WO2014157705A1 - 改変グリシン酸化酵素 - Google Patents
改変グリシン酸化酵素 Download PDFInfo
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- WO2014157705A1 WO2014157705A1 PCT/JP2014/059353 JP2014059353W WO2014157705A1 WO 2014157705 A1 WO2014157705 A1 WO 2014157705A1 JP 2014059353 W JP2014059353 W JP 2014059353W WO 2014157705 A1 WO2014157705 A1 WO 2014157705A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03019—Glycine oxidase (1.4.3.19)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/90605—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4)
- G01N2333/90633—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3) in general
- G01N2333/90638—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3) in general with a definite EC number (1.4.3.-)
Definitions
- the present invention relates to a modified glycine oxidase, a method for analyzing glycine using the same, and the like.
- Amino acids are very important as components of living organisms, and it is known that some amino acids can serve as indicators of health status. For example, plasma glycine concentration has been suggested to be associated with obesity (Non-patent Document 1). In addition to being naturally contained in various foods, glycine is also used, for example, as a food additive. Therefore, measurement of amino acids, particularly glycine, has important significance in a wide range of fields such as biological research, health nutrition, medical care, and food production.
- Non-Patent Document 5 As an enzyme that acts on glycine, glycine oxidase is known (for example, Non-Patent Document 5).
- the enzyme properties eg, enzyme activity, stability, substrate specificity
- the activity when the concentration of the detection target in the specimen is low, such as blood amino acids, the activity is required to be high under the low substrate concentration.
- the use of glycine oxidase is promising for the analysis of glycine-containing specimens, but glycine oxidase, which has been known so far, has a low activity and stability (for example, Non-Patent Document 6). there were.
- the substrate specificity has not been investigated for many types of amino acids. As described above, there is no enzyme that can be practically used for the measurement of glycine, and therefore, a practical glycine measurement method using an enzyme, a measurement kit, and a measurement biosensor have not been realized.
- glycine concentration may be measured by using an enzyme having improved properties related to the measurement of glycine, and glycine having such properties improved.
- the present inventors have succeeded in developing an oxidase and thus completed the present invention.
- the present invention is as follows.
- the mutation is (a) a substitution of the first threonine in the TTS motif in the amino acid sequence of glycine oxidase, (b) a substitution of serine in the TTS motif, (c) a substitution of cysteine in the HCY motif, d) 1 selected from the group consisting of substitution of leucine in the LRP motif, (e) substitution of methionine in the GML motif, (f) substitution of serine in the SG motif, and (g) substitution of g
- [3] The modified enzyme according to [2], wherein the first threonine in the TTS motif is substituted with alanine, serine, cysteine, or glycine.
- [4] The modified enzyme according to [2] or [3], wherein serine in the TTS motif is substituted with lysine.
- [5] The modified enzyme according to any one of [2] to [4], wherein cysteine in the HCY motif is substituted with alanine, aspartic acid, glycine, histidine, asparagine, tryptophan, tyrosine, or serine.
- [6] The modified enzyme according to any one of [2] to [5], wherein leucine in the LRP motif is substituted with isoleucine, valine, cysteine, threonine, or proline.
- [7] The modified enzyme according to any one of [2] to [6], wherein methionine in the GML motif is substituted with isoleucine.
- [8] The modified enzyme according to any one of [2] to [7], wherein serine in the SG motif is substituted with arginine.
- [9] The modified enzyme according to any one of [2] to [8], wherein glycine in the PGT motif is substituted with tyrosine or glutamine.
- a protein comprising an amino acid sequence having an additional mutation and having at least one property selected from the group consisting of the following (a) to (c): (A) the activity of glycine oxidase on glycine; (B) thermal stability of glycine oxidase; and (c) substrate specificity of glycine oxidase for glycine.
- [12] A method for analyzing glycine, comprising measuring glycine contained in a test sample using the modified enzyme according to any one of [1] to [11]. [13] The method according to [12], wherein the measurement of glycine is performed using 4-aminoantipyrine, phenol, and peroxidase in addition to any of the modified enzymes of [1] to [11]. [14] A method for producing glyoxylic acid, comprising producing glyoxylic acid from glycine using the modified enzyme according to any one of [1] to [11]. [15] A polynucleotide encoding the modified enzyme according to any one of [1] to [11]. [16] An expression vector comprising the polynucleotide of [15].
- a transformant comprising the expression vector according to [16]. [18] using the transformant of [17], producing a modified enzyme in which at least one amino acid residue is mutated so as to improve the properties of glycine oxidase related to measurement of glycine.
- a method for producing a modified enzyme [19] A glycine analysis kit comprising the modified enzyme according to any one of [1] to [11]. [20] The glycine analysis kit according to [19], further comprising at least one of a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a glyoxylic acid detection reagent.
- a detection system for glycine analysis comprising (a) the device and (b) the modified enzyme according to any one of [1] to [11].
- the device further comprises at least one of a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a glyoxylic acid detection reagent, and the device is a microchannel chip Detection system for glycine analysis.
- An enzyme sensor for glycine analysis comprising: (a) a detection electrode; and (b) a modified enzyme according to any one of [1] to [11] fixed or arranged on the detection electrode.
- the modified enzyme of the present invention Since the modified enzyme of the present invention has improved activity against glycine, it is useful for rapid and sensitive measurement of glycine and / or production of glyoxylic acid.
- the modified enzyme of the present invention is also excellent in stability because it is excellent in thermal stability in an aqueous solution. Therefore, the modified enzyme of the present invention is particularly useful as a liquid reagent. Furthermore, since the modified enzyme of the present invention is excellent in substrate specificity for glycine, glycine can be specifically measured.
- the analysis method of the present invention is useful in a wide range of fields such as biological research, health nutrition, medical care, and food production.
- FIG. 1 shows the substrate specificity of wild-type glycine oxidase for glycine.
- Gly 1 mM Gly;
- 25mix a mixture containing 1 mM each of 20 types of standard amino acids, cystine, taurine, citrulline, ornithine and ⁇ -aminobutyric acid;
- 25mix-Gly 25 of (2) What removes Gly from the mixed liquid of the kind (the same applies hereinafter).
- FIG. 2 is a diagram showing the substrate specificity of a mutant glycine oxidase (T42A / C245S / L301V) for glycine.
- T42A / C245S / L301V mutant glycine oxidase
- FIG. 3 is a diagram showing the substrate specificity of a mutant glycine oxidase (T42A / C245S / L301V / G304Q) for glycine.
- FIG. 4 is a graph showing the relationship between the absorbance obtained by the endpoint method using a mutant glycine oxidase (T42A / C245S / L301V) and the Gly concentration in the reaction system.
- FIG. 5 is a graph showing the relationship between the absorbance obtained by the endpoint method using a mutant glycine oxidase (T42A / C245S / L301V / G304Q) and the Gly concentration in the reaction system.
- the present invention provides a modified enzyme.
- the modified enzyme of the present invention may be one in which at least one amino acid residue has been mutated to improve the properties of glycine oxidase associated with the measurement of glycine.
- amino acid residue mutations include substitution, deletion, addition and insertion, and substitution is preferred.
- the amino acid residues to be mutated are natural L- ⁇ -amino acids L-alanine (A), L-asparagine (N), L-cysteine (C), L-glutamine (Q), L-isoleucine ( I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W ), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine ( K) or glycine (G).
- L and ⁇ may be omitted in the description of amino acids.
- Glycine oxidase (sometimes referred to as glyoxidase: GlyOX) is an oxidoreductase that catalyzes the following reaction (EC 1.2.3.19). Glycine + H 2 O + O 2 ⁇ glyoxylic acid + NH 3 + H 2 O 2
- glycine oxidase from which the modified enzyme of the present invention is derived for example, an enzyme derived from any organism (eg, microorganisms such as bacteria, actinomycetes and fungi, and insects, fish, animals, and plants) is used. Examples thereof include microorganisms belonging to the genus Bacillus and enzymes derived from organisms belonging to this related genus. Examples of related genera of the genus Bacillus include the genus Geobacillus, the genus Paenibacillus, and the genus Oceanobacillus. These closely related genera of the genus Bacillus, like the genus Bacillus, belong to the family Bacillaceae.
- microorganisms belonging to the genus Bacillus and related genera include, for example, Bacillus aerophilus, Bacillus cereus, Bacillus macauensis, Bacillus pumilus, Bacillus subtilis, Bacillus sphaericus, Bacillus cereus, Bacillus licheniformis, Bacillus guessio s. (Geobacillus stearothermophilus), Geobacillus Kausutofirasu (Geobacillus kaustophilus) and the like.
- the position where the mutation is introduced in glycine oxidase is preferably an amino acid residue near the active center of glycine oxidase.
- Concerning glycine oxidase derived from Bacillus subtilis analysis results of the three-dimensional structure have been reported (for example, see PDB ID: 1NG3, 1NG4, 1RYI, 3IF9). Since those skilled in the art can align the amino acid sequence of glycine oxidase derived from Bacillus subtilis with the amino acid sequence of other glycine oxidase, glycine oxidase derived from organisms other than Bacillus subtilis An amino acid residue near the active center can be easily identified.
- the mutation that improves the properties of glycine oxidase associated with the measurement of glycine is a substitution of the first threonine (T) and / or serine (S) in the TTS motif of the amino acid sequence of wild-type glycine oxidase.
