Enzyme agent and application thereof

Abstract

The present invention addresses the problem of providing an enzyme agent containing a novel aminopeptidase that exhibits substrate specificity different from that of conventional aminopeptidase. The present invention relates to an enzyme agent comprising, as an active ingredient, aminopeptidase comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8 or an amino acid sequence equivalent thereto. Furthermore, the present invention relates to an enzyme agent which contains, as an active ingredient, an aminopeptidase that is derived from Aspergillus nidulans or Aspergillus oryzae and has the following physicochemical properties. (1) the optimum temperature is about 50 DEG C; and (2) the optimum pH is about 7-8. And (3) primer specificity: the primer has the highest specificity on L-alanine residues or L-lysine residues.

Description

Technical Field

[0001] The present invention relates to an enzyme preparation containing a novel aminopeptidase as an active ingredient and its application. Background Art

[0002] Amino acids and peptides are widely used in medicine and food. Amino acids and peptides can be mainly obtained by decomposing proteins. As decomposition methods, an acid hydrolysis method and an enzymatic decomposition method are known, and these decomposition methods have a great influence on taste properties. In recent years, the demand for taste properties has diversified, and there is also a demand for improving the taste properties of plant-based protein foods that pursue health. For example, it is known that valine, leucine, isoleucine, phenylalanine, tyrosine, and tryptophan, which are amino acid taste components, are related to bitterness (Patent Document 1). In addition, in addition to glutamic acid, it has also been reported that glycine and alanine are related to umami, and arginine is related to the rich taste in seafood (Non-Patent Document 1).

[0003] Aminopeptidase is an enzyme (exopeptidase) that catalyzes the cleavage of amino acid units from the amino terminus (N-terminus) of proteins and peptides. Aminopeptidases are widely distributed in all animals, plants, and microorganisms, and are present in many intracellular organelles, the cell matrix, membrane components, and extracellularly.

[0004] As aminopeptidases, aminopeptidases having various substrate specificities have been reported. For example, it is disclosed in Patent Document 1 that an aminopeptidase derived from Aspergillus oryzae can be used for bread improvement and has an effect of removing bitterness. Patent Document 2 discloses a leucine aminopeptidase derived from Aspergillus sojae that can be used for the production of seasonings such as soy sauce. Patent Document 3 discloses a glutamic acid aminopeptidase derived from Aspergillus oryzae that can enhance the umami of seasonings. Patent Document 4 discloses a glycine aminopeptidase derived from Actinomucor elegance that can produce a seasoning with strong taste ability. Patent Document 5 discloses an alanine aminopeptidase derived from Aeromonas that is useful for improving the taste and flavor of edible meat.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 11-509082

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 11-346777

[0009] Patent Document 3: Japanese Patent Application Laid-Open No. 2008-11710

[0010] Patent Document 4: Japanese Patent Application Laid-Open No. 2001-17165

[0011] Patent Document 5: Japanese Unexamined Patent Application Publication No. 7-289256

[0012] Non-Patent Document

[0013] Non-Patent Document 1: Biotechnology, Vol. 89, No. 11, 2011, pp. 679 - 682 Summary of the Invention

[0014] As described above, aminopeptidases with various substrate specificities are known. However, in recent years, the demand for changing the taste properties in protein-containing foods has been increasing, and there is a need for an aminopeptidase that exhibits a substrate specificity different from that of existing aminopeptidases. Therefore, an object of the present invention is to provide an enzyme agent containing a novel aminopeptidase that exhibits a substrate specificity different from that of existing aminopeptidases.

[0015] To solve the above problems, the present inventors focused on a protein with unknown function secreted by microorganisms in response to proteins, prepared a recombinant protein from a protein whose function could not be inferred from the primary sequence, and analyzed its function. Then, through in-depth research, a novel aminopeptidase with a substrate specificity different from that of existing aminopeptidases was discovered, thus completing the present invention.

[0016] Specifically, the present invention has the following configuration.

[0017] <1> An enzyme agent comprising an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 or an equivalent amino acid sequence as an active ingredient.

[0018] <2> The enzyme agent according to <1>, wherein the aminopeptidase composed of the equivalent amino acid sequence has 90% or more homology with the amino acid sequence of SEQ ID NO: 1 and has aminopeptidase activity.

[0019] <3> The enzyme agent according to <1> or <2>, wherein the aminopeptidase is derived from Aspergillus nidulans

[0020] (Aspergillus nidulans).

[0021] <4> An enzyme agent comprising an aminopeptidase derived from Aspergillus nidulans and having the following physicochemical properties as an active ingredient,

[0022] (1) Optimum temperature: around 50°C;

[0023] (2) Optimum pH: around 7 - 8;

[0024] (3) Substrate specificity: highest specificity for L-alanine residues.

[0025] <5> An enzyme preparation comprising an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 8 or an equivalent amino acid sequence as an active ingredient.

[0026] <6> The enzyme preparation according to <5>, wherein the aminopeptidase composed of the equivalent amino acid sequence has a homology of 90% or more with the amino acid sequence of SEQ ID NO: 8 and has aminopeptidase activity.

[0027] <7> The enzyme preparation according to <5> or <6>, wherein the aminopeptidase is derived from Aspergillus oryzae.

[0028] <8> The enzyme preparation according to any one of <5> to <7>, which has the highest specificity for L-lysine residues.

[0029] <9> The enzyme preparation according to any one of <1> to <8>, which is for changing the taste of food.

[0030] <10> A method for producing a food or a food material, comprising the step of allowing the enzyme preparation according to any one of <1> to <8> to act on a raw material containing protein.

[0031] <11> A method for producing a seasoning, comprising the step of allowing the enzyme preparation according to any one of <1> to <8> to act on at least one selected from proteins and peptides.

[0032] <12> A method for changing the taste of a food, comprising the step of allowing the enzyme preparation according to any one of <1> to <8> to act on a raw material containing protein.

[0033] <13> A food comprising the enzyme preparation according to any one of <1> to <8> and at least one selected from proteins and peptides.

[0034] <14> A seasoning comprising the enzyme preparation according to any one of <1> to <8> and at least one selected from proteins and peptides.

[0035] According to the present invention, an enzyme preparation containing a novel aminopeptidase with substrate specificity different from that of existing aminopeptidases can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a graph showing the optimum temperature of the aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1.

[0037] Figure 2 is a graph showing the optimum pH of the aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1.

[0038] Figure 3 is a graph showing the enzyme activity of the aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 in the presence of various metal ions. Detailed implementation mode

[0039] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below is sometimes based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. It should be noted that the numerical range represented by "~" in this specification means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0040] In this specification, the 20 amino acid residues in the amino acid sequence are sometimes represented by single-letter abbreviations. In this case, glycine (Gly) is G, alanine (Ala) is A, valine (Val) is V, leucine (Leu) is L, isoleucine (Ile) is I, phenylalanine (Phe) is F, tyrosine (Tyr) is Y, tryptophan (Trp) is W, serine (Ser) is S, threonine (Thr) is T, cysteine (Cys) is C, methionine (Met) is M, aspartic acid (Asp) is D, glutamic acid (Glu) is E, asparagine (Asn) is N, glutamine (Gln) is Q, lysine (Lys) is K, arginine (Arg) is R, histidine (His) is H, and proline (Pro) is P. In addition, in this specification, the left end of the displayed amino acid sequence is the N-terminus, and the right end is the C-terminus.

[0041] 1. Enzyme agent containing aminopeptidase

[0042] The first aspect of the present invention relates to an enzyme agent containing an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent thereto as an active ingredient. In addition, the second aspect of the present invention relates to an enzyme agent containing an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence equivalent thereto as an active ingredient. The enzyme agent of the present invention (hereinafter, also referred to as "the present enzyme agent") contains an aminopeptidase (hereinafter, also referred to as "the present enzyme") as an active ingredient.

