WO2015020200A1 - 改変型アマドリアーゼ及びその製造法、並びにアマドリアーゼの界面活性剤耐性向上剤及びこれを用いたHbA1c測定用組成物 - Google Patents
改変型アマドリアーゼ及びその製造法、並びにアマドリアーゼの界面活性剤耐性向上剤及びこれを用いたHbA1c測定用組成物 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0032—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
- C12Y105/03—Oxidoreductases acting on the CH-NH group of donors (1.5) with oxygen as acceptor (1.5.3)
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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
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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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)
- G01N2333/9065—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5)
- G01N2333/90672—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with oxygen as acceptor (1.5.3) in general
Definitions
- the present invention relates to an amadoriase excellent in surfactant resistance, its gene and recombinant DNA, and amadoriase excellent in surfactant resistance, which can be advantageously used as a diagnostic enzyme for diabetes and in a kit for measuring a diabetes marker. It relates to the manufacturing method.
- the present invention also relates to an amadoriase stabilizer and / or buffer, and a composition containing the same.
- Glycated proteins are produced by non-enzymatic covalent bond formation between the aldehyde group of aldoses (monosaccharides and derivatives thereof that potentially have aldehyde groups) such as glucose, and Amadori transfer It is.
- aldehyde group of aldoses monosaccharides and derivatives thereof that potentially have aldehyde groups
- Amadori transfer It is.
- the amino group of a protein include an ⁇ -amino group at the amino terminal and an ⁇ -amino group of a side chain of a lysine residue in the protein.
- Known glycated proteins generated in vivo include glycated hemoglobin in which hemoglobin in blood is glycated, glycated albumin in which albumin is glycated, and the like.
- HbA1c glycated hemoglobin
- ⁇ -fructosylvalylhistidine (hereinafter referred to as “ ⁇ FVH”), which is obtained by decomposing HbA1c with a protease or the like and releasing it from its ⁇ -chain amino terminus.
- ⁇ FV ⁇ -fructosyl valine
- Amadoriase catalyzes a reaction that oxidizes iminodiacetic acid or a derivative thereof (also referred to as an “Amadori compound”) in the presence of oxygen to produce glyoxylic acid or ⁇ -ketoaldehyde, an amino acid or peptide, and hydrogen peroxide. .
- Amadoriase has been found from bacteria, yeasts and fungi, and is particularly useful for the measurement of HbA1c.
- Examples of amadoriase having enzyme activity against ⁇ FVH and / or ⁇ FV include, for example, the genus Coniochaeta, Eupenicillium ( Eunicillium genus, Pyrenochaeta genus, Arthrinium genus, Curvularia genus, Neocosmospora genus, Cryptococcus erus sp Emericella genus, urocladium genus, Penicillium genus, Fusarium genus, Achaetomiella genus, Achaetomium genus, Sierravia genus, Chaetomium inc Leptosphaeria, Ophiobolus, Pleospora, Coniochaetidium, Pichia, Corynebacterium, Corynebacterium (Arthrobacter)
- amadoriase may be described by expressions such as ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, and fructosylamine oxidase depending on the literature.
- HbA1c When measuring HbA1c, it is known that an amadoriase is excessively contained in the reagent composition for measurement. For example, when measuring HbA1c with a final concentration of 0.36 ⁇ M, Amadoriase uses 1.4 kU / L, that is, a concentration at which 1.4 mM of substrate can be reacted per minute (see Patent Document 16). .
- a technique using an automatic analyzer is the mainstream, and the reaction time of the amadoriase with a substrate is often measured at about 5 to 25 minutes.
- amadoriase is contained in an excessive amount is that it sufficiently reacts with the substrate in the short measurement time as described above, and can greatly affect the reactivity and stability of the amadoriase. This is because when a substance coexists in the composition for measurement, it is unavoidable to add amadoriase excessively as a countermeasure against this influence.
- Examples of pretreatment methods for measuring HbA1c from whole blood or erythrocytes using amadoriase include cases where hemolysis is performed using a surfactant (see, for example, Patent Document 2, 16 to 18).
- a surfactant may be used as a reaction accelerator (see, for example, Patent Document 19). Therefore, a surfactant is indispensable for measuring HbA1c using amadoriase, but when the HbA1c quantitative reaction is started after the HbA1c solution and the amadoriase solution treated with the surfactant and protease are mixed.
- the current kit for measuring HbA1c is able to make accurate measurements because it prescribes amadoriase more than necessary and prescribes a stabilizer, but it uses a large amount of reagents. High costs are inevitable. Moreover, if a surfactant having a stronger effect than the current one can be used, it is highly possible that the protease decomposition efficiency of HbA1c is further improved and the measurement sensitivity of HbA1c is improved.
- the surfactant also has an effect of solubilizing insoluble peptide fragments derived from hemoglobin or HbA1c, it prevents the occurrence of turbidity and contributes to improvement in measurement accuracy. Accordingly, it can be said that one of the properties required for prescribing Amadoriase as an enzyme for clinical diagnosis of diabetes in a kit reagent is good stability of a liquid containing a surfactant.
- amadoriase has been used in an excessive amount so as to sufficiently react with a substrate during a measurement time.
- a surfactant is a component that can significantly negatively affect the stability of amadoriase. Therefore, if an enzyme with even better surfactant resistance than conventional amadoriase is produced, it can be reduced by reducing the amount of amadoriase and stabilizer prescribed in the kit, and convenience in the distribution of the enzyme and kit. It is expected to greatly contribute to the improvement of the measurement sensitivity of HbA1c due to cost reduction and the ability to formulate a strong surfactant.
