Sequence alignment with fewer gaps (reflecting a higher degree of relatedness between the two compared sequences) The alignment is then adjusted so that the alignment with the most gaps achieves a higher score than the alignment with the most gaps. A "gap penalty" is assigned to each gap that occurs in a It charges a relatively high cost for the presence of a gap and a higher cost for each subsequent residue in the gap. The "affine gap cost" that imposes the smallest penalty possible is used. A commonly used gap scoring system. High gap penalties are It will, however, produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparisons, the default values ​​should be used. It is preferable to use, for example, the GCG Wisconsin Bestfit package. When using a gap page, the default gap penalty for amino acid sequences is The loop is -12 and each extension is -4.

[0149] Therefore, the calculation of maximum homology percentage first involves finding the optimal algorithm taking into account gap penalties. It is required to create an alignment. The computer program is the GCG Wisconsin Bestfit package (Dev ereux et al 1984 Nuc. Acids Research 12 p 387). Other examples of software capable of performing sequence comparisons include, for example, BLA ST package (Ausubel et al., 1999, see above), FASTA (Altschul et al.,1990 J.Mol.Biol.403-410 ) and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searching. (Ausubel et al., 1999, see above). However, some In practice, the GCG Bestfit program is preferably used. 2 A new tool called Sequences also analyzes protein and nucleotide sequences. Can be used to compare columns.

[0150] The final homology percentage can be measured in terms of identity, but the alignment process itself The body is not usually based on all-or-nothing pairwise comparisons. Instead, it is based on chemical A number that assigns a score to each pairwise comparison based on its functional similarity or evolutionary distance. A scaled similarity score matrix is ​​commonly used. An example of such a matrix commonly used is the BLOSUM62 matrix (BLAST programs). The GCG Wisconsin program uses Generally, the public default value or a custom symbol comparison if supplied. Use a table (see the user manual for further details). In some applications, The public default values ​​for the GCG package, or in the case of other software It is preferred to use default matrices such as BLOSUM62.

[0151] Alternatively, the homology ratio can be calculated using the CLUSTAL (Higgins DG & Sharp P DNASIS™ (based on an algorithm similar to that of M (1988), see above) The multiple alignment features are calculated using the Hitachi Software It is possible.

[0152] Once the software has produced an optimal alignment, the percentage of homology, preferably sequence identity, is calculated. It is possible to calculate the ratio of homology between nucleotides to homology between nucleotides; the software typically does this as part of the sequence comparison. The algorithm performs the calculations and produces a numerical result.

[0153] The sequences may be modified by the deletion of amino acid residues which produce a silent change and result in a functionally equivalent substance. Intentional amino acid substitutions may also be made so as to preserve the secondary binding activity of the substance. The polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues may be varied as long as they are maintained. For example, negatively charged amino acids include asparagus, selenium, and arginine. positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include: Leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, selenite Phosphorus, threonine, phenylalanine and tyrosine.

[0154] Conservative substitutions can be made, for example, according to the following table: Preferably, amino acids in the same line in the third column may be substituted for each other:

[0155] [Table 3]

[0156] The present invention also relates to possible homologous substitutions (substitutions and replacements). As used herein, all of the terms "replacement" and "substitution" refer to replacement of existing amino acid residues. is used to mean replacing a basic residue with a non-basic one. This includes homogeneous substitutions such as acidic for acidic, polar for polar, etc. Non-homologous substitutions, i.e., substitutions of one class of residue with another, or ornithine ( (hereinafter referred to as Z), ornithine diaminobutyrate (hereinafter referred to as B), norleucine ornithine Nitin (hereinafter referred to as O), pyridylalanine, thienylalanine and phenylgly Substitutions involving the incorporation of unnatural amino acids such as syn are also possible.

[0157] Replacement can also be with unnatural amino acids, such as , Alpha* and Alpha-disubstituted* Amino Acids, N-Alkyl Amino Acids*, Lactic Acid*, Natural Halogenated derivatives of amino acids, e.g., trifluorotyrosine*, p-Cl-phenyl Lanine*, p-Br-Phenylalanine*, pI-Phenylalanine*, L-Allyl- Glycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α- Aminoisobutyric acid*, L-ε-aminocaproic acid # , 7-aminoheptanoic acid*, L-methionine Ninsulfone * , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine Lanine*, L-Hydroxyproline # , L-thioproline*, phenylalanine (Phe ) methyl derivatives, such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe* e(4-amino) # , L-Tyr(methyl)*, L-Phe(4-isopropyl)*, L -Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-dia amidopropionic acid and L-Phe(4-benzyl)*. The symbol * denotes derivatives. For the purposes of the above discussion (regarding homologous or non-homologous substitutions) it is used to indicate the hydrophobicity of while # is used to indicate the hydrophilicity of the derivatives, and #* indicates amphiphilicity. .

[0158] The mutated amino acid sequence can be inserted between any two amino acid residues of the sequence. spacer groups, for example alkyl groups, such as methyl, ethyl or propyl groups; , in addition to an amino acid spacer, such as a glycine or β-alanine residue. Further forms of the peptide include the presence of one or more amino acid residues in peptoid form, which will be readily apparent to those skilled in the art. It will be appreciated that, for the avoidance of doubt, "peptoid form" refers to a peptide having an α-carbon substituent that is It is used to refer to variant amino acid residues where the residue has a nitrogen atom rather than a carbon. Methods for preparing peptides in peptide form are known in the art, for example, Simon RJ t al., PNAS (1992) 89(20), 9367-9371, and Horw ell DC,Trends Biotechnol.(1995)13(4),132 -134 is known.

[0159] The nucleotide sequences used in the present invention include within their scope synthetic or modified nucleotides. Numerous different types of modifications to oligonucleotides can be made to the oligonucleotide. These include methylphosphonates and phosphorothioates. the oate backbone and / or the acridinyl group at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, the nucleic acids described herein may be modified by the addition of nucleic acid or polylysine chains. It is understood that the nucleotide sequence may be modified by any method available in the art. It should be understood that such modifications may adversely affect the in vivo activity or longevity of the nucleotide sequences of the present invention. It can be implemented to improve lives.

[0160] The present invention also relates to nucleotide sequences complementary to the sequences presented herein, or any of the This includes the use of any derivative, fragment or derivative of the sequence, where the sequence is complementary to a fragment thereof. , using the sequences as probes to identify similar coding sequences in other organisms, etc. It is possible.

[0161] Polynucleotides that are not 100% homologous to the sequences of the present invention but are within the scope of the present invention are Other variants of the sequences described herein can be obtained in a number of ways, for example by isolating a series of individual Probing DNA libraries made from individuals from different populations, for example In addition, other homologues can be obtained, and such homologues can be The antibodies and fragments thereof generally selectively hybridize to the sequences shown in the sequence listing herein. Such sequences can be obtained from cDNA libraries made from other animal species, or from other Genomic DNA libraries from animal species were probed using medium to high stringency PCR. under conditions using a probe containing all or part of any one of the sequences in the attached sequence listing. Similar considerations can be obtained by probing such libraries. Obtaining species homologs and allelic variants of the polypeptide or nucleotide sequences of the invention. This also applies to

[0162] Mutants and strain / species homologs also include variations that encode conserved amino acid sequences within the sequences of the invention. Degenerate PC using primers designed to target sequences within variants and homologs Conserved sequences can be obtained, for example, by comparing the sequence of a sequence from several variants / homologues. This can be predicted by aligning amino acid sequences. This can be implemented using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0163] The primers used in degenerate PCR contain one or more degenerate positions and have a known sequence. Lower than that used for cloning sequences using single sequence primers for sequences Used in stringent conditions.

[0164] Alternatively, such polynucleotides can be prepared by site-specific mutagenesis of the characterized sequences. This can be achieved by induction, for example by inducing a particular Silent codon sequence changes may be required to optimize codon preferences for the host cell. It may be useful to introduce restriction enzyme recognition sites or to synthesize a polynucleotide Other sequence changes may be desired to alter the properties or function of the polypeptide encoded by It may be possible to get caught in the trap.

[0165] The polynucleotides (nucleotide sequences) of the present invention can be used as primers, e.g., PCR primers. ampli?ers, primers for alternative ampli?cation reactions, probes, e.g., radioactive or non-radioactive Use the label to produce a product labeled with the label by conventional means. Alternatively, the polynucleotide may be cloned into a vector. Such primers, probes and other fragments should be at least 15, preferably at least At least 20, e.g., at least 25, 30 or 40 nucleotides in length, are encompassed by the term polynucleotide as used herein.