[0043] The aminopeptidase as the active ingredient, that is, the present enzyme, is composed of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to this amino acid sequence, or the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence equivalent to this amino acid sequence. Here, the "equivalent amino acid sequence" means an amino acid sequence that is partially different from the reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 8), but this difference has no substantial influence on the protein function (here, aminopeptidase activity). In addition, in the present invention, the "equivalent amino acid sequence" means an amino acid sequence with the same substrate specificity.

[0044] An aminopeptidase is an enzyme that cleaves peptide bonds from the amino-terminal (N-terminal) side of a protein or peptide to release amino acids. In particular, in the first mode, this enzyme has the activity of releasing alanine at the amino terminus of a protein or peptide (alanine aminopeptidase activity). It should be noted that the degree of alanine aminopeptidase activity of an enzyme composed of an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 1 is not particularly limited as long as it can function as an alanine aminopeptidase. Among them, the degree of alanine aminopeptidase activity of an enzyme composed of an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 1 is preferably the same as or higher than that of an enzyme composed of a reference amino acid sequence (the enzyme having the amino acid sequence of SEQ ID NO: 1). The same degree of alanine aminopeptidase activity means that it has an alanine-releasing ability of ±20%, preferably ±10%, compared with the alanine aminopeptidase having the reference amino acid sequence.

[0045] In addition, in the second mode, this enzyme has the activity of releasing lysine at the amino terminus of a protein or peptide (lysine aminopeptidase activity). It should be noted that the degree of lysine aminopeptidase activity of an enzyme composed of an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 8 is not particularly limited as long as it can function as a lysine aminopeptidase. Among them, the degree of lysine aminopeptidase activity of an enzyme composed of an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 8 is preferably the same as or higher than that of an enzyme composed of a reference amino acid sequence (the enzyme having the amino acid sequence of SEQ ID NO: 8). The same degree of lysine aminopeptidase activity means that it has a lysine-releasing ability of ±20%, preferably ±10%, compared with the lysine aminopeptidase having the reference amino acid sequence.

[0046] This enzyme preparation contains a novel aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent thereto, or a novel aminopeptidase composed of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence equivalent thereto, and the present invention provides a novel enzyme preparation. In addition, this enzyme is a novel aminopeptidase with a substrate specificity different from that of existing aminopeptidases. The substrate specificity of this enzyme is relatively broad, so it can be expected to have an effect of changing the taste of foods different from that of existing aminopeptidases, for example.

[0047] In the first mode, this enzyme is preferably derived from Aspergillus nidulans. That is, preferably, the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence (mature form) of an aminopeptidase derived from Aspergillus nidulans. It should be noted that the full-length amino acid sequence of an aminopeptidase derived from Aspergillus nidulans having a signal peptide is shown as SEQ ID NO: 2.

[0048] In the second mode, this enzyme is preferably derived from Aspergillus oryzae. That is, preferably, the amino acid sequence of SEQ ID NO: 8 is the amino acid sequence (mature form) of an aminopeptidase derived from Aspergillus oryzae. It should be noted that the full-length amino acid sequence of an aminopeptidase derived from Aspergillus oryzae having a signal peptide is shown as SEQ ID NO: 9.

[0049] The amino acid sequence of the aminopeptidase composed of an equivalent amino acid sequence is partially different from the reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 8). Here, when it is "partially different from the reference amino acid sequence", for example, one or more amino acids in the amino acids constituting the amino acid sequence are deleted, substituted, one or more amino acids are added or inserted into the amino acid sequence, or any combination thereof. As long as the partial difference in the amino acid sequence can maintain the aminopeptidase activity, it is allowed, and the activity may also change slightly. As long as this condition is satisfied, the position where the amino acid sequence is different is not particularly limited. In addition, the difference in the amino acid sequence can occur at multiple sites.

[0050] The number of different sites in the amino acid sequence is preferably 30% or less, more preferably 20% or less, further preferably 15% or less, still further preferably 10% or less, even more preferably 5% or less, yet even more preferably 3% or less, still even more preferably 2% or less, further even more preferably 1% or less, particularly preferably 0.7% or less, more particularly preferably 0.5% or less, further particularly preferably 0.3% or less, and most preferably 0.1% or less of the total number of amino acids constituting the amino acid sequence. Therefore, the aminopeptidase composed of an equivalent amino acid sequence preferably has a homology of 70% or more with the reference amino acid sequence (the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 8), more preferably 80% or more, further preferably 85% or more, still further preferably 90% or more, even more preferably 95% or more, yet even more preferably 97% or more, still even more preferably 98% or more, further even more preferably 99% or more, particularly preferably 99.3% or more, more particularly preferably 99.5% or more, further particularly preferably 99.7% or more, and most preferably 99.9% or more.

[0051] One typical example of "partially different from the reference amino acid sequence" is that the amino acid sequence is mutated (changed) by deletion, substitution of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, further preferably 1 to 7, still further preferably 1 to 5, even more preferably 1 to 3) amino acids in the amino acids constituting the amino acid sequence, addition or insertion of 1 to 40 (preferably 1 to 30, more preferably 1 to 10, further preferably 1 to 7, still further preferably 1 to 5, even more preferably 1 to 3) amino acids into the amino acid sequence, or a combination thereof.

[0052] When it is "partially different from the reference amino acid sequence", it is preferable to introduce conservative amino acid substitutions in amino acid residues that are not essential for aminopeptidase activity, whereby an equivalent amino acid sequence can be easily obtained. Here, "conservative amino acid substitution" means substituting an amino acid residue with an amino acid residue having a side chain of the same property. Amino acid residues are classified into several families according to their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In conservative amino acid substitutions, it is preferable to introduce substitutions between amino acid residues within the same family.

[0053] The homology (%) between two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned in the best comparable manner (for example, gaps can be introduced into the first sequence to optimize the alignment with the second sequence). When the molecule (amino acid residue) at a specific position in the first sequence is the same as the molecule (amino acid residue) at the corresponding position in the second sequence, it can be said that the molecule (amino acid residue) at that position is identical. The homology between the two sequences is calculated from the number of identical positions common to the two sequences (homology (%) = number of identical positions / total number of positions × 100). It should be noted that in this case, it is preferable to also consider the number and size of the gaps required for optimizing the alignment.

[0054] The comparison of two sequences and the determination of homology can be achieved using mathematical algorithms. As a specific example of the mathematical algorithms that can be used in sequence comparison, there is the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 and modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, but it is not limited thereto. The homology of an amino acid sequence can be obtained, for example, by blastp (protein-protein BLAST) of the National Center for Biotechnology Information (NCBI). The default parameters can be used, for example, using the BLOSUM62 matrix and setting the Gap Costs to Existence: 11 and Extension: 1.

[0055] The aminopeptidase, which is the active ingredient of this enzyme preparation, i.e., this enzyme, can also be a part of a larger protein (such as a fusion protein). As the sequence added in the fusion protein, for example, sequences that contribute to purification such as polyhistidine residues, additional sequences that ensure stability during recombinant production, etc. can be cited.

[0056] This enzyme can be obtained by culturing a microorganism that produces this aminopeptidase (aminopeptidase-producing strain). In the first method, for example, it can be obtained by culturing Aspergillus nidulans, specifically Aspergillus nidulans strain A26 (obtained from the Fungal Genetic Stock Center (Kansas State University)). In addition, in the second method, for example, it can be obtained by culturing Aspergillus oryzae. The aminopeptidase-producing strain can be a wild strain or a mutant strain (such as a mutant strain obtained by ultraviolet irradiation).

[0057] This enzyme can be prepared from the culture broth and / or cells of the microorganism that produces this enzyme. The culture conditions and culture methods are not particularly limited as long as this enzyme can be produced. That is, the methods and conditions suitable for culturing the microorganism used can be appropriately set on the condition that this enzyme can be produced. As the culture method, either liquid culture or solid culture can be used, and liquid culture is preferably used. Hereinafter, taking liquid culture as an example, the culture conditions will be described.