- the problem to be solved by the present invention is to provide an amadoriase having superior surfactant resistance as compared with conventional amadoriases, and to quantify HbA1c or a glycated peptide derived therefrom even in the presence of a surfactant. Is to provide a simple reagent composition.
- the problem to be solved by the present invention is to provide a stabilizer and / or a buffer that maintains the residual activity of amadoriase in the presence of a surfactant or reduces the decrease in the residual activity, and a composition containing these. It is to provide.
- the present inventors have conducted extensive research and, as a result, conducted research on specific amino acid residues for amadoriase derived from the genus Coniochaeta. It is found that the above-mentioned problems can be solved by introducing a substitution of the above, and by including in the reagent composition an amadoriase that remains active even in the presence of a surface activity, and further, a specific stabilizer and / or It was found that the residual activity of amadoriase in the presence of a surfactant can be maintained or a decrease in the residual activity can be significantly reduced by using a buffer, and the present invention has been completed.
- the present invention is as follows.
- Amadoriase whose residual activity (%) after 5 minutes from the addition of the surfactant is improved as compared with the amadoriase having the amino acid sequence shown in SEQ ID NO: 1, 3, or 37, (I) having an amino acid sequence in which one or several amino acid deletions, insertions, additions and / or substitutions have been made in the amino acid sequence shown in SEQ ID NO: 1, 3, or 37, and / or (ii) a sequence Having an amino acid sequence having at least 70% identity with the amino acid sequence shown in number 1, 3, or 37; Said amadoriase.
- amino acid of the amino acid sequence shown in SEQ ID NO: 1 or 3 is Substitution from (i) to (xiv): (I) Asparagine at position 262 is replaced with histidine; (Ii) valine at position 257 is substituted with cysteine, serine, threonine; (Iii) glutamic acid at position 249 is substituted with lysine or arginine; (Iv) glutamic acid at position 253 is substituted with lysine or arginine; (V) glutamine at position 337 is replaced with lysine or arginine; (Vi) glutamic acid at position 340 is replaced with proline; (Vii) Aspartic acid at position 232 is substituted with lysine or arginine; (Viii) aspartic acid at position 129 is substituted with lysine or arginine; (Ix) Aspartic acid at position 132 is substituted with lysine or arginine; (Ix)
- amino acid of the amino acid sequence shown in SEQ ID NO: 37 is Substitution from (i) to (ix): (I) glutamic acid at position 247 is substituted with lysine or arginine; (Ii) glutamic acid at position 251 is substituted with lysine or arginine; (Iii) threonine at position 335 is substituted with lysine or arginine; (Iv) Aspartic acid at position 230 is substituted with lysine or arginine; (V) Aspartic acid at position 129 is substituted with lysine or arginine; (Vi) aspartic acid at position 132 is substituted with lysine or arginine; (Vii) glutamic acid at position 133 is substituted with alanine, methionine, lysine, arginine; (Viii) asparagine at position 254 is substituted with lysine and arginine; and (ix): (
- a host cell comprising the recombinant vector according to [9].
- composition for use in measuring glycated hemoglobin comprising the amadoriase according to any one of [1] to [7].
- a composition for measuring glycated hemoglobin comprising one or more surfactants and amadoriase.
- Amadoriase (I) Residual activity (%) 5 minutes after addition of surfactant remains at least 15% as compared to the case where it is not added and / or (ii) Interface with final concentration of 0.04%
- An amadoriase having a difference from the absorbance at 751 nm 5 minutes after addition of a control solution using ion-exchanged water instead of a glycated amino acid solution or a glycated peptide solution is 0.006 or more
- composition according to [13] or [14], wherein the amadoriase is an amadoriase having an amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO: 1, 3, 37 or 40.
- the surfactant is a quaternary ammonium salt represented by the following general formula (I): [Wherein R 1 to R 4 may be the same or different and each represents a substituted or unsubstituted C 1 to C 20 alkyl, alkenyl, aryl, or benzyl, and Z ⁇ represents a monovalent anion. .
- the surfactant is octyltrimethylammonium chloride, octyltrimethylammonium bromide, dioctyldimethylammonium chloride, dioctyldimethylammonium bromide, decyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyl Trimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, octadecyltrimethylammonium bromide, eicosyltrimethylammonium chloride and eicosyltrimethylammonium Moniumuburomido, benzyl do
- composition according to any one of [13] to [18], wherein the surfactant contained is 0.01% (w / v) or more as a final concentration at the time of measurement.
- composition for measuring glycated hemoglobin according to [13], further comprising one or more stabilizers selected from the group consisting of ammonium sulfate and combinations thereof.
- Tricarboxylic acid is citric acid or dicarboxylic acid is fumaric acid, glutaric acid, citraconic acid, mesaconic acid, malonic acid, tartaric acid, succinic acid, adipic acid, maleic acid, malic acid and combinations thereof
- the monocarboxylic acid is acetic acid, or the compound of formula (IV) is selected from the group consisting of MES, MOPS, MOPSO and combinations thereof [ 22].
- the stabilizer is phosphoric acid with a final concentration of 2 mM or more in the measurement solution, citric acid with a final concentration of 0.2 mM or more in the measurement solution, malic acid with a final concentration of 2 mM or more in the measurement solution, measurement Maleic acid with a final concentration of 2 mM or more in the solution, citraconic acid with a final concentration of 2 mM or more in the measurement solution, malonic acid with a final concentration of 2 mM or more in the measurement solution, glutaric acid with a final concentration of 2 mM or more in the measurement solution , Tartaric acid with a final concentration of 2 mM or more in the measurement solution, acetic acid with a final concentration of 10 mM or more in the measurement solution, MES (2- (N-morpholino) ethanesulfonic acid) with a final concentration of 10 mM or more in the measurement solution, measurement MOPS (3- (N-morpholino) propanesulfonic acid) with a final concentration of
- a composition for measuring glycated hemoglobin comprising the buffer according to [20] or [21] and the stabilizer according to [22], [23] or [24].