- the TTS motif is composed of three consecutive amino acid residues of threonine (T) -threonine (T) -serine (S).
- the C-terminal threonine residue of the TTS motif may be referred to as a first threonine, and the central threonine residue of the TTS motif may be referred to as a second threonine.
- the position of the TTS motif in the amino acid sequence of wild-type glycine oxidase may vary depending on the origin of the enzyme, those skilled in the art can appropriately determine the position of the TTS motif in the amino acid sequence of wild-type glycine oxidase.
- the positions of the first threonine (T) and serine (S) should be specified.
- the TTS motif is in the amino acid region at positions 42 to 44, the first threonine (T) is at position 42, and the serine (S) is at position 44 (eg, Tables). 1).
- the mutation that improves the properties of glycine oxidase associated with measurement of glycine is a substitution of cysteine (C) in the HCY motif of the amino acid sequence of wild-type glycine oxidase.
- the HCY motif is composed of three consecutive amino acid residues of histidine (H) -cysteine (C) -tyrosine (Y).
- the HCY motif is in the amino acid region at positions 244 to 252 and cysteine (C) is at positions 245 to 251 (eg, see Table 2).
- the modified enzyme of the present invention is a mutation that improves the properties of glycine oxidase related to measurement of glycine, in addition to the substitution of cysteine (C) in the HCY motif, in addition to the substitution of the first threonine (T) in the TTS motif. You may further have the said substitution.
- the mutation that improves the properties of glycine oxidase associated with the measurement of glycine is a substitution of leucine (L) in the LRP motif of the amino acid sequence of wild-type glycine oxidase.
- the LRP motif is composed of three consecutive amino acid residues of leucine (L) -arginine (R) -proline (P).
- the position of the LRP motif in the amino acid sequence of wild-type glycine oxidase may vary depending on the origin of the enzyme, but those skilled in the art can appropriately determine the position of the LRP motif in the amino acid sequence of wild-type glycine oxidase.
- the position of power leucine (L) can be specified.
- the LRP motif is in the amino acid region at positions 294 to 316, and leucine (L) is at positions 294 to 314 (eg, see Table 3).
- the modified enzyme of the present invention is a mutation that improves the properties of glycine oxidase related to the measurement of glycine, in addition to substitution of leucine (L) in the LRP motif, in addition to the substitution of the first threonine (T) in the TTS motif. It may further have the above substitution and / or the above substitution of cysteine (C) in the HCY motif.
- the mutation that improves the properties of glycine oxidase associated with the measurement of glycine is a substitution of methionine (M) in the GML motif of the amino acid sequence of wild-type glycine oxidase.
- the GML motif is composed of three consecutive amino acid residues of glycine (G) -methionine (M) -leucine (L).
- G glycine
- M methionine
- L leucine
- the position of the GML motif in the amino acid sequence of the wild-type glycine oxidase may vary depending on the origin of the enzyme, but those skilled in the art can substitute the GML motif in the amino acid sequence of the wild-type glycine oxidase, as appropriate.
- the position of power methionine (M) can be specified.
- the GML motif is in the amino acid region at positions 45 to 59, and methionine (M) is at positions 46 to 58 (eg, see Table 4).
- the modified enzyme of the present invention comprises a first threonine (T) in the TTS motif as well as a substitution of methionine (M) in the GML motif as a mutation that improves the properties of glycine oxidase associated with the measurement of glycine. It may further have the above substitution of serine (S) and / or the above substitution of cysteine (C) in the HCY motif and / or the above substitution of leucine (L) in the LRP motif.
- the mutation that improves the properties of glycine oxidase associated with the measurement of glycine is a substitution of serine (S) in the SG motif of the amino acid sequence of wild-type glycine oxidase.
- the SG motif is composed of two consecutive amino acid residues of serine (S) -glycine (G).
- the position of the SG motif in the amino acid sequence of the wild-type glycine oxidase may vary depending on the origin of the enzyme, but those skilled in the art can appropriately determine the position of the SG motif in the amino acid sequence of the wild-type glycine oxidase.
- the position of power serine (S) can be specified.
- the SG motif is in the amino acid region at positions 190 to 204, and serine (S) is at positions 190 to 203 (eg, see Table 5).
- the modified enzyme of the present invention comprises a first threonine (T) in the TTS motif as well as a substitution of serine (S) in the SG motif as a mutation that improves the properties of glycine oxidase associated with the measurement of glycine.
- the mutation that improves the properties of glycine oxidase associated with the measurement of glycine is a substitution of glycine (G) in the PGT motif of the amino acid sequence of wild-type glycine oxidase.
- the PGT motif is composed of three consecutive amino acid residues of proline (P) -glycine (G) -threonine (T).
- P proline
- G G
- T threonine
- the position of the PGT motif in the amino acid sequence of the wild-type glycine oxidase may vary depending on the origin of the enzyme, a person skilled in the art can appropriately determine the position of the PGT motif in the amino acid sequence of the wild-type glycine oxidase.
- the position of power glycine (G) can be specified.
- the PGT motif is in the amino acid region at positions 303 to 317, and glycine (G) is at positions 304 to 316 (see, for example, Table 6).
- the modified enzyme of the present invention comprises a first threonine (T) in the TTS motif as well as a substitution of glycine (G) in the PGT motif as a mutation that improves the properties of glycine oxidase associated with the measurement of glycine.
- the modified enzyme of the present invention can be prepared by introducing a mutation into a wild-type enzyme having one or more motifs selected from the six motifs described above.
- the wild-type enzyme may have two motifs selected from the six motifs described above, may have three motifs, may have four motifs, or may have five motifs. It may have and may have six motifs.
- glycine oxidase that are relevant to the measurement of glycine include the following: (A) the activity of glycine oxidase on glycine; (B) thermal stability of glycine oxidase; and (c) substrate specificity of glycine oxidase for glycine.
- the modified enzyme of the present invention may have only one of the above-described characteristics, but may have two or three of the above-described characteristics.
- a mutation that improves at least one characteristic selected from the characteristics (a) to (c) includes, for example, alanine Examples include substitution to (A), serine (S), cysteine (C), or glycine (G).
- serine (S) in the TTS motif as a mutation that improves at least one characteristic selected from characteristics (a) to (c) (single mutation or a combination with other mutations), for example, lysine (K) The substitution to is mentioned.
- cysteine (C) in the HCY motif as a mutation that improves at least one characteristic selected from the characteristics (a) to (c) (single mutation or a combination with other mutations), for example, alanine (A) , Aspartic acid (D), glycine (G), histidine (H), asparagine (N), tryptophan (W), tyrosine (Y), or serine (S).
- A Aspartic acid
- G glycine
- H histidine
- asparagine N
- W tryptophan
- Y tyrosine
- S serine
- a mutation that improves at least one characteristic selected from the characteristics (a) to (c) includes, for example, isoleucine (I) , Valine (V), cysteine (C), threonine (T), or proline (P).
- methionine (M) in the GML motif as a mutation that improves at least one characteristic selected from characteristics (a) to (c) (single mutation or a combination with other mutations), for example, isoleucine (I) The substitution to is mentioned.
- serine (S) in the SG motif as a mutation that improves at least one characteristic selected from characteristics (a) to (c) (single mutation or a combination with other mutations), for example, arginine (R) The substitution to is mentioned.
- a mutation that improves at least one characteristic selected from the characteristics (a) to (c) includes, for example, tyrosine (Y) Or substitution with glutamine (Q).
- the activity of glycine oxidase on glycine is improved as a property of glycine oxidase related to the measurement of glycine.
- the improvement of the activity of glycine oxidase with respect to glycine means that the activity of the modified enzyme with respect to glycine is improved as compared with that of the wild type enzyme.
- the improvement of the activity of glycine oxidase with respect to glycine is determined when the activity of wild-type glycine oxidase with respect to glycine at a predetermined concentration (eg, either low concentration or high concentration) is defined as 100.
- the degree of improvement of the activity of the modified enzyme with respect to the wild-type enzyme is preferably 1.3 times or more, more preferably 1.5 times or more, still more preferably 1.7 times or more, It is particularly preferable that the number is twice or more.
- the modification of the modified enzyme of the present invention in which the degree of improvement in the activity of the modified enzyme with respect to the wild-type enzyme is 1.3 times or more is, for example, 1)
- Substitution suitable for improving activity 1-1) Amino acid residues after substitution of the first threonine (T) in the TTS motif Alanine (A), Serine (S), Cysteine (C), or Glycine ( G) 1-2) Amino acid residue after substitution of serine (S) in TTS motif Lysine (K) 1-3) Amino acid residue after substitution of cysteine (C) in HCY motif Serine (S) 1-4) Amino acid residue after substitution of leucine (L) in the LRP motif Isoleucine (I), valine (V), cysteine (C), or threonine (T) 1-5) Regarding the glycine (G) in the PGT motif, as a mutation that improves at least one characteristic selected from the characteristics (a) to (c) (single mutation or a combination with other mutations), for example, Substitution with glutamine (Q) is mentioned.
- the thermal stability of glycine oxidase is improved as a property of glycine oxidase related to the measurement of glycine.
- Improvement of the thermal stability of glycine oxidase means that the thermal stability of the modified enzyme is further improved compared to that of the wild-type enzyme.