[0058] As the medium, as long as it is a medium in which the microorganism used can grow and reproduce, there is no particular limitation. As the medium, for example, a medium supplemented with carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerol, dextrin, molasses, organic acids, nitrogen sources such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or gelatin, peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, meat extract, and inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts, zinc salts can be used. In order to promote the growth and reproduction of the microorganism used, vitamins, amino acids, etc. can also be added to the medium. The pH of the medium is preferably about 3 to 8, more preferably about 4 to 7. In addition, the culture temperature is usually preferably about 20 to 40 °C, more preferably about 25 to 35 °C, and cultured for 1 to 20 days, preferably about 3 to 10 days, under aerobic conditions. As the culture method, for example, the shaking culture method or the aerobic deep culture method based on a fermenter can be used.

[0059] After culturing under the above conditions, the target enzyme is recovered from the culture broth or the cells. When recovering from the culture broth, for example, after removing insolubles by filtering the culture supernatant, centrifuging, etc., the enzyme can be obtained by appropriately combining various separation and purification methods such as concentration using an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, and various chromatography methods such as ion exchange resin. On the other hand, when recovering from the cells, for example, after disrupting the cells by pressure treatment, ultrasonic treatment, etc., the enzyme can be obtained by performing separation and purification in the same manner as above. It should be noted that the cells can be recovered from the culture broth in advance by filtration, centrifugation, etc., and then the above series of processes (cell disruption, separation, purification) can be carried out.

[0060] This enzyme can also be easily prepared using genetic engineering techniques. For example, it can be prepared by transforming a suitable host cell (such as Escherichia coli) with the DNA encoding this enzyme and recovering the protein expressed in the transformant. The recovered protein can be appropriately purified according to the purpose. In this way, the target enzyme can be obtained in the form of a recombinant protein, and various modifications can be carried out according to this method. For example, if the DNA encoding this enzyme and other appropriate DNA are inserted into the same vector and the vector is used for the production of the recombinant protein, an enzyme composed of a recombinant protein to which an arbitrary peptide or protein is linked can be obtained. In addition, modifications such as the addition of sugar chains and / or lipids, or the processing of the N-terminus or C-terminus can also be carried out. By the above methods, the extraction and purification of the recombinant protein can be simplified, or biological functions can be added, etc.

[0061] Generally, gene expression is carried out using an appropriate host-vector system as described above, and the expression product (this enzyme) is recovered. The cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system) can also be used. Here, the "cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system)" refers to a system that synthesizes the encoded mRNA and protein in vitro from a nucleic acid (DNA, mRNA) as a template using ribosomes, transcription and translation factors, etc. derived from living cells (or obtained by genetic engineering techniques) without using living cells. In the cell-free synthesis system, a cell extract obtained by purifying the cell lysate as needed is generally used. The cell extract generally contains various factors required for protein synthesis such as ribosomes and initiation factors, and various enzymes such as tRNA. When performing protein synthesis, various amino acids, energy sources such as ATP and GTP, and other substances required for protein synthesis such as phosphocreatine are added to the cell extract. When performing protein synthesis, ribosomes, various factors, and / or various enzymes prepared separately can also be supplemented as needed.

[0062] The development of a transcription / translation system for reconstructing the molecules (factors) required for protein synthesis has also been reported (Shimizu, Y. et al.: Nature Biotech., 19, 751 - 755, 2001). In this synthesis system, genes of 31 factors consisting of 3 initiation factors, 3 elongation factors, 4 factors involved in termination, 20 aminoacyl - tRNA synthetases that bond each amino acid to tRNA, and methionyl - tRNA formyltransferase, which constitute the bacterial protein synthesis system, are amplified from the Escherichia coli genome, and a protein synthesis system is reconstructed in vitro using them. The synthesis system reconstructed in this way can be utilized in the present invention.

[0063] "Cell - free transcription system, cell - free transcription / translation system" can be used interchangeably with cell - free protein synthesis system, in vitro translation system, or in vitro transcription / translation system. In the in vitro translation system, RNA is used as a template to synthesize proteins. As the template RNA, total RNA, mRNA, in vitro transcription products, etc. can be used. In the in vitro transcription / translation system in another aspect, DNA is used as a template. The template DNA preferably contains a ribosome - binding region, and in addition, preferably contains an appropriate terminator sequence. It should be noted that in the in vitro transcription / translation system, conditions are set to continuously carry out the transcription reaction and the translation reaction by adding the factors required for each reaction.

[0064] The purified enzyme obtained as described above can also be provided in a powdered form by, for example, freeze - drying, vacuum - drying, or spray - drying.

[0065] The purified enzyme can be dissolved in water. Preferably, the purified enzyme is dissolved in a buffer. As the buffer, acetic acid buffer, phosphate buffer, triethanolamine buffer, Tris - hydrochloric acid buffer, GOOD's buffer, etc. can be cited. As GOOD's buffer, PIPES, MES, or MOPS can be cited.

[0066] The purification degree of the enzyme is not particularly limited. For example, it can be purified such that the aminopeptidase activity is 1 - 2000 (U / g), preferably 10 - 1500 (U / g), and more preferably 100 - 1000 (U / g). In addition, the final form can be liquid or solid (including powder).

[0067] The third aspect of the present invention relates to an enzyme preparation comprising an aminopeptidase derived from Aspergillus nidulans and having the following physicochemical properties as an active ingredient.

[0068] (1) Optimum temperature: around 50 °C.

[0069] (2) Optimum pH: around 7 - 8.

[0070] (3) Substrate specificity: It has the highest specificity for L - alanine residues.

[0071] Based on the research of the present inventors, the properties of the Aspergillus nidulans-derived aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 were determined as follows. Therefore, in the third aspect of the present invention, this enzyme can also be determined by the above enzymatic chemical properties. It should be noted that the detailed conditions for measuring aminopeptidase activity, measurement procedures, etc. when evaluating each enzymatic chemical property are as shown in the following examples.

[0072] <Action>

[0073] The enzyme in the third aspect is an aminopeptidase that acts on proteins and peptides to cleave the peptide bond at the amino terminus. In particular, it is preferred that this enzyme has the activity of liberating alanine at the amino terminus of a protein or peptide (alanine aminopeptidase activity).

[0074] <Optimum temperature>

[0075] The optimum temperature of the enzyme in the third aspect is around 50 °C. More specifically, the optimum temperature of this enzyme is preferably 40 - 60 °C, more preferably 45 - 55 °C.

[0076] <Optimum pH>

[0077] The optimum pH of the enzyme in the third aspect is 7 - 8. The optimum pH is judged based on the measurement results in Mcllvaine buffer in the pH range of pH 3 - 7, based on the measurement results in Tris - hydrochloride buffer in the pH range of pH 6 - 7, and based on the measurement results in CAPS buffer in the pH range of pH 8 - 11.

[0078] <Substrate specificity>

[0079] The enzyme in the third aspect preferably has the activity of liberating alanine at the amino terminus of a protein or peptide (alanine aminopeptidase activity). For example, with various amino acids - p-nitroanilide

[0080] (Ala - pNA, Gly - pNA, Lys - pNA, Val - pNA, Arg - pNA, Phe - pNA, Pro - pNA, Ile - pNA, Glu - pNA, Leu - pNA, Asp - pNA) as substrates, it shows the highest activity towards alanine - p-nitroanilide (Ala - pNA). Among them, this enzyme preferably has the highest specificity for L-alanine residues.

[0081] In the third mode, this enzyme shows activity against hydrophobic amino acids (alanine, glycine, valine, phenylalanine) and some basic amino acids (lysine, arginine). For example, when the activity value of alanine - p - nitroaniline (Ala - pNA) is set to 100%, the activity value of glycine - p - nitroaniline (Gly - pNA) can be 40% or more, the activity value of lysine - p - nitroaniline (Lys - pNA) can be 30% or more, the activity value of valine - p - nitroaniline (Val - pNA) can be 20% or more, the activity value of arginine - p - nitroaniline (Arg - pNA) can be 20% or more, and the activity value of phenylalanine - p - nitroaniline (Phe - pNA) can be 10% or more.

