Mutant transglutaminase

Abstract

Provided is a mutant transglutaminase (mutant TG) having low acid resistance. This mutant TG has mutation at an amino acid residue such as A261, V271, A322, V326, D46, M16, Y34, W38, and S199.

Description

Technical Field

[0001] The present invention relates to a mutant transglutaminase (mutant TG) with low acid resistance and its use.

Background Art

[0002] Transglutaminase (TG) is known as an enzyme that catalyzes protein cross-linking and is used to improve the physical properties of foods. For example, a method for producing yogurt using TG is known (Patent Document 1).

[0003] Also, various mutant TGs with modified functions are known. Examples of mutant TGs include mutant TGs having properties such as reduced heat resistance, improved heat resistance, improved antioxidant properties (Patent Document 2), mutant TGs with improved heat resistance (Patent Document 3), mutant TGs with improved heat resistance and / or pH stability (Patent Document 4), and mutant TGs with improved specific activity (Patent Document 5). As mutations that improve the heat resistance and / or pH stability of TG, specifically, for example, mutations that introduce a disulfide bond by substituting an amino acid residue such as D46 with a cysteine residue are known (Patent Document 4). As mutations that improve the specific activity of TG, specifically, for example, mutations at M16 such as M16T, mutations at Y34 such as Y34F, mutations at S199 such as S199A, and mutations at W38 such as W38F are known (Patent Document 5). <​​​​​​​​​​​​​​​​​​​​​​​WO2010 / 101256A1 [Patent Document 4] WO2008 / 099898A1 [Patent Document 5] Japanese Patent Publication No. 2008-194004 [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of this invention is to provide a variant TG with low acid resistance. [Means for solving the problem]

[0007] The inventors of this invention discovered a mutation that reduces the acid resistance of TG, and thus completed the present invention.

[0008] In other words, the present invention can be illustrated as follows. [1] A method for manufacturing food, The process includes treating food ingredients with a variant transglutaminase. The aforementioned food is either food (A) or (B) below: (A) Foods whose pH decreases during manufacturing; (B) Food containing the food (A) And, The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. A method wherein the aforementioned specific mutation is a mutation that reduces acid resistance. [2] The method wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 80% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%. [3] The method wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 50% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%. [4] The method wherein the specific mutation is a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46, M16, Y34, W38, S199. [5] The method wherein the specific mutation comprises a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46. [6] The method wherein the specific mutation includes one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P, M16T, Y34F, W38F, S199A. [7] The method wherein the specific mutation includes one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P. [8] The method wherein the specific mutation includes any of the following mutations: A261C / A322C, A261C / A322C / S199A, V271C / V326C, M16T / Y34F, M16T / S199A. [9] The method, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase from a bacterium of the genus Streptomyces.

[10] The method wherein the bacterium of the genus Streptomyces is Streptomyces mobaraensis.

[11] The method, wherein the wild-type transglutaminase is any of the following proteins: (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 2; (b) A protein comprising an amino acid sequence containing substitution, deletion, insertion, and / or addition of 1 to 10 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2; (c) A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 2.

[12] The method, wherein the food (A) is yogurt or cheese, and the food raw material is a milk raw material, the method.

[13] The method, wherein the food (B) is ice cream containing yogurt or cheese.

[14] The method, wherein the mutant transglutaminase is added to the food raw material when the pH of the food raw material is 5.0 or higher.

[15] A composition for producing food, containing mutant transglutaminase, where the food is the following food (A) or (B): (A) Food that undergoes a decrease in pH during production; (B) Food containing the food (A),<0000—100>and<—— the mutant transglutaminase is a protein having specific mutations in the amino acid sequence of wild-type transglutaminase and having transglutaminase activity, where the specific mutation is a mutation that reduces acid resistance, the composition.

[16] [[ID=4—1]] The composition, wherein the relative value of the residual activity of the mutant transglutaminase after treatment at pH 4.0, 37 °C for 1 hour is 80% or less relative to the residual activity after treatment at pH 6.0, 37 °C for 1 hour, which is taken as 100%.

[17] The composition wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 50% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

[18] The composition wherein the specific mutation is a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46, M16, Y34, W38, S199.

[19] The composition comprising the aforementioned specific mutation, which includes a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46.

[20] The composition wherein the specific mutation comprises one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P, M16T, Y34F, W38F, S199A. [twenty one] The composition wherein the specific mutation comprises one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P. [twenty two] The composition comprising any of the following mutations: A261C / A322C, A261C / A322C / S199A, V271C / V326C, M16T / Y34F, M16T / S199A. [twenty three] The composition wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces. [twenty four] The composition wherein the Streptomyces bacterium is Streptomyces mobaraensis. [twenty five] The wild-type transglutaminase is one of the following proteins in the composition: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No. 2.

[26] The aforementioned food (A) is yogurt or cheese, The composition wherein the food ingredient is a dairy ingredient.

[27] The composition wherein the food (B) is ice cream containing yogurt or cheese.

[28] A mutant transglutaminase having a specific mutation in the amino acid sequence of wild-type transglutaminase and possessing transglutaminase activity, The aforementioned specific mutation is a mutation that reduces acid resistance. The aforementioned specific mutation is a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46, M16, Y34, W38, S199 However, the mutations in M16, Y34, W38, and S199 were not selected individually. If the mutation at D46 is selected alone, the mutation at D46 is a mutant transglutaminase other than the D46C mutation.

[29] The mutant transglutaminase wherein the residual activity after treatment at pH 4.0 at 37°C for 1 hour is 80% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is set to 100%.

[30] The mutant transglutaminase wherein the residual activity after treatment at pH 4.0 at 37°C for 1 hour is 50% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

[31] The mutant transglutaminase wherein the specific mutation comprises a mutation in one or more amino acid residues selected from the following: A261, V271, A322, V326, D46.

[32] The mutant transglutaminase wherein the specific mutation includes one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P, M16T, Y34F, W38F, S199A.

[33] The mutant transglutaminase wherein the specific mutation includes one or more mutations selected from the following: A261C, V271C, A322C, V326C, D46P.

[34] The mutant transglutaminase in which the aforementioned specific mutation includes any of the following mutations: A261C / A322C, A261C / A322C / S199A, V271C / V326C, M16T / Y34F, M16T / S199A.

[35] The mutant transglutaminase is a protein in which the wild-type transglutaminase contains the amino acid sequence of mature transglutaminase from a bacterium of the genus Streptomyces.

[36] The mutant transglutaminase wherein the bacterium of the genus Streptomyces is Streptomyces mobaraensis.

[37] The mutant transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins containing an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No. 2.

[38] The gene encoding the aforementioned mutant transglutaminase.

[39] A vector containing the aforementioned gene.

[40] A microorganism possessing the aforementioned gene.

[41] The microorganism, which is a bacterium or yeast.

[42] The aforementioned microorganism is a Corynebacterium or a bacterium of the Enterobacteriaceae family.

[43] The microorganism is a bacterium of the genus Corynebacterium or Escherichia.

[44] The microorganism is Corynebacterium glutamicum or Escherichia coli. [Brief explanation of the drawing]

[0009] [Figure 1] A diagram showing the breaking strength of yogurt produced using wild-type TG or various mutant TG. [Figure 2] This figure shows the TG activity (absorbance by hydroxamate method) in the supernatant of yogurt produced using wild-type TG or various mutant TGs. [Figure 3] This figure shows the time course of TG activity (fluorescence intensity by fluorescence method) when producing yogurt using wild-type TG or mutant TG (A261C / A322C). [Figure 4]This figure shows the time course of pH and TG activity (absorbance by hydroxamate method) when producing yogurt using wild-type TG or mutant TG (A261C / A322C). [Modes for carrying out the invention]

[0010] <1> Mutant transglutaminase (mutant TG) The present invention provides transglutaminase (TG) having the “specific mutations” described herein.

[0011] "Transglutaminase (TG)" may refer to a protein that has the activity to catalyze an acyl transfer reaction between the amide group of a glutamine residue in a protein and a primary amine (EC 2.3.2.13, etc.). This activity is also called "TG activity." The gene that codes for TG is also called the "TG gene." Examples of primary amines include lysine residues in proteins. In other words, "TG activity" may specifically refer to the activity that catalyzes a crosslinking reaction between glutamine residues and lysine residues in a protein. The crosslinking reaction may result in intramolecular crosslinking and / or intermolecular crosslinking. Typically, the crosslinking reaction may result in at least intermolecular crosslinking.

[0012] TG activity can be measured, for example, by hydroxamate (Lorand, L., et al.: Anal. Biochem., 44, 221-213 (1971)) or fluorescence (Takagi, J., et al.: Anal. Biochem., 153, 296-298 (1986)). Unless otherwise specified, "TG activity" may refer to TG activity measured by hydroxamate.

[0013] The procedure for measuring TG activity by the hydroxamate method is as follows: TG activity can be measured by incubating the enzyme with substrates (i.e., benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine) at 37°C and pH 6.0, and measuring the enzyme and substrate-dependent production of hydroxamic acid. Hydroxamic acid production can be measured by forming an iron complex of hydroxamic acid in the presence of trichloroacetic acid, with the increase in absorbance at 525 nm serving as an indicator. Under the above conditions, the amount of enzyme that catalyzes the production of 1 μmol of hydroxamic acid per minute is defined as 1 U (unit).

[0014] The procedure for measuring TG activity by fluorescence is as follows: TG activity can be measured by incubating the enzyme with substrates (i.e., dimethylated casein and monodansylcadaverine (MDC)) at 37°C and pH 7.5, and measuring the enzyme and substrate-dependent incorporation of MDC into dimethylated casein. MDC incorporation can be measured by an increase in fluorescence (excitation wavelength 350 nm, emission wavelength 480 nm).

[0015] A triglyceridyl transgenic (TG) gene possessing a specific mutation is also called a "mutant TG gene." Furthermore, the gene encoding a mutant TG gene is also called a "mutant TG gene."

[0016] TG that does not possess a "specific mutation" is also called "wild-type TG." The gene that codes for wild-type TG is also called the "wild-type TG gene." Here, "wild-type" is a convenient designation to distinguish "wild-type" TG from "mutant" TG, and is not limited to naturally occurring TG as long as it does not possess a "specific mutation." "TG does not possess a 'specific mutation'" can be interpreted as TG not possessing the mutation selected as a "specific mutation." Wild-type TG may or may not possess mutations that were not selected as "specific mutations," as long as it does not possess the mutation selected as a "specific mutation."

[0017] When a certain wild-type TG and a certain mutant TG are identical except for the presence or absence of a "specific mutation," the wild-type TG is also referred to as the "wild-type TG corresponding to a certain mutant TG," and the mutant TG is also referred to as the "mutant TG corresponding to a certain wild-type TG."

[0018] The following explains the wild type TG.

[0019] Wild-type TG may or may not have TG activity, as long as its corresponding mutant TG has TG activity. Wild-type TG usually has TG activity.

