Modified protein glutaminase

Abstract

The purpose of the present invention is to provide a protein glutaminase that has an optimum temperature in a temperature region higher than the optimum temperature of a protein glutaminase derived from C. proteolyticum. A protein glutaminase comprising a polypeptide that comprises an amino acid sequence in which, in the amino acid sequence indicated by SEQ ID NO: 1, at least one substitution has been introduced from among a substitution of the second amino acid residue to a valine residue, a substitution of the 113th amino acid residue to an alanine residue, a substitution of the 154th amino acid residue to an aspartic acid residue, and a substitution of the 158th amino acid residue to a histidine residue has an optimum temperature in a temperature region higher than that of a polypeptide comprising the amino acid sequence indicated by SEQ ID NO: 1.

Description

Modified protein glutaminase

[0001] The present invention relates to modified protein glutaminases. More specifically, the present invention relates to protein glutaminases that have been modified to have an improved optimum temperature.

[0002] Protein glutaminase is an enzyme that acts on high-molecular-weight proteins, catalyzing the decomposition (i.e., deamidation) of amide-containing side chains without cleaving peptide bonds or crosslinking proteins. Protein glutaminase deamidates glutamine residues in proteins, generating negatively charged carboxyl groups, thereby altering various protein properties. For example, increased hydration and electrostatic repulsion due to a decrease in the isoelectric point of proteins reduce protein-protein interactions (i.e., reduce association), thereby increasing the solubility and water dispersibility of proteins. Furthermore, the exposure of internal hydrophobic regions due to changes in the higher-order structure of proteins confers surface activity to proteins, thereby improving their emulsifying power, emulsion stability, foaming ability, and foam stability. Because protein glutaminase can significantly alter protein properties, it has dramatically expanded the applications of proteins. For this reason, protein glutaminase is highly useful and has attracted great interest in the art.

[0003] The first protein glutaminase discovered was found in Chryseobacterium proteolyticum in 2000 (Non-Patent Document 1). Since then, for a long time, protein glutaminase derived from C. proteolyticum has been the only active ingredient in protein glutaminase enzyme preparations that has been used industrially.

[0004] A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil. Applied and Environmental Microbiology 2000; 66(8): 3337-43

[0005] Protein glutaminase derived from C. proteolyticum is highly useful because it has a high optimum temperature of 60°C, but its reactivity drops significantly at temperatures higher than the optimum temperature. Therefore, when using protein glutaminase derived from C. proteolyticum, the temperature must be strictly limited so as not to exceed 60°C, taking into account the inherent thermal properties of the enzyme.

[0006] On the other hand, in view of the possibility of further expanding the applications expected from the high usefulness of protein glutaminase, it is desirable to create a protein glutaminase having an optimum temperature in a higher range than that of protein glutaminase derived from C. proteolyticum.

[0007] Therefore, an object of the present invention is to provide a protein glutaminase having an optimum temperature in a higher range than the optimum temperature of protein glutaminase derived from C. proteolyticum.

[0008] As a result of extensive research, the present inventors have discovered a new mutation that improves the optimum temperature in protein glutaminase derived from C. proteolyticum. The present invention was completed based on this finding.

[0009] That is, the present invention provides the following aspects: Item 1. A modified protein glutaminase consisting of a polypeptide shown in any of (I) to (III) below: (I) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, into which at least one of the following substitutions has been introduced: a valine residue at position 2, an alanine residue at position 113, an aspartic acid residue at position 154, and a histidine residue at position 158; (II) a polypeptide into which one or more amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted in the amino acid sequence into which the substitutions have been introduced, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (III) a polypeptide into which the sequence identity of the portion of the amino acid sequence into which the substitutions have been introduced, excluding the substituted amino acid residue, is 70% or more, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. Item 1. DNA encoding the modified protein glutaminase of Item 1. Item 3. An expression cassette or recombinant vector comprising the DNA of Item 2. Item 4. A transformant obtained by transforming a host with the expression cassette or recombinant vector of Item 3. Item 5. A method for producing a modified protein glutaminase, comprising the step of culturing the transformant of Item 4. Item 6. An enzyme preparation comprising the modified protein glutaminase of Item 1. Item 7. A protein material modifier comprising the modified protein glutaminase of Item 1. Item 8. A method for producing a modified protein material, comprising an enzyme treatment step of allowing the modified protein glutaminase of Item 1 to act on a protein material. Item 9. The production method of Item 8, wherein the protein material is a vegetable protein material. Item 10. The production method of Item 8 or 9, wherein the protein material is provided in the form of a vegetable protein-containing liquid composition prepared using cereals, and wherein the vegetable protein-containing liquid composition is simultaneously treated with the modified protein glutaminase and amylase in the enzyme treatment step.

[0010] According to the present invention, there is provided a protein glutaminase having an optimum temperature in a range higher than the optimum temperature of protein glutaminase derived from C. proteolyticum.

[0011] 1 shows a comparison of the temperature dependence of the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) and the specific activity of the C. proteolyticum-derived protein glutaminase (Comparative Example 1, dashed line) when Z-Gln-Gly is used as a substrate. 1 shows a comparison of the temperature dependence of the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) and the specific activity of the modified protein glutaminase of the present invention (Example 2, dashed dotted line) when Z-Gln-Gly is used as a substrate. 1 shows a comparison of the temperature dependence of the specific activity of the protein glutaminase of the present invention (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) when soybean protein is used as a substrate. 1 shows a comparison of the temperature dependence of the specific activity of the C. proteolyticum-derived protein glutaminase (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) when pea protein is used as a substrate. Figure 1 shows a comparison of the temperature dependence of the specific activity of the protein glutaminase derived from C. proteolyticum (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) when pea protein is used as a substrate. Figure 2 shows a comparison of the temperature dependence of the specific activity of the protein glutaminase derived from C. proteolyticum (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 2, dashed dotted line) when milk casein is used as a substrate. Figure 3 shows a comparison of the temperature dependence of the specific activity of the protein glutaminase derived from C. proteolyticum (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 1, solid line) when milk casein is used as a substrate. 1 shows a comparison of the temperature dependence of the specific activity of the protein glutaminase derived from C. proteolyticum (Comparative Example 1, dashed line) and the specific activity of the modified protein glutaminase of the present invention (Example 2, dashed dotted line).

[0012] The present invention will be described in detail below. The 20 types of amino acid residues in an amino acid sequence may be expressed by single-letter abbreviations: G for glycine (Gly), A for alanine (Ala), V for valine (Val), L for leucine (Leu), I for isoleucine (Ile), F for phenylalanine (Phe), Y for tyrosine (Tyr), W for tryptophan (Trp), S for serine (Ser), T for threonine (Thr), C for cysteine ​​(Cys), M for methionine (Met), D for aspartic acid (Asp), E for glutamic acid (Glu), N for asparagine (Asn), Q for glutamine (Gln), K for lysine (Lys), R for arginine (Arg), H for histidine (His), and P for proline (Pro).

[0013] In the present specification, the left end of an amino acid sequence is the N-terminus and the right end is the C-terminus.

[0014] As used herein, "nonpolar amino acids" include glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0015] As used herein, the term "substitution" refers not only to cases where an amino acid residue substitution is artificially introduced, but also to cases where an amino acid residue substitution is naturally introduced, i.e., cases where the amino acid residue is originally different. As used herein, the amino acid residue substitution may be either an artificial substitution or a natural substitution, with artificial substitution being preferred.