- the improvement of the thermal stability of glycine oxidase can be achieved by a predetermined time (eg, 1 hour) in an aqueous solution under a predetermined high temperature (eg, a temperature of 40 ° C., 50 ° C. or 60 ° C.). It can be achieved when the modified enzyme has a residual activity greater than that of the wild-type enzyme when treated.
- the thermal stability test of glycine oxidase in an aqueous solution can have significance as an accelerated test for evaluating the stability (particularly liquid stability) of glycine oxidase. Therefore, when the thermal stability of the modified enzyme in the aqueous solution is high, the stability (particularly liquid stability) of the modified enzyme tends to be high. Since an enzyme having high liquid stability can be stored in a liquid state for a long time, such a modified enzyme is useful for measuring glycine as a liquid reagent.
- the degree of improvement in the thermal stability of the modified enzyme relative to the wild-type enzyme is preferably 1.1 times or more, and more preferably 1.2 times or more.
- the modification of the modified enzyme of the present invention in which the degree of improvement of the thermal stability of the modified enzyme relative to the wild-type enzyme is 1.1 times or more is, for example, 2) Cysteine in the HCY motif suitable for improving the thermal stability Substitution with the following amino acid residues in (C) may be used.
- the substrate specificity of glycine oxidase for glycine is improved as a property of glycine oxidase related to the measurement of glycine.
- Improvement of the substrate specificity of glycine oxidase for glycine means that the reactivity of the modified enzyme for glycine is improved compared to that of the wild-type enzyme. In other words, it means that the reactivity of the modified enzyme with respect to amino acids other than glycine decreases. Examples of amino acids other than glycine include L- ⁇ -amino acids other than glycine.
- L- ⁇ -amino acids other than glycine examples include 19 L- ⁇ -amino acids other than glycine, and cystine, taurine, citrulline, ornithine and ⁇ -aminobutyric acid that constitute proteins. It is done.
- the mutation of the modified enzyme of the present invention in which the substrate specificity of glycine oxidase for glycine is improved is, for example, 3) the following amino acid of the first threonine (T) in the TTS motif, which is suitable for improving the substrate specificity: Substitution to a residue, substitution of cysteine (C) in the HCY motif to the following amino acid residue, substitution of leucine (L) in the LRP motif to the following amino acid residue, or a combination of these substitutions: Also good.
- the modified enzyme of the present invention may also have other peptide components (eg, tag portion) at the C-terminus or N-terminus.
- Other peptide components that can be added to the modified enzyme of the present invention include, for example, peptide components that facilitate purification of the target protein (eg, tag portions such as histidine tag, Strep-tag II; glutathione-S-transferase, maltose, etc. Proteins commonly used for purification of target proteins such as binding proteins), peptide components that improve the solubility of target proteins (eg, Nus-tag), peptide components that function as chaperones (eg, trigger factors), proteins with other functions Or the peptide component as a linker connecting protein domains or them may be mentioned.
- the modified enzyme of the present invention has an additional mutation (eg, substitution, deletion, insertion) of one or several amino acid residues with respect to the amino acid sequence of the glycine oxidase having the mutation as long as the above-described properties are maintained. And addition).
- the number of additional mutations is, for example, 1 to 100, preferably 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, most preferably 1 to 20 or 1 to 10 (eg 1, 2, 3, 4 or 5).
- Those skilled in the art can appropriately produce such a modified enzyme having the above-mentioned characteristics.
- the degree of improvement of the activity of the modified enzyme relative to the wild-type enzyme is preferably 1.3 times or more, more preferably 1.5 times or more. It is even more preferably 7 times or more, and particularly preferably 2 times or more.
- the degree of improvement in the thermal stability of the modified enzyme relative to the wild-type enzyme is preferably 1.1 times or more, more preferably 1.2 times or more. preferable.
- the modified enzyme of the present invention may be the following (i) or (ii): (I) in the amino acid sequence of glycine oxidase, the first threonine in the TTS motif, the serine in the TTS motif, the cysteine in the HCY motif, the leucine in the LRP motif, the methionine in the GML motif, the serine in the SG motif, And an amino acid sequence in which one or more amino acid residues selected from the group consisting of glycine in the PGT motif are mutated (eg, substituted), and the properties of glycine oxidase related to the measurement of glycine are improved.
- a protein or (ii) a first threonine in the TTS motif, a serine in the TTS motif, a cysteine in the HCY motif, a leucine in the LRP motif, a methionine in the GML motif, an SG motif in the amino acid sequence of glycine oxidase Serine and PGT motif
- the modified enzyme of the present invention has an amino acid sequence having at least 90% or more amino acid sequence identity with the amino acid sequence of the (wild-type) glycine oxidase before the mutation by having both the mutation and the additional mutation described above. It may be included.
- the percent amino acid sequence identity may preferably be 92% or more, more preferably 95% or more, even more preferably 97% or more, and most preferably 98% or more or 99% or more.
- an amino acid residue into which an additional mutation can be introduced in an amino acid sequence is obvious to those skilled in the art.
- the additional mutation can be introduced with reference to the alignment of the amino acid sequence.
- a person skilled in the art compares 1) the amino acid sequences of a plurality of homologs (eg, the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequences of other homologs), and 2) is relatively conserved. Region, and relatively unconserved region, and then 3) regions that can play an important role in function from the relatively conserved region and the relatively unconserved region, respectively Since it is possible to predict areas that cannot play an important role in function, it is possible to recognize the correlation between structure and function.
- the sites where additional mutations are introduced are the first threonine in the TTS motif, serine in the TTS motif, cysteine in the HCY motif, leucine in the LRP motif, methionine in the GML motif, serine in the SG motif, and PGT An amino acid residue other than glycine in the motif, preferably an amino acid residue other than the amino acid residues in the TTS motif, TTS motif, HCY motif, LRP motif, GML motif, SG motif, and PGT motif Good.
- substitution of an amino acid residue may be a conservative substitution.
- conservative substitution refers to substitution of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art.
- such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g.
- the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.
- the modified enzyme of the present invention can be prepared using the transformant of the present invention that expresses the modified enzyme of the present invention, or using a cell-free system or the like.
- the transformant of the present invention can be prepared, for example, by preparing the expression vector of the present invention and then introducing this expression vector into a host.
- the expression vector of the present invention includes the polynucleotide of the present invention (eg, DNA, RNA) encoding the modified enzyme of the present invention.
- the expression vector of the present invention further comprises a region such as a region encoding a promoter, terminator and drug (eg, tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin) resistance gene.
- drug eg, tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin
- the expression vector of the present invention may be a plasmid or an integrative vector.
- the expression vector of the present invention may be a viral vector or a cell-free vector.
- the expression vector of the present invention may further contain a polynucleotide encoding another peptide component that can be added to the modified enzyme of the present invention at the 3 'or 5' terminal side of the polynucleotide of the present invention.
- the polynucleotide encoding another peptide component include a polynucleotide encoding a peptide component that facilitates purification of the target protein as described above, and a peptide component that improves the solubility of the target protein as described above.
- polynucleotide examples include a polynucleotide encoding a peptide component that functions as a chaperone, and a polynucleotide encoding a protein component having another function, a domain of the protein, or a peptide component as a linker connecting them.
- Various expression vectors containing polynucleotides encoding other peptide components are available. Therefore, such an expression vector may be used for preparing the expression vector of the present invention.
- an expression vector containing a polynucleotide encoding a peptide component that facilitates purification of the target protein, a peptide component that improves the solubility of the target protein
- Expression vector containing a polynucleotide encoding a protein eg, pET-50b
- an expression vector containing a polynucleotide encoding a peptide component that acts as a chaperone eg, pCold TF
- a protein having another function or a domain of a protein eg. pCold TF
- the expression vector of the present invention encodes the region encoding the cleavage site by the protease into the region encoding the modified enzyme of the present invention. Between the polynucleotide to be encoded and the polynucleotide encoding the other peptide component.
- Examples of the host for expressing the modified enzyme of the present invention include Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus genus.
- Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (eg, Corynebacterium glutamicum), and Bacillus genus.
- Various prokaryotic cells including bacteria (eg, Bacillus subtilis), bacteria belonging to the genus Saccharomyces (eg, Saccharomyces cerevisiae), bacteria belonging to the genus Pichia (eg, Pichia stipitis) , Including bacteria of the genus Aspergillus (eg, Aspergillus oryzae) Can be used s eukaryotic cells.
- a strain lacking a predetermined gene may be used.
- the transformant include a transformant having an expression vector in the cytoplasm, and
- the transformant of the present invention can be cultured in a medium having the composition described below, for example, using a predetermined culture apparatus (eg, test tube, flask, jar fermenter).
- the culture conditions can be set as appropriate.
- the culture temperature may be 10 ° C. to 37 ° C.
- the pH may be 6.5 to 7.5
- the culture time may be 1 h to 100 h.
- the dissolved oxygen concentration (DO value) in the culture solution may be used as a control index. Control the aeration / stirring conditions so that the relative dissolved oxygen concentration DO value when the oxygen concentration in the atmosphere is 21% does not fall below 1-10%, for example, preferably 3-8%.
- the culture may be batch culture or fed-batch culture.
- a solution serving as a sugar source or a solution containing phosphoric acid can be added continuously or discontinuously to the culture solution to continue the culture.
- the host to be transformed is as described above, but in detail about E. coli, it can be selected from Escherichia coli JM109 strain, DH5 ⁇ strain, HB101 strain, BL21 (DE3) strain, etc. Methods for performing transformation and methods for selecting transformants are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like. Hereinafter, a method for producing transformed E. coli and producing a predetermined enzyme using the same will be described more specifically as an example.