[0166] Polynucleotides, such as DNA polynucleotides and probes, according to the present invention may be recombinantly produced. It can be produced by hand, synthetically, or by any means available to one of skill in the art. They can also be cloned by standard techniques.

[0167] Generally, a primer is used to initiate a synthesis that involves the stepwise production of a desired nucleic acid sequence, one nucleotide at a time. Techniques for achieving this using automated techniques are described in the art. are readily available in the field.

[0168] Longer polynucleotides are generally synthesized using recombinant means, e.g., PCR (polymerase chain reaction). The primers are used to amplify the DNA produced using the cloning technique known as alpha-dimerase chain reaction (AML). A suitable cloning vector may be used so that the DNA can be cloned into a suitable cloning vector. It can be designed to contain restriction enzyme recognition sites.

[0169] Hybridization The present invention also relates to sequences complementary to the nucleic acid sequences of the invention, or to sequences of the invention or to the nucleic acid sequences of the invention. It encompasses sequences that are capable of hybridizing to sequences that are complementary to the sequence of.

[0170] The term "hybridization" as used herein means "the process by which a nucleic acid strand is hybridized with a base "The process of combining with a complementary strand through base pairing," and the polymerase chain reaction (PCR) technique This includes the process of amplification carried out in the procedure.

[0171] The present invention also provides nucleic acids capable of hybridizing to sequences complementary to the sequences presented herein. The present invention encompasses the use of the leutidine sequence, or any derivative, fragment or derivative thereof.

[0172] The term "variant" refers to a mutant that hybridizes to the nucleotide sequences presented herein. The term also includes sequences complementary to the sequences obtained.

[0173] Preferably, the term "variant" refers to a variant that is specific to the nucleotide sequences presented herein. Stringent conditions (e.g., 50°C and 0.2×SSC {1×SSC=0.15M NaCl, 0.015M Na Citrate 3 Can hybridize under pH 7.0 It includes sequences complementary to the sequences.

[0174] More preferably, the term "mutant" refers to a variant of the nucleotide sequence presented herein. High stringency conditions (e.g., 65°C and 0.1x SSC {1x SSC=0. 15M NaCl, 0.015M Na Citrate 3 Hybridization at pH 7.0 The term "antigen" includes sequences complementary to sequences which can be used to express the same or similar sequences.

[0175] The present invention also relates to the nucleotide sequences of the present invention (sequences complementary to the sequences presented herein). The present invention relates to a nucleotide sequence capable of hybridizing to a nucleic acid sequence comprising:

[0176] The present invention also relates to the nucleotide sequences of the present invention (sequences complementary to the sequences presented herein). The term "hybridizable nucleic acid" refers to a nucleotide sequence that is complementary to a sequence that can hybridize to a nucleic acid ... is complementary to a nucleic acid sequence that is complementary to a nucleic acid sequence that is complementary to a nucleic acid sequence that

[0177] The nucleotide sequences presented herein are capable of binding to the nucleic acid sequences under conditions of moderate to maximal stringency. Hybridizable polynucleotide sequences are also included within the scope of the present invention.

[0178] In a preferred embodiment, the present invention provides a method for the preparation of a fermentation product under stringent conditions (e.g., 50°C and A nucleotide sequence capable of hybridizing to the nucleotide sequence of the present invention under conditions of 0.2×SSC and 0.2×SSC. It covers the nucleotide sequence, or its complement.

[0179] In a more preferred embodiment, the present invention provides a method for the preparation of a nucleic acid sequence using high stringency conditions (e.g., 65 0.5° C. and 0.1×SSC) to hybridize to the nucleotide sequence of the present invention. The present invention covers the nucleotide sequence, or its complement, of any nucleotide sequence that can be used to identify a target gene.

[0180] molecular evolution As a non-limiting example, multiple site-specific generating differential or random mutations and then isolating the encoded It is possible to screen for improved functionality of a polypeptide that is

[0181] Furthermore, mutations or naturally occurring variants of a polynucleotide sequence may be referred to as wild type or other variants. Alternatively, the mutants can be recombined with naturally occurring mutants to produce new mutants. New mutants can also be screened for improved functionality of the encoded polypeptide. The generation of new preferred mutants is a well-established procedure in the art. Various methods, such as Error Threshold Mutagenesis, enesis) (International Publication WO 92 / 18645), oligonucleotide mediator mediated random mutagenesis (U.S. Pat. No. 5,723,323), DNA shuffling (U.S. Pat. No. 5,605,793), Exo-Mediated Gene Assembly, International Publication No. These and similar runs can be accomplished by The application of dam-directed molecular evolution methods allows the synthesis of proteins without any prior knowledge of protein structure or function. Thus, it is possible to predictably identify and select mutants of the enzymes of the invention that have favorable characteristics. Although the enzyme is not yet capable of producing a desired product, it is possible to produce beneficial mutations or variants. There are many examples of the application of molecular evolution to optimize or modify activity, such as: These may include, but are not limited to, one or more of the following: Optimizing expression and / or activity in a host cell or in vitro; Increased enzyme activity, altered substrate and / or product specificity; Increase or decrease enzyme stability or structural stability, favorable environmental conditions, e.g. temperature, pH, Alteration of enzyme activity / specificity on substrate.

[0182] Site-directed mutagenesis Once a protein-coding nucleotide sequence has been isolated or a putative protein-coding nucleotide sequence has been isolated, Once the polypeptide sequence has been identified, the sequence can be mutated to prepare the protein of the invention. In some cases, it may be desirable to

[0183] Mutations can be introduced using synthetic oligonucleotides. The nucleotide contains nucleotide sequences flanking the desired mutation site.

[0184] A suitable method is described in Morinaga et al., (Biotechnology (1 984)2, p646-649). Another method for introducing spontaneous mutations is described by Nelson and Long (Analytical Biochemistry (1989), 180, p147-151) do.

[0185] Recombinant In one embodiment, the sequences used in the present invention are recombinant sequences, i.e., recombinant D The sequences were prepared using NA technology.

[0186] These recombinant DNA techniques are within the skill of one of ordinary skill in the art. For example, J. Sambrook, E. Fritsch, and T. Maniat is, 1989, Molecular Cloning: A Laboratory M anual, Second Edition,Books 1-3,Cold Spr This is described in the Journal of Biotechnology and Life Sciences, 2013, 1:131–135.

[0187] compound In one embodiment, the sequences used in the present invention are synthetic sequences, i.e. sequences synthesized in vitro. Sequences prepared by chemical or enzymatic synthesis, including but not limited to However, host organisms such as the methylotrophic yeasts Pichia and Hansenulae are Sequences generated using optimal codon usage for Hansenula are listed. can be done.

[0188] Expression Enzyme expression The nucleotide sequences used in the present invention may be incorporated into a recombinant replicable vector. Vectors are used to replicate nucleotide sequences and express them in protein / enzyme form in compatible Can be used for expression in and / or from compatible host cells. Cut.

[0189] Expression can be controlled using control sequences, e.g., regulatory sequences.

[0190] The protein produced by a host recombinant cell upon expression of the nucleotide sequence may be used Depending on the sequence and / or vector used, it can be secreted or contained intracellularly. The coding sequence may direct the secretion of the substance coding sequence through a particular prokaryotic or eukaryotic cell membrane. The gene can be designed with a signal sequence that

[0191] Expression vector The term "expression vector" refers to a construct capable of expression in vivo or in vitro. It means .

[0192] Preferably, the expression vector is integrated into the genome of a suitable host organism. The term "being incorporated" preferably covers stable incorporation into the genome.

[0193] The nucleotide sequence of the present invention can be present in a vector operably linked to regulatory sequences that can provide expression of the nucleotide sequence by a suitable host organism.

[0194] The vector used in the present invention can be transformed into the following suitable host cells so as to provide expression of the polypeptide of the present invention.

[0195] The choice of vector, such as a plasmid, cosmid, or phage vector, often depends on the host cell into which it is to be introduced.

[0196] The vector used in the present invention can contain one or more selectable marker genes, such as genes conferring antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Alternatively, selection can be achieved by co-transformation (described in WO 91 / 17243).

[0197] The vector can be used, for example, for in vitro production of RNA, or for transfecting, transforming, transducing, or infecting host cells.

[0198] Accordingly, in a further embodiment, the present invention provides a method for producing the nucleotide sequence of the present invention by introducing the nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that result in replication of the vector.