[0082] <Molecular weight>

[0083] In the third mode, the molecular weight of this enzyme, calculated based on the theoretical value of the amino acid sequence, is approximately 47 kDa.

[0084] The content of the active ingredient (this enzyme) in the enzyme agent in the third mode is not particularly limited. For example, it is preferably set or adjusted such that the alanine aminopeptidase activity per 1 g of the enzyme agent is 0.001 U - 10000 U, 0.005 U - 5000 U, 0.01 U - 2000 U, 0.1 U - 1000 U, and preferably 1 U - 500 U.

[0085] In the fourth mode, this enzyme agent preferably has the highest specificity for L - lysine residues. According to the research of the inventors, the properties of the Aspergillus oryzae - derived aminopeptidase composed of the amino acid sequence of SEQ ID NO: 8 were determined as follows. It should be noted that the detailed content of the measurement conditions, measurement procedures, etc. of the aminopeptidase activity when evaluating each enzyme - chemical property is shown in the examples described later.

[0086] <Function>

[0087] The enzyme in the fourth mode is an aminopeptidase that acts on proteins and peptides to cleave the peptide bond at the amino - terminal. In particular, it is preferred that this enzyme has the activity of liberating lysine at the amino - terminal of proteins or peptides (lysine aminopeptidase activity).

[0088] <Substrate specificity>

[0089] Preferably, the enzyme in the fourth mode has the activity of liberating lysine at the amino terminus of a protein or peptide (lysine aminopeptidase activity). For example, when using various amino acid - p-nitroanilides (Ala - pNA, Gly - pNA, Lys - pNA, Val - pNA, Arg - pNA, Phe - pNA, Pro - pNA, Ile - pNA, Glu - pNA, Leu - pNA, Asp - pNA) as substrates, it shows the highest activity towards lysine - p-nitroanilide (Lys - pNA). Among them, the enzyme preferably has the highest specificity for L-lysine residues.

[0090] The enzyme in the fourth mode shows activity towards hydrophobic amino acids (alanine, glycine, valine, phenylalanine) and some basic amino acids (lysine, arginine). For example, when the activity value of lysine - p-nitroanilide (Lys - pNA) is set to 100%, the activity value of glycine - p-nitroanilide (Gly - pNA) can be 40% or more, the activity value of alanine - p-nitroanilide (Ala - pNA) can be 30% or more, the activity value of valine - p-nitroanilide (Val - pNA) can be 15% or more, the activity value of arginine - p-nitroanilide (Arg - pNA) can be 10% or more, and the activity value of leucine - p-nitroanilide (Leu - pNA) can be 10% or more.

[0091] <Molecular weight>

[0092] The molecular weight of the enzyme in the fourth mode is approximately 47 kDa based on the theoretical value according to the amino acid sequence.

[0093] The content of the active ingredient (the enzyme) in the enzyme agent in the fourth mode is not particularly limited. For example, it is preferably set or adjusted such that the lysine aminopeptidase activity per 1 g of the enzyme agent is 0.001 U to 10,000 U, 0.005 U to 5,000 U, 0.01 U to 2,000 U, 0.1 U to 1,000 U, and preferably 1 U to 500 U.

[0094] <Optional components of the enzyme agent containing aminopeptidase>

[0095] The enzyme preparation of the present invention is usually provided in a solid form (for example, an immobilized enzyme in which the enzyme is immobilized on the surface or inside of a material capable of immobilizing the enzyme such as a granule, powder, silica, or porous polymer) or a liquid form. In addition to the active ingredient (the present enzyme), the enzyme preparation may also contain excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, and the like. As the excipient, lactose, sorbitol, D-mannitol, maltodextrin, sucrose, etc. can be used. As the buffer, phosphates, citrates, acetates, etc. can be used. As the stabilizer, propylene glycol, ascorbic acid, etc. can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate, etc. can be used. As the antiseptic, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. can be used.

[0096] In addition to the active ingredient (the present enzyme), the enzyme preparation of the present invention may also contain metal ions. As the metal ions, for example, Cu 2+ , Zn 2+ , Mn 2+ , Mg 2+ , Ca 2+ , Co 2+ , Fe 2+ , etc. can be cited. These metal ions can be added in the form of water-soluble metal salts such as chlorides, sulfates, acetates, nitrates, etc. Among them, the metal ions are preferably at least one selected from Cu 2+ , Mn 2+ and Mg 2 . By making such metal ions coexist, the substrate specificity can be more effectively improved and the enzyme activity can be increased.

[0097] The concentration of metal ions in the reaction solution during the enzyme reaction is preferably 0.001 mM or more, more preferably 0.01 mM or more. In addition, the concentration of metal ions in the reaction solution during the enzyme reaction is preferably 100 mM or less, more preferably 10 mM or less.

[0098] 2. Gene

[0099] The fifth aspect of the present invention relates to a gene, which is a cDNA composed of the base sequence shown in SEQ ID NO: 3, a cDNA composed of the base sequence shown in SEQ ID NO: 4, and a genomic DNA composed of the base sequence shown in SEQ ID NO: 5, encoding an aminopeptidase composed of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent thereto. In addition, the sixth aspect of the present invention relates to a gene, which is a cDNA composed of the base sequence shown in SEQ ID NO: 10, a cDNA composed of the base sequence shown in SEQ ID NO: 11, and a genomic DNA composed of the base sequence shown in SEQ ID NO: 12, encoding an aminopeptidase composed of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence equivalent thereto. The present enzyme can be produced by obtaining the gene encoding the present enzyme and expressing the gene.

[0100] In the fifth mode, the gene encoding the enzyme contains DNA encoding the amino acid sequence of SEQ ID NO: 1. Specific examples of this mode include cDNA consisting of the base sequence shown in SEQ ID NO: 3, cDNA consisting of the base sequence shown in SEQ ID NO: 4, and genomic DNA consisting of the base sequence shown in SEQ ID NO: 5. The DNA of SEQ ID NO: 3 encodes only the amino acid sequence of the mature form (SEQ ID NO: 1), and the DNA of SEQ ID NO: 4 encodes not only the mature form (the amino acid sequence of SEQ ID NO: 1) but also a signal peptide. The DNA of SEQ ID NO: 5 contains an intron in addition to the sequences encoding the mature form and the signal peptide.

[0101] In addition, in the sixth mode, the gene encoding the enzyme contains DNA encoding the amino acid sequence of SEQ ID NO: 8. Specific examples of this mode include cDNA consisting of the base sequence shown in SEQ ID NO: 10, cDNA consisting of the base sequence shown in SEQ ID NO: 11, and genomic DNA consisting of the base sequence shown in SEQ ID NO: 12. The DNA of SEQ ID NO: 10 encodes only the amino acid sequence of the mature form (SEQ ID NO: 8), and the DNA of SEQ ID NO: 11 encodes not only the mature form (the amino acid sequence of SEQ ID NO: 8) but also a signal peptide. The DNA of SEQ ID NO: 12 contains an intron in addition to the sequences encoding the mature form and the signal peptide.

[0102] The gene encoding the enzyme is typically used for the preparation of the enzyme. According to the genetic engineering preparation method using the gene encoding the enzyme, the enzyme in a more homogeneous state can be obtained. In addition, this method can also be said to be a preferred method when preparing a large amount of the enzyme. It should be noted that the use of the gene encoding the enzyme is not limited to the preparation of the enzyme. For example, as an experimental tool for elucidating the action mechanism of the enzyme, etc., or as a tool for designing or making mutants (variants) of the enzyme, this nucleic acid can also be used.