- amadoriase having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 37 or SEQ ID NO: 40, or the amadoriase according to any one of [1] to [7], [20] to [25] A composition according to any one of the above.
- an amadoriase having excellent surfactant resistance, a gene encoding the same, and the like that can be advantageously used as a diagnostic enzyme for diabetes and in a kit for measuring a diabetes marker.
- this amadoriase is used, glycated hemoglobin can be measured even in the presence of a high concentration of a surfactant.
- the stabilizer and / or buffering agent of the present invention the residual activity of amadoriase in the presence of a surfactant can be maintained or reduction in the residual activity can be reduced, and saccharification can be achieved even in the presence of a high concentration of surfactant. Hemoglobin can be measured.
- FIG. 1-1 is an alignment of amino acid sequences of various known amadoriases.
- FIG. 1-2 is an alignment of amino acid sequences of various known amadoriases.
- FIGS. 1-3 are alignments of amino acid sequences of various known amadoriases.
- FIG. 2 shows the results of measuring ⁇ FVH using CFP-T7 under a mixture of 0.01% CTAC.
- FIG. 3 shows the results of measuring ⁇ FVH using CFP-T7 under a mixture of 0.02% CTAC.
- FIG. 4 shows the result of measuring ⁇ FVH using CFP-D7 under a mixture of 0.02% CTAC.
- FIG. 5 shows the results of measuring ⁇ FVH using CFP-D7 under a mixture of 0.2% CTAC.
- Amadoriase is also called ketoamine oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosylamine oxidase, etc., and oxidizes iminodiacetic acid or its derivative (Amadori compound) in the presence of oxygen to give glyoxylic acid or An enzyme that catalyzes a reaction that produces ⁇ -ketoaldehyde, an amino acid or peptide, and hydrogen peroxide.
- Amadoriase is widely distributed in nature and can be obtained by searching for microorganisms and enzymes of animal or plant origin. The microorganism can be obtained from, for example, filamentous fungi, yeast, or bacteria.
- amadoriase of the present invention is a surfactant-resistant product produced based on the amadoriase derived from the genus Coniochaeta having the amino acid sequence shown in SEQ ID NO: 1 or the amadoriase derived from Curvularia clavata having the amino acid sequence shown in SEQ ID NO: 37. Is an improved amadoriase variant.
- variants include high sequence identity with SEQ ID NO: 1 or SEQ ID NO: 37 (eg, 70% or more, preferably 75% or more, preferably 80% or more, more preferably 85% or more, More preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and most preferably 99% or more)
- Amadoriase having an amino acid sequence and the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 37 from 1 to Mention may be made of amadoriase having an amino acid sequence in which several amino acids have been modified or mutated, or deleted, substituted, added and / or inserted.
- the amadoriase of the present invention includes, for example, the genus Eupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Amergillus, Emergillus, , Thielavia genus, Chaetomium genus, Gelasinospora genus, Microascus genus, Leptosphaeria genus, Ophiobolus genus, Pleospora genus, Coniochaetidium genus, Pichia genus, Corynebacterium , Agrobacterium genus, or which has been produced on the basis of amadoriase derived from the species of such Arthrobacter sp.
- those having surfactant resistance and / or having high sequence identity with SEQ ID NO: 1 or SEQ ID NO: 37 as described above are preferable.
- a variant of amadoriase with altered surfactant resistance can be obtained by substituting, adding, or deleting at least one amino acid residue in the amino acid sequence of amadoriase.
- Substitution of aspartic acid at position 129 for example, substitution with lysine or arginine.
- Substitution of aspartic acid at position 132 for example, substitution with lysine or arginine.
- Substitution of glutamic acid at position 133 for example, substitution with alanine, methionine, lysine, arginine.
- Substitution of glutamic acid at position 44 for example, substitution with proline.
- Substitution of glycine at position 256 for example, substitution with lysine or arginine.
- Substitution of glutamic acid at position 231 such as lysine or arginine.
- Substitution of glutamic acid at position 81 for example, substitution with lysine or arginine.
- the variant of amadoriase with improved surfactant resistance only needs to have at least one amino acid substitution as described above, and may have a plurality of amino acid substitutions. For example, it has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the above amino acid substitution.
- a mutant having an amino acid substitution corresponding to the following amino acid position is preferable.
- (11)-(6) Substitution of glutamic acid at position 44 and substitution of glutamic acid at position 340, for example, substitution of amino acid at the position corresponding to glutamic acid at position 44 with proline and amino acid at the position corresponding to glutamic acid at position 340 Mutants with substitution for proline.
- substitution of glutamic acid at position 44 substitution of valine at position 257, substitution of asparagine at position 262, substitution of glutamic acid at position 340, and position 249
- substitution of amino acid at the position corresponding to glutamic acid at position 44 with proline substitution of amino acid at position corresponding to valine at position 257, substitution of amino acid at position corresponding to asparagine at position 262
- the surfactant-resistant amadoriase mutant of the present invention may have an amino acid substitution that brings about the improvement of the surfactant resistance in the amino acid sequence shown in SEQ ID NO: 1.
- the surfactant-resistant amadoriase mutant of the present invention is further one or several (for example, 1 to 15, for example, 1 to 10, preferably 1 to 5, more preferably) at positions other than those substituted amino acids. 1 to 3 (particularly preferably 1) amino acids may be deleted, inserted, added and / or substituted.
- the present invention has an amino acid substitution mutation that improves properties other than the surfactant resistance, such as amino acid substitution mutation and substrate specificity that bring about the improvement of the surfactant resistance, and is represented by SEQ ID NO: 1 or 3.