[0199] ​​​​​​​​​​​ The vector may further comprise a nucleotide sequence that enables the vector to replicate in the host cell. Examples of such sequences include plasmids pUC19, pACYC177, pUB1 10, the origin of replication of pE194, pAMB1 and pIJ702.

[0200] Regulatory Sequences In some applications, the nucleotide sequences used in the present invention may be, for example, operably linked to a regulatory sequence capable of providing for expression of the nucleotide sequence by a host cell By way of example, the present invention provides a nucleotide sequence of the present invention operably linked to such a regulatory sequence. The term "vector" covers a vector containing the codon sequence, i.e., the vector is an expression vector.

[0201] The term "operably linked" refers to a combination of components that are in their intended manner. "Operably linked" to a coding sequence refers to a juxtaposition in which the coding sequence is in a functional relationship. The regulatory sequences are designed to label the coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. It will be igated.

[0202] The term "regulatory sequences" refers to promoters and enhancers and other expression control sequences. Includes signals.

[0203] The term "promoter" is used in its usual sense in the art, e.g. , an RNA polymerase binding site.

[0204] Enhancement of expression of a nucleotide sequence encoding an enzyme of the invention can also be achieved by the use of heterologous regulatory regions, e.g. This can be achieved, for example, by the selection of promoter, secretory leader and terminator regions. can.

[0205] Preferably, the nucleotide sequence according to the invention is operably linked to at least a promoter. They are combined.

[0206] Other promoters can also be used to direct expression of the polypeptides of the invention. .

[0207] A promoter suitable for directing transcription of the nucleotide sequence in a bacterial, fungal or yeast host. Examples are well known in the art.

[0208] The promoter may further comprise features to ensure or increase expression in a suitable host. For example, the features may include Pribnow boxes or TATA boxes. and the like.

[0209] construct The term "construct" (including "conjugate," "cassette," and "hybrid (synonymous with terms such as "promoter") refers to a molecule that is directly or indirectly attached to a promoter. The invention includes nucleotide sequences used in the invention.

[0210] An example of an indirect attachment is an intron between the promoter and the nucleotide sequence of the present invention. A suitable spacer group, such as the sequence, for example, the Sh1-intron or the ADH intron. The same applies to "fused" in the context of the present invention, including direct or indirect attachment. In some cases, these terms also refer to the wild-type gene promoter. A naturally occurring combination of nucleotide sequences encoding a protein normally associated with a promoter is It does not cover any of them occurring in their natural environment.

[0211] The construct may also contain or express a marker that allows for selection of the genetic construct. It can appear.

[0212] For some applications, the constructs of the invention preferably comprise at least a promoter. The present invention further comprises a nucleotide sequence of the present invention operably linked to

[0213] host cell The term "host cell" in the context of the present invention refers to a cell that is capable of expressing the above-mentioned nucleotide sequences or The vectors are used for the recombinant production of proteins having specific properties as defined herein. The term "cell" includes any cell that can be used in culture.

[0214] Thus, a further embodiment of the present invention is a nucleic acid encoding a nucleotide sequence expressing a protein of the present invention. The present invention provides a host cell transformed or transfected with the vector. and is selected to be compatible with, for example, prokaryotic (e.g., bacterial), fungal, yeast or plant cells. It could be.

[0215] Examples of suitable bacterial host organisms are gram-positive or gram-negative bacterial species.

[0216] The properties of the nucleotide sequence encoding the polypeptide of the present invention and / or the expression protein Depending on the desirability of further processing of the protein, yeast or other fungi may be used. Yeast hosts may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are poorly secreted from yeast cells. In some cases, they are not properly processed (e.g., the hyperglycosylation of yeast In these instances, a different fungal host organism should be selected.

[0217] The use of suitable host cells, such as yeast, fungal and plant host cells, is useful in the production of recombinant expression vectors of the present invention. Post-translational modifications (e.g., For example, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine phosphorylation of threonine or threonine.

[0218] The host cell is an aminopeptidase-deficient or aminopeptidase-minus strain. The present invention also provides a method for producing a mutant cell of a parent cell, comprising: The nucleic acid sequence that encodes the polypeptide or its control sequence is destroyed or deleted, thereby The method further comprises reducing the amount of the polypeptide produced by the host cell compared to the parent cell.

[0219] The construction of a strain with reduced aminopeptidase activity was carried out by By modifying or inactivating a nucleic acid sequence required for the expression of a polypeptide which It can be done.

[0220] living thing The term "organism" in the context of the present invention refers to an organism that encodes a polypeptide according to the invention. It may comprise a nucleotide sequence and / or a product derived therefrom, and / or Any gene that, when present in an organism, can allow expression of the nucleotide sequence according to the invention. This includes living beings.

[0221] Suitable organisms may include prokaryotes, fungi, yeasts or plants.

[0222] The term "transgenic organism" in the context of the present invention refers to a polypeptide according to the invention. and / or products derived therefrom, and / or a promoter capable of allowing expression of the nucleotide sequence according to the invention in the organism. This includes any organism. Preferably, the nucleotide sequence is incorporated into the genome of the organism.

[0223] The term "transgenic organism" refers to an organism in which a natural nucleotide coding sequence is reproduced from its natural promoter. When a gene is under the control of a promoter (which is also present in its natural environment), It does not cover the nucleotide coding sequence.

[0224] Thus, the transgenic organism of the present invention comprises a polypeptide encoding the polypeptide of the present invention. The nucleotide sequence according to the present invention, the construct according to the present invention, the vector according to the present invention, a plasmid according to the invention, a cell according to the invention, a tissue according to the invention, or one of their products, or a combination thereof.

[0225] For example, transgenic organisms can also contain a polypeptide of the invention under the control of a heterologous promoter. The polypeptide may include a nucleotide sequence encoding the polypeptide.

[0226] Transformation of host cells / organisms As mentioned above, the host organism can be a prokaryotic or eukaryotic organism. Examples of suitable prokaryotic hosts include The following bacteria are considered to be pathogenic: Escherichia coli (E. coli) and Bacillus subtilis (Bacil lus subtilis).

[0227] Techniques for transformation of prokaryotic hosts are well described in the art, see, for example, S. See Ambrook et al. (supra). When prokaryotic hosts are used, The nucleotide sequence must be suitably modified prior to transformation, for example by removal of introns. is possible.

[0228] Filamentous fungal cells can be cultured by a variety of methods known in the art, for example, by protoplast culture in a known manner. A process involving plastogenesis and transformation of protoplasts, followed by cell wall regeneration. The host microorganism can be transformed by the method of the genus Aspergillus. The use of Streptococcus gillus is described in EP 0238023.

[0229] Other host organisms may be plants. A summary of the general techniques used in plant transformation can be found in Potrykus(Annu Rev Plant Physiol Plant Mo l Biol

[1991] 42:205-225) and Christou (Agro- Food-Industry Hi-Tech March / April 1994 1 7-27). Further teachings on plant transformation can be found in the European Patent This can be found in U.S. Pat. No. 0449375.

[0230] General teachings regarding transformation of fungi, yeast and plants are presented in the following sections. .

[0231] The fungus to be transformed The host organism may be a fungus, such as a mold. Examples of suitable such hosts include Serratia cerevisiae. Thermomyces, Acremonium, Aspergillus, Penicillinium um), Mucor, Neurospora, Trichomonas Examples include members of the genus Trichoderma.

[0232] In one embodiment, the host organism may be a filamentous fungus.

[0233] Transformation of filamentous fungi can be carried out using U.S. Pat. No. 5,741,696, which describes standard techniques for the transformation of filamentous fungi. 65, and fungal culture is well known in the art. For a comprehensive review of the techniques applied to Neurospora crassa, see, for example, Dav is and de Serres,Methods Enzymol(1971)17 A:79-143.

[0234] Further teachings, which may also be utilized for transforming filamentous fungi, are set forth in U.S. Pat. No. 5,674,700. This is outlined in specification no. 7.

[0235] Furthermore, gene expression in filamentous fungi was studied by Punt et al. (2002) Tren ds Biotechnol 2002 May;20(5):200-6,Arche r & Peberdy, Crit Rev Biotechnol (1997) 17( 4):273-306.

[0236] The present invention relates to transgenic plants according to the invention prepared by the use of these standard techniques. This includes the production of filamentous fungi.

[0237] In one embodiment, the host organism is a member of the genus Aspergillus, e.g. It may be Aspergillus niger.