- E. promoters used for heterologous protein production in E. coli can be used, such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, T5 promoter, etc.
- a strong promoter is mentioned, and PhoA, PhoC, and lac are preferable.
- the vector examples include pUC (eg, pUC19, pUC18), pSTV, pBR (eg, pBR322), pHSG (eg, pHSG299, pHSG298, pHSG399, pHSG398), RSF (eg, RSF1010), pACYC (eg, pACYC177, pACYC184), pMW (eg, pMW119, pMW118, pMW219, pMW218), pQE (eg, pQE30), and derivatives thereof may be used.
- phage DNA vectors may be used.
- an expression vector containing a promoter and capable of expressing the inserted DNA sequence may be used.
- the vector may be pUC, pSTV, pMW.
- a terminator that is a transcription termination sequence may be linked downstream of the polynucleotide of the present invention.
- examples of such terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and E. coli trpA gene terminator.
- a so-called multicopy type is preferable, and a plasmid having a replication origin derived from ColE1, such as a pUC-type plasmid, a pBR322-type plasmid, or a derivative thereof can be mentioned. It is done.
- the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion and / or addition.
- the vector has a marker such as an ampicillin resistance gene in order to select transformants.
- a marker such as an ampicillin resistance gene
- an expression vector having a strong promoter is commercially available [eg, pUC system (manufactured by Takara Bio Inc.), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)].
- the modified enzyme of the present invention can be obtained by transforming E. coli with the obtained expression vector of the present invention and culturing the E. coli.
- the medium a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used.
- the culture medium may contain a predetermined carbon source, nitrogen source, and coenzyme (eg, pyridoxine hydrochloride).
- coenzyme eg, pyridoxine hydrochloride
- peptone, yeast extract, NaCl, glucose, MgSO 4 , ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like may be used.
- Culture conditions and production induction conditions are appropriately selected according to the type of the vector marker, promoter, host fungus and the like used.
- the modified enzyme of the present invention is disrupted (eg, sonication, homogenization) or lysed (eg, lysozyme treatment) to obtain a crushed product and a lysate.
- the modified enzyme of the present invention can be obtained by subjecting such crushed material and lysate to techniques such as extraction, precipitation, filtration, and column chromatography.
- the present invention provides a method for analyzing glycine.
- the analysis method of the present invention can include measuring glycine contained in a test sample using the modified enzyme of the present invention.
- the test sample is not particularly limited as long as it is a sample suspected of containing glycine.
- biological samples eg, blood, urine, saliva, tears, etc.
- food and beverage products eg, nutrition drinks and amino acid drinks
- Etc. The glycine in the test sample may be at a low concentration (eg, a concentration of less than 1 mM such as 1 ⁇ M or more and less than 1 mM) or a high concentration (eg, a concentration of 1 mM or more such as 1 mM or more but less than 1 M).
- the analysis method of the present invention is not particularly limited as long as glycine can be measured using the modified enzyme of the present invention.
- the generated glyoxylic acid may be detected, and NH 3 produced as a by-product with the production of glyoxylic acid or H 2 O 2 may be detected.
- the product of the conjugation reaction may be detected by conjugation with another reaction. Examples of such a conjugation reaction include the following conjugation reactions.
- glycine can be measured using 4-aminoantipyrine, phenol, and peroxidase in addition to the modified enzyme of the present invention. Specifically, a test sample is mixed with 4-aminoantipyrine (4-aminoantipyrine) and phenol and peroxidase in an aqueous solution (eg, a buffer solution), and then the mixed sample is subjected to the enzyme reaction described above. In addition, glycine is measured by detecting the absorbance (about 500 nm) of the produced quinoneimine dye. Measurements can be made qualitatively or quantitatively.
- the measurement may be performed, for example, based on an endpoint method in which measurement is performed until all the substrates have reacted, or may be performed based on a rate method (initial velocity method).
- a rate method initial velocity method
- the modified enzyme of the present invention does not react with amino acids other than glycine (eg, L- ⁇ -amino acid) or has very low reactivity with it. Therefore, even when not only glycine but also other amino acids are contained in the test sample, the amount of glycine in the test sample can be specifically evaluated by using the modified enzyme of the present invention. .
- amino acids other than glycine eg, L- ⁇ -amino acid
- the amount of glycine in the test sample can be specifically evaluated.
- the present invention includes (A) a glycine analysis kit containing the modified enzyme of the present invention.
- the kit of the present invention may further comprise at least one of (B) a reaction buffer or buffer salt, (C) a hydrogen peroxide detection reagent, (D) an ammonia detection reagent, and (E) a glyoxylic acid detection reagent. .
- reaction buffer or buffer salt is used to maintain the pH of the reaction solution at a value suitable for the target enzyme reaction.
- the hydrogen peroxide detection reagent is used when hydrogen peroxide is detected by, for example, color development or fluorescence.
- a combination of a peroxidase and a color former that can serve as a substrate thereof can be mentioned, and specific examples include, for example, a combination of horseradish peroxidase, 4-aminoantipyrine and phenol, but are not limited to this combination.
- an ammonia detection reagent for example, an indophenol method combining phenol and hypochlorous acid can be used.
- Examples of the glyoxylic acid detection reagent include a combination of reagents described in Non-Patent Document 6.
- the present invention also provides a detection system for glycine analysis comprising (a) a device and (b) the modified enzyme of the present invention.
- the modified enzyme of the present invention may exist as a unit independent of the microdevice that can be supplied into the device at the time of use, but may be previously injected, fixed, or arranged in the device.
- the modified enzyme of the present invention is provided in a form pre-injected, fixed or arranged in the device. Immobilization or arrangement of the modified enzyme of the present invention on the device is performed directly or indirectly.
- a microdevice such as a microchannel chip having a channel can be suitably used.
- the detection system for glycine analysis of the present invention may further include (c) at least one component of a reaction buffer or buffer salt, a hydrogen peroxide detection reagent, an ammonia detection reagent, and a glyoxylic acid detection reagent.
- all the components of (c) may be provided in a form accommodated in the device.
- a part of the component of (c) may be provided in a form accommodated in the device, and the rest may be provided in a form not accommodated in the device (eg, a form accommodated in a different container).
- the component (c) that is not accommodated in the device may be used by being injected into the device during measurement of the target substance.
- Examples of the device include 1) a first zone for preparing a mixed solution by mixing the sample and the component of (c), and contacting the prepared mixed solution with the modified enzyme of the present invention, A device comprising a second zone for detecting glycine (a device in which the mixing and detection steps are carried out in different zones); 2) mixing the sample, the component of (c) and the modified enzyme of the invention , A device comprising an area for detecting glycine by the modified enzyme of the present invention (a device in which the mixing and detection steps are carried out in the same area); and 3) a sample and a component of (c) (and optionally A device having a flow path that allows mixing with the modified enzyme of the present invention and a zone for detecting glycine by the modified enzyme of the present invention (when a sample is injected into the inlet of the device, Sending Is sample and the like are mixed automatically to, glycine resulting mixture in the device) and the like is automatically detected in the detection zone.
- the device 3 particularly the device 3) in the form of a microchannel device is preferable.
- the modified enzyme of the present invention may be provided in the liquid flowing through the flow path, or may be provided in a form fixed or arranged in the detection zone. Provided in a deployed form.
- the present invention also provides an enzyme sensor for glycine analysis comprising (a) a detection electrode, and (b) a modified enzyme of the present invention fixed or arranged on the detection electrode.
- the modified enzyme of the present invention is fixed or disposed directly or indirectly on the electrode.
- a hydrogen peroxide detection electrode for example, a hydrogen peroxide detection electrode can be used, and more specifically, an enzyme-type hydrogen peroxide detection electrode, a diaphragm-type hydrogen peroxide detection electrode, and the like.
- glycine can be analyzed by detecting hydrogen peroxide generated when glycine is oxidized by glycine oxidation activity.
- Other configurations can be used as they are, or can be appropriately modified from those used in known sensors.
- Example 1 Synthesis of GlyOX and purification of GlyOX using a cell-free synthesis system Using a wild-type gene of GlyOX derived from Bacillus subtilis 168 strain or a desired mutant type gene as a template, 2-Step PCR method, A linear DNA of a construct in which a histidine tag and a TEV protease recognition sequence were fused to the N-terminal side and a target mutation was introduced was prepared. Using this DNA as a template, proteins were synthesized in a cell-free synthesis reaction system derived from E. coli.
- the product synthesized for 6 hours by dialysis on a 1 mL reaction scale was centrifuged, and the supernatant fraction was eluted with histidine tag affinity purification using Ni Sepharose High Performance (GE Healthcare Japan, Inc.). A fraction was obtained. Subsequently, the presence of the protein considered to be the target enzyme was confirmed by staining with SDS-PAGE and SYPRO ORANGE Protein Gel Stain (Life Technologies Japan Co., Ltd.), and the protein was quantified by the Bradford method, followed by evaluation. .
- the wild-type enzyme was also subjected to the same procedure for the preparation of linear DNA, cell-free synthesis, enzyme purification and analysis. The following buffer solution was used for purification.