[0103] In this specification, the "gene encoding the enzyme" refers to a nucleic acid that gives the enzyme when expressed, and includes not only a nucleic acid having a base sequence corresponding to the amino acid sequence of the enzyme, but also a nucleic acid obtained by adding a sequence that does not encode an amino acid sequence to such a nucleic acid. In addition, codon degeneracy is taken into account.

[0104] The nucleic acid can be prepared in an isolated state with reference to the sequence information disclosed in this specification or the attached sequence listing and using standard genetic engineering techniques, molecular biology techniques, biochemical techniques, chemical synthesis, PCR methods (such as overlap PCR), or combinations thereof.

[0105] In the fifth or sixth embodiment of the present invention, a nucleic acid (hereinafter also referred to as "equivalent nucleic acid") is provided, which has a different partial base sequence while having the same function of the encoded protein when compared with the base sequence of the gene encoding the present enzyme. Examples of the equivalent nucleic acid include: DNA having a base sequence formed by substitution, deletion, insertion, addition or inversion of one or more bases based on the base sequence of the nucleic acid encoding the present enzyme and encoding a protein having the characteristic enzyme activity (i.e., aminopeptidase activity) of the present enzyme. Substitutions, deletions, etc. of bases can occur at multiple sites. Here, "multiple" also varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid, and is, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases. The equivalent nucleic acid preferably has a homology of 70% or more, more preferably 80% or more, further preferably 90% or more, still more preferably 92% or more, even more preferably 94% or more, yet more preferably 96% or more, further even more preferably 98% or more, and particularly preferably 99% or more with respect to the reference base sequence (SEQ ID NO: 3 or SEQ ID NO: 4, or SEQ ID NO: 10 or SEQ ID NO: 11).

[0106] The above equivalent nucleic acid can be obtained, for example, by restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., mutagenesis based on site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), etc. In addition, equivalent nucleic acid can also be obtained by other methods such as ultraviolet irradiation.

[0107] When preparing the present enzyme, a nucleic acid having a base sequence complementary to the base sequence of the gene encoding the present enzyme can be used. In the fifth or sixth embodiment of the present invention, a nucleic acid is provided, which has a base sequence that is at least 70%, 80%, 90%, 92%, 94%, 96%, 98% or 99% or more identical to the base sequence of the gene encoding the present enzyme or its complementary base sequence.

[0108] When preparing the present enzyme, a nucleic acid having a base sequence that hybridizes with a base sequence complementary to the base sequence of the gene encoding the present enzyme or its equivalent base sequence under stringent conditions can be used. Here, the "stringent conditions" refer to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Such stringent conditions are well known to those skilled in the art and can be set, for example, with reference to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York), Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). As the stringent conditions, for example, incubation can be carried out at about 50 °C using a hybridization solution (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5×Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)), and then washing can be carried out at about 65 °C using 0.1×SSC and 0.1% SDS. As more preferred stringent conditions, for example, conditions using 50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) as the hybridization solution can be cited.

[0109] When preparing the present enzyme, a nucleic acid (nucleic acid fragment) having a part of the base sequence of the gene encoding the present enzyme or its complementary base sequence can also be used. Such a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having the base sequence of the gene encoding the present enzyme, etc. The nucleic acid fragment is designed to contain, for example, a part that hybridizes with a continuous nucleotide part (for example, about 10 to about 100 bases in length, preferably about 20 to about 100 bases in length, more preferably about 30 to about 100 bases in length) in the base sequence of the gene encoding the present enzyme. When used as a probe, the nucleic acid fragment can be labeled. For the labeling, for example, a fluorescent substance, an enzyme, or a radioactive isotope can be used.

[0110] When preparing the present enzyme, a recombinant DNA containing the above gene (the gene encoding the present enzyme) can be used. The recombinant DNA is provided, for example, in the form of a vector. In this specification, the term "vector" refers to a nucleic acid molecule capable of transporting the nucleic acid inserted therein into a target such as a cell.

[0111] An appropriate vector is selected according to the purpose of use (cloning, protein expression), and the type of host cell is additionally considered. Examples of vectors for Escherichia coli as the host include M13 phage or its variants, λ phage or its variants, pBR322 or its variants (pB325, pAT153, pUC8, etc.). Examples of vectors for yeast as the host include pYepSec1, pMFa, pYES2, pPICZ, etc. Examples of vectors for insect cells as the host include pAc, pVL, etc. Examples of vectors for mammalian cells as the host include pCDM8, pMT2PC, etc.

[0112] The vector is preferably an expression vector. An "expression vector" refers to a vector that can introduce the inserted nucleic acid into a target cell (host cell) and can be expressed in the cell. An expression vector usually contains a promoter sequence required for expressing the inserted nucleic acid, an enhancer sequence for promoting expression, etc. An expression vector containing a selection marker can also be used. In the case of using the above expression vector, the selection marker can be used to confirm the presence (and its degree) of the introduction of the expression vector.

[0113] Insertion of nucleic acid into the vector, insertion of the selection marker gene (if necessary), insertion of the promoter (if necessary), etc. can be carried out using standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York, and the known methods using restriction enzymes and DNA ligases can be referred to).

[0114] As the host cell, from the aspect of ease of operation, microorganisms such as Escherichia coli, Bacillus subtilis, yeast (Saccharomyces cerevisiae, Pichia pastoris), etc. are preferably used. As long as it is a host cell in which recombinant DNA can replicate and express the gene of this enzyme, there is no particular limitation. As an example of Escherichia coli, Escherichia coli BL21(DE3)pLysS can be cited when using the T7 series promoter, and Escherichia coli JM109 can be cited when it is not the case. In addition, as examples of yeast, budding yeast SHY2, budding yeast AH22 or budding yeast INVSc1, Pichia pastoris KM-71H (Invitrogen) can be cited.

[0115] When preparing this enzyme, a microorganism carrying recombinant DNA (i.e., a transformant) can also be used. The microorganism can be obtained by transfection or transformation using the above-mentioned vector. For example, it can be carried out by the calcium chloride method (J. Mol. Biol., Vol. 53, p. 159 (1970)), the Hanahan method (J. Mol. Biol., Vol. 166, p. 557 (1983)), the SEM method (Gene, Vol. 96, p. 23 (1990)), the method of Chung et al. (Proceedings of the National Academy of Sciences USA, Vol. 86, p. 2172 (1989)), the calcium phosphate coprecipitation method, the electroporation method (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161 - 7165 (1984)), the lipofection method (Felgner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7413 - 7417 (1984)), etc. The above-mentioned microorganism can be used to produce this enzyme.

[0116] 3. Use of this enzyme preparation

[0117] The seventh aspect of the present invention relates to the use of this enzyme preparation. This enzyme preparation is preferably for changing the taste of food. In the seventh aspect of the present invention, a method for manufacturing food or a food material is provided, which includes the following steps: allowing this enzyme preparation to act on a raw material containing protein. In addition, in the seventh aspect of the present invention, a method for manufacturing a flavoring agent is provided, which includes the following steps: allowing this enzyme preparation to act on at least one selected from proteins and peptides.

[0118] In the method for manufacturing food or a food material of the present invention, this enzyme preparation is allowed to act on a raw material containing protein. For example, this enzyme preparation is added to a solution containing protein, and reacted for a specified time (for example, 1 hour to 12 hours) under the conditions of 30 to 70 °C, preferably 40 to 60 °C. Based on the decomposition reaction of the active ingredient aminopeptidase of this enzyme preparation, specific amino acids (such as alanine, glycine, etc.) are generated as a result. The composition, ratio, etc. of free amino acids in the product can vary depending on the type, source, etc. of the substrate used, but according to the manufacturing method of the present invention, food or a food material containing particularly more alanine and glycine can be obtained.