[0238] The transgenic Aspergillus according to the present invention also comprises: For example, Turner, G. 1994 (Vectors for genetic ma nipulation.In:Martinelli SD,Kinghorn J .R.(Editors)Aspergillus:50 years on.Prog ress in industrial microbiology vol 29.E Based on the teachings of the paper "P. L. Amsterdam 1994, pp. 641-666" It can be prepared accordingly.

[0239] In one embodiment, the host organism is a member of the genus Trichoderma, e.g. It may be Trichoderma Reesei.

[0240] Yeast to be transformed In other embodiments, the transgenic organism may be a yeast.

[0241] For an overview of the principles of heterologous gene expression in yeast, see, for example, Methods Mol Biol. (1995), 49:341-54, and Curr Opin Biotechnol. (1997) Oct;8(5):554-60.

[0242] In this regard, yeasts such as Saccharomyces cerevisiae es cerevisiae or Pichia pastoris is) (FEMS Microbiol Rev (2000 24(1):45-66 Species such as Escherichia coli (see below) can be used as vehicles for heterologous gene expression.

[0243] Saccharomyces cerevisiae A review of the principles of heterologous gene expression and secretion of gene products in Escherichia coli is provided by E. Hinchcliff. e E Kenny(1993,“Yeast as a vehicle for t he expression of heterologous genes”,Yea sts,Vol 5,Anthony H Rose and J Stuart Ha rrison, eds, 2nd edition, Academic Press Lt. d.) is shown.

[0244] Several transformation protocols have been developed for the transformation of yeast. The transgenic Saccharomyces of the invention is nnen et al.,(1978,PNAS USA 75,1929);Begg s, JD (1978, Nature, 275, 104); and Ito, H et al. Based on the teachings of I (1983, J Bacteriology 153, 163-168) It can be prepared accordingly.

[0245] The transformed yeast cells may be modified with various selectable markers, e.g., auxotrophic markers, domi. Selection can be performed using a nano-antibiotic resistance marker.

[0246] Cultivation and production Host cells transformed with the nucleotide sequences of the present invention can be used to express the encoded polypeptides. and facilitating recovery of the polypeptide from the cells and / or culture medium. The cells can be cultured under conditions as described above.

[0247] The medium used to culture the cells is suitable for the growth of the host cells and the expression of polypeptides. The medium may be any conventional medium suitable for obtaining the present invention.

[0248] Proteins produced by recombinant cells can be displayed on the surface of the cells. Cut.

[0249] Proteins can be secreted from the host cells and removed from the culture medium by well-known procedures. It can be easily collected.

[0250] secretion The protein may be more easily recovered from the expression host by eluting it into the culture medium. In accordance with the present invention, a secretory leader sequence is provided to the desired The selection can be based on the expression host. Hybrid signal sequences are also relevant to the present invention. can be used.

[0251] A typical example of a heterologous secretory leader sequence is the fungal amyloglucosidase (AG) gene ( laA - e.g., 18 amino acid type from Aspergillus and 24 amino acid forms), the a-factor gene (yeast, e.g., Saccharomyces charomyces, Kluyveromyces and Ha Hansenula), or α-amylase genes (Bacillus It is derived from the fungus Cillus.

[0252] As an example, secretion of heterologous proteins in Escherichia coli (E. coli) is mediated by the Met Hods Enzymol (1990) 182:132-43.

[0253] detection A variety of protocols for detecting and measuring the expression of amino acid sequences are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RA), and These include immunoassays (RIA) and fluorescence activated cell sorting (FACS).

[0254] A wide variety of labeling and conjugation techniques are known by those skilled in the art and include various nucleic acid and and can be used in amino acid assays.

[0255] Many companies, such as Pharmacia Biotech (Piscataway, (New Jersey, United States), Promega (Madison, Wisconsin, United States), and US Biochemica Commercially available kits and their procedures are available from I.L. Corp (Cleveland, OH). The protocol is provided by

[0256] Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent agents, chemical These include luminescent or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, etc. Patents teaching the use of such labels include U.S. Pat. No. 3,817,837; U.S. Patent No. 850,752; U.S. Patent No. 3,939,350; U.S. Patent No. 3,996,34 No. 5; U.S. Patent No. 4,277,437; U.S. Patent No. 4,275,149 and U.S. Patent No. See, for example, US Pat. No. 4,366,241.

[0257] Recombinant immunoglobulins may also be used as described in U.S. Pat. No. 4,816,567. It can be produced as follows.

[0258] Fusion proteins The amino acid sequences used in the present invention may, for example, be used as fusion proteins to aid in extraction and purification. Examples of fusion protein partners include glutamate, glycerol, and glycerol. Thione-S-transferase (GST), 6×His, GAL4 (DNA binding and and / or transcription activation domains) and (β-galactosidase). The fusion tag contains a proteolytic cleavage site between the protein partner and the protein of interest. It may also be advantageous to allow removal of protein sequences.

[0259] Preferably, the fusion protein does not interfere with the activity of the protein sequence.

[0260] Gene fusion expression systems in Escherichia coli (E. coli) Reviewed in Biotechnol (1995) 6(5):501-6.

[0261] In another embodiment of the invention, the amino acid sequence is ligated to a heterologous sequence to form a fusion protein. For example, proteins can be encoded for drugs that can affect the activity of a substance. For screening of peptide libraries, heterologous epitopes recognized by commercially available antibodies were used. It may be useful to encode a chimeric entity that expresses the polypeptide.

[0262] Additional Proteins of Interest (POI) The sequences used in the present invention may also be used to encode one or more additional proteins of interest (POIs) or or in combination with a nucleotide sequence of interest (NOI).

[0263] Non-limiting examples of POIs include proteins or enzymes involved in starch metabolism, Proteins or enzymes involved in the metabolism of glycogen, including acetylesterase and aminopeptidase Glutamate, amylase, arabinase, arabinofuranosidase, carboxypeptidase ase, catalase, cellulase, chitinase, chymosin, cutinase, deoxyribonuclease ase, epimerase, esterase, α-galactosidase, β-galactosidase, α-glucanase, glucan lyase, endo-β-glucanase, glucoamylase, Glucose oxidase, α-glucosidase, β-glucosidase, glucuronidase , hemicellulase, hexose oxidase, hydrolase, invertase, isomerase tripeptidyl exopeptidase, laccase, lipase, lyase, mannosylase idase, oxidase, oxidoreductase, pectate lyase, pectin acetylase Esterase, pectin depolymerase, pectin methylesterase, pectin decomposition enzyme enzymes, peroxidase, phenol oxidase, phytase, polygalacturonase, Proline endoprotease, rhamnogalacturonase, ribonuclease, thaumatin , transferase, exoprotease, transport protein, transglutaminase Taminase, aminopeptidase, hexose oxidase (D-hexose:O 2 -O oxidoreductase, EC 1.1.3.5), or a combination thereof. The NOI may even be an antisense sequence of any of these sequences.

[0264] The POI may even be a fusion protein, for example to aid in extraction and purification.

[0265] The POI may even be fused to a secretory sequence.

[0266] Other sequences can also facilitate secretion or increase the yield of secreted POI. Such sequences can be found, for example, in the aspartate sequences described in UK Patent Application No. 9821198.0. Aspergillus niger cypB gene product The chaperone protein can be encoded by any of the following:

[0267] The NOI may be, for example but not limited to, involved in the processing of its expression product and / or They can be engineered to alter their activity for many reasons, including modifications that alter their expression. By way of further example, the NOI may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired for introducing restriction enzyme recognition sites.

[0268] NOIs may include synthetic or modified nucleotides, such as methylphosphonates and phospho It may contain a thiolate backbone.

[0269] The NOI may be modified to improve its intracellular stability and half-life. Possible modifications include: Examples of such methods include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends of the molecule. or phosphorothioate or phosphorothioate bonds rather than phosphodiesterase linkages in the molecular backbone. Examples include the use of 2'O-methyl.

[0270] General recombinant DNA technology The present invention relates to methods, techniques and methods of chemistry, molecular biology, microbiology, and the like, which are within the skill of those in the art, unless otherwise indicated. Conventional techniques of immunology, recombinant DNA and immunology are used. Such techniques are explained in the literature. For example, J. Sambrook, EF Fritsch, and T. Ma niatis,1989,MolecularCloning:A Laborator y Manual,Second Edition,Books1-3,Cold Sp ring Harbor Laboratory Press;Ausubel,FM .etal.(1995 and periodic supplements;Cur rent Protocols in Molecular Biology,ch.9 ,13,and,16,John Wiley & Sons,New,York,N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:Essential Tech niques,John Wiley & Sons;MJGait(Editor ),1984,Oligonucleotide Synthesis:A Pract ical Approach, Irish Press; and D.M. J. Lilley ,and JEDahlberg,1992,Methods of Enzymo. logy:DNA Structure Part A:Synthesis and Physical Analysis of DNA in Methods in E See also Enzymology, Academic Press. each of which is incorporated herein by reference. EXAMPLES

[0271] The present disclosure is further described in the following examples, which are provided to illustrate the principles of the present disclosure. The accompanying drawings are incorporated in the specification and description of this disclosure and are not intended to limit the scope in any way. The following examples are to be considered as part of the claimed disclosure. They are presented for purposes of clarification and are not intended to limit the disclosure.