[0016] 1. Modified Protein Glutaminase The modified protein glutaminase of the present invention comprises a polypeptide shown in any one of (I) to (III) below:

[0017] (I) A polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 1, in which at least one of the following substitutions has been introduced: a valine residue at position 2, an alanine residue at position 113, an aspartic acid residue at position 154, and a histidine residue at position 158; (II) A polypeptide in which one or more amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted in the amino acid sequence in which the substitutions have been introduced, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (III) A polypeptide in which the sequence identity of the portion excluding the substituted amino acid residue in the amino acid sequence in which the substitutions have been introduced is 70% or more, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0018] The amino acid sequence shown in SEQ ID NO: 1 is the mature sequence of protein glutaminase derived from Chryseobacterium proteolyticum.

[0019] The polypeptide of (I) may have at least one of four substitutions (hereinafter also referred to as "predetermined substitution") introduced: a valine residue at amino acid residue position 2, an alanine residue at amino acid residue position 113, an aspartic acid residue at amino acid residue position 154, and a histidine residue at amino acid residue position 158. A preferred embodiment of the polypeptide of (I) consists of the amino acid sequence shown in SEQ ID NO: 2 in which all four substitutions have been introduced.

[0020] In the polypeptide (II), the amino acid modification introduced may include only one type of modification (e.g., substitution only) from substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide (II), the number of amino acid differences at any difference site may be one or several, for example, 1 to 55, 1 to 37, or 1 to 27, preferably 1 to 18, more preferably 1 to 10, even more preferably 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1 or 2, or 1.

[0021] Furthermore, in the polypeptide (III), the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 may be 70% or more, preferably 80% or more, or 85% or more, more preferably 90% or more, or 93% or more, even more preferably 95% or more, even more preferably 98% or more, still more preferably 98.5% or more, or 99% or more, and particularly preferably 99.3% or more, or 99.5% or more.

[0022] Here, in the polypeptide (III), the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 refers to the sequence identity calculated by comparing the sequence with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the term "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0023] In the polypeptides (II) and (III), the amino acid residues corresponding to positions 42 (cysteine ​​residue), 83 (histidine residue), and 103 (aspartic acid residue) in the amino acid sequence shown in SEQ ID NO: 1 are considered to be active catalytic residues, and therefore it is desirable not to introduce substitutions or deletions at these positions.

[0024] In the polypeptides (II) and (III), when an amino acid substitution is introduced into SEQ ID NO: 1, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of substitutions in the polypeptides (II) and (III) include substitution of a nonpolar amino acid with another nonpolar amino acid, substitution of an uncharged amino acid with another uncharged amino acid, substitution of an acidic amino acid with another acidic amino acid, and substitution of a basic amino acid with another basic amino acid.

[0025] From the viewpoint of further improving the specific activity at a temperature higher than the optimum temperature (60°C) of the C. proteolyticum-derived protein glutaminase consisting of the amino acid sequence shown in SEQ ID NO: 1, and more preferably from the viewpoint of further improving the specific activity at said high temperature when a protein is used as a substrate, preferred examples of the polypeptides (II) and (III) above include polypeptides in which, in addition to the specified substitution, the amino acid residue at position 26 (serine residue) in the amino acid sequence shown in SEQ ID NO: 1 has been substituted with a proline residue (hereinafter also referred to as "other specified substitutions"). More preferred examples include polypeptides containing all of the specified substitutions (all of the above four substitutions) and the other specified substitutions.

[0026] The polypeptides shown in (I) to (III) above have protein glutaminase activity and have optimum temperatures in a higher range than the optimum temperature (60°C) of the protein glutaminase derived from C. proteolyticum comprising the amino acid sequence shown in SEQ ID NO: 1. Specific optimum temperatures of the polypeptides shown in (I) to (III) when Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) is used as a substrate are, for example, 61 to 70°C, preferably 62 to 68°C, more preferably 63 to 67°C, and even more preferably 64 to 66°C.

[0027] The modified protein glutaminase of the present invention may also be used as an active ingredient in the enzyme preparations described below, or may be used to constitute a part of a larger protein integrated with peptides or proteins consisting of other amino acid sequences (hereinafter also referred to as "other proteins, etc.") in the form of a fusion protein, etc. Examples of such other proteins, etc. include peptides derived from additional sequences, such as polyhistidine residues, used for protein purification to ensure the stability of mRNA during recombinant production.

[0028] 2. DNA The DNA of the present invention is a DNA encoding the modified protein glutaminase described above in "1. Modified Protein Glutaminase."

[0029] The DNA of the present invention is not particularly limited as long as it has a nucleotide sequence encoding a modified protein glutaminase consisting of the polypeptides shown in (I) to (III) described above in "1. Modified Protein Glutaminase." An example of a nucleotide sequence of DNA encoding the amino acid sequence shown in SEQ ID NO: 1 (protein glutaminase derived from Chryseobacterium proteolyticum), which is the reference sequence for the polypeptides shown in (I) to (III), is SEQ ID NO: 3. Therefore, the DNA of the present invention can be appropriately designed by those skilled in the art using SEQ ID NO: 3 as the reference sequence.

[0030] Examples of the DNA of the present invention include the DNAs shown in any of [i] to [iii] below.

[0031] [i] DNA consisting of a nucleotide sequence in which at least one of the following substitutions has been introduced into the nucleotide sequence shown in SEQ ID NO: 3: substitutions at positions 4 to 6 with a nucleotide sequence encoding a valine residue; substitutions at positions 337 to 339 with a nucleotide sequence encoding an alanine residue; substitutions at positions 460 to 462 with a nucleotide sequence encoding an aspartic acid residue; and substitutions at positions 472 to 474 with a nucleotide sequence encoding a histidine residue; [ii] DNA encoding a polypeptide having an optimum temperature higher than that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which hybridizes under stringent conditions with DNA having a nucleotide sequence complementary to the DNA shown in [i] above; and [iii] DNA encoding a polypeptide having an optimum temperature higher than that of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which has a homology of 70% or more to the DNA shown in [i] above.

[0032] The specific sequence of the DNA of [i] above can be easily determined by a person skilled in the art. For example, an example of a DNA sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, into which all four substitutions have been introduced, which is a preferred embodiment, is SEQ ID NO: 4.

[0033] With regard to the DNA of [ii] above, "under stringent conditions" refers to conditions in which the DNA is incubated at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400) and 100 μg / ml salmon sperm DNA.

[0034] Specifically, hybridization under stringent conditions is carried out by the following method: a nylon membrane onto which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6x SSC, 0.5% SDS, 5x Denhardt's buffer, and 100 µg / ml salmon sperm DNA. 32Each P-labeled probe is added and incubated overnight at 65° C. The nylon membrane is washed in 6×SSC at room temperature for 10 minutes, in 2×SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2×SSC containing 0.1% SDS at 45° C. for 30 minutes, and then autoradiography is performed to detect DNA that has specifically hybridized with the probe.

[0035] For the DNA of [iii], the homology may be 70% or more, preferably 80% or more, or 85% or more, more preferably 90% or more, or 93% or more, even more preferably 95% or more, still more preferably 98% or more, even more preferably 98.5% or more, or 99% or more, and particularly preferably 99.3% or more, or 99.5% or more.

[0036] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, GENETYX (manufactured by Genetics Corporation), or the like can be used, and these may be used with default parameters.

[0037] The DNA of the present invention can be obtained, for example, by introducing the above-mentioned predetermined substitution into DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (i.e., protein glutaminase derived from C. proteolyticum). The DNA of the present invention can also be artificially synthesized by total gene synthesis.