[0119] In the method for manufacturing the flavoring agent of the present invention, the present enzyme agent is allowed to act on at least one selected from proteins and peptides. For example, the present enzyme agent is added to a solution containing at least one selected from proteins and peptides, and the reaction is carried out for a specified time (for example, 1 hour to 12 hours) under the conditions of 30 to 70 °C, preferably 40 to 60 °C. As a result of the decomposition reaction of the active ingredient aminopeptidase of the present enzyme agent, specific amino acids (such as alanine and glycine) are generated. The composition, ratio, etc. of the free amino acids in the product can vary depending on the type, source, etc. of the substrate used, but according to the manufacturing method of the present invention, a flavoring agent containing particularly a large amount of alanine and glycine can be obtained.

[0120] In addition, the seventh aspect of the present invention relates to foods and flavoring agents produced by the above manufacturing method. The food produced by the above manufacturing method contains the present enzyme agent and at least one selected from proteins and peptides. It should be noted that the enzyme contained in the food can be inactivated. Moreover, the food preferably contains amino acids liberated from at least one selected from proteins and peptides. Similarly, the flavoring agent produced by the above manufacturing method contains the present enzyme agent and at least one selected from proteins and peptides. It should be noted that the enzyme contained in the flavoring agent can be inactivated. Moreover, the flavoring agent preferably contains amino acids liberated from at least one selected from proteins and peptides.

[0121] The source of the raw material containing protein used is not particularly limited. For example, it can include animal sources (such as milk, livestock meat, fish meat, etc.), plant sources (such as grains like barley, rice, wheat, rye, oats, buckwheat, sorghum, panicum miliaceum, millet, teff, quinoa, corn, etc.; beans like soybeans, lentils, broad beans, peas, chickpeas, mung beans, lupins, kidney beans, etc.; and fruits like canary seeds, flaxseeds, almonds, cashews, hazelnuts, pecans, macadamia nuts, pistachios, walnuts, brazil nuts, peanuts, coconuts, pili nuts, chestnuts, sesame seeds, and pine nuts, etc.), insect sources, and raw materials of microbial sources. In addition, the source of the protein and peptide used is not particularly limited, and proteins and peptides derived from the above raw materials can be used, or peptides obtained by synthesis, etc. can also be used.

[0122] After the enzymatic reaction using the present enzyme agent, purification treatment (such as filtration, ion exchange, activated carbon treatment) can be carried out as needed to remove insoluble components, improve purity, or decolorize and deodorize.

[0123] The eighth aspect of the present invention relates to a method for changing the taste properties of food, which comprises the following steps: allowing the present enzyme preparation to act on a raw material containing protein. By allowing the present enzyme preparation to act on a raw material containing protein, a decomposition reaction is produced by the active ingredient aminopeptidase of the present enzyme preparation, and as a result, specific amino acids (such as alanine and glycine) are generated. The taste properties of food can be changed by the amino acids thus generated. It should be noted that the "food" in this specification also includes food and beverages.

[0124] Examples

[0125] <Example 1>

[0126] 1. Exploration of aminopeptidase

[0127] Aspergillus nidulans strain A26 was liquid-cultured using tryptone as the sole carbon and nitrogen source. After cultivation, the protein secreted into its culture medium was subjected to SDS-PAGE. Comprehensive analysis (secretome) of the detected extracellular proteins was performed, and proteins with unknown functions (HPs) were identified using MALDI-TOF / MS together with known peptidases.

[0128] mRNA was extracted from Aspergillus nidulans using RNeasy Plant Mini Kit (QIAGEN), and cDNA (SEQ ID NO: 3) was prepared using PrimeScript TM II 1st strand cDNA Synthesis Kit (TAKARA). Based on the publicly available genomic information, the cDNA (SEQ ID NO: 3) encoding HP was amplified by PCR and fused to pPICZα-A (Invitrogen). The resulting vector was introduced into Pichia pastoris KM71H (Invitrogen). Screening of the transformants was carried out by screening for Zeocin-resistant strains. The transformants were cultured to secrete the target recombinant protein, which was concentrated and desalted to obtain an HP sample.

[0129] Using alanine (Ala)-pNA ((Peptide Institute, Inc.)) as a substrate and allowing the HP sample to act, a decomposition reaction could be confirmed. Since it did not act on the substrate of carboxypeptidase, it was shown that HP was aminopeptidase.

[0130] (Enzyme activity assay method)

[0131] React the reaction solution (50 mM buffer (tris-HCl) pH 8.0, 1.0 mM substrate (Ala-pNA), 0.014 mg / mL enzyme) at 45 °C for 25 minutes. After the enzyme reaction, calculate the amount of free pNA by measuring the absorbance at a wavelength of 405 nm. The enzyme amount that increases the absorbance at a wavelength of 405 nm by 27.5 in 1 minute (optical path 5.56 mm) is defined as 1 unit (U) (the enzyme amount that generates 1 μmol of pNA in 1 minute is set as 1 U).

[0132] 2. Enzymatic Chemical Properties of Aminopeptidase

[0133] (1) Substrate Specificity

[0134] Investigate the substrate specificity of this enzyme. As substrates, use various amino acid-pNAs (Ala-pNA, Gly-pNA, Lys-pNA, Val-pNA, Arg-pNA, Phe-pNA, Pro-pNA, Ile-pNA, Glu-pNA, Leu-pNA, Asp-pNA) and measure the activity. Evaluate the substrate specificity with the relative activity when the activity value of Ala-pNA showing the highest activity is set as 100%.

[0135] The results are shown in Table 1. As shown in Table 1, the aminopeptidase contained in the HP sample has the highest reactivity with alanine. In addition, it is shown that high reactivity is also exhibited towards glycine, lysine, valine, arginine, and phenylalanine in addition to alanine.

[0136] [Table 1]

[0137] substrate Relative activity (%) Ala-pNA 100 Gly-pNA 58 Lys-pNA 45 Val-pNA 35 Arg-pNA 33 Phe-pNA 15 Pro-pNA 7 Ile-pNA 7 Glu-pNA 6 Leu-pNA 4 Asp-pNA 3

[0138] (2) Optimum Temperature

[0139] Investigate the optimum temperature of the aminopeptidase obtained above. Use Ala-pNA as the substrate and measure the activity while changing the reaction temperature from 10 °C to 80 °C. Calculate the relative amount of activity at each temperature condition with the activity under the temperature condition showing the maximum activity set as 100% as the relative activity (%).

[0140] The results are shown in Figure 1 . As Figure 1 shown, the optimum temperature of the aminopeptidase is 50 °C.

[0141] (3) Optimum pH

[0142] Investigate the optimal pH of this enzyme. Using Ala-pNA as the substrate, adjust the pH of the reaction solution from pH 3 to 11, and measure the activity at each pH. As the buffer solution, 50 mM Mcllvaine buffer is used when the pH is from 3 to 7, 50 mM tris-hydrochloride buffer is used when the pH is from 7 to 8, and 50 mM CAPS buffer is used when the pH is from 8 to 11. Calculate the relative amount of the activity under each pH condition as the relative activity (%) with the activity under the pH condition showing the maximum activity set as 100%.

[0143] The results are shown in Figure 2 . As Figure 2 shown, the optimal pH of the aminopeptidase is pH 7 - 8.

[0144] (4) Peptide decomposition activity

[0145] Investigate the peptide decomposition activity of this enzyme. As the substrates, use Ala-Ala peptide, Ala-Ala-Ala peptide, Ala-Ala-Ala-Ala-Ala peptide (SEQ ID NO: 6), Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg peptide (SEQ ID NO: 7), and let the aminopeptidase act on them. Analyze the reaction products by TLC or MALDI-TOF-MS.

[0146] As a result, when any of Ala-Ala peptide, Ala-Ala-Ala peptide, and Ala-Ala-Ala-Ala-Ala peptide is used as the substrate, the generation of Ala can be confirmed. In addition, when Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg peptide is used as the substrate, the cleavage between Lys-Arg at the N-terminus and the cleavage between Arg-Pro at the N-terminus after the release of Lys can be confirmed. Thus, it can be confirmed that this enzyme also has aminopeptidase activity in actual peptides.