[0020] Examples of wild-type triglycerides (TGs) include those of actinomycetes (Appl. Environ. Microbiol., 2003, 69(1), 358-366). Examples of actinomycetes include bacteria of the genus Streptomyces. Examples of Streptomyces bacteria include Streptomyces mobaraensis, Streptomyces cinnamoneus, and Streptomyces griseocarneus. Note that the genus Streptomyces also includes bacteria that were formerly classified under the genus Streptoverticillium. For example, "Streptomyces mobaraensis" includes bacteria that were formerly classified as Streptoverticillium mobaraense or Streptoverticillium ladakanum. Similarly, "Streptomyces cinnamoneus" includes bacteria that were formerly classified as Streptoverticillium cinnamoneum. Furthermore, "Streptomyces griseocarneus" includes bacteria that were formerly classified as Streptoverticillium griseocarneum. TG can be expressed in a form including the pro-structure, and the pro-structure can be removed to become a mature protein. The mature protein of TG is also called "mature TG." Specifically, the mature TG of the organisms exemplified above can be cited as an example of TG from those organisms. Sequence ID 1 shows the nucleotide sequence of the portion of the TG gene in Streptoverticillium mobaraense that codes for mature TG, and Sequence ID 2 shows the amino acid sequence of the mature TG encoded by the same gene.In other words, the wild-type TG gene may be, for example, a gene having the nucleotide sequence of the TG gene of the organism exemplified above (for example, the nucleotide sequence shown in Sequence ID No. 1). Also, the wild-type TG may be, for example, a protein having the amino acid sequence of the TG of the organism exemplified above (for example, the amino acid sequence shown in Sequence ID No. 2). Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the said nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the said nucleotide sequence or amino acid sequence.

[0021] A wild-type TG gene may be a variant of the wild-type TG gene exemplified above (for example, a gene with the nucleotide sequence shown in SEQ ID NO: 1), as long as the encoding TG does not have a "specific mutation." Similarly, a wild-type TG may be a variant of the wild-type TG gene exemplified above (for example, a protein with the amino acid sequence shown in SEQ ID NO: 2), as long as it does not have a "specific mutation." In other words, the term "wild-type TG gene" may encompass not only the wild-type TG gene exemplified above (for example, a gene with the nucleotide sequence shown in SEQ ID NO: 1) but also its variants. Similarly, the term "wild-type TG" may encompass not only the wild-type TG gene exemplified above (for example, a protein with the amino acid sequence shown in SEQ ID NO: 2) but also its variants. Note that the gene identified in the species of origin is not limited to the gene itself found in that species, but also includes genes having the nucleotide sequence of the gene found in that species and their variants. Furthermore, the protein identified in the species of origin is not limited to the protein itself found in that species, but also includes proteins having the amino acid sequence of the protein found in that species and their variants. These variants may or may not be found in the species in question. That is, for example, "TG of Streptoverticillium bacteria" is not limited to TG itself found in Streptoverticillium bacteria, but includes proteins having the amino acid sequence of TG found in Streptoverticillium bacteria and their variants. Examples of variants include homologs and artificially modified versions of the genes and proteins exemplified above.

[0022] Homologs of the wild-type TG gene or homologs of wild-type TG can be easily determined from public databases, for example, by BLAST or FASTA searches using the nucleotide sequence of the wild-type TG gene or the amino acid sequence of the wild-type TG gene exemplified above as query sequences. Alternatively, homologs of the wild-type TG gene can be obtained, for example, by PCR using the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequences of the wild-type TG gene exemplified above as primers.

[0023] A wild-type TG gene may encode a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2), unless the encoding TG has a "specific mutation." For example, the encoded protein may have an elongated or shortened N-terminus and / or C-terminus. The above "one or several" will vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically, it may be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3.

[0024] The above substitutions, deletions, insertions, or additions of one or more amino acids are conservative mutations that maintain the original function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered conservative include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include e to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. Furthermore, such amino acid substitutions, deletions, insertions, or additions may also result from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organisms from which the genes originate.

[0025] Furthermore, the wild-type TG gene may also be a gene that encodes a protein having an amino acid sequence that is identical to, for example, 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the entire amino acid sequence, as long as the encoding TG does not have a "specific mutation".

[0026] Furthermore, the wild-type TG gene may also be a gene, such as DNA, that hybridizes under stringent conditions with a probe, such as a complementary sequence to all or part of the above base sequence, which can be prepared from the above base sequence (for example, the base sequence shown in Sequence ID No. 1), as long as the encoding TG does not have a "specific mutation." "Stringent conditions" may mean conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. For example, conditions can be described as those in which DNA with high identity, such as 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, hybridize with each other, while DNA with lower identity does not hybridize. Alternatively, conditions can be described as washing once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0027] As described above, the probe used in the hybridization may be a part of the complementary sequence of the gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known gene sequences as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment approximately 300 bp in length can be used as a probe. When using a DNA fragment approximately 300 bp in length as a probe, the conditions for hybridization washing may be 50°C, 2×SSC, and 0.1% SDS.

[0028] Furthermore, since codon degeneracy differs depending on the host, the wild-type TG gene may be one in which any codon is replaced with an equivalent codon. In other words, the wild-type TG gene may be a variant of the wild-type TG gene exemplified above due to the degeneracy of the genetic code. For example, the wild-type TG gene may be modified to have the optimal codons depending on the codon usage frequency of the host being used.

[0029] Note that "identity" between amino acid sequences refers to the identity between amino acid sequences calculated using the default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence=11, Extension=1; Compositional Adjustments: Conditional compositional score matrix adjustment) by blastp. Furthermore, "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated using the default Scoring Parameters (Match / Mismatch Scores=1,-2; Gap Costs=Linear) by blastn.

[0030] The following describes mutant TG.

[0031] Mutant TG has TG activity. The degree of TG activity of mutant TG is not particularly limited, as long as mutant TG can be used for the desired application. The TG activity of mutant TG may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 120% or more, 150% or more, or 200% or more of the TG activity of the wild-type TG corresponding to the mutant TG and / or the TG activity of the wild-type TG consisting of the amino acid sequence shown in Sequence ID No. 2, or 10000% or less, 1000% or less, 200% or less, 150% or less, 120% or less, or 100% or less, or any non-inconsistent combination thereof.

[0032] Mutant TG has "specific mutations" in the amino acid sequence of wild-type TG.

[0033] In other words, mutant TG may be a protein having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in Sequence ID No. 2, for example. Alternatively, mutant TG may be a protein having an amino acid sequence that has a "specific mutation" in the amino acid sequence shown in Sequence ID No. 2, and also includes one or more additional amino acid substitutions, deletions, insertions, and / or additions at locations other than the "specific mutation", and possessing TG activity.

[0034] In other words, mutant TG may be a protein having the same amino acid sequence as wild-type TG except for having a "specific mutation." That is, mutant TG may be a protein having the amino acid sequence shown in Sequence ID No. 2 except for having a "specific mutation." Also, mutant TG may be a protein having an amino acid sequence that includes one or more amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence shown in Sequence ID No. 2, except for having a "specific mutation," and possessing TG activity. Furthermore, mutant TG may be a protein having an amino acid sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, identity with the amino acid sequence shown in Sequence ID No. 2, except for having a "specific mutation," and possessing TG activity.

[0035] Mutant TG may contain other amino acid sequences in addition to the mutant TG amino acid sequence exemplified above. That is, mutant TG may be a fusion protein with other amino acid sequences. Furthermore, mutant TG may be expressed in a form containing other amino acid sequences (i.e., as a fusion protein with other amino acid sequences) and may ultimately lose part or all of those other amino acid sequences. "Mutant TG contains other amino acid sequences" means, unless otherwise specified, that the ultimately obtained mutant TG contains other amino acid sequences. On the other hand, "Mutant TG is expressed in a form containing other amino acid sequences" means, unless otherwise specified, that mutant TG contains other amino acid sequences at least during expression, and does not necessarily mean that the ultimately obtained mutant TG contains other amino acid sequences. The same applies to wild-type TG. "Other amino acid sequences" are not particularly limited as long as mutant TG has TG activity. "Other amino acid sequences" can be appropriately selected according to various conditions such as the intended use. Examples of "other amino acid sequences" include peptide tags, signal peptides (also called signal sequences), pro-structures, and protease recognition sequences. The "other amino acid sequence" may be, for example, ligated to the N-terminus, C-terminus, or both of the mutant TG. The "other amino acid sequence" may be a single amino acid sequence, or a combination of two or more amino acid sequences.

[0036] Peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphate), and the Fc region of antibodies. A 6xHis tag is an example of a His tag. Peptide tags can be used, for example, to detect and purify expressed mutant TG.

[0037] The signal peptide is not particularly limited as long as it functions in a host expressing the mutant TG. Examples of signal peptides include those recognized by the Sec secretory pathway and those recognized by the Tat secretory pathway. Specifically, examples of signal peptides recognized by the Sec secretory pathway include signal peptides of cell surface proteins of Corynebacteria. Specifically, examples of signal peptides of cell surface proteins of Corynebacteria include the PS1 signal sequence and PS2 (CspB) signal sequence of C. glutamicum (Japanese Patent Publication No. 6-502548) and the SlpA (CspA) signal sequence of C. stationis (Japanese Patent Publication No. 10-108675). Specifically, signal peptides recognized by the Tat secretory pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013 / 118544). Signal peptides can be used, for example, in the secretory production of mutant TG. When mutant TG is secreted using signal peptides, the signal peptide is cleaved during secretion, and mutant TG without the signal peptide may be secreted outside the bacterial cell. In other words, typically, the mutant TG that is ultimately obtained does not need to have the signal peptide.

[0038] Specifically, examples of pro-structures include the pro-structures of each wild-type TG as illustrated above. Sequence ID 3 shows the nucleotide sequence of the Streptoverticillium mobaraense TG gene containing the portion encoding the pro-structure, and Sequence ID 4 shows the amino acid sequence of TG containing the pro-structure encoded by the same gene. In Sequence ID 3, positions 1 to 135 correspond to the portion encoding the pro-structure, and position 136 onwards corresponds to the portion encoding mature TG (i.e., Sequence ID 1). In Sequence ID 4, positions 1 to 45 correspond to the pro-structure, and position 46 onwards corresponds to mature TG (i.e., Sequence ID 2). Mutant TG may be expressed in a form containing the pro-structure, and the pro-structure may be removed to form a mature protein. When mutant TG is expressed in a form containing the pro-structure, the removal of the pro-structure can activate the mutant TG. Therefore, typically, the final mutant TG does not need to have a pro-structure. Removal of the pro-structure can be carried out, for example, using a processing enzyme. Examples of processing enzymes include proteases such as SAM-P45 (Appl. Environ. Microbiol., 2003, 69(1), 358-366) and alcalase. Furthermore, as will be described later, when expressing mutant TG by inserting a protease recognition sequence into the junction between the pro structure and the mutant TG, the pro structure can be removed using the corresponding protease.

[0039] Specific examples of protease recognition sequences include those for Factor Xa protease and proTEV protease. Protease recognition sequences can be used, for example, to cleave expressed mutant TG. Specifically, for example, when expressing mutant TG as a fusion protein with other amino acid sequences such as peptide tags or pro-structures, inserting a protease recognition sequence into the linkage between the mutant TG and the other amino acid sequence allows the expressed mutant TG to use the corresponding protease to cleave the other amino acid sequence, thereby obtaining a mutant TG that does not contain the other amino acid sequence.