[0038] DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 can be obtained by a standard method using PCR from a nucleic acid construct such as a plasmid incorporating the nucleotide sequence shown in SEQ ID NO: 3. In addition to the nucleotide sequence shown in SEQ ID NO: 3, the nucleic acid construct can also incorporate a sequence in which a signal sequence and a pro-sequence are added to the 5'-end of the nucleotide sequence.

[0039] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known methods such as the Kunkel method and the Gapped duplex method, and site-directed mutagenesis kits such as QuikChange. TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), Takara Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.), etc. can be used.

[0040] The DNA of the present invention encompasses various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are used infrequently in the host, the expression level of the protein may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0041] The sum of the host-optimal codon usage frequencies for each codon can be used as an index of codon usage frequency. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for E. coli: F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0042] The base sequence of DNA into which a mutation has been introduced can be confirmed by conventional sequencing. Specific sequencing methods include the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463) and sequence analysis using an appropriate DNA sequencer. Methods for confirming whether a DNA encodes a polypeptide of interest include comparing the determined base sequence with an unsubstituted base sequence such as the base sequence shown in SEQ ID NO: 3, or comparing the amino acid sequence deduced from the determined base sequence with an unsubstituted amino acid sequence such as the amino acid sequence shown in SEQ ID NO: 1.

[0043] 3. Expression Cassette or Recombinant Vector The expression cassette or recombinant vector of the present invention comprises the DNA of the present invention described above in "2. DNA." The expression cassette or recombinant vector of the present invention can be obtained by linking a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette of the present invention or the DNA of the present invention into an expression vector.

[0044] The expression cassette or recombinant vector of the present invention may contain, as control elements, a promoter and a terminator, as well as transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary. These control elements may be operably linked to the DNA of the present invention. "Operably linked" means that the DNA of the present invention is linked to various control elements that regulate the DNA of the present invention in a state that allows it to operate in a host cell.

[0045] Regarding the recombinant vector of the present invention, an expression vector constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host is preferred. Such expression vectors are known, and commercially available expression vectors include pQE-based vectors (Qiagen, Inc.), pDR540, pRIT2T (GE Healthcare Biosciences, Inc.), and pET-based vectors (Merck & Co., Inc.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21(DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain.

[0046] 4. Transformant The transformant of the present invention is obtained by transforming a host with the expression cassette or recombinant vector of the present invention described above in "3. Expression cassette or recombinant vector."

[0047] The host used for producing the transformant of the present invention is not particularly limited, as long as it allows gene introduction, the expression cassette or recombinant vector is stable, is capable of autonomous replication, and is capable of expressing the traits of the gene comprising the DNA of the present invention. Suitable examples include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, and the genus Chryseobacterium such as Chryseobacterium proteolyticum; yeast; and the like. Other examples include animal cells, insect cells, and plants.

[0048] The transformant of the present invention can be obtained by introducing the expression cassette of the present invention or the recombinant vector of the present invention into a host. The location of introduction of the DNA of the present invention is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, a recombinant vector method and a genome editing method. Conditions for introducing the expression cassette or recombinant vector into a host may be appropriately determined depending on the type of host, etc. When the host is a bacterium, examples of methods include a method using competent cells treated with calcium ions and an electroporation method. When the host is a yeast, examples of methods include electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of methods include electroporation, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of methods include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of methods include electroporation, the Agrobacterium method, the particle gun method, and the PEG method.

[0049] Whether or not the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0050] When confirming by PCR whether the expression cassette of the present invention or the recombinant vector of the present invention has been incorporated into a host, for example, genomic DNA, the expression cassette, or the recombinant vector may be isolated and purified from the transformant.

[0051] For example, when the host is a bacterium, the expression cassette or recombinant vector is isolated and purified using a lysate obtained by lysing the bacterium. Lysis can be achieved by treating the bacterium with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS).

[0052] Furthermore, physical disruption methods such as freeze-thawing and French press treatment may be combined. DNA can be separated and purified from the lysate by, for example, an appropriate combination of deproteinization treatments using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0053] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, a type II restriction enzyme that acts on a specific nucleotide sequence can be used. DNA can be ligated to an expression cassette or expression vector using, for example, DNA ligase.

[0054] Then, PCR is performed using the isolated and purified DNA as a template and primers specific to the DNA of the present invention. The PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band, thereby confirming transformation.

[0055] Alternatively, PCR may be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzyme reaction, or the like.

[0056] 5. Method for Producing Modified Protein Glutaminase The method for producing the modified protein glutaminase of the present invention is a method for producing the enzyme described above in "1. Modified Protein Glutaminase," and includes a step of culturing the transformant of the present invention. Note that, when the predetermined substitution contained in the modified protein glutaminase is naturally introduced, the modified protein glutaminase can be obtained by a production method including a step of culturing a microorganism that produces the modified protein glutaminase.

[0057] The culture conditions may be appropriately set taking into consideration the nutritional and physiological properties of the transformant or microorganism, but liquid culture is preferred. For industrial production, aeration and agitation culture is preferred. The nutrient source for the medium may be any nutrient required for the growth of the transformant or microorganism. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to the carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, may also be used as needed.

[0058] The culture temperature can be appropriately set within a range in which the transformant or microorganism of the present invention can grow and the transformant or microorganism can produce the modified protein glutaminase, and is preferably about 15 to 37° C. The culture may be completed at an appropriate time when the modified protein glutaminase reaches its maximum yield, and the culture time is usually about 12 to 48 hours.

[0059] After culturing the transformant or the microorganism, the culture medium is subjected to a method such as centrifugation, and the culture supernatant and / or bacterial cells are recovered. The bacterial cells are treated with a mechanical method such as ultrasound or a French press, or with a lytic enzyme such as lysozyme, and then solubilized, if necessary, using an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), to obtain a water-soluble fraction containing the desired modified protein glutaminase. Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed modified protein glutaminase can be secreted into the culture medium.

[0060] The water-soluble fraction containing the modified protein glutaminase obtained as described above may be subjected to a purification treatment as is, or the modified protein glutaminase in the water-soluble fraction may be concentrated and then subjected to a purification treatment. Concentration can be performed by, for example, vacuum concentration, membrane concentration, salting out, fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.), etc.

[0061] The modified protein glutaminase can be purified by, for example, an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. The purified modified protein glutaminase may be powdered, if necessary, by freeze-drying, vacuum drying, spray drying, etc.

[0062] 6. Enzyme Preparation The modified protein glutaminase can be provided in the form of an enzyme preparation. Accordingly, the present invention also provides an enzyme preparation comprising the modified protein glutaminase described above in "1. Modified Protein Glutaminase" as an active ingredient.

[0063] The content of modified protein glutaminase in the enzyme preparation of the present invention is not particularly limited, but the lower limit of the content can be, for example, 1 U / g or more, 5 U / g or more, or 10 U / g or more, preferably 50 U / g or more, 100 U / g or more, 500 U / g or more, or 800 U / g or more. The upper limit of the content can be, for example, 10,000 U / g or less, 5,000 U / g or less, 2,000 U / g or less, 1,500 U / g or less, or 1,000 U / g or less. Specific contents can be 1 to 10,000 U / g, 5 to 10,000 U / g, 10 to 10,000 U / g, 50 to 5,000 U / g, 100 to 2,000 U / g, 500 to 1,500 U / g, or 800 to 1,000 U / g.

[0064] One unit (1 U) of protein glutaminase activity is defined as the enzyme activity that produces 1 μmol of ammonia per minute using Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) as a substrate.