- Binding buffer 750 mM NaCl, 20 mM NaPi, pH 8.0
- Wash buffer 750 mM NaCl, 20 mM NaPi, pH 8.0
- Collection / measurement buffer 300 mM NaCl, 50 mM NaPi, 34 mM EDTA, pH 7.0, 10% D 2 O, 0.01% NaN 3
- mutation is divided
- T42A / C245S means a mutant GlyOX having two mutations
- WT means wild type.
- Example 2 Activity measurement The activity evaluation of the wild-type GlyOX and the mutant GlyOX synthesized in Example 1 was performed according to the following procedure. First, the following reaction solution A and reaction solution B were prepared. Reaction solution A: 4 mM phenol, 100 mM potassium phosphate, pH 8.0 Reaction solution B: 50 mM 4-aminoantipyrine, 500 U / ml peroxidase, and then using a 96-well microplate at 25 ° C., reaction solution A 49 ⁇ l, reaction solution B 1 ⁇ l, 2.5 mM Gly or 100 mM Gly 10 ⁇ l The change after 3 minutes of the absorbance at a wavelength of 500 nm of a liquid obtained by mixing 20 ⁇ l of ultrapure water and 20 ⁇ l of 0.5 mg / ml GlyOX was measured using a microplate reader (SpectraMax M2e, Molecular Device Japan Co., Ltd.).
- Tables 7 and 8 show the activity of each mutant GlyOX as a ratio to the activity of wild-type GlyOX. Each value was calculated from an average value when three experiments were performed on the same sample. Table 7 shows the results for proteins synthesized using a wild-type gene as a template when preparing linear DNA, and Table 8 shows a protein synthesized using a target mutant-type gene as a template.
- Example 3 Evaluation of stability The stability of the wild-type enzyme and the mutant enzyme synthesized in Example 1 was evaluated according to the following procedure. 0.5 mg / ml GlyOX was dispensed into a microtube and incubated at 4 ° C, 40 ° C, 50 ° C, 60 ° C for 1 hour. Thereafter, the activity of these enzyme solutions was measured from the change in absorbance after 10 minutes of reaction under the condition that the substrate solution added in Example 2 was 100 mM Gly, and remained as a ratio to the activity of the enzyme solution incubated at 4 ° C. The stability was evaluated by determining the activity. The results are shown in Tables 9 and 10.
- Table 9 shows the results relating to proteins synthesized using a wild-type gene as a template when preparing linear DNA, and Table 10 using a target mutant-type gene as a template.
- Example 4 GlyOX expression system using E. coli and purification of GlyOX
- a recombinant expression system of GlyOX using E. coli was constructed.
- a plasmid for recombinant expression was constructed.
- the target gene was amplified by a standard PCR method using DNA primer 1 (TAATTCCCATGGCTAAAAGGCATTTGAAGCAGTGGGTGATTG: SEQ ID NO: 3) and DNA primer 2 (TAATACTCGAGTATCTGACCCGCTCCTCTGGATC: SEQ ID NO: 4).
- coli DH5 ⁇ was obtained using the ligation product by a standard method.
- a plasmid was extracted from the obtained transformant of DH5 ⁇ , and insertion of the gene of interest into the plasmid was confirmed using a standard DNA sequence analysis method.
- the plasmid in which the target gene is inserted is called pET28a-GlyOX
- the transformant of BL21 (DE3) by pET28a-GlyOX is called pET28a-GlyOX-BL21 (DE3).
- GlyOX was prepared as follows. First, an LB plate containing 25 ⁇ g / ml kanamycin was inoculated from a glycerol stock of pET28a-GlyOX-BL21 (DE3), and statically cultured at 37 ° C. overnight. 50 ml of LB medium containing 25 ⁇ g / ml kanamycin was placed in a 250 ml baffled flask, inoculated from a single colony on an LB plate, and cultured overnight at 37 ° C. with swirling shaking.
- the disrupted solution was centrifuged at 12000 ⁇ g for 30 minutes to recover the supernatant, and then Ni Sepharose 6 Fast equilibrated with Wash buffer (50 mM HEPES, 500 mM NaCl, 20 mM imidazole, 0.02 ⁇ M Flavin adenine dinucleotide, pH 7.5). After adding to Flow (GE Healthcare Japan Co., Ltd.) and gently mixing by inversion for 5 minutes at room temperature, the solution was removed by natural falling using an Econopack column (Bio-Rad Laboratories Co., Ltd.).
- the target protein GlyOX was eluted with an elution buffer (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, 0.02 ⁇ M Flavin adenine dinucleotide, pH 7.5).
- the GlyOX solution was prepared by substituting the solvent into a stock buffer (50 mM potassium phosphate, 0.02 ⁇ M Flavin adenine dinucleotide, pH 8.0) by ultrafiltration to a concentration of 0.5 mg / ml.
- T42A / C245S / L301V mutant using E. coli T42A / C245S / L301V mutant was prepared as follows. Using QuikChange Lightening Site-Directed Mutagenesis Kits (Agilent Technology Co., Ltd.), mutation was introduced into the GlyOX gene using pET28a-GlyOX as a template according to the protocol attached to the product. Multiple mutations were introduced by adding mutations using a plasmid containing the mutation as a template. Using this mutated plasmid, recombinant expression, purification, and solvent replacement were performed according to the method described in Example 4 to obtain a T42A / C245S / L301V mutant.
- Example 6 Preparation of mutant using Escherichia coli
- the target mutant was prepared as follows. Using QuikChange Lightening Site-Directed Mutagenesis Kits (Agilent Technology Co., Ltd.), mutation was introduced into the GlyOX gene using pET28a-GlyOX as a template according to the protocol attached to the product. Multiple mutations were introduced by adding mutations using a plasmid containing the mutation as a template. Using this mutated plasmid, recombinant expression, purification, and solvent replacement were performed according to the method described below to obtain the target mutant.
- GlyOX was prepared as follows. First, an LB plate containing 25 ⁇ g / ml kanamycin was inoculated from a glycerol stock of pET28a-GlyOX-BL21 (DE3), and statically cultured at 37 ° C. overnight. 50 ml of LB medium containing 25 ⁇ g / ml kanamycin was placed in a 250 ml baffled flask, inoculated from a single colony on an LB plate, and cultured overnight at 37 ° C. with swirling shaking.
- the disrupted solution was centrifuged at 12000 ⁇ g for 30 minutes to collect the supernatant, and then the HisTrap FF crude (50 mM HEPES, 500 mM NaCl, 20 mM imidazole, 0.02 ⁇ M Flavin adenine dinucleotide, pH 7.5) equilibrated with Wash Buffer ( GE Healthcare Japan Ltd.). Subsequently, after washing with a Wash buffer, the target protein GlyOX was eluted with an elution buffer (50 mM HEPES, 500 mM NaCl, 500 mM imidazole, 0.02 ⁇ M Flavin adenine dinucleotide, pH 7.5).
- the GlyOX solution was prepared by substituting the solvent into a stock buffer (50 mM potassium phosphate, 0.02 ⁇ M Flavin adenine dinucleotide, pH 8.0) by ultrafiltration to a concentration of 0.5 mg / ml.
- a stock buffer 50 mM potassium phosphate, 0.02 ⁇ M Flavin adenine dinucleotide, pH 8.0
- Example 7 Activity measurement Activity was evaluated for the wild type and the mutant GlyOX synthesized in Example 6. Specifically, among the compositions shown in Example 2, the enzyme solution was GlyOX prepared in Example 6. The results are shown in Table 11.
- Example 8 Evaluation of stability The stability of the wild type and the mutant GlyOX synthesized in Example 6 was evaluated. Specifically, among the compositions shown in Example 3, the enzyme solution was GlyOX prepared in Example 6. The results are shown in Table 12.
- Example 9 Preparation of T42A / C245S / L301V / G304Q mutant using E. coli T42A / C245S / L301V / G304Q was prepared in the same manner as in Example 5.
- Example 10 Evaluation of substrate specificity Substrate specificity was confirmed for the wild type, the T42A / C245S / L301V mutant, and the T42A / C245S / L301V / G304Q mutant.
- the enzyme solution is the GlyOX solution prepared in Example 4 or Example 5 or Example 9, and (1) 1 mM Gly, (2 ) One of 20 standard amino acids, a mixture containing 1 mM of cystine, taurine, citrulline, ornithine and ⁇ -aminobutyric acid, respectively, and (3) (2) 25 mixtures obtained by removing Gly The activity was evaluated.
- Tyr and cystine were dissolved in 2M HCl, and other amino acids were dissolved in 0.1M HCl to prepare a 100 mM solution. After mixing each amino acid solution, an amino acid solution was prepared by diluting 100 times with ultrapure water. . At this time, in (1) and (3), 2M HCl or / and 0.1M HCl was mixed instead of the amino acid solution not mixed. The activity was determined by detecting the change in absorbance ( ⁇ Abs 500 ) at a wavelength of 500 nm at 37 ° C. with a microplate reader. The results for the wild type are shown in FIG. 1, the results for the T42A / C245S / L301V mutant are shown in FIG.
- the results for the T42A / C245S / L301V / G304Q mutant are shown in FIG.
- the substrates were (1), (2), and (3), they were expressed as Gly, 25mix, and 25mix-Gly, respectively.
- No catalytic activity was observed for amino acids other than Gly, wild type, T42A / C245S / L301V mutant, and T42A / C245S / L301V / G304Q mutant.