[0147] In addition, use BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu (SEQ ID NO: 13: 1424 Da)), angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 14: 931 Da)) as the substrates, and let the aminopeptidase act on them. As the reaction solution, use 50 mM buffer (tris-HCl) pH 8.0, 0.1 mM substrate (each peptide), 1.0 mM CuSO 4 and 0.1 μM enzyme, and react at 50 °C for 180 minutes. Analyze the reaction products by TLC or MALDI-TOF-MS.

[0148] As a result, cleavage between Tyr-Gly, Gly-Gly, and Gly-Phe at the N-terminus was confirmed when BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu) was used as the substrate. In addition, cleavage between Arg-Val and Val-Tyr at the N-terminus was confirmed when angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) was used as the substrate. Based on the above results, it was also confirmed that this enzyme has aminopeptidase activity in actual peptides.

[0149] (5) Metal ions

[0150] The enzyme activity was measured by the following method when various metal ions or EDTA as a metal ion chelator were added to the reaction solution. As the metal ions, CuCl 2 , ZnCl 2 , MnCl 2 , MgCl 2 , CaCl 2 , CoCl 2 , FeCl 2 were used.

[0151] The substrate solution (50 mM buffer (tris-HCl) pH 8.0, 1.0 mM substrate (Ala-pNA), 1.0 mM various metal ions) was incubated at 45 °C for 5 minutes. Enzyme was added thereto to a final concentration of 0.3 mg / mL, and the reaction was carried out at 45 °C for 20 minutes. After the addition of the enzyme, the amount of free pNA was calculated by measuring the absorbance at a wavelength of 405 nm.

[0152] The results are shown in Figure 3 . As Figure 3 shown, compared with the sample without added metal ions (non-denatured, native) and the sample added with EDTA, the relative activity was higher in the samples added with metal ions. It should be noted that Figure 3 in, the activity value of the sample using Ala-pNA as the substrate and adding CuCl 2 as the metal ion was set as 100%.

[0153] <Example 2>

[0154] 1. Exploration of aminopeptidase

[0155] The Aspergillus oryzae strain was cultured in liquid medium with tryptone as the sole carbon and nitrogen source. After cultivation, the proteins secreted into its culture medium were subjected to SDS-PAGE. Comprehensive analysis (secretome) of the detected extracellular proteins was performed, and MALDI-TOF / MS was used to identify proteins with unknown functions (HP) together with known peptidases.

[0156] Total RNA was extracted from Aspergillus oryzae using the RNeasy Plant Mini Kit (QIAGEN), and cDNA (Accession No. 10) was prepared using the PrimeScript TM II 1st strand cDNA Synthesis Kit (TAKARA). According to the publicly available genomic information, the cDNA (Accession No. 10) encoding HP was amplified by PCR and fused to pPICZα-A (Invitrogen). The resulting vector was introduced into Pichia pastoris KM71H (Invitrogen). Screening of the transformants was carried out by screening for Zeocin-resistant strains. The transformants were cultured to secrete the target recombinant protein, which was then concentrated and desalted to obtain the HP sample.

[0157] Using alanine (Ala)-pNA (Peptide Institute, Inc.) as the substrate, the HP sample was allowed to act, and as a result, a decomposition reaction could be confirmed. Since it did not act on the substrates of carboxypeptidases, it was indicated that HP was an aminopeptidase.

[0158] 2. Enzymatic properties of aminopeptidase

[0159] (Method for measuring enzyme activity)

[0160] A reaction solution (50 mM buffer (tris-HCl) pH 8.0, 2.5 mM substrate (various amino acid-pNA (Ala-pNA, Gly-pNA, Lys-pNA, Val-pNA, Arg-pNA, Phe-pNA, Pro-pNA, Ile-pNA, Glu-pNA, Leu-pNA, Asp-pNA)), 0.014 mg / mL enzyme, 1 mM CuCl 2 ) was prepared and reacted at 45 °C for 60 minutes. After the enzyme reaction, the amount of free pNA was calculated by measuring the absorbance at a wavelength of 405 nm. The amount of enzyme that increased the absorbance at a wavelength of 405 nm by 27.5 (optical path length 5.56 mm) in 1 minute was defined as 1 unit

[0161] (U) (the amount of enzyme that generates 1 μmol of pNA in 1 minute was set as 1 U).

[0162] The results are shown in Table 2. The substrate specificity was evaluated using the relative activity when the activity value in Lys-pNA, which shows the highest activity, was set to 100%. As shown in Table 2, the aminopeptidase contained in the HP sample has the highest reactivity with lysine. In addition, it was shown that high reactivity was also exhibited towards glycine, alanine, valine, arginine, and leucine in addition to lysine.

[0163] [Table 2]

[0164]

[0165] Industrial Applicability

[0166] The enzyme preparation of the present invention contains a novel aminopeptidase as an active ingredient. The enzyme preparation of the present invention is suitable for uses such as changing the taste of foods, and has high industrial utility value.

[0167] The present invention is not limited by any of the descriptions of the above embodiments and examples of the invention. Various modification methods within the range that can be easily conceived by those skilled in the art without departing from the scope of the claims are also included in the invention. The contents of papers, published patent gazettes, and patent gazettes explicitly stated in this specification are incorporated by reference in their entirety.

[0168] Sequence No. 1: Amino acid sequence of this enzyme (mature sequence)

[0169] Sequence No. 2: Amino acid sequence of this enzyme (full-length sequence)

[0170] Sequence No. 3: Base sequence of cDNA of this enzyme (mature sequence)

[0171] Sequence No. 4: Base sequence of cDNA of this enzyme (full-length sequence)

[0172] Sequence No. 5: Base sequence of the genome of this enzyme (full-length sequence, including introns)

[0173] Sequence No. 6: Peptide (substrate)

[0174] Sequence No. 7: Peptide (substrate)

[0175] Sequence No. 8: Amino acid sequence of this enzyme (mature sequence)

[0176] Sequence No. 9: Amino acid sequence of this enzyme (full-length sequence)

[0177] Sequence No. 10: Base sequence of cDNA of this enzyme (mature sequence)

[0178] Sequence No. 11: Base sequence of cDNA of this enzyme (full-length sequence)

[0179] Serial number 12: Base sequence of the genome of this enzyme (full-length sequence, including introns)

[0180] Serial number 13: Peptide (substrate)

[0181] Serial number 14: Peptide (substrate)

[0182] Serial number 1

[0183] AETMEYLMSLKQQSRERARSQGLFDINRYPDEGAKKCKNGKAGEYSCENVDLLSFLSHQALGSVTREGNDVWGWTSAEGREFGIVGQTDGVAFVEILEDGSLEYVGRLGSQTEPSTWRDIKVIGDHAYIGSEAAGHGLQIFDLNKLTTASSSKPTVFSTKKDLTAWYRGFGSSHNIVAHEETNMIYAVGTARNLSCAGGLWMVDVSDPANPTSPGCVNEDGYVHDAQCVIYKGPDEKYIGQEICFNFNEDTLTIADVTDKKNPIQISKTPYVGASYTHQGWLVDENDHSYLLLDDELDEMDGTGSAANGHTTTYIFDIKDLSAPKHTGTYQSPVRSIDHNQYVVAGLSYQSNYGSGLRVVDVSSVFEDPTASSFKEVGSFDVHPEDDAVGGEVEFVGSWSVYPFFASGHILLNSIERGIYSLKYTGPAAEN