[0065] The enzyme preparation of the present invention may contain other components in addition to the modified protein glutaminase to the extent that the effects of the present invention are not affected. Examples of other components include enzymes other than the modified protein glutaminase, additives, and culture residues generated in the above-mentioned production method.

[0066] Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidase, β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), proteases (acid proteases, neutral proteases, alkaline proteases), peptidases (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatases (acid phosphatases, alkaline phosphatases), nucleases, deaminases, oxidases, dehydrogenases, glutaminase, pectinases, catalases, dextranases, transglutaminases, protein deamidase (other than the above-mentioned modified protein glutaminases), pullulanases, etc. One of these other enzymes may be contained alone, or multiple types may be contained in combination.

[0067] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, and glycerol. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of preservatives include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. These additives may be contained alone or in combination.

[0068] Culture residues include components derived from the culture medium, contaminating proteins, bacterial components, and the like.

[0069] The form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid and solid forms (powder, granules, etc.) Enzyme preparations in these forms can be prepared by generally known methods.

[0070] 7. Modifier for Protein Materials The modified protein glutaminase can be used for known applications of protein glutaminases. For example, the modified protein glutaminase can be used for the purpose of modifying protein materials. Therefore, the present invention also provides a modifier for protein materials containing the modified protein glutaminase.

[0071] The specific manner of modification of a protein material is not particularly limited, and any change in the properties of the protein may be brought about by deamidation of the γ-amide and β-amide groups of glutamine and asparagine residues in the protein to generate carboxyl groups. Specific modifications of the protein material include increased protein solubility, increased water dispersibility, improved emulsifying power, improved emulsion stability, improved foaming ability, and improved foam stability. Specific methods for using the modifier for a protein material are described below in "8. Method for producing modified protein material."

[0072] 8. Method for Producing Modified Protein Materials As described above, modified protein glutaminase can be used for the purpose of modifying protein materials. Accordingly, the present invention also provides a method for producing a modified protein material, which comprises an enzymatic treatment step in which the modified protein glutaminase acts on a protein material.

[0073] In the enzyme treatment step, for example, a mixture containing a protein material and modified protein glutaminase is subjected to conditions for the action of the modified protein glutaminase, thereby allowing the reaction to modify the protein to proceed.

[0074] The protein material is not particularly limited as long as it contains protein, and may be either edible or non-edible. Edible protein materials can be used as food or beverages, or as ingredients for producing food or beverages. Non-edible protein materials can be used as materials for protein experiments, medical materials, textile materials, cosmetic materials, etc.

[0075] Specific examples of protein materials include the protein source itself (i.e., an organ or part thereof of an organism, or a body fluid of an organism, etc.), and a preparation obtained by treating a protein source by a known method to concentrate the protein by removing at least one component other than the protein, and these can be selected appropriately by those skilled in the art. Furthermore, the protein may be either a plant protein or an animal protein, and either one of these may be used, or both may be used in combination.

[0076] When the protein is a vegetable protein, examples of the vegetable protein include proteins from cereals such as soybeans, peas, lentils, chickpeas, black beans, broad beans, mung beans, lupin beans, and kidney beans; proteins from cereals such as wheat, barley, oats, sorghum, rice, rye, buckwheat, barnyard millet, foxtail millet, teff, corn, and potato; proteins from nuts and seeds such as almonds, coconuts, peanuts, cashew nuts, hazelnuts, pecan nuts, macadamia nuts, pistachios, walnuts, Brazil nuts, pili nuts, chestnuts, sesame seeds, pine nuts, hemp seeds (hemp refers to industrial hemp containing less than 0.3% by weight, preferably less than 0.2% by weight, more preferably less than 0.01% by weight, and most preferably no tetrahydrocannabinol, per dry weight), chia seeds, chia seeds, amaranth, canary seeds, and flaxseed. These vegetable proteins may be used alone or in combination of two or more. Among these vegetable proteins, soybeans, peas, lentils, chickpeas, broad beans, mung beans, wheat, oats, rice, and almonds are preferred, soybeans, peas, lentils, chickpeas, broad beans, mung beans, wheat, oats, and almonds are more preferred, soybeans, lentils, chickpeas, broad beans, mung beans, wheat, oats, and almonds are even more preferred, and soybeans, broad beans, mung beans, wheat, oats, and almonds are even more preferred.

[0077] When the protein is an animal protein, examples of the animal protein include proteins of livestock meat, fish meat, eggs, milk, serum, hair, insects, etc., such as casein (α-casein, β-casein, κ-casein, etc.), whey protein, albumin (ovalbumin, α-lactalbumin, serum albumin, etc.), globulin (β-lactoglobulin, immunoglobulin, etc.), lactoferrin, wool, silk, myofibrillar proteins (myosin, actin, troponin, tropomyosin, etc.), muscle matrix proteins (collagen, etc.). These animal proteins may be used alone or in combination of two or more. Among these animal proteins, egg protein and milk protein are preferred, casein, whey protein, and ovalbumin are more preferred, and casein is even more preferred.

[0078] The protein material may be in a non-structured or structured form. When the protein material is in a non-structured form, its form may be a fluid form such as a liquid, a slurry, or a paste.

[0079] When the protein material is a non-structured protein material containing a vegetable protein, the protein material can be used in the form of the following vegetable protein-containing liquid composition when treated with modified protein glutaminase. (i) A liquid obtained by using a dried powder of a plant raw material (an organ or part of a plant from which plant protein is derived) as the plant material, dispersing the plant material or a water-soaked product thereof in water, and, if necessary, removing water-insoluble matter derived from the plant raw material by any means such as centrifugal filtration, filtration, a filter bag, or a sieve; (ii) A liquid obtained by using the plant raw material itself or a dried product thereof as the plant material, crushing and dispersing the plant material or a water-soaked product thereof in water, and, if necessary, removing water-insoluble matter derived from the plant raw material by any means such as centrifugal filtration, filtration, a filter bag, or a sieve; (iii) A liquid obtained by removing at least a portion of components other than plant protein from the liquid (i) or (ii) above to increase the plant protein content; (iv) A liquid obtained by using a dried powder prepared from any of the liquids (i) to (iii) above as the plant material, and dissolving and / or dispersing the plant material or a water-soaked product thereof in water, etc.

[0080] Protein glutaminase without any modification (i.e., protein glutaminase derived from C. proteolyticum) maintains relatively high protein glutaminase activity even at temperatures about 5°C higher than the optimum temperature when using animal protein as a substrate, but when using low-molecular-weight molecules such as Z-Gln-Gly or plant protein as substrates, activity drops sharply at temperatures about 5°C higher than the optimum temperature. In contrast, the modified protein glutaminase of the present invention can maintain relatively high protein glutaminase activity even at temperatures about 5°C higher than the optimum temperature when using plant protein as a substrate. Therefore, it can exhibit significantly high activity even against plant proteins that have previously been barely able to exhibit protein glutaminase activity when treated at high temperatures (e.g., about 70°C, specifically 67-73°C, 68-72°C, or 69-71°C). From this perspective, suitable protein materials include materials containing plant proteins.

[0081] Furthermore, the modified protein glutaminase of the present invention may have a higher optimum temperature when the substrate is an animal protein than when the substrate is a plant protein. Thus, in a preferred embodiment of the present invention, a material containing an animal protein that allows protein treatment at higher temperatures (e.g., 65 to 77°C, 65 to 76°C, or 65 to 75°C) is used.