- Amadoriase variants having amino acid sequence identity of 99% or more, having amadoriase activity, and modified surfactant resistance are included.
- the amadoriase having the amino acid sequence shown in SEQ ID NO: 1 is produced by Escherichia coli carrying a recombinant plasmid (deposit number: FERM BP-10593) called pKK223-3-CFP-T7 in International Publication No. 2007/125797.
- This is an amadoriase derived from the genus Coniochaeta (CFP-T7), which is a modified amadoriase with excellent thermal stability previously found by the applicant.
- This CFP-T7 is a triple mutant obtained by sequentially introducing artificial mutations at positions 272, 302 and 388 with respect to the natural type Amadoriase derived from the genus Coniochaeta.
- SEQ ID NO: 3 is a substrate specificity-improving mutation (E98A) shown in International Publication No. 2012/18094, and a thermal stability-improving mutation shown in International Publication Nos. 2007/12579 and 2013/100006. It is an amino acid sequence of Amadoriase derived from the genus Coniochaeta, into which (F43Y, G184D, deletion of 3 amino acid residues at the carboxyl terminus) was introduced.
- the amino acid position represents the position in the amino acid sequence of the amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1, but in the amino acid sequence of the amadoriase derived from other species, it is shown in SEQ ID NO: 1.
- the amino acid at the position corresponding to the position in the amino acid sequence to be substituted is substituted. The meaning of “corresponding position” will be described later.
- amino acid substitution that brings about the improvement of the surfactant resistance include substitution of amino acids at positions corresponding to the following amino acids in the amino acid sequence shown in SEQ ID NO: 37.
- substitution of glutamic acid at position 247 for example, substitution with lysine or arginine
- substitution of glutamic acid at position 251 such as lysine or arginine
- substitution of threonine at position 335 for example, substitution with lysine or arginine
- substitution of aspartic acid at position 230 for example, lysine, arginine
- V substitution of aspartic acid at position 129, for example, substitution with lysine, arginine
- substitution of aspartic acid at position 132 for example, lysine, arginine
- substitution of glutamic acid at position 133 for example, alanine, methionine, lysine, arginine
- the variant of amadoriase with improved surfactant resistance only needs to have at least one amino acid substitution as described above, and may have a plurality of amino acid substitutions. For example, it has 1, 2, 3, 4, 5, 6, 7, 8, or 9 of the above amino acid substitution.
- a mutant having an amino acid substitution corresponding to the following amino acid position is preferable.
- amadoriase gene (Acquisition of gene encoding amadoriase)
- amadoriase gene In order to obtain the gene of the present invention encoding these amadoriases (hereinafter also simply referred to as “amadoriase gene”), generally used gene cloning methods are used.
- chromosomal DNA or mRNA can be extracted from microbial cells having the ability to produce amadoriase and various cells by a conventional method, for example, a method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989).
- cDNA can be synthesized using mRNA as a template.
- a chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA thus obtained.
- a suitable probe DNA is synthesized, and using this, a method for selecting the amadoriase gene from a chromosomal DNA or cDNA library, or a suitable primer DNA based on the amino acid sequence.
- PCR method polymerase chain reaction
- amadoriase As a preferred example of the gene encoding amadoriase thus obtained, an example of an amadoriase gene derived from the genus Coniochaeta (Japanese Patent Laid-Open No. 2003-235585) and the like can be mentioned.
- amadoriase genes are linked to various vectors as usual.
- Coniochaeta sp An example is a recombinant plasmid pKK223-3-CFP (Japanese Patent Laid-Open No. 2003-235585) in which DNA encoding an amadoriase gene derived from NISL 9330 strain is inserted into pKK223-3 Vector (manufactured by GE Healthcare).
- the vector that can be used in the present invention is not limited to the above-mentioned plasmid, and any other vector known to those skilled in the art, such as bacteriophage and cosmid, can be used. Specifically, for example, pBluescript II SK + (manufactured by STRATAGENE) is preferable.
- amadoriase gene mutation treatment The mutation process of the amadoriase gene can be performed by any known method depending on the intended mutant form. That is, a wide range of methods such as a method of contacting and acting an amadoriase gene or a recombinant DNA into which the gene is incorporated and a mutagen drug; an ultraviolet irradiation method; a genetic engineering method; or a method using a protein engineering method Can be used.
- Examples of the mutagen used in the above mutation treatment include hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfite, hydrazine, formic acid, 5-bromouracil and the like. Can do.
- the various conditions for the contact and action are not particularly limited as long as conditions according to the type of drug used can be taken and a desired mutation can actually be induced in the amadoriase gene.
- a desired mutation can be induced by contact and action at a reaction temperature of 20 to 80 ° C. for 10 minutes or more, preferably 10 to 180 minutes, preferably at a drug concentration of 0.5 to 12M.
- a reaction temperature 20 to 80 ° C. for 10 minutes or more, preferably 10 to 180 minutes, preferably at a drug concentration of 0.5 to 12M.
- Even in the case of performing ultraviolet irradiation it can be carried out according to a conventional method as described above (Hyundai Kagaku, p24-30, June 1989 issue).
- a method generally known as Site-Specific Mutagenesis can be used.
- Kramer method Nucleic Acids Res., 12, 9441 (1984): Methods Enzymol., 154, 350 (1987): Gene, 37, 73 (1985)
- Eckstein method Nucleic Acids Res., 13, 8749 ( (1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res, 14, 9679 (1986)
- Kunkel method Proc. Natl. Acid. Sci. USA, 82, 488 (1985).