[0040] The mutant TG gene is not particularly limited as long as it encodes the mutant TG described above. In this invention, the term "gene" is not limited to DNA, but may include any polynucleotide as long as it encodes the target protein. That is, "mutant TG gene" may mean any polynucleotide that encodes mutant TG. The mutant TG gene may be DNA, RNA, or a combination thereof. The mutant TG gene may be single-stranded or double-stranded. The mutant TG gene may be single-stranded DNA or single-stranded RNA. The mutant TG gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The mutant TG gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the mutant TG gene contains RNA, the descriptions of DNA such as the exemplified base sequences above may be appropriately read in accordance with RNA. The form of the mutant TG gene can be appropriately selected according to various conditions such as its intended use.

[0041] The following describes "specific mutations."

[0042] Certain mutations are those that reduce acid tolerance. That is, mutant TG has low acid tolerance. Mutant TG may have lower acid tolerance than wild-type TG. For example, mutant TG may have lower acid tolerance than the wild-type TG corresponding to the mutant TG and / or wild-type TG consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0043] "Acid resistance" can mean resistance to deactivation under acidic conditions. In other words, "mutant TG has low acid resistance" can mean that mutant TG is deactivated to a greater degree when treated under acidic conditions, or in other words, that the residual activity of mutant TG after treatment under acidic conditions is low. Also, "mutant TG has lower acid resistance than wild-type TG" can mean that the degree of deactivation of mutant TG when treated under acidic conditions is greater than the degree of deactivation of wild-type TG when treated under acidic conditions, or in other words, that the residual activity of mutant TG after treatment under acidic conditions is lower than that of wild-type TG after treatment under acidic conditions.

[0044] "Having low acid resistance in mutant TG" may mean, for example, that the residual activity of mutant TG after treatment under acidic conditions is 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0%. "Having low acid resistance in mutant TG" may also mean, for example, that the residual activity of mutant TG after treatment under acidic conditions is 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% as a relative value with the residual activity after treatment under control conditions being 100%.

[0045] Examples of treatment under acidic conditions include treatment at pH 5.0, pH 4.5, or pH 4.0 at 37°C for 1 hour. An example of treatment under control conditions is treatment at pH 6.0 at 37°C for 1 hour. "Residual activity after treatment of TG under certain conditions" refers to the ratio of the TG activity after treatment under those conditions to the TG activity before treatment under those conditions. "Treatment of TG under certain conditions" means placing TG under those conditions.

[0046] For example, the residual activity of mutant TG after treatment at pH 4.0 at 37°C for 1 hour may be 80% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%. Alternatively, for example, the residual activity of mutant TG after treatment at pH 4.0 at 37°C for 1 hour may be 50% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

[0047] Examples of "specific mutations" include mutations in the following amino acid residues: A261, V271, A322, V326, D46, M16, Y34, W38, S199.

[0048] A "specific mutation" may be a mutation in a single amino acid residue, or a combination of mutations in two or more amino acid residues. That is, a "specific mutation" may include, for example, a mutation in one or more amino acid residues selected from these amino acid residues. A "specific mutation" may be, for example, a mutation in a single amino acid residue selected from these amino acid residues, or a combination of mutations in two or more amino acid residues selected from these amino acid residues.

[0049] Any mutation at any of the amino acid residues may be selected individually or not. For example, the mutations at M16, Y34, W38, and S199 do not necessarily have to be selected individually.

[0050] Examples of "specific mutations" include mutations in A261, V271, A322, V326, and D46. That is, a "specific mutation" may include, for example, a mutation in one or more amino acid residues selected from A261, V271, A322, V326, and D46. A "specific mutation" may be, for example, a mutation in one or more amino acid residues selected from A261, V271, A322, V326, and D46, or a combination of a mutation in one or more amino acid residues selected from M16, Y34, W38, and S199.

[0051] "Specific mutations" can be more specifically defined as mutations at A322 and D46. That is, "specific mutations" may include, for example, mutations at A322 and / or D46. "Specific mutations" may be, for example, mutations at A322 and / or D46, or a combination of mutations at A322 and / or D46 and mutations at one or more amino acid residues selected from A261, V271, V326, M16, Y34, W38, and S199.

[0052] In the above notation used to identify amino acid residues, the number indicates the position in the amino acid sequence shown in Sequence ID No. 2, and the letter to the left of the number indicates the amino acid residue at each position in the amino acid sequence shown in Sequence ID No. 2 (i.e., the amino acid residue at each position before modification). For example, "A261" indicates the A (Ala) residue at position 261 in the amino acid sequence shown in Sequence ID No. 2.

[0053] In any wild-type TG, these amino acid residues each represent "the amino acid residue corresponding to the amino acid residue shown in the amino acid sequence shown in Sequence ID No. 2." That is, for example, "A261" in any wild-type TG represents the amino acid residue corresponding to the A(Ala) residue at position 261 in the amino acid sequence shown in Sequence ID No. 2.

[0054] Each of the above mutations may be an amino acid residue substitution. In each of the above mutations, the modified amino acid residue may be any amino acid residue other than the original amino acid residue, as long as it reduces the acid resistance of TG. In other words, the modified amino acid residue should be one that reduces the acid resistance of TG. Specifically, examples of modified amino acid residues include amino acid residues selected from K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln), other than the original amino acid residue. Note that the modified amino acid residue in D46 may be selected from amino acid residues other than C (Cys), for example. The modified amino acid residue at D46 may be selected from amino acid residues other than C(Cys), for example, when the "specific mutation" consists of a mutation at D46 (i.e., the mutation at D46 is selected alone).

[0055] Specifically, the following mutations can be cited as examples of "specific mutations": A261C, V271C, A322C, V326C, D46P, M16T, Y34F, W38F, S199A.

[0056] In other words, a "specific mutation" may include, for example, one or more mutations selected from these mutations. A "specific mutation" may be, for example, one mutation selected from these mutations, or a combination of two or more mutations selected from these mutations. Alternatively, a "specific mutation" may be, for example, a combination of one or more mutations selected from these mutations and one or more mutations in amino acid residues selected from A261, V271, A322, V326, D46, M16, Y34, W38, S199.

[0057] In other words, the mutations in A261, V271, A322, V326, D46, M16, Y34, W38, and S199 may be, for example, A261C, V271C, A322C, V326C, D46P, M16T, Y34F, W38F, and S199A, respectively.

[0058] Examples of "specific mutations" include A261C, V271C, A322C, V326C, and D46P. That is, a "specific mutation" may include, for example, one or more mutations selected from A261C, V271C, A322C, V326C, and D46P. A "specific mutation" may be, for example, one mutation selected from A261C, V271C, A322C, V326C, and D46P, or a combination of two or more mutations selected from A261C, V271C, A322C, V326C, and D46P. Furthermore, a “specific mutation” may be a combination of, for example, one or more mutations selected from A261C, V271C, A322C, V326C, and D46P, and one or more mutations in other amino acid residues selected from A261, V271, A322, V326, D46, M16, Y34, W38, and S199.

[0059] "Specific mutations" include, more specifically, A322C and D46P. That is, "specific mutations" may include, for example, A322C and / or D46P. "Specific mutations" may also be, for example, A322C and / or D46P. Furthermore, "specific mutations" may be, for example, a combination of A322C and / or D46P and a mutation in one or more amino acid residues selected from A261, V271, A322, V326, D46, M16, Y34, W38, and S199.

[0060] In the above notation for identifying mutations, the meaning of the numbers and the letters to their left is the same as described above. In the above notation for identifying mutations, the letters to the right of the numbers indicate the modified amino acid residue at each position. For example, "A261C" indicates a mutation in which the A (Ala) residue at position 261 in the amino acid sequence shown in Sequence ID No. 2 is replaced with a C (Cys) residue.

[0061] In any wild-type TG, these mutations each represent "a mutation corresponding to the mutation in the amino acid sequence shown in SEQ ID NO: 2". In any wild-type TG, "a mutation corresponding to a mutation in which the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue" should be read as "a mutation in which the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a certain amino acid residue". That is, for example, in any wild-type TG, "A261C" represents a mutation in which the amino acid residue corresponding to the A (Ala) residue at position 261 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a C (Cys) residue.

[0062] There are no particular restrictions on the combinations of mutations. Possible combinations of mutations include: A261C / A322C, A261C / A322C / S199A, V271C / V326C, M16T / Y34F, M16T / S199A.

[0063] Notable mutation combinations include A261C / A322C, A261C / A322C / S199A, and V271C / V326C.

[0064] More specifically, the mutation combinations A261C / A322C and A261C / A322C / S199A are examples of this combination.

[0065] In other words, a "specific mutation" may include, for example, any combination of these. A "specific mutation" may also be, for example, any combination of these. Furthermore, a "specific mutation" may also be, for example, a combination of any combination of these and a mutation in one or more amino acid residues selected from A261, V271, A322, V326, D46, M16, Y34, W38, and S199.

[0066] In the above notation used to identify combinations, the meaning of the numbers and the letters to their left and right is the same as described above. In the above notation used to identify combinations, the listing of two or more mutations separated by " / " indicates a double mutation or multiple mutation. For example, "A261C / A322C" indicates a double mutation of A261C and A322C.

[0067] The positions of the amino acid residues mentioned in each of the above mutations are for convenience in identifying the amino acid residues being modified and do not need to indicate their absolute positions in wild-type TG. In other words, the positions of the amino acid residues in each of the above mutations indicate relative positions based on the amino acid sequence shown in SEQ ID NO: 2, and their absolute positions may change due to deletions, insertions, or additions of amino acid residues. For example, if one amino acid residue is deleted or inserted at a position closer to the N-terminus than position X in the amino acid sequence shown in SEQ ID NO: 2, the original amino acid residue at position X becomes the (X-1) or (X+1) amino acid residue from the N-terminus, respectively, but is considered to be "the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2". Furthermore, the pre-modification amino acid residues mentioned in each of the above mutations are for convenience in identifying the amino acid residues being modified and do not need to be conserved in wild-type TG. In other words, if wild-type TG does not have the amino acid sequence shown in SEQ ID NO: 2, the pre-modification amino acid residues mentioned in each of the above mutations may not be conserved. In other words, each of the above mutations may include mutations in which the amino acid residue before modification mentioned in each mutation is replaced with another amino acid residue (for example, the modified amino acid residue mentioned in each mutation) if that amino acid residue is not conserved. For example, "mutation at A261" is not limited to mutations in which the amino acid residue corresponding to A261 is replaced with another amino acid residue when that amino acid residue is conserved (i.e., it is an A(Ala) residue), but may also include mutations in which the amino acid residue corresponding to A261 is replaced with another amino acid residue when that amino acid residue is not conserved (i.e., it is not an A(Ala) residue). The amino acid residues before and after modification are selected so as not to be identical to each other. Furthermore, for any mutant TG, the corresponding wild-type TG may be one in which the amino acid residue before modification mentioned in each of the above mutations is conserved.