[0082] The protein content of these protein materials is not particularly limited, and may be, for example, 10 to 100% by weight, 20 to 100% by weight, 30 to 100% by weight, 40 to 100% by weight, or 50 to 100% by weight. The lower limit of the range of these protein contents may be, for example, 40% by weight or more, 45% by weight or more, 50% by weight or more, 55% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, or 90% by weight or more. The upper limit of the range of these protein contents may be, for example, 95% by weight or less, 90% by weight or less, 85% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less.

[0083] The content of the protein material in the mixture is, for example, an amount such that the concentration of the protein contained in the protein material in the mixture is 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, 0.5 wt% or more, or 1 wt% or more, preferably 2 wt% or more, 3 wt% or more, and more preferably 4 wt% or more. The upper limit of the concentration of the protein contained in the mixture in the protein material is not particularly limited, but examples include 80 wt% or less, 60 wt% or less, 40 wt% or less, 30 wt% or less, 20 wt% or less, 15 wt% or less, 10 wt% or less, 8 wt% or less, or 5.5 wt% or less. Specific ranges for the content of the protein material in the mixture include 0.01 to 80 wt%, 0.05 to 60 wt%, 0.1 to 40 wt%, 0.5 to 30 wt%, or 1 to 20 wt%, preferably 2 to 15 wt% or 3 to 10 wt%, more preferably 4 to 8 wt% or 4 to 5.5 wt%.

[0084] The amount of modified protein glutaminase used is not particularly limited, and examples of the amount of modified protein glutaminase used per gram of protein contained in the protein material include 0.01 U or more, or 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, even more preferably 3.5 U or more, and even more preferably 4.5 U or more. The upper limit of the amount of modified protein glutaminase used per gram of protein contained in the protein material is not particularly limited, and examples include 50 U or less, 45 U or less, 35 U or less, 20 U or less, 10 U or less, 8 U or less, and 5.5 U or less.

[0085] The amount of modified protein glutaminase used per 1 g of protein material is, for example, 0.01 U or more, or 0.1 U or more, preferably 0.5 U or more, more preferably 1 U or more, even more preferably 2 U or more, still more preferably 3 U or more, and even more preferably 4 U or more. There are no particular limitations on the upper limit of the amount of modified protein glutaminase used per 1 g of protein material, and examples include 50 U or less, 40 U or less, 30 U or less, 20 U or less, 10 U or less, 7 U or less, and 5 U or less.

[0086] The conditions for the action of the modified protein glutaminase (temperature conditions, pH conditions, etc.) are appropriately determined based on the optimum temperature, optimum pH, etc. of the protein glutaminase to be used.

[0087] The optimum temperature of the modified protein glutaminase is higher than 60°C, which is the optimum temperature of protein glutaminase, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 without any specific substitutions. Therefore, when acting on a vegetable protein, the temperature conditions include, for example, 61°C or higher, preferably 62°C or higher, more preferably 63°C or higher, and even more preferably 64°C or higher or 65°C or higher, with the upper limit being, for example, 70°C or lower, 69°C or lower, 68°C or lower, 67°C or lower, or 66°C or lower. Furthermore, when acting on an animal protein, suitable temperature conditions include 66°C or higher, 67°C or higher, 68°C or higher, 69°C or higher, or 70°C or higher, with the upper limit being, for example, 77°C or lower, 76°C or lower, 75°C or lower, 74°C or lower, 73°C or lower, 72°C or lower, or 71°C or lower.

[0088] Furthermore, when a modified protein glutaminase uses a protein as a substrate, it can maintain relatively high enzymatic activity even at temperatures that are, for example, about 5°C higher than the optimum temperature, compared to when Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) is used as the substrate. Therefore, when acting on a vegetable protein, the temperature conditions include not only the above-mentioned temperature conditions (i.e., 61°C or higher, preferably 62°C or higher, more preferably 63°C or higher, even more preferably 64°C or higher or 65°C or higher), but also more preferably 66°C or higher, 67°C or higher, 68°C or higher, 69°C or higher, or 70°C or higher, and the upper limit thereof is not only the above-mentioned temperature conditions (i.e., 70°C or lower, 69°C or lower, 68°C or lower, 67°C or lower, or 66°C or lower), but also, for example, 73°C or lower, 72°C or lower, or 71°C or lower. Furthermore, when acting on animal proteins, the temperature conditions include not only the above-mentioned temperature conditions (i.e., 66°C or higher, 67°C or higher, 68°C or higher, 69°C or higher, or 70°C or higher), but also 71°C or higher, 72°C or higher, 73°C or higher, 74°C or higher, or 75°C or higher, and the upper limit of the temperature conditions includes not only the above-mentioned temperature conditions (i.e., 77°C or lower, 76°C or lower, 75°C or lower, 74°C or lower, 73°C or lower, 72°C or lower, or 71°C or lower), but also, for example, 80°C or lower, 79°C or lower, 78°C or lower, 77°C or lower, or 76°C or lower.

[0089] Therefore, specific ranges of temperature conditions for the modified protein glutaminase to act on plant proteins include, for example, 61 to 73°C, 62 to 73°C, 63 to 73°C, 64 to 73°C, 65 to 73°C, 66 to 73°C, 67 to 73°C, 68 to 73°C, 69 to 73°C, 70 to 73°C, 61 to 72°C, 61 to 71°C, 61 to 70°C, 61 to 69°C, 61 to 68°C, and 61 to 67°C. or 61 to 66°C; when acting on animal proteins, examples include 66 to 80°C, 67 to 80°C, 68 to 80°C, 69 to 80°C, 70 to 80°C, 71 to 80°C, 72 to 80°C, 73 to 80°C, 74 to 80°C, 75 to 80°C, 66 to 79°C, 66 to 78°C, 66 to 77°C, 66 to 76°C, 66 to 75°C, 66 to 74°C, 66 to 73°C, 66 to 72°C, or 66 to 71°C.

[0090] Among the conditions for the action of the modified protein glutaminase, the pH condition at 25°C is, for example, 2 to 12, preferably 3 to 10, more preferably 4 to 9, even more preferably 5.5 to 7, and even more preferably 6 to 7.

[0091] The time for which the modified protein glutaminase is allowed to act is not particularly limited and may be determined appropriately depending on factors such as the preparation scale, and examples include 10 minutes or more, 30 minutes or more, 1 hour or more, 8 hours or more, 16 hours or more, or 20 hours or more. The upper limit of this time range is not particularly limited, and examples include 40 hours or less, 30 hours or less, 25 hours or less, 20 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less. Specific ranges for the time for which the modified protein glutaminase is allowed to act include 10 minutes to 40 hours, 30 minutes to 40 hours, 1 to 40 hours, 8 to 40 hours, 16 to 40 hours, 20 to 40 hours, 10 minutes to 30 hours, 10 minutes to 25 hours, 10 minutes to 20 hours, 10 minutes to 10 hours, 10 minutes to 8 hours, 10 minutes to 6 hours, 10 minutes to 4 hours, 10 minutes to 2 hours, or 10 minutes to 1 hour.

[0092] The protein material can be treated not only with the modified protein glutaminase, but also with an enzyme other than the modified protein glutaminase, if necessary. The treatment with the other enzyme may be carried out simultaneously with the treatment with the modified protein glutaminase, or may be carried out before or after the treatment with the modified protein glutaminase. If the optimum temperature of the other enzyme overlaps with the optimum temperature range of the modified protein glutaminase, the treatment with the modified protein glutaminase and the treatment with the other enzyme can be carried out simultaneously in the enzyme treatment step. In this treatment, both the treatment with the modified protein glutaminase and the treatment with the other enzyme can be carried out under the same temperature conditions. Specific temperature conditions can be selected from the temperature conditions described above.