- a technique known as a general PCR method polymerase chain reaction
- a desired modified amadoriase gene can also be directly synthesized by an organic synthesis method or an enzyme synthesis method.
- the amadoriase gene obtained as described above is incorporated into a vector such as a bacteriophage, a cosmid, or a plasmid used for transformation of prokaryotic cells or eukaryotic cells by a conventional method, and a host corresponding to each vector is usually used. Transformation or transduction can be performed by the method.
- an arbitrary host for example, a microorganism belonging to the genus Escherichia, specifically E. coli K-12 strain, preferably E. coli JM109 strain, E. coli DH5 ⁇ strain (both manufactured by Takara Bio Inc.).
- E. coli B strain preferably E. coli BL21 strain (manufactured by Nippon Gene), etc., can be transformed or transduced to obtain the respective strains.
- amino acid sequence identity The identity of the amino acid sequence is determined according to GENETYX Ver. 11 (manufactured by Genetics) such as maximum matching and search homology, or DNASIS Pro (manufactured by Hitachi Software) such as maximum matching and multiple alignment.
- the “position corresponding to an amino acid” refers to a position in the amino acid sequence of an amadoriase derived from another species corresponding to the amino acid at a specific position in the amino acid sequence of the amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1.
- amino acid sequences are compared using a known algorithm such as Lippmann-Person method, and the maximum conserved amino acid residue present in the amino acid sequence of each amadoriase is determined. It can be done by giving sex.
- aligning the amino acid sequences of the amadoriases in this way it is possible to determine the positions of the homologous amino acid residues in the sequence of each amadoriase sequence regardless of insertions or deletions in the amino acid sequences.
- the homologous position is considered to exist at the same position in the three-dimensional structure, and it can be estimated that the homologous position has a similar effect on the specific function of the target amadoriase.
- FIGS. 1-1, 1-2, and 1-3 illustrate examples of amadoriase sequences derived from various known species.
- the amino acid sequence represented by SEQ ID NO: 1 is shown at the top.
- Each of the various sequences shown in FIG. 1 has 70% or more identity with the sequence of SEQ ID NO: 1, and was aligned using a known algorithm.
- mutation points in the mutant of the present invention are shown. 1-1, 1-2, and 1-3, the position in the amino acid sequence of the amadoriase derived from another species corresponding to the amino acid at the specific position of the amino acid sequence of the amadoriase derived from the genus Coniochaeta can be known.
- Amadoriase (SEQ ID NO: 1) derived from Coniochaeta genus, Amadoriase (SEQ ID NO: 34) derived from Eupenicillium terrenum, Pyrenochaeta sp. Derived ketoamine oxidase (SEQ ID NO: 35), Arthrinium sp.
- Ketoamine oxidase derived from Curvularia clavata (SEQ ID NO: 37), ketoamine oxidase derived from Neocosmospora vasinfecta (SEQ ID NO: 38), fructosyl amino acid oxidase derived from Cryptococcus neoformans (SEQ ID NO: 39) , Phaeosphaeria nodorum-derived fructosyl peptide oxidase (SEQ ID NO: 40), Aspergillus nidulans-derived fructosyl amino acid oxidase (SEQ ID NO: 41), Ulocladium sp.
- the amino acid sequences of the fructosyl amino acid oxidase (SEQ ID NO: 42) derived from Penicillium crysogenum and the fructosyl amino acid oxidase (SEQ ID NO: 43) derived from Penicillium are shown.
- the “position corresponding to glutamic acid at position 44 of the amino acid sequence described in SEQ ID NO: 1” refers to the confirmed amino acid sequence of amadoriase and the amino acid sequence of the amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. When compared, it means an amino acid corresponding to glutamic acid at position 44 of the amadoriase of SEQ ID NO: 1.
- the amino acid sequence can be identified by FIG. 1-1 in which the amino acid sequences are aligned by the above-described method of identifying “corresponding amino acid residue”.
- Proline at position 44 in the ketoamine oxidase derived from proline at position 44 in the ketoamine oxidase derived from Curvularia clavata, proline at position 44 in the ketoamine oxidase derived from Neocosmospora vasinfecta, and position 44 in the fructosyl amino acid oxidase derived from Cryptococcus neoformans
- the proline at position 44, and in the fructosyl amino acid oxidase derived from Aspergillus nidulans the proline at position 43, Ulocladium sp.
- the 44th position is proline, and in the case of the fructosyl amino acid oxidase derived from Penicillium crysogenum, it is the proline position 44.
- position corresponding to glutamic acid at position 81 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means an amino acid corresponding to glutamic acid at position 81 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-1 in which the amino acid sequences are aligned by the above method.
- the “position corresponding to glutamic acid at position 133 in the amino acid sequence described in SEQ ID NO: 1” means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means an amino acid corresponding to glutamic acid at position 133 in the amino acid sequence described in SEQ ID NO: 1. This can also be identified from FIG. 1-1 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from alanine at position 133 in the ketoamine oxidase derived from Curvularia clavata in position 133, glutamic acid in position 133, in ketoamine oxidase derived from Neocosmospora vasinfecta, in alanine at position 133, in the fructosyl amino acid oxidase from fructosyl amino acid oxidase 13 in the form of Cryptococcus neoformans.
- fructosyl peptide oxidase derived from Phaeosphaeria nodorum glutamic acid at position 131
- fructosyl amino acid oxidase derived from Aspergillus nidulans glutamic acid at position 132
- Ulocladium sp In the fructosyl amino acid oxidase derived from lysine, lysine at position 133, and in the fructosyl amino acid oxidase derived from Penicillium crysogenen, it is aspartic acid at position 133.