[0272] Example 1 - TRI031, TRI032, TRI033, TRI034, TRI035, Cloning and transformation of TRI036, TRI037, and TRI038 For expression in Trichoderma reesei As a codon-optimized gene, fungal type 2 aminopeptidase (pepN_2, Merops family M28.008.; merops.sanger.ac.uk / ) Synthetic genes encoding (TRI031, TRI032, TRI033, TRI034, T RI035, TRI036, TRI037, TRI038), Geneart (Lif The order was received from Neo Sartoria Technologies. NCBI derived from Neosartorya fischeri NRRL181 Accession numbers: XP_001258675; TRI032 corresponds to Myceliophore Myceliophthora thermophila ATC NCBI accession number derived from C42464: XP_00366735 4; TRI033 corresponds to Fusarium oxysporum porum) Fo5176 NCBI accession number: EGU74500 TRI034 is a Fusarium oxysporum cubense specialised xysporum f.sp.cubense) race 1 NCBI accession Number: ENH69875; TRI035 corresponds to Aspergillus clavatus (Asp ergillus clavatus) NRRL1 NCBI accession number: XP_001273779; TRI036 corresponds to Chaetomium thermophilum (C Haetomium thermophilum var. thermophilum hilum) DSM1495 to NCBI accession number: EGS23402 TRI037 is a strain of Aspergillus terreus. us) NCBI accession number: XP_001217759 derived from NIH2624 and TRI038 is a mutant of Aspergillus nidulans. nidulans) FGSC A4 NCBI accession number: XP_6817 Corresponds to 14.

[0273] The genes contain Gateway-specific recombination sites (att The samples were ordered with attB1 and attB2 (Life Technologies). , pDonr221 Gateway vector (Life Technologies) Delivered as plasmid stocks in TRI031, TRI032, and TRI033. , TRI034, TRI035, TRI036, TRI037, and TRI038 amino acids The sequence is a predicted secretory signal sequence (SignalP 4.0: from the transmembrane domain to the signal peptide). Thomas Nordahl Petersen, Soren B Runak, Gunnar von Heijne & Henrik Nielse n.Nature Methods,8:785-786,2011), These endogenous signal sequences were replaced with leader sequences containing the Kozak sequence, Trichoderma lignin. from Trichoderma reesei acid fungal protease (AFP) and the secretory signal sequence of Trichoderma reesei ei) was replaced with an intron from the glucoamylase gene (TrGA1) (see Figure 1). (see).

[0274] LR CLONASE™ Enzyme Mix (Life Technologies) The synthetic gene in the pDonr221 vector was inserted into the destination vector tor pTrex8gM, and the expression vector pTrex8gM_TRI031( SEQ ID NO: 9), pTrex8gM_TRI032 (SEQ ID NO: 10), pTrex8gM _TRI033 (SEQ ID NO: 11), pTrex8gM_TRI034 (SEQ ID NO: 12), pTrex8gM_TRI035 (SEQ ID NO: 13), pTrex8gM_TRI036 ( SEQ ID NO: 14), pTrex8gM_TRI037 (SEQ ID NO: 15), and pTrex 8gM_TRI038 (sequence number 16) was obtained.

[0275] Basically, as described in U.S. Pat. No. 8,592,194 B2, PEG intermediates are used. Using the mediated protoplast transformation method, 1.5-17 μg of expression vector was transformed into Trichoderma Trichoderma reesei strain Cellulight™ were transformed individually. As described, a slightly modified PEG-protoplast was used for transformation. The protoplast method was used. Protoplasts were prepared by inducing pyr auxotrophy with 10 mM uridine. Supplemented Trichoderma germination medium allowed spores to grow for approximately 1 h at 26°C. Grow for 8 hours. (Trichoderma) Germination medium: 40 ml 50% glucose, 2g / L peptone, 15g / L KH 2 PO 4 , 5g / L (NH 4 )2 SO 4 , 2.4 ml of 1 M MgSO 4 , 4.1 ml of 1 M CaCl 2 , 1 ml 400x Trichoderma reesei (T. reesei) trace element solution {200g / L FeSO 4 x7H 2 O, 16 g / L ZnSO 4 x7H 2 O, 1.4 g / L MnSO 4 ×H 2 O, 3.2 g / L CuSO 4 ×5H 2 O, 0.8 g / L H 3 BO 3 , 175g 1 / L citric acid) at a shaking speed of 200 rpm. The germinated spores were harvested by centrifugation and washed. , 45mg / ml lytic enzyme solution (Trichoderma harzianum A. harzianum, Sigma Cat. No. L1412) to treat the fungal cells. Furthermore, Penttilae et al. (Gene 61 (1987) 155 Protoplasts were prepared by standard methods as described by (-164).

[0276] Typically, 1.5-17 μg of DNA and approximately 5 × 10 7 Professional The transformation mixture containing the toplasts was treated with 2 mL of 25% PEG solution and 2 volumes of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl 2 solution Dilute with 3% selective top agarose, 1 M sorbitol, 10 mM NH 4 Cl , 1x MM solution (2x MM solution: 30 g / L KH 2 PO 4 , 20mL of 1M acetamide 20 ml of 1M CsCl, 6 ml of 20% MgSO 4 x7H 2 O, 20% of 6 ml CaCl 2 ×2H 2 O, 2mL of Trichoderma reesei (T.reesei) trace elements Solution (400x), 80mL of 50% glucose, total volume 1L, pH 4.5) Mix with 10 mM NH 4 MM plates containing Cl (MM plates 2% agar, 1x M The transformants were then poured into MM solution (uridine-deficient) plates. Incubate the plates at 28 °C for 5 to 7 days until spores form. For better sporulation, transformants that appeared stable were cultured in 10 mM NH 4 Cl Once spores were formed, the plates were transferred to fresh MM plates containing 0.85% NaCl, 0.0 Spores were harvested using a solution of 15% Tween® 80. The liquid cultures were incubated for 24 h. Well MTP format (for screening) or shake flask (validation testing) The spore suspension was used to inoculate a 3 mL YEG medium (5 g / L). Yeast extract, 22 g / L glucose, H 2 The main culture is in the next production medium (production medium : 35g / L of 61% glucose / sophorose mixture, 9g / L of casamino acids, 5g / L (NH 4 ) 2 SO 4 , 4.5g / L KH 2 PO 4 , 1g / L CaCl 2 ×2H 2 O , 1g / L MgSO 4 x7H 2 O, 33 g / L PIPPS buffer, pH 5.5; 2.5mL / L of 400x Trichoderma reesei (T. reesei) trace elements (17 5g / L citric acid, 200g / L FeSO 4 x7H 2 O, 16 g / L ZnSO 4 × 7H 2 O, 3.2 g / L CuSO 4 ×5H 2 O, 1.4 g / L MnSO 4 ×H 2 O. 0.8 g / L boric acid) pH 5.5. To generate mutants, 3 mL of production medium was added to 24 Add to well MTP. Volumes were scaled up for shake flasks.

[0277] Cultures were grown for 7 days at 28°C and 80% humidity with shaking at 180 rpm. Culture supernatants were harvested by vacuum filtration and their performance and expression levels were tested. was used to

[0278] The amino acid and nucleotide sequences of the pepN_2 enzyme are shown below:

[0279] SEQ ID NO:1 = amino acid sequence of TRI031. Encoded by the leader sequence shown in FIG. The secretion signals shown are underlined: [ka] The underlined sequence without the leader sequence is SEQ ID NO:18.

[0280] SEQ ID NO:2 = amino acid sequence of TRI032. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined leader-less sequence is SEQ ID NO:19.

[0281] SEQ ID NO:3 = amino acid sequence of TRI033. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:20.

[0282] SEQ ID NO:4 = Amino acid sequence of TRI034. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:21.

[0283] SEQ ID NO:5 = Amino acid sequence of TRI035. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:22.