[0068] The amino acid residue that corresponds to the amino acid residue at position X in the amino acid sequence shown in Sequence ID No. 2 in any given TG amino acid sequence can be determined by aligning the amino acid sequence of the given TG with the amino acid sequence shown in Sequence ID No. 2. Alignment can be performed, for example, using known gene analysis software. Specific examples of such software include DNASIS from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0069] <2> Manufacturing of mutant TG Mutant TG can be produced, for example, by expressing the mutant TG gene in a host that possesses the mutant TG gene.

[0070] Furthermore, mutant TG can also be produced, for example, by expressing the mutant TG gene in a cell-free protein synthesis system.

[0071] The following details the production of mutant TG using a host with a mutant TG gene.

[0072] <2-1>Host A host possessing a mutant TG gene can be obtained by introducing the mutant TG gene into a suitable host. "Introducing a mutant TG gene into a host" may also include modifying the host's existing TG gene, such as the wild-type TG gene, to encode the mutant TG gene. "Possessing a mutant TG gene" is also referred to as "having mutant TG."

[0073] The host is not particularly restricted as long as it can express a functional mutant TG. Examples of hosts include microorganisms, plant cells, insect cells, and animal cells. Microorganisms are particularly noteworthy as hosts. Examples of microorganisms include bacteria and yeast. Examples of microorganisms include bacteria.

[0074] Examples of bacteria include those belonging to the family Enterobacteriaceae, Corynebacteria, and Bacillus.

[0075] Bacteria belonging to the Enterobacteriaceae family include those belonging to genera such as Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Photorhabdus, Providencia, Salmonella, and Morganella. Specifically, bacteria classified under the Enterobacteriaceae family according to the classification method used in the NCBI (National Center for Biotechnology Information) database (http: / / www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. While there are no particular restrictions on Escherichia bacteria, those classified under the Escherichia genus according to classifications known to microbiology experts are generally considered. Examples of Escherichia bacteria include those described in the book by Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). An example of Escherichia bacteria is Escherichia coli. Examples of Escherichia coli include Escherichia coli K-12 strains such as strain W3110 (ATCC 27325) and strain MG1655 (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as strain BL21 (DE3); and their derivative strains.Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Examples of Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Examples of Klebsiella bacteria include Klebsiella planticola. It should be noted that bacteria belonging to the Enterobacteriaceae family have recently been reclassified into multiple families through comprehensive comparative genomic analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599). However, in this invention, bacteria that were conventionally classified under the Enterobacteriaceae family will be treated as bacteria belonging to the Enterobacteriaceae family.

[0076] Examples of coryne-type bacteria include those belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

[0077] Examples of Corynebacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0078] Examples of Corynebacterium strains include the following: Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium molassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354

[0079] Furthermore, the genus Corynebacterium includes bacteria that were previously classified under the genus Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). Additionally, the genus Corynebacterium statyonis includes bacteria that were previously classified under Corynebacterium ammoniagenes but have been reclassified as Corynebacterium statyonis based on 16S rRNA sequencing analysis (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

[0080] Examples of bacteria belonging to the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, and Bacillus stearothermophilus. Specific examples of Bacillus subtilis include Bacillus subtilis strain 168 Marburg (ATCC 6051) and Bacillus subtilis strain PY79 (Plasmid, 1984, 12, 1-9). Examples of Bacillus amyloricephasiens include Bacillus amyloricephasiens strain T (ATCC 23842) and Bacillus amyloricephasiens strain N (ATCC 23845).

[0081] Examples of yeasts include those belonging to genera such as Saccharomyces cerevisiae, Candida utilis, Pichia, Pichia pastoris, Hansenula, Hansenula polymorpha, and Schizosaccharomyces pombe.

[0082] These strains can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a registration number, which can be used to obtain them (see http: / / www.atcc.org / ). The registration numbers for each strain are listed in the American Type Culture Collection catalog. These strains can also be obtained, for example, from the depositary institutions where each strain is deposited.

[0083] A mutant TG gene can be obtained, for example, by modifying a wild-type TG gene so that the encoded TG has a "specific mutation." The wild-type TG gene used as the basis for modification can be obtained, for example, by cloning from an organism that possesses the wild-type TG gene, or by chemical synthesis. Alternatively, a mutant TG gene can be obtained without going through the wild-type TG gene. The mutant TG gene may be obtained directly, for example, by chemical synthesis. The obtained mutant TG gene may be used as is or after further modification. For example, a mutant TG gene of one form may be obtained by modifying a mutant TG gene of another form.

[0084] Gene modification can be performed using known methods. For example, site-directed mutagenesis (SMU) can be used to introduce a desired mutation at a target site in DNA. That is, for example, SMU can be used to modify the coding region of a gene so that the encoded protein includes substitution, deletion, insertion, and / or addition of amino acid residues at a specific site. Examples of SMU methods include methods using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0085] The method for introducing the mutant TG gene into a host is not particularly limited. The mutant TG gene only needs to be held in the host in an expressible state. That is, in the host, the mutant TG gene only needs to be held in an expressible state under the control of a promoter that functions in that host. In the host, the mutant TG gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or it may be introduced onto a chromosome. The host may have only one copy of the mutant TG gene, or it may have two or more copies. The host may have only one type of mutant TG gene, or it may have two or more types of mutant TG genes.

[0086] The promoter used to express the mutant TG gene is not particularly limited as long as it functions in the host. A "promoter that functions in the host" means a promoter that has promoter activity in the host. The promoter may be a promoter of host origin or a promoter of heterologous origin. The promoter may be the intrinsic promoter of the TG gene or a promoter of another gene. The promoter may be a more potent promoter than the intrinsic promoter of the TG gene. Examples of potent promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and the Bifidobacterium-derived Pm1 promoter, PR promoter, and PL promoter. Furthermore, potent promoters that function in Corynebacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnolo., 53, 674-679 (2000)), the pta, aceA, aceB, adh, and amyE promoters that can be induced in Corynebacteria with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, and tuf((EF-Tu)) promoters, which are potent promoters with high expression levels in Corynebacteria (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005). Examples include the P2 promoter (WO2018 / 079684), P3 promoter (WO2018 / 079684), lac promoter, tac promoter, trc promoter, and F1 promoter (WO2018 / 179834). Furthermore, as a strong promoter, highly active versions of conventional promoters may be obtained and used by using various reporter genes. For example, promoter activity can be increased by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00 / 18935).Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and the pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)), among others.

[0087] Furthermore, a terminator for transcription termination can be placed downstream of the mutant TG gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be of host origin or of heterologous origin. The terminator may be a terminator specific to the TG gene or a terminator of another gene. Specific examples of terminators include, for example, the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0088] A mutant TG gene can be introduced into a host, for example, using a vector containing the gene. A vector containing a mutant TG gene is also called a mutant TG gene expression vector or recombinant vector. A mutant TG gene expression vector can be constructed, for example, by ligating a DNA fragment containing the mutant TG gene with a vector that functions in the host. By transforming the host with a mutant TG gene expression vector, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication within the host cell can be used. The vector is preferably a multicopy vector. Furthermore, the vector is preferably equipped with a marker such as an antibiotic resistance gene for selecting transformants. The vector may also be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA (Takara Bio), pACYC vector, and the broad-host-range vector RSF1010.Specifically, examples of vectors capable of autonomous replication in Corynebacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids containing improved drug resistance genes; pCRY30 (JP-A-3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, U.S. Patent No. 5,185,262); pCRY2 and pCRY Examples include 3 (JP-A-1-191686); pAJ655, pAJ611, and pAJ1844 (JP-A-58-192900); pCG1 (JP-A-57-134500); pCG2 (JP-A-58-35197); pCG4 and pCG11 (JP-A-57-183799); pPK4 (US Patent No. 6,090,597); pVK4 (JP-A-9-322774); pVK7 (JP-A-10-215883); pVK9 (WO2007 / 046389); pVS7 (WO2013 / 069634); and pVC7 (JP-A-9-070291). Furthermore, specific examples of vectors capable of autonomous replication in Corynebacteria include variants of pVC7, such as pVC7H2 (WO2018 / 179834). When constructing an expression vector, for example, the mutant TG gene containing a unique promoter region may be directly incorporated into the vector, the coding region of the mutant TG may be bound downstream of the promoter as described above before being incorporated into the vector, or the coding region of the mutant TG may be incorporated downstream of the promoter originally present on the vector.

[0089] Vectors, promoters, and terminators usable in various microorganisms are described in detail in, for example, "Basic Microbiology Course 8: Genetic Engineering," Kyoritsu Shuppan, 1987, and these can be utilized.

[0090] Furthermore, mutant TG genes can be introduced, for example, onto host chromosomes. Gene introduction into chromosomes can be carried out, for example, using homologous recombination (Miller I, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include methods using linear DNA such as Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), methods using plasmids containing temperature-sensitive origins of replication, methods using conjugate-transferable plasmids, methods using suicide vectors that do not have origins of replication that function in the host, and phage-based transduction methods. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of the gene can be introduced into the chromosome by performing homologous recombination targeting sequences that have many copies on the chromosome. Sequences with multiple copies on a chromosome include repetitive DNA sequences and inverted repeats located at both ends of transposons. Homologous recombination may also be performed targeting appropriate sequences on the chromosome, such as genes that are not necessary for carrying out the present invention. Genes can also be randomly introduced onto the chromosome using transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, a mutant TG gene containing a unique promoter region may be directly incorporated into the chromosome, the coding region of the mutant TG may be bound downstream of the promoter as described above before being incorporated into the chromosome, or the coding region of the mutant TG may be incorporated downstream of a promoter that is originally present on the chromosome.

[0091] The introduction of a gene onto a chromosome can be confirmed, for example, by Southern hybridization using a probe with a nucleotide sequence complementary to all or part of the gene, or by PCR using primers created based on the nucleotide sequence of the gene.

[0092] The transformation method is not particularly limited, and conventionally known methods can be used. Examples of transformation methods include the method of treating receptor bacterial cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A.,J. Mol. Biol. 1970, 53, 159-162), and the method of preparing competent cells from cells in the growth stage and introducing DNA, as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE., 1977. Gene 1: 153-167). Furthermore, as a transformation method, a method in which the cells of DNA-receiving bacteria are made into protoplasts or spheroplasts that readily incorporate recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeasts, and recombinant DNA is introduced into the DNA-receiving bacteria (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933) can also be applied. Additionally, as a transformation method, the electropulse method (Japanese Patent Publication No. Hei 2-207791), as reported for Corynebacteria, can also be used.

[0093] Furthermore, if a host already possesses a TG gene, such as a wild-type TG gene, on its chromosomes, the host can be modified to possess a mutant TG gene by altering the TG gene to encode a mutant TG gene. "Introduction of a mutant TG gene" may also include altering the host's existing TG gene to a mutant TG gene. Modification of TG genes present on chromosomes can be carried out, for example, by spontaneous mutation, mutation treatment, or genetic engineering.

[0094] The host may or may not have the wild-type TG gene. In particular, the host does not need to have the wild-type TG gene.

[0095] The host may have any properties as long as it can produce mutant TG.

[0096] <2-2> Host culture By culturing a host containing a mutant TG gene, mutant TG can be expressed.