- the wild type and T42A / C245S / L301V / G304Q mutants showed inhibition of the reaction by other amino acids, and ⁇ Abs 500 10 minutes after the start of the wild type reaction was Gly.
- the ⁇ Abs500 10 minutes after the start of the reaction of the T42A / C245S / L301V / G304Q mutant type was 52% when only Gly was used as the substrate, but the T42A / C245S / L301V mutation was 85%. As for the type, the reaction was not inhibited and the properties were improved.
- reaction solution C (4 mM phenol, 100 mM HEPES, pH 8.0) was prepared, and using a 96-well microplate at 37 ° C., reaction solution C was 49 ⁇ l, 50 mM 4-aminoantipyrine 1 ⁇ l, 500 U / ml peroxidase.
- Example 12 Quantitative analysis of plasma Gly Gly in human plasma was quantitatively analyzed using T42A / C245S / L301V mutant or T42A / C245S / L301V / G304Q mutant. Reaction and measurement were performed at 37 ° C. using a cuvette.
- the calibration curve was prepared using the average value when three experiments were performed at each Gly concentration, and the human plasma sample of the same lot was analyzed six times and compared with the analysis value (332 ⁇ M) in the amino acid analyzer.
- the above is method 1, and the results are shown in Table 13 or 14.
- Table 13 or 14 also shows the results of obtaining the Gly concentration by the method of obtaining the concentration from the ratio with the absorbance value based on the target component (Method 2). Whichever method was used, both accuracy and precision were good, and it was shown that the Gly concentration in human plasma can be quantitatively analyzed with high accuracy by using a Gly measurement system using mutant GlyOX.
- the modified enzyme of the present invention is useful for rapid and sensitive measurement of glycine and / or production of glycine oxylate.
- the modified enzyme of the present invention is also useful as a liquid reagent.
- the modified enzyme of the present invention is further useful for specific measurement of glycine.
- the analysis method of the present invention is useful in a wide range of fields such as biological research, health nutrition, medical care, and food production.
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Abstract
Description
〔1〕以下:
(a)グリシンに対するグリシン酸化酵素の活性;
(b)グリシン酸化酵素の熱安定性;および
(c)グリシンに対するグリシン酸化酵素の基質特異性;
からなる群より選ばれる、グリシンの測定に関連するグリシン酸化酵素の1以上の特性を改善するようにその少なくとも1つのアミノ酸残基を変異させた、改変酵素。
〔2〕変異が、グリシン酸化酵素のアミノ酸配列における(a)TTSモチーフ中の第1のスレオニンの置換、(b)TTSモチーフ中のセリンの置換、(c)HCYモチーフ中のシステインの置換、(d)LRPモチーフ中のロイシンの置換、(e)GMLモチーフ中のメチオニンの置換、(f)SGモチーフ中のセリンの置換、および(g)PGTモチーフ中のグリシンの置換からなる群より選ばれる1以上の置換である、〔1〕の改変酵素。
〔3〕TTSモチーフ中の第1のスレオニンが、アラニン、セリン、システイン、またはグリシンに置換されている、〔2〕の改変酵素。
〔4〕TTSモチーフ中のセリンが、リジンに置換されている、〔2〕または〔3〕の改変酵素。
〔5〕HCYモチーフ中のシステインが、アラニン、アスパラギン酸、グリシン、ヒスチジン、アスパラギン、トリプトファン、チロシン、またはセリンに置換されている、〔2〕~〔4〕のいずれかの改変酵素。
〔6〕LRPモチーフ中のロイシンが、イソロイシン、バリン、システイン、スレオニン、またはプロリンに置換されている、〔2〕~〔5〕のいずれかの改変酵素。
〔7〕GMLモチーフ中のメチオニンが、イソロイシンに置換されている、〔2〕~〔6〕のいずれかの改変酵素。
〔8〕SGモチーフ中のセリンが、アルギニンに置換されている、〔2〕~〔7〕のいずれかの改変酵素。
〔9〕PGTモチーフ中のグリシンが、チロシン、またはグルタミンに置換されている、〔2〕~〔8〕のいずれかの改変酵素。
〔10〕グリシン酸化酵素がバチルス属に由来する、〔1〕~〔9〕のいずれかの改変酵素。
〔11〕以下(1)あるいは(2)のタンパク質である、〔1〕~〔10〕のいずれかの改変酵素:
(1)配列番号2のアミノ酸配列において、TTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が、下記(i)~(vii)に示されるアミノ酸残基に置換されたアミノ酸配列を含む、タンパク質:
(i)TTSモチーフ中の第1のスレオニンの置換
アラニン、セリン、システイン、またはグリシン;
(ii)TTSモチーフ中のセリンの置換
リジン;
(iii)HCYモチーフ中のシステインの置換
アラニン、アスパラギン酸、グリシン、ヒスチジン、アスパラギン、トリプトファン、チロシン、またはセリン;
(iv)LRPモチーフ中のロイシンの置換
イソロイシン、バリン、システイン、スレオニン、またはプロリン
(v)GMLモチーフ中のメチオニンの置換
イソロイシン;
(vi)SGモチーフ中のセリンの置換
アルギニン;
(vii)PGTモチーフ中のグリシンの置換
チロシン、またはグルタミン。
(2)配列番号2のアミノ酸配列におけるTTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が、上記(i)~(vii)に示されるアミノ酸残基に置換されたアミノ酸配列において、1または数個のアミノ酸残基の追加変異を有するアミノ酸配列を含み、かつ、下記(a)~(c)からなる群より選ばれる1以上の特性が改善されている、タンパク質:
(a)グリシンに対するグリシン酸化酵素の活性;
(b)グリシン酸化酵素の熱安定性;および
(c)グリシンに対するグリシン酸化酵素の基質特異性。
〔12〕〔1〕~〔11〕のいずれかの改変酵素を用いて被検試料中に含まれるグリシンを測定することを含む、グリシンの分析方法。
〔13〕グリシンの測定が、〔1〕~〔11〕のいずれかの改変酵素に加えて、4-アミノアンチピリンおよびフェノール、ならびにペルオキシダーゼを用いて行われる、〔12〕の方法。
〔14〕〔1〕~〔11〕のいずれかの改変酵素を用いてグリシンからグリオキシル酸を生成することを含む、グリオキシル酸の製造方法。
〔15〕〔1〕~〔11〕のいずれかの改変酵素をコードするポリヌクレオチド。
〔16〕〔15〕のポリヌクレオチドを含む発現ベクター。
〔17〕〔16〕の発現ベクターを含む形質転換体。
〔18〕グリシンの測定に関連するグリシン酸化酵素の特性を改善するようにその少なくとも1つのアミノ酸残基を変異させた改変酵素を、〔17〕の形質転換体を用いて生成することを含む、改変酵素の製造方法。
〔19〕〔1〕~〔11〕のいずれかの改変酵素を含む、グリシン分析用キット。
〔20〕反応用緩衝液または緩衝塩、過酸化水素検出試薬、アンモニア検出試薬およびグリオキシル酸検出試薬の少なくとも一つをさらに含む、〔19〕のグリシン分析用キット。
〔21〕(a)デバイス、および(b)〔1〕~〔11〕のいずれかの改変酵素を含む、グリシン分析用検出系。
〔22〕(c)反応用緩衝液または緩衝塩、過酸化水素検出試薬、アンモニア検出試薬およびグリオキシル酸検出試薬の少なくとも一つをさらに含み、かつデバイスがマイクロ流路チップである、〔21〕のグリシン分析用検出系。
〔21〕(a)検出用電極、および(b)検出用電極に固定または配置された、〔1〕~〔11〕のいずれかの改変酵素を含む、グリシン分析用酵素センサー。