[0093] For example, when the protein material is used in the form of the above-mentioned vegetable protein-containing liquid composition, and the vegetable protein-containing liquid composition is prepared using cereals as the vegetable raw material, amylase, for example, can be used as the other enzyme. Amylase can be used to improve the solubility of the vegetable protein-containing liquid composition prepared using cereals. Examples of amylase include α-amylase and β-amylase. The α-amylase is not particularly limited, and examples thereof include α-amylases derived from the genus Aspergillus and Bacillus, and preferred examples include α-amylases derived from the genus Bacillus, such as Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis. The β-amylase is not particularly limited, but examples include β-amylases derived from plants (wheat, soybean) and the genus Bacillus, preferably β-amylases derived from the genus Bacillus, and more preferably β-amylases derived from the species Bacillus flexus. These amylases may be used alone or in combination of two or more. In a preferred embodiment of the present invention, in the enzyme treatment step, a vegetable protein-containing liquid composition prepared using cereals can be simultaneously treated with a modified protein glutaminase and an amylase. In this treatment, both the treatment with the modified protein glutaminase and the treatment with the amylase can be carried out under the same temperature conditions. Specific temperature conditions can be selected from the temperature conditions described above (i.e., the temperature conditions when acting on vegetable proteins).

[0094] After the reaction is complete, the enzyme is inactivated, followed by cooling and, if necessary, post-treatment, to obtain a modified protein material.

[0095] Specific examples of modified protein materials include those obtained from materials containing animal protein, such as milk, cream (e.g., coffee creamer), dried milk or cream (e.g., skim milk powder), fermented milk products (e.g., cheese, yogurt), and processed meat foods (e.g., processed meat, poultry, and fish paste foods, more specifically, hamburgers, meatballs, patties, meatloaf, minced meat cutlets, dim sum, etc.); and those obtained from materials containing plant protein, such as plant-based milk, plant-based cream (e.g., plant-based coffee creamer), dried plant-based milk or plant-based cream (e.g., powdered plant-based milk), fermented plant-based milk (e.g., cheese substitutes, yogurt substitutes), and meat-like processed foods (foods that imitate processed meat, poultry, and / or fish paste foods using meat substitutes, such as foods that imitate hamburgers, meatballs, patties, meatloaf, minced meat cutlets, dim sum, etc.) using meat substitutes.

[0096] The present invention will be specifically described below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0097] (1) Preparation of modified protein glutaminase (A2V / T113A / N154D / N158H and A2V / S26P / T113A / N154D / N158H) Using the base sequence (SEQ ID NO: 3) encoding the mature amino acid sequence (SEQ ID NO: 1) of wild-type C. proteolyticum-derived protein glutaminase introduced into a pET21 vector as a template, mutations were randomly introduced into the protein glutaminase to prepare a library consisting of approximately 8,000 mutant strains.

[0098] Specifically, PCR was performed using the following primers and Diversify PCR Random Mutagenesis Kit (Takara Bio) in a standard manner to prepare a gene product in which random mutations were introduced into wild-type C. proteolyticum protein glutaminase (SEQ ID NO: 1). (Primers for random mutation) Fw: 5'-GATCCTGACAAAAGAAGTAAAAGGGCAAACCAATAAA-3' (SEQ ID NO: 5) Rv: 5'-CTCAGTGGTGGTGGTGGTGGTGCTCGAG-3' (SEQ ID NO: 6)

[0099] Furthermore, PCR was performed in a standard manner using the following primers and PrimeSTAR GXL DNA Polymerase (manufactured by Takara Bio) to prepare a gene product of the expression vector. (Primers for expression vector) Fw: 5'-CTCGAGCACCACCACCACCACCACTGAG-3' (SEQ ID NO: 7) Rv: 5'-TTTATTGGTTTGCCCTTTTACTTCTTTTGTCAGGATC-3' (SEQ ID NO: 8)

[0100] The resulting gene products were ligated using an In-Fusion HD Cloning Kit (manufactured by Takara Bio) and then transformed into E. coli BL21 (DE3) by standard methods to obtain transformants. The transformants were cultured in LB medium at 37 ° C for 16 hours with shaking. They were then subcultured in TB medium and cultured at 37 ° C for 4 hours, after which IPTG (final concentration 0.5 mM) was added and the cultured at 33 ° C for 20 hours with shaking. The cells were collected from the culture medium and lysed using B-PER Bacterial Cell Lysis Reagent (manufactured by Thermo Scientific) according to standard methods, followed by centrifugation at 15,000 rpm for 10 minutes. The collected centrifuged supernatant was used as a crude enzyme solution, and trypsin was added to a final concentration of 0.05 mg / mL, followed by treatment at 37° C. for 1 hour to prepare a mutant library.

[0101] As a result of screening this mutant library, two mutant strains were narrowed down to ones whose optimum temperature was shifted to a higher temperature range than that of wild-type C. proteolyticum-derived protein glutaminase: specifically, a polypeptide A2V / T113A / N154D / N158H quadruple mutant (Example 1) consisting of the amino acid sequence shown in SEQ ID NO: 1, in which the amino acid residue at position 2 was substituted with a valine residue, the amino acid residue at position 113 with an alanine residue, the amino acid residue at position 154 with an aspartic acid residue, and the amino acid residue at position 158 with a histidine residue (SEQ ID NO: 2); and a polypeptide A2V / S26P / T113A / N154D / N158H quintuple mutant (Example 2) consisting of the amino acid sequence of the quadruple mutant further comprising a substitution of the amino acid residue at position 26 with a proline residue.

[0102] The target transformant (mutant) was cultured in 5 mL of TB medium for 16 hours at 37°C. 1 mL of the culture solution was inoculated into 200 mL of TB medium and cultured at 27°C for 48 hours. After 24 hours of culture, IPTG was added to a final concentration of 0.1 mM. After culture was completed, the cells were collected and disrupted with glass beads. After disruption, the cells were centrifuged at 15,000 rpm for 10 minutes. The collected supernatant was used as a crude enzyme solution, and trypsin was added to a final concentration of 0.05 mg / mL. After treatment at 37°C for 1 hour, TALON (R) Purification was carried out by a standard method using Spin Columns (manufactured by Takara Bio). The resulting enzyme solution was buffer-exchanged into 50 mM phosphate buffer (pH 6.5) using Prepacked Disposable PD-10 Columns (manufactured by Cytiva).

[0103] (2) Protein Content Test Method—BCA Method (Bicinchoninic Acid Method) Pierce BCA Protein Assay Kits (Pierce) were used. 50 mL of BCA Protein Assay Reagent (Reagent A Pierce) and 1 mL of BCA Protein Assay Reagent (Reagent B Pierce) were mixed in a 50 mL Erlenmeyer flask with a stopper to prepare a BCA reagent.

[0104] Four mL of BCA reagent was weighed into a test tube (15 x 150 mm) and left at 37°C for exactly 10 minutes. Then, 0.2 mL of a sample solution containing protein glutaminase was added and immediately shaken. This solution was left at 37°C for exactly 30 minutes and then cooled under running water. Absorbance (AT) at a wavelength of 562 nm was measured using water as a control. Separately, 4 mL of BCA reagent was left at 37°C for exactly 10 minutes, and then 0.2 mL of water was added and shaken. Absorbance (AB) was measured in the same manner. A calibration curve showing the relationship between albumin concentration and absorbance (562 nm) was also prepared. The protein concentration (mg / g or mg / mL) in the sample solution was calculated from the difference in absorbance (AT-AB) between the calibration curve prepared with albumin and the sample solution.