[0097] The culture medium used is not particularly limited, as long as it allows the host to grow and express a functional mutant TG. For example, a standard culture medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain, as needed, a carbon source, nitrogen source, phosphate source, sulfur source, and other various organic and inorganic components. The types and concentrations of the culture medium components may be appropriately set according to various conditions, such as the type of host.

[0098] Specific carbon sources include, for example, sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, hydrolyzed starch, and hydrolyzed biomass; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. Plant-derived raw materials are preferably used as carbon sources. Examples of plants include corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds; plant bodies containing these organs; and decomposition products of these plant organs. The form of use of plant-derived raw materials is not particularly limited and can be used in any form, such as unprocessed products, juices, pulverized products, or refined products. In addition, pentoses such as xylose, hexoses such as glucose, or mixtures thereof can be obtained and used, for example, from plant biomass. Specifically, these sugars can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis by enzymes such as cellulase, and alkaline treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed beforehand to release pentoses, and then cellulose may be hydrolyzed to produce hexoses. Xylose may also be supplied by converting hexoses, such as glucose, into xylose, for example, by providing the host with a conversion pathway from hexoses. As a carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0099] The concentration of the carbon source in the culture medium is not particularly limited, as long as the host can grow and functional mutant TG is expressed. The concentration of the carbon source in the culture medium may be as high as possible, for example, without inhibiting the production of mutant TG. The initial concentration of the carbon source in the culture medium may be, for example, usually 5-30 w / v%, preferably 10-20 w / v%. In addition, the carbon source may be supplied to the culture medium as appropriate. For example, the carbon source may be supplied to the culture medium in response to the decrease or depletion of the carbon source as the culture progresses. The carbon source may be temporarily depleted as long as mutant TG is eventually produced, but it is preferable in some cases to carry out the culture in a way that prevents the carbon source from being depleted or to prevent a state of carbon source depletion from continuing.

[0100] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; peptone; organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may also be used as a nitrogen source. One nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0101] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. One phosphate source may be used, or two or more phosphate sources may be used in combination.

[0102] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. A single sulfur source may be used, or a combination of two or more sulfur sources may be used.

[0103] Other various organic and inorganic components include, specifically, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other organic and inorganic components may be used individually, or in combination of two or more components.

[0104] Furthermore, when using a nutrient-dependent mutant strain that requires nutrients such as amino acids for growth, it is preferable to supplement the culture medium with such required nutrients.

[0105] The culture conditions are not particularly limited, as long as the host can grow and a functional mutant TG is expressed. Culturing can be carried out under the usual conditions used for culturing microorganisms such as bacteria and yeast. The culture conditions may be set appropriately depending on various factors, such as the type of host. Furthermore, the expression of the mutant TG gene can be induced as needed.

[0106] Culturing can be carried out using a liquid medium. During culturing, for example, the host may be cultured on a solid medium such as agar medium and then directly inoculated into the liquid medium, or the host may be seed cultured on a liquid medium and then inoculated into the liquid medium for the main culture. In other words, culturing may be carried out separately as seed culture and main culture. In this case, the culture conditions for seed culture and main culture may be the same or different. Mutant TG should be expressed at least in the main culture. The amount of host contained in the medium at the start of culturing is not particularly limited. For example, a seed culture solution with OD660 = 4 to 100 may be added to the medium for the main culture at the start of culturing in an amount of 0.1% to 100% by mass, preferably 1% to 50% by mass.

[0107] Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium used at the start of culture is also called the "initial medium." The culture medium supplied to the culture system (e.g., a fermenter) in fed-batch or continuous culture is also called the "fed-batch medium." The act of supplying fed-batch medium to the culture system in fed-batch or continuous culture is also called "fed-batch." When culture is carried out separately as a seed culture and a main culture, the culture forms of the seed culture and the main culture may or may not be the same. For example, both the seed culture and the main culture may be carried out as batch cultures, or the seed culture may be carried out as a batch culture and the main culture as a fed-batch or continuous culture.

[0108] In the present invention, various components such as carbon sources may be contained in the initial culture medium, the fed-batch medium, or both. That is, during the culture process, various components such as carbon sources may be supplied to the medium individually or in any combination. These components may be supplied once, multiple times, or continuously. The types of components contained in the initial culture medium may be the same as, or different from, the types of components contained in the fed-batch medium. Furthermore, the concentration of each component contained in the initial culture medium may be the same as, or different from, the concentration of each component contained in the fed-batch medium. In addition, two or more types of fed-batch media with different types and / or concentrations of components may be used. For example, if multiple feedings are performed intermittently, the types and / or concentrations of components contained in each fed-batch medium may be the same or different.

[0109] Culturing can be carried out, for example, under aerobic conditions. "Aerobic conditions" may mean that the dissolved oxygen concentration in the culture medium is 0.33 ppm or higher, preferably 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 50% of the saturated oxygen concentration, preferably about 5%. Culturing can be carried out, for example, by aeration culture or shaking culture. The pH of the culture medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During cultivation, the pH of the culture medium can be adjusted as needed. The pH of the culture medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The culture temperature may be, for example, 20 to 45°C, preferably 25 to 37°C. The culture period may be, for example, 10 to 120 hours. The culture may be continued, for example, until the carbon source in the culture medium is consumed or until the host becomes inactive.

[0110] By culturing the host in this manner, a culture containing mutant TG is obtained. The mutant TG may accumulate, for example, within the host's cells. "Bacterial cells" may be appropriately replaced with "cells" depending on the type of host. Depending on the type of host and / or the design of the mutant TG gene, the mutant TG may accumulate, for example, in the periplasm, or be secreted outside the cell.

[0111] Mutant TG may be used as is contained in the culture (specifically, the culture medium or bacterial cells), or it may be used after purification from the culture (specifically, the culture medium or bacterial cells). Purification can be carried out to any desired degree. That is, mutant TG may be purified mutant TG or a fraction containing mutant TG. In other words, mutant TG may be used in the form of a purified enzyme, in the form of such a fraction (i.e., in the form contained in such a fraction), or in a combination thereof. Such fractions are not particularly limited as long as they contain mutant TG so that it can act on their substrate. Such fractions may be cultures of hosts having the mutant TG gene (i.e., hosts having mutant TG), bacterial cells recovered from the same culture, culture supernatant recovered from the same culture, processed products thereof (e.g., processed products of bacterial cells such as cell lysates, cell lysates, cell extracts, and immobilized cells), partially purified products thereof (i.e., crude products), or combinations thereof. Furthermore, "purified mutant TG" may include crude products. These fractions may be used individually or in combination with purified mutant TG. Mutant TG may be used, for example, in a form not contained within bacterial cells. Mutant TG may also be used, for example, in the form of the compositions of the present invention described later.

[0112] If mutant TG accumulates in the culture medium, the culture supernatant can be obtained by, for example, centrifugation, and the mutant TG can be purified from the culture supernatant. Alternatively, if mutant TG accumulates within the host bacterial cells, the bacterial cells can be subjected to processes such as disruption, lysis, or extraction, and the mutant TG can be purified from the treated material. Purification of mutant TG can be carried out, for example, by known methods used for protein purification. Such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. These methods can be used individually or in appropriate combinations.

[0113] The uses of mutant TG are not particularly limited.

[0114] Mutant TG may be used, for example, to modify food. Furthermore, mutant TG may be used, for example, in the manufacture of food. Food manufactured using mutant TG may be modified food. In other words, mutant TG may be specifically used, for example, in the manufacture of modified food. To put it another way, modified food may be obtained through food modification.

[0115] Examples of foods include those whose pH decreases during manufacturing. Examples of foods include those produced by fermentation. Fermentation can be carried out using microorganisms such as lactic acid bacteria and yeast. pH may decrease during fermentation, for example. In other words, foods whose pH decreases during manufacturing can be an example of foods produced by fermentation. Also, foods produced by fermentation can be an example of foods whose pH decreases during manufacturing. Specifically, examples of such foods include yogurt and cheese. Yogurt is a particularly good example of such a food. Both yogurt and cheese can be examples of foods whose pH decreases during manufacturing and / or foods produced by fermentation.

[0116] Furthermore, food products manufactured using mutant TG may be used as raw materials to manufacture other food products. For the sake of explanation, food products manufactured using mutant TG may also be called "intermediate products," and other food products manufactured using intermediate products as raw materials may also be called "final products." In other words, such final products can also be considered as food products. Examples of final products include foods containing the examples given above (for example, yogurt or cheese). Examples of foods containing yogurt or cheese include ice cream containing yogurt or cheese. The final product may also be modified by manufacturing the final product using the intermediate product as a raw material. The type of modification of the final product may be the same as the type of modification of the intermediate product, or it may not be. "Using mutant TG in the modification or manufacture of food products" may also include cases where mutant TG is indirectly used in the modification or manufacture of the final product by manufacturing the final product using the intermediate product as a raw material.

[0117] Food modification can include improving physical properties. In the case of yogurt or cheese, improvements in physical properties include increasing tensile strength, preventing syneresis, and imparting a smooth texture. In particular, improvements in yogurt or cheese can include increasing tensile strength. That is, for example, by using mutant TG, improvements in physical properties can be obtained in foods such as yogurt and cheese compared to when mutant TG is not used. Specifically, for example, by manufacturing foods such as yogurt and cheese using mutant TG, foods with improved physical properties such as yogurt and cheese can be obtained, which have increased tensile strength, prevent syneresis, and / or have a smooth texture, compared to when yogurt and cheese are manufactured without mutant TG.

[0118] <3> Composition of the present invention The composition of the present invention is a composition containing mutant TG.

[0119] The composition of the present invention may, for example, be a composition used for the applications of mutant TG as illustrated above. That is, the composition of the present invention may, for example, be a composition for modifying foods such as yogurt and cheese. Furthermore, the composition of the present invention may, for example, be a composition for manufacturing foods such as yogurt and cheese. More specifically, the composition of the present invention may, for example, be a composition for manufacturing modified foods such as modified yogurt and cheese.

[0120] The composition of the present invention may consist of mutant TG, or it may contain components other than mutant TG.

[0121] Other components besides mutant TG are not particularly limited, as long as they do not impair the function of mutant TG. Other components can be those acceptable depending on the intended use of the composition of the present invention. Examples of other components include those incorporated into foods or pharmaceuticals.

[0122] As a composition of the present invention, for example, a variant TG in the embodiment exemplified above may be used as is or after being appropriately formulated. In formulation, appropriate additives may be used depending on the intended use of the composition of the present invention. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, and solvents. Additives can be appropriately selected, for example, depending on various conditions such as the shape of the composition of the present invention.

[0123] The form of the composition of the present invention is not particularly limited. The composition of the present invention may be provided in any form, such as powder, flakes, tablets, paste, or liquid.

[0124] <4> Method of the present invention The method of the present invention utilizes mutant TG.

[0125] In the method of the present invention, mutant TG may be used for applications of mutant TG as exemplified above. That is, the method of the present invention may be a method for modifying foods such as yogurt and cheese. The method of the present invention may also be a method for producing foods such as yogurt and cheese. More specifically, the method of the present invention may be a method for producing modified foods such as modified yogurt and cheese.