[0184] Serial number 2

[0185] MKLSALSLLTVACVATASSHGAETMEYLMSLKQQSRERARSQGLFDINRYPDEGAKKCKNGKAGEYSCENVDLLSFLSHQALGSVTREGNDVWGWTSAEGREFGIVGQTDGVAFVEILEDGSLEYVGRLGSQTEPSTWRDIKVIGDHAYIGSEAAGHGLQIFDLNKLTTASSSKPTVFSTKKDLTAWYRGFGSSHNIVAHEETNMIYAVGTARNLSCAGGLWMVDVSDPANPTSPGCVNEDGYVHDAQCVIYKGPDEKYIGQEICFNFNEDTLTIADVTDKKNPIQISKTPYVGASYTHQGWLVDENDHSYLLLDDELDEMDGTGSAANGHTTTYIFDIKDLSAPKHTGTYQSPVRSIDHNQYVVAGLSYQSNYGSGLRVVDVSSVFEDPTASSFKEVGSFDVHPEDDAVGGEVEFVGSWSVYPFFASGHILLNSIERGIYSLKYTGPAAEN

[0186] Serial number 3

[0187]

[0188] Serial number 4

[0189]

[0190] Serial number 5

[0191]

[0192] Serial number 6

[0193] AAAAA

[0194] Serial number 7

[0195] KRPPGFSPFR

[0196] Serial number 8

[0197] APIMSHLMSIKTEHRERARAQGLFKPNSYIDLAKTPCVDGKAGEYSCENVDLLGFLSHQAMGSTTREGNDIWGWTSADGREFGIVGQTDGTAFVEVLDDGSLQYVGRLPTQTTATIWRDMKVIGDHAYIGSESPGHGLQIFDLKKLLETDSNNPTNFSTTEDLTAWYSGFGSSHNIVAHEETNMIFAVGTARNLSCAGGLWMIDVSDPANPTSPGCVSEDGYVHDAQCVIYTGPDKEYTNREICFNYNEDTLTIVDITDRASPIQISKTPYVGASYTHQGWIAVSDMSYLLLDDELDEQDGTGEAANGHTTTYIFDIKDLANPKHTGTYQSPVRSIDHNQYVIDGLTYQANYGSGLRIVDVSSVKDDPTGKGFKQVGFFDCHPEDDAQGGEVEFVGAWSVYPYFRSGNILLNSIERGVYSLKYTGKA

[0198] Serial number 9

[0199] MKPSFVSLLSLACLGAASREGAPIMSHLMSIKTEHRERARAQGLFKPNSYIDLAKTPCVDGKAGEYSCENVDLLGFLSHQAMGSTTREGNDIWGWTSADGREFGIVGQTDGTAFVEVLDDGSLQYVGRLPTQTTATIWRDMKVIGDHAYIGSESPGHGLQIFDLKKLLETDSNNPTNFSTTEDLTAWYSGFGSSHNIVAHEETNMIFAVGTARNLSCAGGLWMIDVSDPANPTSPGCVSEDGYVHDAQCVIYTGPDKEYTNREICFNYNEDTLTIVDITDRASPIQISKTPYVGASYTHQGWIAVSDMSYLLLDDELDEQDGTGEAANGHTTTYIFDIKDLANPKHTGTYQSPVRSIDHNQYVIDGLTYQANYGSGLRIVDVSSVKDDPTGKGFKQVGFFDCHPEDDAQGGEVEFVGAWSVYPYFRSGNILLNSIERGVYSLKYTGKA

[0200] Serial number 10

[0201]

[0202] Serial number 11

[0203]

[0204] Serial number 12

[0205] atgaaaccatcatttgtctcccttttgtccctggcttgcctcggggcagcctcccgcgagggcgctccgattatgagccatttaatgtctattaaaacagaacatcgtgagcgagctcgcgcccagggcttattcaagccaaacagctacattgacctcgcaaagacgccttgcgtggatggcaaagcaggagaatattcctgtgagaacgttgatcttcttggcttcctgagtcatcaggccatgggcagtacgacccgggaggggaatgatatctggggtatgttaaccatactatgcatctgttccgtggacataagatttaacatgaacaggatggacatcagccgatggccgtgaattcggcatcgttggccaaacggacggaacggcctttgtagaagttctggacgatggcagtctgcagtatgtcggccgtctaccgacgcagacaacggcaactatttggagagatatgaaggtgattggggatcatgcctatattggatcagagtcaccgggacatggtcttcagatctttgacttgaagaaggtatattatcgtaacaatattgtgctcaatacttgagtacgcccaa

[0206] ggctaa

[0207] cggagtccagcttctcgagacggacagcaataacccgaccaatttctcgaccacggaagacctaacggcttggt

[0208] acagcggttttggtagctcacacaacatcgttgcgcatgaagagactaacatgatcttcgctgtcggcacagcca

[0209] ggaacctatcctgcgctggcggcctgtggatgatcgatgtgtctgatcccgcgaacccgacgtcgcctggatgt

[0210] gtcagtgaagacggttatgtgcacgatggtaggtctctatggttcaatgggtcagctcacagtccaatactaacgc

[0211] aaaccagcacaatgtgtgatctacaccggccccgacaaggaatacacaaaccgtgagatctgcttcaactacaa

[0212] cgaagacaccctcaccatcgtcgacataacagacagggcctcgccaatccagatctccaaaaccccctatgttg

[0213] gagcaagctacacgcaccaaggctggatagcagtctccgacatgtcctacctcctcctggacgacgaattggac

[0214] gaacaagacgggacaggcgaagccgctaacggacacacaacaacgtacattttcgacattaaggacctagcg

[0215] aatccgaaacatacagggacgtaccagtctcccgtgcgatcaatcgaccataaccagtatgttatcgatgggttg

[0216] acataccaagctaactacgggagcggattgcgcatcgtcgatgttagctctgttaaggatgatccgacaggcaa

[0217] ggggttcaaacaggtgggtttctttgactgccacccagaagatgatgcccagggcggggaagttgagtttgtgg

[0218] gtgcttggagtgtctatccatatttccggagcgggaatattttgttgaacagtatcgagaggggtgtctattcgttga

[0219] agtacaccggaaaggcttga

[0220] Sequence No. 13

[0221] YGGFMRRVGRPE

[0222] Serial number 14

[0223] RVYIHPF

Claims

1. An enzyme preparation comprising an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence equivalent thereto as an active ingredient.

2. The enzyme preparation according to claim 1, wherein the aminopeptidase composed of the equivalent amino acid sequence has a homology of 90% or more with the amino acid sequence of SEQ ID NO: 1 and has aminopeptidase activity.

3. The enzyme preparation according to claim 1, wherein the aminopeptidase is derived from Aspergillus nidulans.

4. An enzyme preparation comprising an aminopeptidase derived from Aspergillus nidulans and having the following physicochemical properties as an active ingredient, (1) Optimum temperature: around 50 °C, (2) Optimum pH: around 7 - 8, (3) Substrate specificity: having the highest specificity for L-alanine residues.

5. An enzyme preparation comprising an aminopeptidase composed of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence equivalent thereto as an active ingredient.

6. The enzyme preparation according to claim 5, wherein the aminopeptidase composed of the equivalent amino acid sequence has a homology of 90% or more with the amino acid sequence of SEQ ID NO: 8 and has aminopeptidase activity.

7. The enzyme preparation according to claim 5, wherein the aminopeptidase is derived from Aspergillus oryzae.

8. The enzyme preparation according to claim 5, wherein it has the highest specificity for L-lysine residues.

9. The enzyme preparation according to any one of claims 1 - 8, which is used for changing the taste of food.

10. A method for manufacturing a food or food material, comprising the step of: allowing the enzyme preparation according to any one of claims 1 - 8 to act on a raw material containing protein.

11. A method for manufacturing a flavoring, comprising the step of: allowing the enzyme preparation according to any one of claims 1 - 8 to act on at least one selected from proteins and peptides.

12. A method for changing the taste of food, comprising the step of: allowing the enzyme preparation according to any one of claims 1 - 8 to act on a raw material containing protein.

13. A food comprising the enzyme preparation according to any one of claims 1 - 8 and at least one selected from proteins and peptides.

14. A flavoring comprising the enzyme preparation according to any one of claims 1 - 8 and at least one selected from proteins and peptides.

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