[0284] SEQ ID NO:6 = Amino acid sequence of TRI036. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:23.

[0285] SEQ ID NO:7 = Amino acid sequence of TRI037. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:24.

[0286] SEQ ID NO:8 = Amino acid sequence of TRI038. Encoded by the leader sequence shown in FIG. The secretion signals that are involved are underlined. [ka] The underlined sequence without the leader sequence is SEQ ID NO:25.

[0287] SEQ ID NO:9 = Nucleotide sequence of pTrex8gM_TRI031 expression construct . [ka] [ka] [ka]

[0288] SEQ ID NO:10 = Nucleotide sequence of pTrex8gM_TRI032 expression construct Column. [ka] [ka] [ka]

[0289] SEQ ID NO:11 = Nucleotide sequence of pTrex8gM_TRI033 expression construct Column. [ka] [ka] [ka]

[0290] SEQ ID NO:12 = Nucleotide sequence of pTrex8gM_TRI034 expression construct Column. [ka] [ka] [ka]

[0291] SEQ ID NO:13 = Nucleotide sequence of pTrex8gM_TRI035 expression construct Column. [ka] [ka] [ka]

[0292] SEQ ID NO: 14 = Nucleotide sequence of pTrex8gM_TRI036 expression construct Column. [ka] [ka] [ka]

[0293] SEQ ID NO: 15 = Nucleotide sequence of pTrex8gM_TRI037 expression construct Column. [ka] [ka] [ka]

[0294] SEQ ID NO: 16 = Nucleotide sequence of pTrex8gM_TRI038 expression construct Column. [ka] [ka]

[0295] Example 2 - Neosartoria fischeri (Neosartoria fischeri) in gluten hydrolysis orya fischeri) derived PepN2 (TRI031) Optimal value of degree The release of glutamic acid is due to the addition of vegetable proteins, such as the hydrolysis of soy or gluten. It is an important quality parameter in water splitting. The liberated glutamic acid is the so-called "umami" taste. In terms of the release of glutamic acid and glutamine, To investigate the effect of the initial pH on gluten hydrolysis, gluten was lysed for 15–30 min. The gluten was then hydrolyzed with 1M HCl immediately afterwards to adjust the initial pH of the gluten hydrolysis. The gluten was further treated with glutaminase (Amano, Japan). Alkaline Protease, FoodPro PNL and PepN 2 (Ne Treated with Neosartorya fischeri After 20 hours, the hydrolysis was stopped by ultrafiltration (cutoff 10 kDa, Sar torius Stedium Biotech, Goettingen, Germany y). Permeabilization was used for enzymatic glutamate analysis (enzymatic L-glutamate analysis). (Analysis kit, Roche, Mannheim, Germany). As shown in Figure 2, pN2 showed higher efficiency in the pH range of 7.0-9.0 compared with pH 6.0.

[0296] Temperatures applied to the hydrolysis of gluten in terms of the release of glutamic acid and glutamine To investigate the effect of HC1, the gluten suspension was liquefied for 15–30 min and then immediately added 1 M HC1. The initial pH of gluten hydrolysis was adjusted to pH 7.0 with 100 mL of water. As shown in Figure 3, A temperature range of 0°C was used. Gluten was mixed with glutaminase (Amano, Japan). In addition, gluten is reduced by using FoodPro® Alkaline Proteases. e. FoodPro® PNL and PepN 2 (Neosartoria Ficus The hydrolysis was started by mixing it with Neosartorya fischeri. After 20 h, hydrolysis was stopped by ultrafiltration (cut-off 10 kDa, Sarto (Rius Stadium, Goettingen, Germany). Permeabilization was used for glutamate analysis (enzymatic L-glutamate assay kit, Roche , Mannheim, Germany). PepN2 from A. torya fischeri showed the highest efficiency at 60°C. .

[0297] Example 3 - The effect of hydrolysis on the hydrolysis degree of Neosartoria fischeri PepN 2 (TRI031) from Aspergillus fischeri and PepN 2 (TRI) from Aspergillus clavatus 035) The degree of hydrolysis (DH) is approximately 12 for the same amount of protein in, for example, 6M HCl. The relative amount of peptide bonds cleaved is expressed as compared to acid hydrolysis carried out at 0° C. for approximately 24 hours. Therefore, the amino acid sequence of common PepNs, such as PepN type 1 or PepN type 2, A higher DH can be applied to evaluate the isolation efficiency. Offset 10 kDa, Sartorius, Goettingen, Germany), Disposable desalting columns (PD10, GE, Munich, Germany) were used as described by the manufacturer. The substrate was prehydrolyzed with 10% (w / w) Na-calcium sulfate. Zeinate (DMK, Germany), whey protein isolate (WPI; Arla, V iby, Denmark), soy protein isolate (SPI; SUPRO® 760, DuPont, Brabrand, Denmark) and gluten suspension. Prehydrolysis was performed at 1% ( w / w タンパク質 )'s FOODPRO® Alkaline Protease , 1% (w / w タンパク質 The experiment was carried out using FOODPRO® PNL. All hydrolysis was carried out at 55°C for 18 h, followed by inactivation at 95°C for 20 min. Each PepN2 used had a carryover of amino acids from the culture broth to the final hydrolysis. As a control for the removal of β-amino acids and to fully inactivate the endopeptidase used, The inactivation was performed at 5°C for 15 minutes. The experiment was carried out in a 150 μL flask mixed with 50 μL of PepN2 stock solution as shown in Table 1 below. The mixture consisted of a prehydrolyzed protein suspension of L. Hydrolysis was carried out at 50°C for 20 h. Then, 20 μL of 2 M TCA (trichloroacetic acid, Sigma-Aldrich, Sc Hydrolysis was stopped by the addition of Neosalt (H.Nelldorf, Germany). Neosartorya fischeri and Aspergillus PepN type 2 from Aspergillus clavatus The amino acid release efficiency is shown in Table 1 (B) and (A). In particular, Na-caseinate, WPI and In the SPI substrate, Neosartoria fischeri (N. fischeri) and Using PepN type 2 from Aspergillus clavatus Furthermore, the DH achieved with the PepN type 2 stock solution was This is considered a significant increase. In the case of gluten hydrolysis, the increase was 0.02-0.3 mg / m DH was found to increase over the protein range of L PepN 2.

[0298] [Table 4]

[0299] Example 4: Two endopeptidases, TRI032 and TRI0, which are candidates for PepN2, were 33, TRI034, TRI035, TRI037 and TRI038. Hydrolysis of gluten using FOODPRO® Alkaline Protease (FPAP) and Prehydrolyzed glutamate was obtained by adding FOODPRO® PNL (FPPNL). A suspension of ten was prepared. Hydrolysis was carried out at 55°C. After about 18 hours, it was heated to 90°C for 10 min. Hydrolysis was stopped by heating. After cooling to 50°C, glutaminase was added to the gluten. Then, 150 μL of prehydrolyzed gluten was added to a 96-well microplate. 50 μL (protein concentration: 0.5 mg / mL) of the solution was transferred to each well of the iterator plate. TRI032, TRI033, TRI034, TRI035, TRI037 and TRI Hydrolysis was carried out for 18 hours using 038. The hydrolysis was stopped by the addition of M TCA. The stopped hydrolysate was filtered (0.22 mm) and Enzymatic glutamate assay kit (Roche, Mannheim, Germany) The concentration of released glutamic acid is shown in FIG.

[0300] Example 5 - Hydrolysis of proline-containing peptides Peptide WHWLQLKPGQPMY was hydrolyzed with TRI031 and TRI035. 20 mM CPB-buffer (20 mM citrate, 20 mM phosphate, 20 mM phosphate) was used. The peptide (1 mg / mL) was incubated with 1 μg / mL aminopeptidase in 100 mM ethanol at 55 °C. Aliquots (50 μl) were added with 50 μl of 5% TFA at the indicated times. L) was stopped and subjected to LC-MS analysis.

[0301] The results show that TRI031 and TRI035 are Y (Figure 5) can be hydrolyzed to KPGQPMY (Figure 6) and then to QPMY (Figure 7). This indicates that Aspergillus oryzae ae) PepN 2 has been previously described (AMBlinkovsk y et al., Biochimica et Biophysica Acta, 1 480(2000)171-181, in which Aspergillus oryzae PepN 2 from P. illus oryzae is a genus of the peptide WHWLQLKPGQPMY. It was claimed that it did not hydrolyze any X-Pro bonds examined, including those However, we also found that these enzymes cleave the peptide to QPMY. We have found that (Figure 7).