- the “position corresponding to glutamic acid at position 253 of the amino acid sequence described in SEQ ID NO: 1” means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means an amino acid corresponding to glutamic acid at position 253 of the amino acid sequence described in SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from glutamic acid at position 253 in ketoamine oxidase derived from Curvularia clavata, glutamic acid in position 251; in ketoamine oxidase derived from Neocosmospora vasinfecta, valine at position 253; in fructosyl amino acid derived from cructococcus neoformans oxidase; In the case of fructosyl peptide oxidase derived from Phaeosphaeria nodorum, arginine at position 249, fructosyl amino acid oxidase derived from Aspergillus nidulans, alanine at position 253, Ulocladium sp. It is glutamic acid at position 251 in the fructosyl amino acid oxidase derived from it, and glutamine at position 253 in the fructosyl amino acid oxidase derived from Penicillium crysogenum.
- the “position corresponding to glycine at position 256 of the amino acid sequence described in SEQ ID NO: 1” means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means an amino acid corresponding to glycine at position 256 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- asparagine Pyrenochaeta sp., which is 256th in the amadoriase derived from Eupenicillium terrenum.
- ketoamine oxidase derived from aspartic acid at position 254, Arthrium sp. Glycine at position 256 in the ketoamine oxidase derived from, 254th asparagine in the ketoamine oxidase derived from Curvularia clavata, glycine in the 256th position in ketoamine oxidase derived from Neocosmospora vasinfecta, and fructosyl amino acid glutamine glutamin oxidase derived from Cryptococcus neoformans in the 56th position.
- fructosyl amino acid oxidase derived from asparagine, asparagine at position 254, and in the fructosyl amino acid oxidase derived from Penicillium crysogenum it is aspartic acid at position 256.
- the position corresponding to valine at position 257 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to the valine at position 257 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- fructosyl peptide oxidase derived from Phaeosphaeria nodorum serine at position 253, fructosyl amino acid oxidase derived from Aspergillus nidulans, threonine at position 257, Ulocladium sp.
- the fructosyl amino acid oxidase derived from Penicillium crysogenum is valine at position 257.
- the position corresponding to asparagine at position 262 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to asparagine at position 262 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from asparagine at position 260 Arthrinium sp.
- the ketidine oxidase derived from histidine at position 262 the ketoamine oxidase derived from Curvularia clavata asparagine at position 260, the ketoamine oxidase derived from Neocosmospora vasinfecta histidine at position 262, and the fructosyl oxidase derived from ascites oxidase 26 at the position Cryptococcus neoformans oxidase.
- the aspartic acid at position 260, and in the fructosyl amino acid oxidase derived from Penicillium crysogenum aspartic acid at position 262.
- the position corresponding to glutamine at position 337 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to glutamine at position 337 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- fructosyl peptide oxidase derived from Phaeosphaeria nodorum lysine at position 333
- aspartic acid fructosyl amino acid oxidase derived from Aspergillus nidulans asparagine at position 337, Ulocladium sp.
- the fructosyl amino acid oxidase derived from Penicillium crysogenum is lysine in position 337
- the position corresponding to glutamic acid at position 340 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means an amino acid corresponding to glutamic acid at position 340 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from Eupenicillium terrenum glutamic acid at position 340, Pyrenochaeta sp.
- ketoamine oxidase derived from glutamic acid at position 338 Arthrium sp.
- glutamic acid in position 340 in ketoamine oxidase derived from Neocosmospora vasinfecta, in the form of proline at position 340 in fructosyl amino acid derived from Cryptococcus neoformans.
- fructosyl peptide oxidase derived from Phaeosphaeria nodorum lysine at position 336
- fructosyl amino acid oxidase derived from Aspergillus nidulans glutamic acid at position 340, Ulocladium sp.
- the fructosyl amino acid oxidase derived from Penicillium crysogenum is glutamic acid in position 340.
- the position corresponding to aspartic acid at position 129 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1 And amino acid corresponding to aspartic acid at position 129 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-1 in which the amino acid sequences are aligned by the above method.
- the position corresponding to aspartic acid at position 132 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1 Further, it means an amino acid corresponding to aspartic acid at position 132 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-1 in which the amino acid sequences are aligned by the above method.
- the position corresponding to glutamic acid at position 231 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to glutamic acid at position 231 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from glutamic acid at position 231 In the case of ketoamine oxidase derived from glutamic acid at position 231; in the case of ketoamine oxidase derived from Curvularia clavata, glutamic acid in position 229; in ketoamine oxidase derived from Neocosmospora vasinfecta; glutamic acid in position 231; In the case of fructosyl peptide oxidase derived from Phaeosphaeria nodorum, histidine at position 227, and as fructosyl amino acid oxidase derived from Aspergillus nidulans, glutamic acid at position 231 and Ulocladium sp. In the derived fructosyl amino acid oxidase, it is glutamine at position 229, and in the fructosyl amino acid oxidase derived from Penicillium crysogenum, it is glutamic acid at position 231.
- the position corresponding to aspartic acid at position 232 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of the amadoriase derived from Coniochaeta genus shown in SEQ ID NO: 1 Further, it means an amino acid corresponding to aspartic acid at position 232 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- ketoamine oxidase derived from glutamic acid at position 232 in ketoamine oxidase derived from Curvularia clavata aspartic acid in position 230, in ketoamine oxidase derived from Neocosmospora vasinfecta, glutamic acid in position 232 from position 2 of fructosylamino acid derived from Cryptococcus neoformans oxidase Glycine, Phthaosphaeria nodorum-derived fructosyl peptide oxidase, glutamic acid at position 228, and Aspergillus nidulans-derived fructosyl amino acid oxidase, glutamic acid at position 232, Ulocladium sp.