グリシン+H2O+O2 → グリオキシル酸+NH3+H2O2
(a)グリシンに対するグリシン酸化酵素の活性;
(b)グリシン酸化酵素の熱安定性;および
(c)グリシンに対するグリシン酸化酵素の基質特異性。
本発明の改変酵素は、上述した特性のうち1つのみを有していてもよいが、上述した特性のうち2つまたは3つの特性を併有していてもよい。
1-1)TTSモチーフ中の第1のスレオニン(T)の置換後のアミノ酸残基
アラニン(A)、セリン(S)、システイン(C)、またはグリシン(G)
1-2)TTSモチーフ中のセリン(S)の置換後のアミノ酸残基
リジン(K)
1-3)HCYモチーフ中のシステイン(C)の置換後のアミノ酸残基
セリン(S)
1-4)LRPモチーフ中のロイシン(L)の置換後のアミノ酸残基
イソロイシン(I)、バリン(V)、システイン(C)、またはスレオニン(T)
1-5)PGTモチーフ中のグリシン(G)について、特性(a)~(c)から選ばれる少なくとも1つの特性を改善する変異(単独の変異または他の変異との組合せ)としては、例えば、グルタミン(Q)への置換が挙げられる。
2-1)TTSモチーフ中の第1のスレオニン(T)の置換後のアミノ酸残基
アラニン(A)、セリン(S)、グリシン(G)
2-2)TTSモチーフ中のセリン(S)の置換後のアミノ酸残基
リジン(K)
2-3)HCYモチーフ中のシステイン(C)の置換後のアミノ酸残基
アラニン(A)、アスパラギン酸(D)、グリシン(G)、ヒスチジン(H)、アスパラギン(N)、トリプトファン(W)、チロシン(Y)、またはセリン(S)
2-4)LRPモチーフ中のロイシン(L)の置換後のアミノ酸残基
イソロイシン(I)、バリン(V)、プロリン(P)、またはシステイン(C)
2-5)GMLモチーフ中のメチオニン(M)について、特性(a)~(c)から選ばれる少なくとも1つの特性を改善する変異(単独の変異または他の変異との組合せ)としては、例えば、イソロイシン(I)への置換が挙げられる。
2-6)SGモチーフ中のセリン(S)について、特性(a)~(c)から選ばれる少なくとも1つの特性を改善する変異(単独の変異または他の変異との組合せ)としては、例えば、アルギニン(R)への置換が挙げられる。
2-7)PGTモチーフ中のグリシン(G)について、特性(a)~(c)から選ばれる少なくとも1つの特性を改善する変異(単独の変異または他の変異との組合せ)としては、例えば、チロシン(Y)、またはグルタミン(Q)への置換が挙げられる。
3-1)TTSモチーフ中の第1のスレオニン(T)の置換後のアミノ酸残基
アラニン(A)
3-2)HCYモチーフ中のシステイン(C)の置換後のアミノ酸残基
セリン(S)
3-3)LRPモチーフ中のロイシン(L)の置換後のアミノ酸残基
バリン(V)
(i)グリシン酸化酵素のアミノ酸配列において、TTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が変異(例、置換)したアミノ酸配列を含み、かつ、グリシンの測定に関連するグリシン酸化酵素の特性が改善されている、タンパク質;または
(ii)グリシン酸化酵素のアミノ酸配列におけるTTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が変異(例、置換)したアミノ酸配列において、1または数個のアミノ酸残基の追加変異を有するアミノ酸配列を有し、かつ、グリシンの測定に関連するグリシン酸化酵素の改善された特性が保持されている、タンパク質。
グリシン+H2O+O2 → グリオキシル酸+NH3+H2O2
共役反応)ペルオキシダーゼにより触媒される反応
2H2O2+4-アミノアンチピリン+フェノール → キノンイミン色素+4H2O
本発明のキットは、(B)反応用緩衝液または緩衝塩、(C)過酸化水素検出試薬、(D)アンモニア検出試薬および(E)グリオキシル酸検出試薬の少なくとも一つをさらに含むことができる。
Bacillus subtilis 168株由来GlyOXの野生型の遺伝子または目的とする変異型の遺伝子を鋳型に用いて、2-StepPCR法で、ヒスチジンタグおよびTEVプロテアーゼ認識配列をN末端側に融合させ、かつ目的の変異が導入されたコンストラクトの直鎖状DNAを調製した。このDNAを鋳型として用いて、大腸菌由来のセルフリー合成反応系でタンパク質を合成した。1mLの反応スケールにて、透析法で6時間合成を行った産物を遠心し、その上清画分について、Ni Sepharose High Performance(GEヘルスケア・ジャパン株式会社)を用いたヒスチジンタグアフィニティ精製の溶出画分を得た。続いて、SDS-PAGEおよびSYPRO ORANGE Protein Gel Stain(ライフテクノロジーズジャパン株式会社)を用いた染色により目的酵素と考えられるタンパク質の存在を確認し、Bradford法によるタンパク質定量を行った後、評価に供した。なお、野生型酵素についても同様の手順で、直鎖状DNAの調製、セルフリー合成、酵素の精製および分析を行った。精製には、下記の緩衝液を用いた。
結合バッファ:750mM NaCl、20mM NaPi、pH 8.0
洗浄バッファ:750mM NaCl,20mM NaPi、pH 8.0
回収・測定バッファ:300mM NaCl、50mM NaPi、34mM EDTA、pH 7.0、10% D2O、0.01% NaN3
なお、変異を複数導入した変異型GlyOXを示す場合、導入した変異を/で区切り、続けて記載する。例えば、T42A/C245Sは、T42AとC245Sの2つの変異を有する変異型GlyOXであることを意味する。WTは野生型であることを意味する。
実施例1で合成された野生型GlyOXおよび変異型GlyOXの活性評価は、以下の手順にて行った。まず、下記の反応液Aおよび反応液Bを調製した。
反応液A:4mM フェノール、100mM リン酸カリウム、pH8.0
反応液B:50mM 4-アミノアンチピリン、500U/ml ペルオキシダーゼ
続いて、96穴マイクロプレートを用い、25℃にて、反応液Aを49μl、反応液Bを1μl、2.5mM Glyまたは100mM Glyを10μl、超純水を20μl、0.5mg/ml GlyOXを20μl混合した液の、波長500nmにおける吸光度の3分後の変化をマイクロプレートリーダー(SpectraMax M2e、モレキュラーデバイス ジャパン株式会社)を用いて測定した。表7および8に、各変異型GlyOXの活性を、野生型GlyOXの活性に対する比で示した。各値は、同一サンプルについて3回実験を行った際の平均値より算出した。なお、表7は直鎖状DNAを調製する際に野生型の遺伝子を鋳型として用いて、表8は目的とする変異型の遺伝子を鋳型として用いて合成したタンパク質に関する結果である。
実施例1で合成された野生型酵素および変異型酵素の安定性評価は、下記手順にて行った。0.5mg/ml GlyOXをマイクロチューブへ分注し、4℃、40℃、50℃、60℃にて1時間インキュベーションした。その後、これら酵素液について、実施例2にて添加する基質溶液を100mM Glyとした条件で、反応10分後の吸光度変化から活性を測定し、4℃でインキュベーションした酵素液の活性に対する比として残存活性を求めることで、安定性を評価した。その結果を表9および10に示す。各値は、表9のL301を変異したGlyOXについては同一サンプルを用いた2回の実験から、それ以外のGlyOXについては3回の実験から求めた平均値より算出した。なお、表9は直鎖状DNAを調製する際に野生型の遺伝子を鋳型として用いて、表10は目的とする変異型の遺伝子を鋳型として用いて合成したタンパク質に関する結果である。
大腸菌を用いたGlyOXの組換え発現系を構築した。まず、組換え発現用のプラスミドを構築した。DNAプライマー1(TAATTCCATGGCTAAAAGGCATTATGAAGCAGTGGTGATTG:配列番号3)およびDNAプライマー2(TAATACTCGAGTATCTGAACCGCCTCCTTGCGATC:配列番号4)を用いて、標準的なPCR法により目的遺伝子を増幅した。続いて、NcoI(タカラバイオ株式会社)とXhoI(タカラバイオ株式会社)を用いて、PCR産物とpET-28a(メルク株式会社)を制限酵素消化し、プラスミドの消化物はさらにAlkaline Phosphatase(E.Coli C75)(タカラバイオ株式会社)で脱リン酸処理した後、QIAquick PCR Purification Kit(株式会社キアゲン)を用いて除タンパク及び不要な消化断片の除去を行った。得られた両産物を、Ligation High Ver.2(東洋紡績株式会社)を用いてライゲーションし、標準的な方法でライゲーション産物による大腸菌DH5αの形質転換体を取得した。得られたDH5αの形質転換体からプラスミドを抽出し、標準的なDNA配列の解析法を用いて、プラスミドへの目的遺伝子の挿入を確認した。この目的遺伝子が挿入されたプラスミドを、以後、pET28a-GlyOX、pET28a-GlyOXによるBL21(DE3)の形質転換体をpET28a-GlyOX-BL21(DE3)と呼ぶ。
T42A/C245S/L301V変異型を、下記のようにして調製した。QuikChange Lightning Site-Directed Mutagenesis Kits(アジレント・テクノロジー株式会社)を用いて、pET28a-GlyOXを鋳型として製品添付のプロトコルに従い、GlyOX遺伝子への変異導入を行った。変異の複数導入については、変異を入れたプラスミドを鋳型として用い変異を追加していくことで行った。この変異導入済みプラスミドを用いて、実施例4に記載の方法に従い組換え発現、精製および溶媒置換を行うことで、T42A/C245S/L301V変異型を取得した。
目的とする変異型を、下記のようにして調製した。QuikChange Lightning Site-Directed Mutagenesis Kits(アジレント・テクノロジー株式会社)を用いて、pET28a-GlyOXを鋳型として製品添付のプロトコルに従い、GlyOX遺伝子への変異導入を行った。変異の複数導入については、変異を入れたプラスミドを鋳型として用い変異を追加していくことで行った。この変異導入済みプラスミドを用いて、以下に記載の方法に従い組換え発現、精製および溶媒置換を行うことで、目的とする変異型を取得した。