[0302] Data collection: LTQ Orbitrap Classic hybrid mass spectrometer (T Hermo Scientific, Bremen, Germany A Lent 1100 LC system (Agilent Technologies) was used. Capillary LC-MS / MS analysis was performed using a 15 cm Phenomenon column. ix Jupiter 4μ Proteo 90A, C4 analytical column. ONMAX® ion source (Thermo Scientific, San Joa flow rate of 16 μL / min, 0-40% solvent BH for 10 min. 2 O / CH 3 CN The separation was performed using a gradient of LT / HCOOH (50 / 950 / 0.65, v / v / v). The Q Exactive Orbitrap was operated in data-dependent MS / MS mode. Peptide masses were measured by the Orbitrap (MS scans were performed at a resolution of 60,000 at m / z 400), and the m / z of the two most intense peptides were selected and fragmented by CID in a linear ion trap (LTQ). Dynamic exclusion was enabled with a list size of 500 masses, a duration of 40 s, and an exclusion mass width of ±10 ppm around the masses on the list.

[0303] Label-free quantification: The raw files were accessed using MS1 intensities to build chromatograms with the program Skyline 2.6.0.7176 (MacLean, B., et al., “Skyline: an open source document editor for creating and analyzing targeted proteomic s experiments”, Bioinformatics, 2010, 26(7) : p. 966–8). The precursor isotope import filter was set to three counts (M, M+1, and M+2) at a resolution of 60,000, and the most intense et al., “Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in Skyline: application to protein acetylation and phosphorylation”, Mol Cell Proteomics, 2012.11(5): p. 202–14). ​ The peptide sequences of the substrate and cleavage products were entered into Skyline. The intensity of each sample was calculated. The figures were copied directly from the program (see Figure 8). ).

[0304] Example 6: PepN2 from Aspergillus clavatus Degree of hydrolysis of Aspergillus oryzae and Lactobacillus hepatitis Dehydration of PepN from L. helveticus ATCC 12046 Comparison with resolution According to Stressler, Eisele et al. (2013, see below), PepN 1 Lactobacillus helveticus eticus) ATCC® 12046, PepN 1 Aspergillus oryzae (Aspergillus oryzae), and Aspergillus clavatus (As Enzyme activity of PepN2 derived from Pergillus clavatus (TRI035) The activity of Lactobacillus helveticus was measured. pN1 was expressed and the results were analyzed by Stressler, Eisele et al. (2013, see below). The product was purified as described above.

[0305] PepN 1 derived from Aspergillus oryzae was analyzed by FLAVOUR ZYME® 500L (Sigma-Aldrich, Schnelldorf PD (Fujitsu Limited, Germany) equilibrated with 20 mM Bis-Tris, pH 6.5 10 mosquito After desalting with hexane, the product was purified by anion exchange chromatography. Q15, XK26 / 15 (SQ15) column (GE-Lifesciences, USA) ) and equilibrated with 20 mM Bis / Tris, pH 6.5 (buffer A). The sample (30 mL) was injected into the column at a flow rate of 7 mL / min. The bound proteins were washed with PhA and eluted with 0–0.5 M NaCl (20 mM Bis Elution was performed with a linear gradient of 0.1% ethanol (in Tris, pH 6.5) (50 min). Approximately 13 mL of each fraction was collected and stored on ice. The fraction with the highest aminopeptidase activity The eluates were pooled, desalted in PD10 (20 mM Bis-Tris (pH 6.0)) and diluted with 20 Poros Q equilibrated with 1 mM Bis / Tris, pH 6.0 (Buffer A). 20 HR26 / 10, XK26 / 10 columns (GE-Lifesciences, US A) was subjected to a second purification. The PepN1 sample was injected onto the column at a flow rate of 4 mL / min. The column was washed with buffer A, and the bound proteins were eluted with 0–0.25 M NaCl. The solution was eluted with a linear gradient of 30 μl (in 20 mM Bis / Tris (pH 6.0)). Fractions of approximately 8 mL were collected during the entire procedure and stored on ice. PepN 1 from A. oryzae was identified by SDS-PAGE electrophoresis. It was found to have a molecular weight of approximately 40 kDa.

[0306] PepN 1 and PepN 2 aminopeptidases at 2.5-200 nkat*mL -1 For protein hydrolysis, 1% (w / w WPI ) FOODPRO® Alkaline Protease (DuPont, Br abrand, Denmark) and 1% (w / w WPI )FOODPRO (registered trademark) ) PNL (DuPont, Brabrand, Denmark) was diluted with HO at an initial pH of 7.0. Whey protein isolate suspension (WPI; LACPRODAN® 9224, Arla Ingredients, Viby, Denmark). After addition of lysidase, WPI was added and 200 μL was transferred to each well of a 96-well microtiter plate. Then, 5 μL of PepN solution was added to each well as shown in Table 2. The reaction was carried out at 50°C without pH control and stopped after 18 hours by adding 20 μL of 2M TCA. Measurement of DH (Nielsen, Petersen et al., 2001, see below) All samples were 0.22 μm filtered prior to chromatography.

[0307] PepN2 from Aspergillus clavatus (A. clavatus) PepN 1 from A. oryzae had higher DH at all doses. The PepN from Lactobacillus helveticus (L. helveticus) 1 showed that the four most active doses tested showed high DH. At the maximum dose, PepN from Aspergillus clavatus 2 exhibited at least 25% higher DH than PepN 1 aminopeptidase.

[0308] [Table 5]

[0309] Example 7: Effect of chloride on the release of glutamic acid during hydrolysis of soybean and gluten Effects of sodium In the food industry, to reduce the risk of microbial contamination during protein hydrolysis, Sodium chloride is often used. 1% (w / w タンパク質 )FOODPRO(Registered Trademark Alkaline Protease (DuPont, Brabrand, Denm ark) and 1% (w / w タンパク質 ) FOODPRO® PNL (DuPo nt, Brabrand, Denmark) was added to the prehydrolyzed 1 0% (w / w) soybean (SuPro® 760, DuPont, Brabran d, Denmark) and gluten (Sigma-Aldrich, Schnelld orf, Germany) was prepared. Hydrolysis was carried out at 50°C and pH 7.0 (pH uncontrolled). After 18 hours, the mixture was inactivated by heat at 90°C for 10 minutes. The product was divided into a sodium chloride-containing (185 mM) hydrolysate and a salt-free hydrolysate. 150 μL of each prehydrolyzed protein suspension was purified in different ways as shown in Table 3. Standardized Aspergillus clavatus Mix with PepN 2. Hydrolysis was carried out at 50°C without pH control, and 20 μL was added after 18 hours. The reaction was stopped by adding 2M TCA. All samples were filtered at 0.22 μm prior to analysis (Table 3). As shown, the addition of 185 mM NaCl significantly reduced the hydrolysis of soybean and gluten. There was no effect on the measured degree of hydrolysis.

[0310] [Table 6]

[0311] Nielsen, PM, et al. (2001), Journal of Fo od Science 66(5):642-646.

[0312] Stressler, T., et al. (2013), PLoS ONE 8(7) .

[0313] Example 8: PepN2 TRI031 and TRI035 versus TRI063 (Aspergillus Inhibition by products of A. oryzae In this test, enzyme activity was measured by hydrolysis of the substrate H-Ala-nitroanilide (pNA). The absorbance of the released pNA at a wavelength of 405 nm was measured using a microtiter plate. The measurements were taken over time using a rate reader.

[0314] As a buffer for these substances, 20 mM CPB buffer (20 mM NaCl) was used. The enzyme samples used were: Na-citrate, Na-phosphate and Na-borate (pH 9.0). PepN 2 TRI031 and TRI035 were used. pNA substrate (BACHEM, L-1070) was dissolved in 1 mL of DMSO (dimethyl sulfoxide) The cells were solubilized in Costa Rica 96 well plates (Sigma catalog number D2650). The AR assay plate 9017 (Corning Inc.) was used. The plates were placed in a Molecular Devices VERSAMAX® Reader. For inhibition studies, each amino acid, namely lysine, histidine, leucine, , tryptophan, proline, glycine, serine, asparagine, threonine, asparagine 10 mL of 20 mg / mL solutions of acetic acid and glutamic acid were prepared. Proline was dissolved in DMSO. All other amino acids were dissolved in buffer. The final inhibition mixture was prepared by mixing 1 L of each amino acid solution.