[0126] Mutant TG can be used in the modification or manufacture of food products to process food ingredients. "Food ingredients" may refer to materials used as raw materials for manufacturing food products. Food ingredients can be appropriately selected according to various conditions such as the type of food product. Examples of food ingredients include dairy ingredients. For example, if the food product is yogurt or cheese, the food ingredients may be dairy ingredients. The method of the present invention will be described below mainly with reference to the case where the food product is yogurt. This description can also be applied to other foods. In that case, "dairy ingredients" in the modification or manufacture of yogurt can be read as "food ingredients" in the modification or manufacture of other foods.

[0127] In the modification or manufacture of yogurt, mutant TG can be used to process dairy raw materials. That is, one use of mutant TG is to process dairy raw materials with mutant TG. In other words, the method of the present invention may be a method for modifying yogurt, for example, including a step of processing dairy raw materials with mutant TG. The method of the present invention may also be a method for producing yogurt, for example, including a step of processing dairy raw materials with mutant TG. Furthermore, the method of the present invention may specifically be a method for producing modified yogurt, for example, including a step of processing dairy raw materials with mutant TG. Note that "processing dairy raw materials with mutant TG" is also called "applying mutant TG to dairy raw materials." The step of "processing dairy raw materials with mutant TG" is also called the "processing step." In other words, the method of the present invention may include a processing step.

[0128] Mutant TG may be used in the treatment of dairy raw materials in any manner that allows it to act on the dairy raw materials. Mutant TG may be used in the treatment of dairy raw materials in the form exemplified above, for example. Specifically, mutant TG may be used in the treatment of dairy raw materials in the form of the composition of the present invention, for example. That is, "treating dairy raw materials with mutant TG" also includes treating dairy raw materials with the composition of the present invention.

[0129] The modification or manufacture of yogurt may be carried out in the same manner as the modification or manufacture of ordinary yogurt, except, for example, the use of mutant TG. The modification or manufacture of yogurt may be carried out in the same manner as the modification or manufacture of yogurt using TG as described in WO2018 / 079687, WO2018 / 079686, WO2018 / 079685, WO2018 / 079684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, or US2019-0161776A, etc., except, for example, the use of mutant TG as TG.

[0130] The modification or production of yogurt may be carried out using the same dairy ingredients and under the same production conditions as for regular yogurt, except for the use of mutant TG. Furthermore, the dairy ingredients and production conditions may be modified as appropriate for use in the modification or production of yogurt. The method of the present invention may include a step of producing yogurt from dairy ingredients. This step is also referred to as the "yogurt production step." The processing step may also be a step of producing yogurt by treating the dairy ingredients with mutant TG.

[0131] "Milk raw material" may mean a raw material containing milk protein. The milk raw material is not particularly limited as long as it can be used to produce yogurt by the method of the present invention. Examples of milk proteins include casein and whey protein. Examples of whey protein include α-lactalbumin and β-lactoglobulin. The milk raw material may contain one or more of these milk proteins, or all of them. The milk protein content (concentration) in the milk raw material can be measured, for example, by measuring the total nitrogen amount using the Kjeldahl method and multiplying the total nitrogen amount by the nitrogen-protein conversion factor. In the case of milk protein, the nitrogen-protein conversion factor may be 6.38. Examples of milk include cow's milk, goat's milk, sheep's milk, buffalo milk, reindeer milk, donkey's milk, and camel's milk. Cow's milk is particularly noteworthy as a milk ingredient. As a milk raw material, for example, milk can be used as is or modified as appropriate. That is, the milk raw material may be raw milk (unprocessed milk) or not. Dairy raw materials may or may not undergo processing such as heating, homogenization, or drying. Dairy raw materials may or may not have their components adjusted. Specifically, examples of dairy raw materials include whole milk, skim milk, partially skim milk, buttermilk, processed products thereof, and their component-adjusted products. An example of a component-adjusted product is calcium-fortified milk. Dairy raw materials may be used as a single ingredient, or in combination of two or more ingredients.

[0132] Yogurt can be produced by fermentation. Fermentation can be carried out, for example, using a starter. Specifically, fermentation can be carried out, for example, by coexisting the starter with dairy ingredients. The pH of the dairy ingredients at the start of fermentation may be, for example, 6.0 or higher, 7.4 or lower, 7.2 or lower, or 7.0 or lower, or a combination thereof. Specifically, the pH of the dairy ingredients at the start of fermentation may be, for example, 6.0 to 7.0.

[0133] "Starter" refers to microorganisms used in fermentation. The starter is not particularly limited as long as it can produce yogurt by the method of the present invention. Examples of starters include lactic acid bacteria and yeast. Lactic acid bacteria are particularly recommended as starters.

[0134] Examples of lactic acid bacteria include bacteria of the genera Lactobacillus, Lactococcus, Streptococcus, and Bifidobacterium.

[0135] Examples of bacteria belonging to the genus Lactobacillus include Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus gasseri, and Lactobacillus acidophilus. Lactobacillus delbrueckii includes strains classified as any of the Lactobacillus delbrueckii subspecies, such as Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, and Lactobacillus delbrueckii subsp. lactis.

[0136] Lactococcus lactis is an example of a bacterium belonging to the genus Lactococcus. Lactococcus lactis includes strains classified as any of the subspecies of Lactococcus lactis, such as Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. hordniae.

[0137] An example of a bacterium belonging to the genus Streptococcus is Streptococcus thermophilus.

[0138] Bifidobacterium species include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, and Bifidobacterium pseudolongum. Examples include Bifidobacterium pseudolongum and Bifidobacterium thermophilum.

[0139] As a starter, one type of microorganism may be used, or a combination of two or more types of microorganisms may be used.

[0140] As a starter, you may use, for example, a commercially available starter. Alternatively, you may use microorganisms obtained from a depositary or distribution institution as a starter.

[0141] Fermentation may be carried out, for example, until the pH of the fermented product decreases to a desired level. The pH of the fermented product at the completion of fermentation may be, for example, 5.0 or less, 4.8 or less, 4.6 or less, 4.4 or less, or 4.2 or less, or 3.8 or more, 4.0 or more, 4.2 or more, or 4.4 or more, or any non-contradictory combination thereof. Specifically, the pH of the fermented product at the completion of fermentation may be, for example, 4.0 to 4.6. "Fermented product" may mean the dairy raw material after the start of fermentation, and may also include the yogurt that is ultimately obtained. Also, "fermented product at the completion of fermentation" may mean the yogurt that is ultimately obtained. The fermentation time may be, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 7 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more, or 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, or 7 hours or less, or any non-contradictory combination thereof. The fermentation temperature may be, for example, 25°C or higher, 30°C or higher, 35°C or higher, or 40°C or higher, or 45°C or lower, 40°C or lower, or 35°C or lower, or any non-contradictory combination thereof.

[0142] By carrying out fermentation in this way, yogurt is produced.

[0143] Mutant TG may be applied to the dairy raw materials at any stage of the yogurt manufacturing process, as long as the desired effect (e.g., yogurt modification effect) is obtained. Mutant TG can be applied to the dairy raw materials by coexisting with them, either as is or after being prepared in a desired form such as a solution. For example, mutant TG may be added to the dairy raw materials, or a treatment solution containing mutant TG may be mixed with the dairy raw materials. Such operations of coexisting mutant TG with dairy raw materials are collectively referred to as "addition" of mutant TG. Mutant TG may be added, for example, before, at the start of, or after the start of fermentation. Specifically, mutant TG may be added, for example, before, at the time of, or after the addition of the starter. Mutant TG may be added, for example, before, at the time of, or after the completion of fermentation. In particular, mutant TG may be added before the completion of fermentation. Specifically, mutant TG may be added before the pH of the fermented product drops to a desired level. The pH of the fermented product when mutant TG is added may be, for example, 5.0 or higher, 5.2 or higher, 5.4 or higher, 5.6 or higher, 5.8 or higher, or 6.0 or higher, or 7.4 or lower, 7.2 or lower, 7.0 or lower, 6.8 or lower, 6.6 or lower, or 6.4 or lower, or a combination thereof. Specifically, the pH of the fermented product when mutant TG is added may be, for example, 6.0 to 7.0. Depending on various conditions such as the timing of the addition of mutant TG and the remaining activity period of mutant TG, fermentation of the milk raw material may proceed during treatment with mutant TG. Therefore, the "milk raw material" treated with mutant TG is not limited to milk raw material before the start of fermentation, but may also include the fermented product.

[0144] The conditions for carrying out the processing step are not particularly limited, as long as the desired effect (e.g., yogurt modification effect) is obtained. The conditions for carrying out the processing step can be set appropriately according to various conditions such as the properties and amount of mutant TG added. The processing step may be carried out in conjunction with fermentation, for example. That is, the yogurt manufacturing process may also serve as the processing step. However, depending on various conditions such as the timing of the addition of mutant TG and the remaining activity period of mutant TG, some or all of the processing step may be carried out before fermentation, or some or all of the processing step may be carried out after fermentation. For the temperature and time of the processing step, for example, the descriptions for fermentation temperature and fermentation time can be applied mutatis mutandis.

[0145] Mutant TG may be inactivated, for example, during yogurt production. More specifically, mutant TG may be inactivated, for example, during fermentation. More specifically, mutant TG may be inactivated, for example, by a decrease in the pH of the fermented product. The residual activity of mutant TG in the fermented product at the completion of fermentation may be, for example, 20% or less, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, or 0%. In other words, fermentation may be carried out until the residual activity of mutant TG decreases to the above range. "Residual activity of mutant TG in the fermented product at the completion of fermentation" means the ratio of the TG activity of mutant TG in the fermented product at the completion of fermentation to the TG activity of mutant TG at the time of addition. According to the method of the present invention, mutant TG may be inactivated without heat treatment. Therefore, the method of the present invention does not have to include, for example, a treatment to inactivate mutant TG by heating. By sufficiently inactivating mutant TG, it may be possible to prevent unnecessary alteration of the yogurt's physical properties by mutant TG after fermentation is complete (for example, during storage or distribution of the final yogurt product). Furthermore, when enzymes are added to produce food, any enzymes that remain inactive in the final product must be labeled as food additives. However, by sufficiently inactivating mutant TG, it may become unnecessary to label "enzyme" as a food additive in the final product.

[0146] In the method of the present invention, ingredients other than those exemplified above (e.g., dairy ingredients, starter, and mutant TG) may be used, as long as the desired effect (e.g., yogurt modification effect) is obtained. Such ingredients include ingredients other than those exemplified above that are commonly used in the production of yogurt. Such ingredients may be mixed with the dairy ingredients beforehand, or may be added to the dairy ingredients during the production of yogurt as appropriate.

[0147] The amount and ratio of each component added in the method of the present invention are not particularly limited, as long as the desired effect (for example, yogurt modification effect) is obtained. The amount and ratio of each component used in the method of the present invention can be appropriately set according to various conditions such as the type of each component.

[0148] The amount of mutant TG added may be, for example, 0.00033U or more, 0.001U or more, 0.0033U or more, 0.01U or more, 0.033U or more, 0.1U or more, 0.33U or more, or 1U or more per gram of milk protein, or 100U or less, 33U or less, 10U or less, 3.3U or less, or 1U or less, or any non-consistent combination thereof. Specifically, the amount of mutant TG added may be, for example, 0.00033U to 33U, or 0.0033U to 3.3U per gram of milk protein. [Examples]

[0149] The present invention will be described in more detail below with reference to non-limiting embodiments.