- the position corresponding to glutamic acid at position 249 of the amino acid sequence described in SEQ ID NO: 1 means that the confirmed amino acid sequence of amadoriase is compared with the amino acid sequence of Amadoriase derived from the genus Coniochaeta shown in SEQ ID NO: 1. Means the amino acid corresponding to glutamic acid at position 249 of the amadoriase of SEQ ID NO: 1. This can also be identified from FIG. 1-2 in which the amino acid sequences are aligned by the above method.
- fructosyl peptide oxidase derived from Phaeosphaeria nodorum glutamic acid at position 245, fructosyl amino acid oxidase derived from Aspergillus nidulans, alanine at position 249, Ulocladium sp. It is serine at position 247 in the fructosyl amino acid oxidase derived from it, and glutamine at position 249 in the fructosyl amino acid oxidase derived from Penicillium crysogenum.
- this strain may be cultured by a normal solid culture method, It is preferable to employ the liquid culture method as much as possible.
- Examples of the medium for culturing the above strain include one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor or soybean or wheat bran leachate, sodium chloride, phosphoric acid first Add one or more inorganic salts such as potassium, dibasic potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and add sugar raw materials, vitamins, etc. as necessary. Used.
- nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor or soybean or wheat bran leachate, sodium chloride, phosphoric acid
- inorganic salts such as potassium, dibasic potassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and add sugar raw materials, vitamins, etc. as necessary. Used.
- the culture can be carried out under any conditions.
- the culture temperature is 20 to 42 ° C., preferably about 30 ° C. for 4 to 24 hours, more preferably about 30 ° C. for about 8 to 8 hours. It can be carried out for 16 hours by aeration and agitation deep culture, shaking culture, stationary culture or the like.
- amadoriase After completion of the culture, in order to collect amadoriase from the culture, it can be obtained using a normal enzyme collecting means.
- the bacterial cells are subjected to ultrasonic disruption treatment, grinding treatment, or the like, or the enzyme is extracted using a lytic enzyme such as lysozyme, or shaken or left in the presence of toluene or the like to lyse the bacteria. This enzyme can be discharged out of the cells. Then, this solution is filtered, centrifuged, etc.
- nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate or the like, and then ammonium sulfate, alcohol, acetone or the like is added thereto. Fractionation and collecting the precipitate to obtain a crude enzyme of amadoriase.
- an amadoriase purified enzyme preparation further from the above crude amadoriase enzyme, for example, gel filtration method using Sephadex, Superdex, Ultrogel, etc .; adsorption elution method using ion exchanger; electrophoresis using polyacrylamide gel, etc.
- Method Adsorption elution method using hydroxyapatite; Precipitation method such as sucrose density gradient centrifugation; Affinity chromatography method; Fractionation method using molecular sieve membrane or hollow fiber membrane, etc.
- the surfactant in the present invention is not particularly limited as long as it is a surfactant that enables the method for measuring HbA1c of the present invention.
- the surfactant include an anionic surfactant, an amphoteric surfactant, and the like, and a cationic surfactant and an anionic surfactant are particularly preferable.
- this expression when referring to a surfactant, this expression includes one or more surfactants unless otherwise specified.
- the surfactant in the present invention has a critical micelle concentration (CMC) of 70 mM or less, 50 mM or less, 20 mM or less, 10 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4.5 mM or less, 4 mM or less, 3.5 mM or less, 3 mM.
- CMC critical micelle concentration
- it may be 2.5 mM or less, 2 mM or less, 1.5 mM or less, 1.3 mM or less, or 1 mM or less.
- the critical micelle concentration of the surfactant of the present invention can be 0.1 mM or 0.01 mM or more.
- the critical micelle concentration is 50 mM or less, more preferably 20 mM or less, more preferably 10 mM or less, more preferably 7 mM or less, more preferably 6 mM or less, and most preferably 5 mM or less. is there.
- the critical micelle concentration is a critical concentration at which the surfactant forms micelles in the solution, and no micelle is formed at a concentration lower than this. Generally, when the critical micelle concentration is low, micelles are formed even with a low concentration surfactant, and the action as a surfactant tends to be strong.
- a person skilled in the art can determine the critical micelle concentration of the desired surfactant by conventional techniques.
- a commercially available kit for measuring the critical micelle concentration of a surfactant can be used by utilizing the fluorescence change of a fluorescent reagent that interacts with the surfactant (for example, Depent Critical Micelle Concentration (CMC) Assay of PFP). Kit et al.).
- CMC Depent Critical Micelle Concentration
- the CMC of octyltrimethylammonium bromide (C8, OTAB) is about 140 mM
- the CMC of decyltrimethylammonium chloride (C10) is about 65 mM
- the CMC of decyltrimethylammonium bromide (C10) is about 70 mM
- dodecyl CMC of trimethylammonium chloride (C12) is about 20 mM
- CMC of dodecyltrimethylammonium bromide (C12, DTAB) is about 16 mM
- CMC of tetradecyltrimethylammonium chloride (C14, TTAC) is about 4.5 mM.
- Tetradecyltrimethylammonium bromide (C14, TTAB) has a CMC of about 5 mM
- hexadecyltrimethylammonium chloride (C16, CTAC) has a CMC of about 1 CMC of hexadecyltrimethylammonium bromide (C16) is about 1 mM
- CMC of octadecyltrimethylammonium chloride (C18, STAC) is about 0.3 mM
- CMC of octadecyltrimethylammonium bromide (C18, STAB) Is about 0.3 mM (for example, J. PHYS. COLLIDE. CHEM., 52, 130 (1948), J.