[0105]

[0106] [Test Example 1] Reactivity to Z-Gln-Gly [1-1] Using Z-Gln-Gly (benzyloxycarbonyl-L-glutaminylglycine) as a substrate, the specific activities of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2) and the unmodified wild-type C. proteolyticum-derived protein glutaminase (Comparative Example 1, SEQ ID NO: 1) were measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C).

[0107] Specifically, 0.1 mL of a sample solution containing protein glutaminase was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and the mixture was left at each test temperature for 10 minutes. Then, 1 mL of 0.4 M TCA solution was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly to stop the reaction. As a blank, 1 mL of 0.4 M TCA solution was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of a sample solution containing protein glutaminase was added. The mixture was then left at the test temperature for 10 minutes. The amount of ammonia generated in the reaction solution was measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries). The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0108] The enzyme activity of protein glutaminase was calculated using the following formula, where 1 unit (1 U) is the amount of enzyme required to produce 1 μmol of ammonia per minute. In the formula, the reaction solution volume is 2.1, the enzyme solution volume is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0109]

[0110] The specific activity (U / mg protein) for Z-Gln-Gly was calculated at each test temperature. The relative value (relative activity (%)) of the specific activity at each test temperature was calculated, with the maximum specific activity being set at 100%. The relationship between the relative activity and temperature is shown in Figure 1a.

[0111] 1a, the optimum temperature for Z-Gln-Gly of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1: solid line) was 65°C, which was shifted to a higher temperature range by about 5°C than the optimum temperature (60°C) of the wild-type protein glutaminase derived from C. proteolyticum before modification (Comparative Example 1: dashed line).

[0112] [1-2] As in [1-1] above, Z-Gln-Gly was used as a substrate, and the specific activities of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2) and the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2) were measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C). The results showing the relationship between the relative value of specific activity (relative activity (%)) and temperature are shown in Figure 1b.

[0113] As is clear from Figure 1b, the optimum temperature for Z-Gln-Gly of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1: solid line) and the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2: dashed-dotted line) was 65 ° C. Taking into account the results of Figure 1a, it can be seen that the modified protein glutaminase of Example 2 was shifted to a higher temperature range by about 5 ° C. than the optimum temperature (60 ° C) of the wild-type C. proteolyticum-derived protein glutaminase before modification. Furthermore, the modified protein glutaminase of Example 2 had a higher relative activity at 75 ° C. than the modified protein glutaminase of Example 1, and at other test temperatures, it showed almost the same relative activity as the modified protein glutaminase of Example 1.

[0114] Test Example 2 Reactivity to Soybean Protein Using a soybean protein material (product name Fujipro, manufactured by Fuji Oil Co., Ltd., protein content 86.9% by weight) as a substrate, the specific activities of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2) and the unmodified wild-type C. proteolyticum-derived protein glutaminase (Comparative Example 1, SEQ ID NO: 1) were measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C).

[0115] The test procedure was the same as in Test Example 1, except that the substrate was changed to the above soy protein material. In this case, the enzyme was reacted so that the final concentration of the soy protein material was 5% by weight.

[0116] The specific activity (U / mg protein) for soybean protein at each test temperature was calculated. The relative value (relative activity (%)) of the specific activity at each test temperature was calculated, with the maximum specific activity being set at 100%. The relationship between the relative activity and temperature is shown in Figure 2.

[0117] 2, the optimum temperature for soybean protein of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1: solid line) was 65°C, which was shifted to a higher temperature range by about 5°C than the optimum temperature (60°C) of the wild-type C. proteolyticum-derived protein glutaminase before modification (Comparative Example 1: dashed line). Furthermore, the relative activity of the protein glutaminase of Comparative Example 1 dropped sharply to 40% at a temperature 5°C higher (65°C) than the optimum temperature (60°C), whereas the protein glutaminase of Example 1 maintained a high relative activity of 86% even at 70°C, which is 5°C higher than the optimum temperature (65°C).

[0118] [Test Example 3] Reactivity to pea protein [3-1] Using a pea protein material (product name LYSAMINE GPS, manufactured by Roquette, protein content 85% by weight) as a substrate, the specific activities of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2) and the unmodified wild-type protein glutaminase derived from C. proteolyticum (Comparative Example 1, SEQ ID NO: 1) were measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C).

[0119] The test procedure was the same as in Test Example 1, except that the substrate was changed to the above-mentioned pea protein material, in which the enzyme was reacted so that the final concentration of the pea protein material was 5% by weight.

[0120] The specific activity (U / mg protein) for pea protein at each test temperature was calculated. The relative value (relative activity (%)) of the specific activity at each test temperature was calculated, with the maximum specific activity being set at 100%. The relationship between the relative activity and temperature is shown in Figure 3a.

[0121] 3a, the optimum temperature for pea protein of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1: solid line) was 65°C, which was shifted to a higher temperature range by about 5°C than the optimum temperature (60°C) of the wild-type protein glutaminase derived from C. proteolyticum before modification (Comparative Example 1: dashed line). Furthermore, the relative activity of the protein glutaminase of Comparative Example 1 dropped sharply to 40% at a temperature 5°C higher (65°C) than the optimum temperature (60°C), whereas the protein glutaminase of Example 1 maintained a high relative activity of 85% even at 70°C, which is 5°C higher than the optimum temperature (65°C).

[0122] [3-2] As in [3-1] above, the pea protein material was used as a substrate, and the specific activities of the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2) and the unmodified wild-type protein glutaminase derived from C. proteolyticum (Comparative Example 1, SEQ ID NO: 1) were measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C). The results showing the relationship between the relative value of specific activity (relative activity (%)) and temperature are shown in Figure 3b.

[0123] As is clear from Figure 3b, the optimum temperature for pea protein of the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2: dashed-dotted line) was 65°C, which was shifted to a higher temperature range by about 5°C than the optimum temperature (60°C) of the wild-type protein glutaminase derived from C. proteolyticum before modification (Comparative Example 1: dashed line). Furthermore, as shown in Figure 3a, the relative activity of the protein glutaminase of Example 1 at 70°C, which is 5°C higher than the optimum temperature for pea protein, was 85%, whereas the relative activity of the protein glutaminase of Example 2 was further improved to 90%.

[0124] [Test Example 4] Reactivity to casein [4-1] Using milk casein (product name: Casein, Bovine Milk, Carbohydrate and Fatty Acid Free, manufactured by Merck, protein content 99% by weight) as a substrate, the specific activity of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2) and the unmodified wild-type C. proteolyticum-derived protein glutaminase (Comparative Example 1, SEQ ID NO: 1) was measured at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C).

[0125] The test procedure was the same as in Test Example 1, except that the substrate was changed to the above milk casein. In this case, the enzyme was reacted so that the final concentration of milk casein was 5% by weight.

[0126] The specific activity (U / mg protein) against milk casein at each test temperature was calculated. The relative value (relative activity (%)) of the specific activity at each test temperature was calculated, with the maximum specific activity being set at 100%. The relationship between the relative activity and temperature is shown in Figure 4a.

[0127] 4a, the optimum temperature for milk casein of the modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1: solid line) was 70°C, which was shifted to a higher temperature range by about 10°C than the optimum temperature (60°C) of the wild-type protein glutaminase derived from C. proteolyticum before modification (Comparative Example 1: dashed line). When milk casein was used as a substrate, not only the protein glutaminase of Example 1 but also the protein glutaminase of Comparative Example 1 maintained relatively high relative activity even in a temperature range 5°C higher than the optimum temperature (unlike low-molecular-weight proteins such as Z-Gln-Gly and plant proteins such as soybean protein and pea protein).