[0150] Example 1: Construction and analysis of mutant transglutaminase (mutant TG) In this embodiment, mutant TG was constructed and analyzed.

[0151] <1> Experimental methods In this embodiment, the quantification of TG and the measurement of TG activity were performed according to the following procedure unless otherwise specified.

[0152] <1-1>Quantitative determination of TG TG was quantified by HPLC analysis using BSA as the standard. The analytical conditions are as follows.

[0153] Mobile phase A: 0.1% trifluoroacetic acid (TFA) Mobile phase B: 0.1% TFA, 80% acetonitrile Flow rate: 1.0 ml / min Column temperature: 40℃ Detection: UV 280 nm Column: Proteonavi, 4.6 × 150 mm, 5 μm (manufactured by Osaka Soda Co., Ltd.) Gradient: 0 min (B: 30%), 0-20 min (B: 30-50%), 20-25 min (B: 50-100%), 25-26 min (B: 100%), 26-27 min (B: 100-30%), 27-30 min (B: 30%)

[0154] <1-2> Measurement of TG activity TG activity was measured by the hydroxamate method. 500 μL of solution A (50 mM MES, 100 mM NH2OH, 10 mM reduced glutathione, 30 mM CBZ-Gln-Gly (Z-QG), adjusted to pH 6.0 with NaOH) was added to a 1.5 mL tube and incubated at 37°C for 5 minutes. 50 μL of enzyme solution was added, and after reacting at 37°C for 10 minutes, 500 μL of solution B (1N HCl, 4% TCA, 1.67% FeCl3·6H2O) was added to stop the reaction. 200 μL of the stop solution was added to a 96-well plate, and the absorbance at 525 nm was measured using a plate reader. As a control, the absorbance of a similar reaction using 20 mM MES pH 6.0 was measured, and the absorbance difference from the sample solution was determined. Separately, a calibration curve was created using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the absorbance difference. An enzyme activity of 1 U was defined as the amount of hydroxamic acid produced per minute of 1 μmol.

[0155] <2> Mutation introduction into TG Mutations were introduced into TG using PrimeSTAR(R) Max DNA Polymerase (Takara Bio Inc.) with the TG expression plasmid pPSPTG1 (Appl. Environ. Microbiol., 2003, 69(1), 358-366) as a template. pPSPTG1 is a secretory expression plasmid of TG with the pro-structure of Streptoverticillium mobaraense. Two PCR fragments were prepared using the primers listed in Table 1, and the TG expression plasmid with the desired mutation was obtained by ligating the PCR fragments using the In-Fusion(R) HD Cloning Kit (Takara Bio Inc.). Double mutants were constructed by introducing additional mutations using the constructed single mutant as a template in the same procedure. Triple mutants were constructed by introducing additional mutations using the constructed double mutant as a template in the same procedure.

[0156] [Table 1]

[0157] <3> Construction of mutant TG expression strains the above <2> The mutant TG expression plasmid constructed using the method described above was introduced into Corynebacterium glutamicum YDK010 (WO 2002 / 081694 A1) by electroporation. YDK010 is a deficient strain of the cell surface protein PS2 from C. glutamicum AJ12036 (FERM BP-734). AJ12036 was originally deposited as an international deposit on March 26, 1984, at the National Institute of Microbial Science, Agency of Industrial Science and Technology (now the Patent Organism Depositary Center, National Institute of Technology and Evaluation, postal code: 292-0818, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan), and was assigned accession number FERM BP-734. The bacterial cells after electroporation were cultured at 30°C in a CM-Dex plate containing 25 mg / L kanamycin (glucose 5 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.4 g / L, urea 3 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·5H2O 0.01 g / L, biotin 10 μg / L, soy hydrochloride hydrolysate (as total nitrogen) 1.2 g / L, pH adjusted to 7.0 with KOH, agar 20 g / L). The obtained colonies were purified in a CM-Dex plate containing 25 mg / L kanamycin to obtain mutant TG-expressing strains. The obtained strains were cultured in 3 mL of CM-Dex liquid medium containing 25 mg / L kanamycin at 30°C for approximately 16 hours. 0.6 mL of the culture solution was mixed with 0.6 mL of 40% glycerol and stored as a glycerol stock at -80°C.

[0158] <4> Culture of mutant TG-expressing strains the above <3> A small amount of glycerol stock of the mutant TG-expressing strain obtained was scraped off and spread onto a CM-Dex plate containing 25 mg / L kanamycin, and incubated at 20°C for 3 days. The cells grown on the plate were inoculated into 10 mL of CM2G medium containing 25 mg / L kanamycin (5 g / L glucose, 10 g / L polypeptone, 10 g / L yeast extract, 5 g / L NaCl, 0.2 g / L DL-methionine, pH adjusted to 7.0 with KOH), and incubated with shaking at 30°C for 24 hours using a large test tube. 2.5 mL of the obtained culture medium was inoculated into 50 mL of TG production medium containing 25 mg / L kanamycin and 50 g / L CaCO3 (glucose 60 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·5H2O 0.01 g / L, (NH4)2SO4 30 g / L, KH2PO4 1.5 g / L, DL-methionine 0.15 g / L, thiamine hydrochloride 0.45 mg / L, biotin 0.45 mg / L, pH adjusted to 7.5 with KOH), and incubated at 30°C for 48 hours using a Sakaguchi flask. For the A322C-expressing strain, V271C / V326C-expressing strain, A261C / A322C-expressing strain, and A261C / A322C / S199A-expressing strain, DTT was added to a final concentration of 3 mM at 5 hours after the start of culture. After the culture was completed, the culture medium was collected in plastic bottles and stored at -80°C.

[0159] <5> Purification of mutant TG the above <4> After thawing the culture medium obtained, the sterilized solution was obtained by centrifugation (8000 rpm, 4°C, 20 min) and filtration with a 0.45 μm filter. The sterilized solution was replaced with 20 mM acetate buffer (pH 5.5) at room temperature using Sephadex G25(M) (GE Healthcare). After adjusting the pH to 7.0 with NaOH, alcalase (Sigma-Aldrich, P4860-50ML) was added to a weight ratio of 0.5% relative to mutant TG, and the reaction was carried out at 30°C for 17 hours to activate mutant TG. After the reaction was complete, the pH was adjusted to 5.5 with 10% acetic acid, and the entire volume was applied to a cation exchange column (Resource S 6 mL, GE Healthcare) that had been fully equilibrated with 20 mM acetate buffer (pH 5.5). After re-equilibriumization with the same buffer, the protein fraction in which NaCl elutes to around 200 mM was fractionated using UV absorption at a wavelength of 280 nm as an indicator, following a linear concentration gradient from 0 to 0.5 M NaCl. <1> The TG activity and TG content of each fraction were measured using the method described, and fractions with low specific activity were removed, with fractions near the peak top with nearly equivalent specific activity being collected. The collected fractions were desalted with 20 mM phosphate buffer (pH 6.0) using HiPrep 26 / 10 Desalting (GE Healthcare), and the mutant TG was used in the following experiments. Both cation exchange chromatography and buffer replacement were performed at 4°C.

[0160] <6> Evaluation of specific activity of mutant TG the above <5> The mutant TG obtained was diluted in 20 mM MES buffer (pH 6.0) to a concentration of 0.05 mg / mL (BSA equivalent). <1> The TG activity of the diluted solution was measured using the method described, and the specific activity of the mutant TG was calculated. As a control, wild-type TG (prepared from Activa(R)) was analyzed using the same procedure. The results are shown in Table 2.

[0161] [Table 2]

[0162] <7> Acid resistance evaluation of mutant TG the above <5> The mutant TG obtained was diluted to 0.02 mg / mL (BSA equivalent) with 20 mM sodium acetate buffer (pH 4.0, 4.5, 5.0, or 5.5) or 20 mM MES buffer (pH 6.0) and treated at 37°C for 1 hour. The residual TG activity after treatment at each pH was measured by hydroxamate. 500 μL of solution A (50 mM MES, 100 mM NH2OH, 10 mM reduced glutathione, 30 mM CBZ-Gln-Gly (Z-QG), adjusted to pH 6.0 with NaOH) was added to a 1.5 mL tube and incubated at 37°C for 5 minutes. 150 μL of the enzyme solution treated at each pH was added, and the reaction was incubated at 37°C for 30 minutes. Then, 500 μL of solution B (1N HCl, 4% TCA, 1.67% FeCl3·6H2O) was added to stop the reaction. 200 μL of the stop solution was added to a 96-well plate, and the absorbance at 525 nm was measured using a plate reader. As a control, the absorbance of a sample reacted similarly using 20 mM MES pH 6.0 was measured, and the absorbance difference from the sample solution was determined. The absorbance difference in the samples treated at each pH was calculated as a relative value, with the absorbance difference in the sample treated at pH 6.0 set to 100%. This relative absorbance difference was considered the relative value of the residual TG activity after treatment at each pH, ​​with the residual TG activity after treatment at pH 6.0 set to 100%. Wild-type TG (prepared from Activa(R)) was also analyzed as a control using the same procedure. The results are shown in Table 3. While no decrease in residual TG activity was observed in wild-type TG after acid treatment, a decrease in residual TG activity was observed in mutant TG after acid treatment. Therefore, it was revealed that mutant TG has reduced acid resistance.

[0163] [Table 3]

[0164] Example 2: Preparation and analysis of yogurt using mutant transglutaminase (mutant TG). <1> Making yogurt Pasteurized milk, water, and low-heat powder were mixed to prepare a modified milk (protein: 3.8 wt%, lipid: 1.0 wt%). The prepared modified milk was heated at 95°C for 3 minutes, then cooled to 43°C and dispensed into 200g portions. 1 mL of lactic acid bacteria dispersed in the modified milk (Nozawa Group, YF-L811 lactic acid bacteria starter) was added to the dispensed modified milk (final concentration 0.006%). Furthermore, wild-type TG (prepared from a pPSPTG1 expressing strain) or the product from Example 1 was added. <5> The mutant TG (W38F, S199A, M16T / A199A, M16T / Y34F, V271C / V326C, or A261C / A322C) obtained was added to aliquot-prepared milk at a concentration of 1 unit per gram of milk protein. After addition, the mixture was quickly divided into approximately 30 mL portions and incubated at 43°C for approximately 6 hours until the pH was 4.6 or lower. After incubation, the pH of the ferment was confirmed to be 4.5-4.6, and the mixture was further cooled at 5°C overnight.

[0165] <2> Measuring the breaking strength of yogurt It is known that the reaction of triglycerides (TGs) leads to the formation of covalent bonds between lysine or glutamine residues, improving the tensile strength of yogurt. Therefore, as an indicator of the reactivity of each TG, the tensile strength of the prepared yogurt was measured using a texture analyzer. The analytical conditions are shown below. Four small samples were used per sample, and the analysis was performed with n=4.