[0315] Place a 96-well plate on ice and add 180 μL of buffer, 15 μL of diluted enzyme. , and 20 μL of inhibition mix was added to the wells of the plate and mixed. 0.017, 0.085, 0.128, 0.170, 0.426, 0.851, 1.70 2. Containing all four enzymes at an inhibitor mixture concentration of 2.553 mg / mL. 20 μL The reaction was started by adding one substrate concentration to each plate. After the reaction was started, the plates were placed in a microplate reader set at 30°C as soon as possible. The plate was then loaded with a plate-type filter and the absorbance at 405 nm was measured at 30-second intervals for 30 minutes. One substrate concentration (0.017, 0.170, 0.426, 0.851, 1.702, 2 The apparent inhibition was measured on six plates containing either 0.5, 0.5 or 0.53 mg / mL. Export data to GraphPad Prism to determine constants (Ki) This allows fitting of the Michaelis-Menten curve for each inhibitor concentration. The average molecular weight of the inhibitor was 133.9 g / mL. The units were converted to mM using mol. The inhibition by product for different enzymes was The apparent Ki values ​​and their respective standard deviations (SD) are shown in Table 4.

[0316] [Table 7]

[0317] From Table 4, PepN 2 TRI031 and TRI035 were resistant to inhibition by the product. It can be seen that Aspergillus oryzae has the largest apparent Ki value. Compared with TRI063 from A. oryzae, they were inhibited by the product. It means that it is difficult.

[0318] Example 9: PepN2 and PepN on Glu-pNA and Gln-pNA substrates Activity of 1 H-Glu-nitroanilide (pNA) and H-Gln-nitroanilide as substrates (pNA) was used, and the release of p-nitroanilide (pNA) was measured by absorbance at 405 nm. The measurement was performed using 20 mM CPB-buffer (20 mM citric acid , 20 mM Na-phosphate, 20 mM boric acid) pH 9.0 was used. -Glu-pNA and H-Gln-pNA (BACHEM or Schafer N; Copenhagen, Denmark) in 1 ml of DMSO (Sigma The solution was dissolved in 100 mL of ethyl sulfoxide (catalog number D2650).

[0319] To perform the test, a Costar assay plate 9017 (Corn 80 μL of buffer, 10 μL of H-Glu-pNA or H-Gln-pNA substrate and 10 μL of appropriately diluted PepN2 were incubated at 45 °C. VERSAMA was incubated and run on SoftMaxPro 5.4.1 software. 1. Using a .X® microplate reader (Molecular Devices), Measurements were taken at 405 nm every 30 seconds for 30 minutes. The linear portion of the curve was determined from 10 time points. Activity was determined as the maximum slope in minutes.

[0320] The study was carried out on purified samples of PepN 2 TRI031 and TRI035, as well as purified P epN 1 Lactobacillus helveticus (Stres sler, Eisele et al., supra), and non-purified PepN 1 aspergillus. A. sojae (COROLASE® LAP, AB En zymes), but these were not related to Ala-pNA or Leu-pNA. All had equivalent activity (although similar to that described for Ala-pNA in Example H). (The experiment was performed in the absence of inhibitors.) As shown in Table 5, two PepN 2-aminopeptides The Glu-pN1 enzyme was highly cleavable, in contrast to the two PepN1 enzymes, which did not show any significant activity. A and Gln-pNA, which is a PepN 2 aminopeptide. The enzyme is much more efficient than PepN 1 aminopeptidase in releasing Glu and Gln. This shows that it is effective for

[0321] [Table 8]

[0322] Example 10 - Hydrolysis of peptides containing proline at position 2 The peptide library XPAAAR (where X is any amino acid other than cysteine) was TRI032, TRI035, and TRI063 (Aspergillus oryzae e)) and COROLASE® LAP. 3 × 20 mM CP in B-buffer (20 mM citric acid, 20 mM phosphate, 20 mM boric acid) at 55°C The peptide library XPAAAR (1 mg) was incubated with 1 μg / mL of aminopeptidase. 50 μL of the aliquot was incubated with 50 μl of 5% TFA at the indicated times. The cot was stopped and subjected to LC-MS analysis.

[0323] Data collection: LTQ Orbitrap Classic hybrid mass spectrometer (T Hermo Scientific, Bremen, Germany A Lent 1100 LC system (Agilent Technologies) was used. Capillary LC-MS / MS analysis was performed using a 15 cm Phenomenon column. ex Jupiter 4μ Proteo 90A, C4 analytical column. ONMAX® ion source (Thermo Scientific, San Joa flow rate of 16 μL / min, 0-40% solvent BH for 10 min. 2 O / CH 3 CN The separation was performed using a gradient of LT / HCOOH (50 / 950 / 0.65, v / v / v). The Q Orbitrap Classic instrument was operated in data-dependent MS / MS mode. Peptide mass was analyzed by Orbitrap (MS scan: m / z 400 = 60,000 The m / z of the peptides with the highest intensity were selected and linearized. The ions were subjected to fragmentation using CID in a LTQ ion trap. Dynamic exclusion was performed using 500 mass aliquots. The list size is 40 seconds, and the emission is within ±10 ppm of the mass on the list. It was deemed effective depending on the removal mass width.

[0324] Label-free quantification: Use MS1 intensities to construct chromatograms, program Sk Using yline2.6.0.7176 (MacLean, B., et al., supra) (Schilling, B., et al., supra) ). The precursor isotope import filter was set to 3 counts ( The charge states of the substrate and cleavage products were set to M, M+1 and M+2, respectively, and the maximum intensity charge state was used. The peptide sequences of the products were entered into Skyline, and the intensities of each sample were calculated. Figure 10 was generated based on the sum of the peak areas of the species isotopes (M, M+1, and M+2). A substrate standard curve (successive two-fold increase in concentration from Std3.125 to Std50) was included. This indicates that there is a linear relationship between the substrate concentration and the peak area.

[0325] The LC-MS results (Figure 10) showed that in TRI032 and TRI035, peptide TP AAAR was hydrolyzed over time, with the concentration decreasing by more than half after 2 hours of incubation. However, TRI063 (Aspergillus oryzae (A. oryzae)) was not able to inhibit the oxidative stress. e)) and COROLASE® LAP, TPAAR hydration in 12 hours This indicates that it does not cause decomposition.

[0326] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that It should be apparent that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It should be understood that the following claims define the scope of the invention, and It is intended that methods and structures within the scope of these claims and their equivalents be included within the scope of the present invention. It is intended to be more covered.

Claims

1. 1. A method for producing a protein hydrolysate comprising exposing a proteinaceous substrate to an isolated polypeptide having aminopeptidase activity, The method of claim 1, wherein the isolated polypeptide having aminopeptidase activity is an isolated polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5, and hydrolyzing a polypeptide having a proline residue at position 2, as numbered from the N-terminus.

2. The method of claim 1, further comprising exposing the proteinaceous substrate to an endopeptidase.

3. 3. The method according to claim 1 or 2, wherein the hydrolysate is enriched in Leu, Gly, Glu, Ser, Asp, Asn, Pro, Cys, Ala and / or Gln.

4. The method according to claim 1 or 2, wherein the hydrolysate is rich in Glu and / or Gln.

5. 10. The method of claim 1, wherein the protein substrate is selected from whey protein isolate, casein, soy protein isolate, and gluten.

6. A method for obtaining a protein hydrolysate enriched in free glutamic acid and / or peptides carrying glutamic acid residues from a proteinaceous substrate, the method comprising subjecting said substrate to a deamidation process and exposing it to an isolated polypeptide having aminopeptidase activity, The isolated polypeptide having aminopeptidase activity is an isolated polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5, and hydrolyzing a polypeptide having a proline residue at position 2, as numbered from the N-terminus.

7. 7. The method of claim 6, further comprising exposing the substrate to one or more non-specifically acting endopeptidase and / or exopeptidase enzymes.

8. a) providing grain; b) adding an effective amount of an isolated polypeptide having aminopeptidase activity to said grain during the malt production process; and c) Obtaining malted grains Including, 13. A method for producing malted grain, wherein the isolated polypeptide having aminopeptidase activity is an isolated polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5, and hydrolyzing a polypeptide having a proline residue at position 2, as numbered from the N-terminus.

9. 1. A method for producing free amino nitrogen (FAN) during brewing, comprising the step of adding, during a brewing process, an effective amount of an isolated polypeptide having aminopeptidase activity, The method of claim 1, wherein the isolated polypeptide having aminopeptidase activity is an isolated polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:5, and hydrolyzing a polypeptide having a proline residue at position 2, as numbered from the N-terminus.

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