[0128] [4-2] As in [4-1] above, the milk casein was used as a substrate, and the specific activities of the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2) and the unmodified wild-type C. proteolyticum -derived protein glutaminase (Comparative Example 1, SEQ ID NO: 1) at each test temperature (37°C, 50°C, 60°C, 65°C, 70°C, 75°C, or 80°C) were measured. The results showing the relationship between the relative value of specific activity (relative activity (%)) and temperature are shown in Figure 4b.

[0129] As is clear from Figure 4b, the optimum temperature for milk casein of the modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2: dashed-dotted line) was 75°C, which was shifted to a higher temperature range by about 15°C than the optimum temperature (60°C) of the wild-type C. proteolyticum-derived protein glutaminase before modification (Comparative Example 1: dashed line). Furthermore, the optimum temperature for milk casein of the protein glutaminase of Example 2 was shifted to a higher temperature range by about 5°C than the optimum temperature of the protein glutaminase of Example 1 shown in Figure 4a.

[0130] Test Example 5 In a 30 mL glass bottle, 1.2 g of oat flour (product name: Premium oat flour (manufactured by Slow Food), protein content: 13.1 wt%) was dispersed in 10 g of water to prepare a suspension (a vegetable protein-containing liquid composition prepared using cereals). 10 U / g-starch of α-amylase (α-amylase derived from Bacillus amyloliquefaciens, Amano Enzyme Inc.) and the amounts of wild-type protein glutaminase (wild-type PG, Comparative Example 1, SEQ ID NO: 1) or the modified protein glutaminase of the present invention (PG-D4, Example 1, SEQ ID NO: 2) shown in Tables 1 and 2 were added, and the mixture was reacted at 65°C or 70°C for 2 hours. In other words, treatment with α-amylase and treatment with various PGs were carried out simultaneously under the same temperature conditions. After the reaction, the mixture was heated at 95°C for 5 minutes to inactivate the enzyme. The mixture was then centrifuged at 15,000 rpm for 10 minutes, and the supernatant was recovered. The amount of released autoprotein in the recovered supernatant was quantified using the BCA method described above in (2), and the quantitative value was obtained as a protein concentration (mg / mL). For Examples 3-1 to 3-5 and Examples 4-1 to 4-4, the relative protein concentration, when the protein concentration in the corresponding comparative example (Comparative Examples 3-1 to 3-5 and Comparative Examples 5-1 to 5-4, respectively) was set to 1, was calculated as a solubility improvement index. The results are shown in Tables 1 and 2.

[0131]

[0132]

[0133] Since the modified protein glutaminase of the present invention (Example 1, SEQ ID NO: 2) has a higher optimum temperature than the wild-type protein glutaminase (Comparative Example 1, SEQ ID NO: 1), more efficient protein solubilization was possible by simultaneously treating it under the same temperature conditions as α-amylase, which has a higher optimum temperature than the wild-type protein glutaminase. Specifically, as shown in Examples 3-1 to 3-5 and Examples 4-1 to 4-4, which used the modified protein glutaminase of the present invention, protein solubility was improved compared to Comparative Examples 3-1 to 3-5 and Comparative Examples 5-1 to 5-4, which used wild-type protein glutaminase, respectively. Furthermore, in Examples 3-1 to 3-5, which were treated at a temperature condition of 65°C, protein solubility was improved up to 1.33-fold compared to when the wild-type protein glutaminase was used, whereas in Examples 4-1 to 4-4, which were treated at a higher temperature condition of 70°C, protein solubility was improved up to 2.04-fold compared to when the wild-type protein glutaminase was used.

[0134] Similarly, a vegetable protein-containing liquid composition was prepared using oat flour, and simultaneously treated with α-amylase and wild-type PG (Comparative Example 1) or PG-D4 (Example 1), followed by inactivation of the enzyme. The resulting treated product (oat milk containing modified oat protein material) was stirred and foamed without centrifugation. As a result, the oat milk treated with PG-D4 (Example 1) had improved foaming properties and foam stability compared to the oat milk treated with wild-type PG (Comparative Example 1). Furthermore, the treated product (oat milk containing modified oat protein material) was emulsified by adding oil and homogenizing without centrifugation. As a result, the oat milk treated with PG-D4 (Example 1) had improved emulsifying properties and emulsion stability compared to the oat milk treated with wild-type PG (Comparative Example 1).

[0135] [Test Example 6] Various proteins or Z-Gln-Gly shown in Table 3 were used as substrates (final concentration 5% by weight), and as protein glutaminases, wild-type C. proteolyticum-derived protein glutaminase (Comparative Example 1, SEQ ID NO: 1), modified protein glutaminase A2V / T113A / N154D / N158H of the present invention (Example 1, SEQ ID NO: 2), or modified protein glutaminase A2V / S26P / T113A / N154D / N158H of the present invention (Example 2) was used, the reaction temperature was set to 70 ° C., and the specific activity of the protein glutaminase was measured in the same manner as in Test Examples 1 to 4. The specific activity at 70 ° C. is shown in Table 4. Table 4 also shows the relative values ​​of the specific activity of the protein glutaminase of Examples 1 and 2 when the specific activity of the protein glutaminase of Comparative Example 1 is set to 1.

[0136]

[0137]

[0138] It was confirmed that, regardless of which protein was used as a substrate, the optimum temperatures of the protein glutaminase of Example 1 and the protein glutaminase of Example 2 were higher than the optimum temperature of the protein glutaminase of Comparative Example 1. Furthermore, as shown in Table 4, the specific activity at 70°C was significantly improved for the protein glutaminase of Example 1 and the protein glutaminase of Example 2 compared to the protein glutaminase of Comparative Example 1. Furthermore, the specific activity at 70°C was improved for the protein glutaminase of Example 2 compared to the protein glutaminase of Example 1.

Claims

1. A modified protein glutaminase consisting of a polypeptide shown in any one of (I) to (III) below: (I) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, into which at least one of the following substitutions has been introduced: a valine residue at position 2, an alanine residue at position 113, an aspartic acid residue at position 154, and a histidine residue at position 158; (II) a polypeptide into which one or more amino acid residues other than the substituted amino acid residue have been substituted, added, inserted, or deleted in the amino acid sequence into which the substitutions have been introduced, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; and (III) a polypeptide into which the sequence identity of the portion excluding the substituted amino acid residue in the amino acid sequence into which the substitutions have been introduced is 70% or more, and which has an optimum temperature higher than that of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. DNA encoding the modified protein glutaminase of claim 1.

3. An expression cassette or recombinant vector containing the DNA of claim 2.

4. A transformant obtained by transforming a host with the expression cassette or recombinant vector described in claim 3.

5. A method for producing a modified protein glutaminase, comprising the step of culturing the transformant according to claim 4.

6. An enzyme preparation comprising the modified protein glutaminase of claim 1.

7. A modifying agent for protein materials, comprising the modified protein glutaminase according to claim 1.

8. A method for producing a modified protein material, comprising an enzyme treatment step in which the modified protein glutaminase of claim 1 acts on a protein material.

9. The method of claim 8, wherein the protein material is a vegetable protein material.

10. The method of claim 8, wherein the protein material is provided in the form of a vegetable protein-containing liquid composition prepared using cereals, and in the enzyme treatment step, the vegetable protein-containing liquid composition is simultaneously treated with modified protein glutaminase and amylase.

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