[0166] Plunger used: Cylindrical (1cm in diameter) Return Distance: 50mm Return Speed: 10mm / sec Contact Force: 0.5g Test Mode: Compression Pre-test speed: 0.5mm / sec Test speed: 0.5mm / sex Post test speed: 10mm / sec Target mode: Chiin Strain: 30% Trigger type: Auto Trigger force: 0.5g Advanced option: OFF

[0167] The analysis results are shown in Figure 1. All mutant TGs showed roughly the same level of fracture strength improvement as wild-type TG.

[0168] <3> Measurement of TG activity in yogurt (hydroxamate method) The TG activity in the prepared yogurt was measured using the hydroxamate method according to the following procedure.

[0169] The prepared yogurt was centrifuged at 5°C, 10,000g, and 30 minutes to obtain the supernatant. The obtained supernatant was ultrafiltered and concentrated using a UF membrane (Amicon(R) Ultra15 10k) at 5°C, 1,000g, and 40 minutes to obtain a concentrated enzyme solution approximately 10 times its original concentration. The concentration ratio of each concentrated enzyme solution was calculated from the weight of the solution before and after concentration.

[0170] Substrate solution A was prepared by adding 12.1 g of Tris, 3.5 g of hydroxylammonium chloride, 1.55 g of reduced glutathione, and 5.05 g of CBZ-Gln-Gly to 500 mL of distilled water and adjusting the pH to 6.0 with 3 M hydrochloric acid. Reaction stop solution B was prepared by mixing equal parts of 3 M hydrochloric acid, 12% trichloroacetic acid, and 5% ferric chloride. 350 μL of solution A was added to a 1.5 mL tube and heated at 37°C for approximately 5 minutes. 35 μL of each concentrated enzyme solution was added to solution A and reacted at 37°C for 120 or 240 minutes, after which 350 μL of solution B was added to stop the reaction. 200 μL of the reaction solution after stopping was added to a 96-well plate and the absorbance at 525 nm was measured using a plate reader.

[0171] The measurement results are shown in Figure 2. Note that the absorbance of each sample has been corrected to the absorbance at a 10-fold concentration from each concentration factor. All mutant TG showed lower absorbance than wild-type TG. Therefore, it was considered that all mutant TG had lower residual activity than wild-type TG. Furthermore, A261C / A322C showed absorbance roughly equivalent to that of the control. Therefore, it was considered that the residual activity of A261C / A322C was almost zero.

[0172] <4> Measurement of TG activity in yogurt (fluorescence method) Example 2 <3> In this study, A261C / A322C showed the most promising results in terms of inactivation in the product, so TG activity was measured by fluorescence method for both A261C / A322C and wild-type TG. Example 2 <2> Using a similar method, the yogurt supernatant was concentrated approximately 15 times by ultrafiltration concentration using a UF membrane to obtain a concentrated enzyme solution. 50 μL of the concentrated enzyme solution was added to 200 μL of substrate solution (79 mM Tris-HCl buffer, 150 μM Monodancyl cadaverin, 0.5 M DTT, 1 wt% N,N-dimethyl casein), and the reaction time was 80 min. EX 350nm, λ EM The fluorescence spectrum was measured at 480 nm.

[0173] Figure 3 shows the change in fluorescence intensity over time. An increase in fluorescence was observed in wild-type TG, while no increase in fluorescence was observed in A261C / A322C. Therefore, it was concluded that wild-type TG retains residual activity, while A261C / A322C does not.

[0174] Example 2 <2> ~ <4> From this, it was revealed that A261C / A322C has a yogurt-modifying effect that is roughly equivalent to that of wild-type TG, and that it is inactivated in the product and does not show residual activity.

[0175] <5> Measurement of pH and TG activity during incubation Except for the addition of wild-type TG or A261C / A322C at a concentration of 3 U per gram of milk protein, the method is the same as in Example 2. <1> Yogurt was prepared using the same method as in Example 2. After adding lactic acid bacteria, fermentation was started in an incubator at 43°C. Samples were taken every 1.5 hours, starting from the time of lactic acid bacteria addition, and the pH was measured before being cooled on ice and stored. Fermentation was stopped when the pH of the yogurt fell below 4.6, and it was cooled and stored overnight at 5°C. Samples from 4.5 hours of fermentation onward had solidified, so they were separated using a centrifuge at 10,000 g for 30 min, and the supernatant was obtained. The supernatant was then concentrated approximately 10 times by ultrafiltration using a UF membrane at 10,000 g for 30 min. Samples from less than 4.5 hours of fermentation had not solidified, so filtration and concentration were not performed, and they were used in subsequent analytical steps. All obtained samples were analyzed according to Example 2. <3> Activity measurements were performed using the hydroxyamate method with a reaction time of 240 minutes, following the same procedure. For the samples after 3 hours of fermentation, only pH was measured because solidification varied from sample to sample, making direct comparison difficult.

[0176] Figure 4 shows the changes in pH and TG activity over time. Compared to wild-type TG, A261C / A322C deactivated more rapidly as the pH decreased during fermentation. In particular, while wild-type TG showed absorbance and residual activity even at pH 4.6 or below, A261C / A322C did not show absorbance at pH 4.6 or below, suggesting that it was completely deactivated in the product. [Industrial applicability]

[0177] According to the present invention, a variant TG with low acid resistance can be provided. This variant TG with low acid resistance is useful, for example, in the modification or manufacture of foods such as yogurt.

[0178] <Explanation of Sequence Listings> Sequence ID 1: Base sequence of the portion of the TG gene in Streptoverticillium mobaraense that codes for mature TG. Sequence ID 2: Amino acid sequence of mature TG from Streptoverticillium mobaraense Sequence ID 3: Base sequence of the TG gene in Streptoverticillium mobaraense Sequence ID 4: Amino acid sequence of TG containing the pro-structural component of Streptoverticillium mobaraense Sequence IDs 5-24: Primers

Claims

1. A method for manufacturing food, The process includes treating food ingredients with a variant transglutaminase. The aforementioned food is either food (A) or (B) below: (A) Foods whose pH decreases during manufacturing; (B) Food containing the above food (A) And, The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The aforementioned specific mutation is a mutation that reduces acid resistance. The method wherein the aforementioned specific mutation includes one or more mutations selected from the following: A261C, A322C, D46P, M16T, W38F, S199A, M16T / Y34F, V271C / V326C.

2. The method according to claim 1, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 80% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

3. The method according to claim 1 or 2, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 50% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

4. The method according to any one of claims 1 to 3, wherein the specific mutation includes any of the following mutations: A261C / A322C, A261C / A322C / S199A, M16T / S199A.

5. The method according to any one of claims 1 to 4, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

6. The aforementioned Streptomyces bacteria, Streptomyces movalensis The method according to claim 5, wherein the object is Streptomyces mobaraensis.

7. The method according to any one of claims 1 to 6, wherein the wild-type transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No.

2.

8. The aforementioned food (A) is yogurt or cheese, The method according to any one of claims 1 to 7, wherein the food ingredient is a dairy ingredient.

9. The method according to claim 8, wherein the food (B) is ice cream containing yogurt or cheese.

10. The method according to any one of claims 1 to 9, wherein the mutant transglutaminase is added to the food ingredient when the pH of the food ingredient is 5.0 or higher.

11. A composition for the manufacture of food products, It contains mutant transglutaminase, The aforementioned food is either food (A) or (B) below: (A) Foods whose pH decreases during manufacturing; (B) Food containing the above food (A) And, The mutant transglutaminase is a protein that has a specific mutation in the amino acid sequence of wild-type transglutaminase and possesses transglutaminase activity. The aforementioned specific mutation is a mutation that reduces acid resistance. The composition wherein the aforementioned specific mutation comprises one or more mutations selected from the following: A261C, A322C, D46P, M16T, W38F, S199A, M16T / Y34F, M16T / S199A, V271C / V326C.

12. The composition according to claim 11, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 80% or less, relative to the residual activity after treatment at pH 6.0 at 37°C for 1 hour, which is considered 100%.

13. The composition according to claim 11 or 12, wherein the residual activity of the mutant transglutaminase after treatment at pH 4.0 at 37°C for 1 hour is 50% or less as a relative value, with the residual activity after treatment at pH 6.0 at 37°C for 1 hour being 100%.

14. The composition according to any one of claims 11 to 13, wherein the specific mutation includes any of the following mutations: A261C / A322C, A261C / A322C / S199A, M16T / S199A.

15. The composition according to any one of claims 11 to 14, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

16. The composition according to claim 15, wherein the Streptomyces bacterium is Streptomyces mobaraensis.

17. The composition according to any one of claims 11 to 16, wherein the wild-type transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No.

2.

18. The aforementioned food (A) is yogurt or cheese, The composition according to any one of claims 11 to 17, wherein the food ingredient is a dairy ingredient.

19. The composition according to claim 18, wherein the food (B) is ice cream containing yogurt or cheese.

20. A composition according to any one of claims 11 to 19, for adding to the food ingredients of the food when the pH of the food ingredients is 5.0 or higher during the production of the food.

21. A mutant transglutaminase having a specific mutation in the amino acid sequence of wild-type transglutaminase and possessing transglutaminase activity, The aforementioned specific mutation is a mutation that reduces acid resistance, and includes a mutation selected from the following: A261C, A322C, D46P A261C / A322C, A261C / A322C / S199A, V271C / V326C, M16T / Y34F, M16T / S199A, Mutant transglutaminase.

22. The mutant transglutaminase according to claim 21, wherein the residual activity after treatment at pH 4.0 at 37°C for 1 hour is 80% or less as a relative value, with the residual activity after treatment at pH 6.0 at 37°C for 1 hour being 100%.

23. The mutant transglutaminase according to claim 21 or 22, wherein the residual activity after treatment at pH 4.0 at 37°C for 1 hour is 50% or less as a relative value, with the residual activity after treatment at pH 6.0 at 37°C for 1 hour being 100%.

24. The mutant transglutaminase according to any one of claims 21 to 23, wherein the wild-type transglutaminase is a protein containing the amino acid sequence of mature transglutaminase of a bacterium of the genus Streptomyces.

25. The mutant transglutaminase according to claim 24, wherein the Streptomyces bacterium is Streptomyces mobaraensis.

26. The mutant transglutaminase according to any one of claims 21 to 25, wherein the wild-type transglutaminase is one of the following proteins: (a) Proteins containing the amino acid sequence shown in Sequence ID No. 2; (b) Proteins comprising an amino acid sequence in which 1 to 10 amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 2; (c) A protein containing an amino acid sequence that has 90% or more identity with the amino acid sequence shown in Sequence ID No.

2.

27. Encodes a mutant transglutaminase according to any one of claims 21 to 26 gene.

28. A vector carrying the gene described in claim 27.

29. A microorganism having the gene described in claim 27.

30. The microorganism according to claim 29, which is a bacterium or yeast.

31. The microorganism according to claim 29 or 30, which is a Corynebacterium or a bacterium of the Enterobacteriaceae family.

32. The microorganism according to any one of claims 29 to 31, which is a bacterium of the genus Corynebacterium or Escherichia.

33. The microorganism according to any one of claims 29 to 32, which is Corynebacterium glutamicum or Escherichia coli.

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