野生型と実施例6で合成された変異型GlyOXについて、活性を評価した。具体的には、実施例2に示す組成のうち、酵素液を実施例6で調製したGlyOXとした。その結果を表11に示す。
野生型と実施例6で合成された変異型GlyOXについて、安定性を評価した。具体的には、実施例3に示す組成のうち、酵素液を実施例6で調製したGlyOXとした。その結果を表12に示す。
T42A/C245S/L301V/G304Qを実施例5と同様の方法で調製した。
野生型とT42A/C245S/L301V変異型とT42A/C245S/L301V/G304Q変異型について、基質特異性を確認した。具体的には、実施例2に示す組成のうち、酵素液を実施例4または実施例5または実施例9で調製したGlyOX溶液とし、基質としてGly溶液の代わりに(1)1mM Gly、(2)標準アミノ酸20種類、シスチン、タウリン、シトルリン、オルニチンおよびα-アミノ酪酸をそれぞれ1mM含有する混合液、(3)(2)の25種類の混合液からGlyを除いたもの、のいずれかを用いて活性を求めることで評価した。なお、Tyrとシスチンは2M HCl、それ以外のアミノ酸は0.1M HClで溶解して100mM溶液を調製し、各アミノ酸溶液を混合した後に超純水で100倍希釈することでアミノ酸溶液を調製した。この際、(1)および(3)では、混合しないアミノ酸溶液の代わりに2M HClまたは/および0.1M HClを混合した。活性は、37℃で波長500nmにおける吸光度の変化(ΔAbs500)をマイクロプレートリーダーで検出することで求めた。野生型に関する結果を図1に、T42A/C245S/L301V変異型に関する結果を図2に、T42A/C245S/L301V/G304Q変異型に関する結果を図3に示す。基質が上記(1)、(2)、(3)の場合を、それぞれGly、25mix、25mix-Glyと表現した。野生型、T42A/C245S/L301V変異型、T42A/C245S/L301V/G304Q変異型すべて、Gly以外のアミノ酸に対する触媒活性は見られなかった。Glyと共に他のアミノ酸が共存した条件下においては、野生型およびT42A/C245S/L301V/G304Q変異型では他のアミノ酸による反応阻害が見られ、野生型の反応開始より10分後のΔAbs500はGlyのみを基質とした場合の85%、T42A/C245S/L301V/G304Q変異型の反応開始より10分後のΔAbs500 はGlyのみを基質とした場合の52%であったが、T42A/C245S/L301V変異型については反応阻害が見られず性質が改善されていた。
T42A/C245S/L301V変異型またはT42A/C245S/L301V/G304Q変異型を用いて、反応系中のGly濃度とエンドポイント法で得られる吸光度との関係を確認した。まず、反応液C(4mM フェノール、100mM HEPES、pH8.0)を調製し、96穴マイクロプレートを用いて37℃にて、反応液Cを49μl、50mM 4-アミノアンチピリンを1μl、500U/ml ペルオキシダーゼを1μl、各種濃度のGly水溶液10μl、超純水を19μl、4.0mg/ml GlyOXを20μl混合した液の、10分後の波長500nmにおける吸光度をマイクロプレートリーダーを用いて測定した。この実験を3回行い、平均値を求めた。酵素液には実施例5または実施例9で調製したGlyOX溶液を、Gly水溶液は0M(すなわち超純水)、0.025mM、0.05mM、0.1mM、0.25mM、0.5mM、1mM、2.5mMの濃度に調製したものを用いた。反応系中のGly濃度と吸光度の関係は、図4または5に示す通り良好な正の相関を示し、本酵素を用いたGlyの測定が可能であることが示された。なお、図4または5におけるエラーバーは標準偏差を示す。
T42A/C245S/L301V変異型またはT42A/C245S/L301V/G304Q変異型を用いて、ヒト血漿中のGlyを定量分析した。反応、測定はキュベットを用いて37℃にて行った。実施例11の反応液Cを441μl、50mM 4-アミノアンチピリンを9μl、500U/ml ペルオキシダーゼを9μl、超純水を171μl、測定検体として各種濃度のGly水溶液またはヒト血漿を90μl混合した液(合計720μl)へ4.0mg/ml GlyOXを180μl混合させる前および混合10分後の波長500nmおよび800nmにおける吸光度(Abs500nm(前)、Abs500nm(後)、Abs800nm(前)、Abs800nm(後))を測定した。酵素液には実施例5または実施例9で調製したGlyOX溶液を、Gly水溶液には0M(すなわち超純水)、0.25mM、0.5mMの濃度に調製したものを用いた。
Claims (23)
- 以下:
(a)グリシンに対するグリシン酸化酵素の活性;
(b)グリシン酸化酵素の熱安定性;および
(c)グリシンに対するグリシン酸化酵素の基質特異性;
からなる群より選ばれる、グリシンの測定に関連するグリシン酸化酵素の1以上の特性を改善するようにその少なくとも1つのアミノ酸残基を変異させた、改変酵素。 - 変異が、グリシン酸化酵素のアミノ酸配列における(a)TTSモチーフ中の第1のスレオニンの置換、(b)TTSモチーフ中のセリンの置換、(c)HCYモチーフ中のシステインの置換、(d)LRPモチーフ中のロイシンの置換、(e)GMLモチーフ中のメチオニンの置換、(f)SGモチーフ中のセリンの置換、および(g)PGTモチーフ中のグリシンの置換からなる群より選ばれる1以上の置換である、請求項1記載の改変酵素。
- TTSモチーフ中の第1のスレオニンが、アラニン、セリン、システイン、またはグリシンに置換されている、請求項2記載の改変酵素。
- TTSモチーフ中のセリンが、リジンに置換されている、請求項2または3記載の改変酵素。
- HCYモチーフ中のシステインが、アラニン、アスパラギン酸、グリシン、ヒスチジン、アスパラギン、トリプトファン、チロシン、またはセリンに置換されている、請求項2~4のいずれか一項記載の改変酵素。
- LRPモチーフ中のロイシンが、イソロイシン、バリン、システイン、スレオニン、またはプロリンに置換されている、請求項2~5のいずれか一項記載の改変酵素。
- GMLモチーフ中のメチオニンが、イソロイシンに置換されている、請求項2~6のいずれか一項記載の改変酵素。
- SGモチーフ中のセリンが、アルギニンに置換されている、請求項2~7のいずれか一項記載の改変酵素。
- PGTモチーフ中のグリシンが、チロシン、またはグルタミンに置換されている、請求項2~8のいずれか一項記載の改変酵素。
- グリシン酸化酵素がバチルス属に由来する、請求項1~9のいずれか一項記載の改変酵素。
- 以下(1)あるいは(2)のタンパク質である、請求項1~10のいずれか一項記載の改変酵素:
(1)配列番号2のアミノ酸配列において、TTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が、下記(i)~(vii)に示されるアミノ酸残基に置換されたアミノ酸配列を含む、タンパク質:
(i)TTSモチーフ中の第1のスレオニンの置換
アラニン、セリン、システイン、またはグリシン;
(ii)TTSモチーフ中のセリンの置換
リジン;
(iii)HCYモチーフ中のシステインの置換
アラニン、アスパラギン酸、グリシン、ヒスチジン、アスパラギン、トリプトファン、チロシン、またはセリン;
(iv)LRPモチーフ中のロイシンの置換
イソロイシン、バリン、システイン、スレオニン、またはプロリン
(v)GMLモチーフ中のメチオニンの置換
イソロイシン;
(vi)SGモチーフ中のセリンの置換
アルギニン;
(vii)PGTモチーフ中のグリシンの置換
チロシン、またはグルタミン。
(2)配列番号2のアミノ酸配列におけるTTSモチーフ中の第1のスレオニン、TTSモチーフ中のセリン、HCYモチーフ中のシステイン、LRPモチーフ中のロイシン、GMLモチーフ中のメチオニン、SGモチーフ中のセリン、およびPGTモチーフ中のグリシンからなる群より選ばれる1以上のアミノ酸残基が、上記(i)~(vii)に示されるアミノ酸残基に置換されたアミノ酸配列において、1または数個のアミノ酸残基の追加変異を有するアミノ酸配列を含み、かつ、下記(a)~(c)からなる群より選ばれる1以上の特性が改善されている、タンパク質:
(a)グリシンに対するグリシン酸化酵素の活性;
(b)グリシン酸化酵素の熱安定性;および
(c)グリシンに対するグリシン酸化酵素の基質特異性。 - 請求項1~11のいずれか一項記載の改変酵素を用いて被検試料中に含まれるグリシンを測定することを含む、グリシンの分析方法。
- グリシンの測定が、請求項1~11のいずれか一項記載の改変酵素に加えて、4-アミノアンチピリンおよびフェノール、ならびにペルオキシダーゼを用いて行われる、請求項12記載の方法。
- 請求項1~11のいずれか一項記載の改変酵素を用いてグリシンからグリオキシル酸を生成することを含む、グリオキシル酸の製造方法。
- 請求項1~11のいずれか一項記載の改変酵素をコードするポリヌクレオチド。
- 請求項15記載のポリヌクレオチドを含む発現ベクター。
- 請求項16記載の発現ベクターを含む形質転換体。
- グリシンの測定に関連するグリシン酸化酵素の特性を改善するようにその少なくとも1つのアミノ酸残基を変異させた改変酵素を、請求項17記載の形質転換体を用いて生成することを含む、改変酵素の製造方法。
- 請求項1~11のいずれか一項記載の改変酵素を含む、グリシン分析用キット。
- 反応用緩衝液または緩衝塩、過酸化水素検出試薬、アンモニア検出試薬およびグリオキシル酸検出試薬の少なくとも一つをさらに含む、請求項19記載のグリシン分析用キット。
- (a)デバイス、および(b)請求項1~11のいずれか一項記載の改変酵素を含む、グリシン分析用検出系。
- (c)反応用緩衝液または緩衝塩、過酸化水素検出試薬、アンモニア検出試薬およびグリオキシル酸検出試薬の少なくとも一つをさらに含み、かつデバイスがマイクロ流路チップである、請求項21記載のグリシン分析用検出系。
- (a)検出用電極、および(b)検出用電極に固定または配置された、請求項1~11のいずれか一項記載の改変酵素を含む、グリシン分析用酵素センサー。
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