- the CMC of 1-dodecylpyridinium bromide (C12) is about 12 mM
- the CMC of 1-dodecylpyridinium chloride (C12, 1-DPC) is about 14 mM
- 1-tetradecylpyridinium bromide (C14) CMC is about 2.9 mM
- CMC of 1-hexadecylpyridinium chloride (C16,1-CPC) is about 0.6 mM
- CMC of 1-hexadecylpyridinium bromide (C16,1-CPB) is about 0
- N-cetyl-4-methylpyridinium chloride (C16, 4Me-1-CPC) has a CMC of about 1.9 mM
- 1-octadecylpyridinium bromide has a CMC of about 0.6 mM
- the CMC of octadecylpyridinium chloride is about 0.24
- the CMC of benzyldodecyldimethylammonium chloride is about 2.8 mM
- the CMC of benzyltetradecyldimethylammonium chloride (C14, BDTAC) is about 0.37 mM
- the CMC of benzylcetyldimethylammonium chloride (C16, BCDAC) is (See, for example, Surfactant Handbook, 131 (1960), J. COLLIDE. INTERFACE. SCI., 22, 430 (1966), J. COLLIDE. SCI., 8, 385 (1953)) .
- nonionic surfactant examples include polyoxyethylene alkyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether and the like.
- Cationic surfactants include, for example, alkyl trimethyl ammonium salts, dialkyl dimethyl ammonium salts, alkyl benzyl dimethyl ammonium salts, pyridinium salts such as alkyl pyridinium salts, phosphonium salts such as alkyl phosphonium salts, imidazolium salts such as alkyl imidazolium. Salts, isoquinonium salts, such as alkylisoquinonium salts.
- Examples of the cationic surfactant of the present invention include quaternary ammonium salts (I), pyridinium salts (II) and phosphonium salts (III) represented by the following general formula.
- R 1 to R 4 may be the same or different and each represents a substituted or unsubstituted C 1 to C 20 alkyl, alkenyl, aryl, or benzyl, and Z ⁇ represents a monovalent anion. .
- R 5 represents a substituted or unsubstituted C 1 -C 20 alkyl
- each Ra may be the same or different, and is a hydrogen atom or a substituted or unsubstituted C 1 -C 20 alkyl, alkenyl.
- Aryl or benzyl n represents an integer of 1 to 5
- Z ⁇ represents a monovalent anion.
- R 6 to R 9 may be the same or different and each represents a substituted or unsubstituted C 1 to C 20 alkyl, alkenyl, aryl, or benzyl, and Z ⁇ represents a monovalent anion. .
- Z ⁇ represents a monovalent anion.
- Examples of the quaternary ammonium salt include octyltrimethylammonium chloride (OTAC), octyltrimethylammonium bromide (OTAB), decyltrimethylammonium chloride, decyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetra Decyltrimethylammonium chloride (TTAC), tetradecyltrimethylammonium bromide (TTAB), hexadecyltrimethylammonium chloride (CTAC), hexadecyltrimethylammonium bromide, octadecyltrimethylammonium chloride, octadecyltrimethylammonium bromide (STAB), eicosyltrimethylammonium chloride , Icosyltrimethylammonium bromide, benzyldodecyldimethylammoni
- Examples of the pyridinium salt include 1-decylpyridinium chloride, 1-decylpyridinium bromide, 1-dodecylpyridinium chloride (1-DPC), 1-dodecylpyridinium bromide, 1-tetradecylpyridinium chloride, 1-tetradecylpyridinium bromide, 1-hexadecylpyridinium chloride (1-CPC), 1-hexadecylpyridinium bromide (1-CPB), N-cetyl-2-methylpyridinium chloride, N-cetyl-3-methylpyridinium chloride, N-cetyl-4- Examples include methylpyridinium chloride (4Me-1-CPC), 1-octadecylpyridinium chloride, 1-octadecylpyridinium bromide, 1-eicosylpyridinium chloride and 1-eicosylpyridinium bromide. It is.
- Examples of the phosphonium salt include tetraethylphosphonium chloride, tetraethylphosphonium bromide, tributylmethylphosphonium chloride, tributylmethylphosphonium bromide, tributylmethylphosphonium iodide, tetrabutylphosphonium chloride, tetrabutylphosphonium bromide, tetra-n-octylphosphonium chloride, tetra N-octylphosphonium bromide, tributyldodecylphosphonium chloride, tributyldodecylphosphonium bromide, tributylhexadecylphosphonium chloride, tributylhexadecylphosphonium bromide (TBCPB), methyltriphenylphosphonium chloride, methyltriphenylphosphonium bromide, methyltriphenylphosphonium io
- Anion paired cationic surfactants Z - is for example Cl -, Br -, I - it may be like.
- anionic surfactant examples include linear alkylbenzene sulfonate, alkyl sulfate, alpha-olefin sulfonate, polyoxyethylene alkyl ether sulfate, ⁇ -sulfo fatty acid ester salt, and alkali metal salt of natural fatty acid. Is mentioned.
- An example of such a surfactant is sodium dodecyl sulfate (SDS).
- amphoteric surfactants examples include alkyl dimethylamine oxide and alkyl carboxybetaine.
- the present invention provides a kit for measuring glycated hemoglobin comprising amadoriase and a surfactant.
- the surfactant can be a nonionic or ionic surfactant.
- the amadoriase and the surfactant can be included in the kit as the same or separate components.
- the surfactant is preferably included in a concentration that does not deactivate the amadoriase.
- a surfactant having a concentration higher than the final concentration at the time of measurement may be used as the surfactant.
- This stock solution is appropriately diluted to prepare a solution used for measurement.
- the kit containing the amadoriase of the present invention and a surfactant may further contain a reagent for measuring ⁇ FVH, a protease or peptidase for cutting out ⁇ FVH, and other known stabilizers and buffer solutions.
- Techniques used in various kits for measuring ⁇ FVH can be appropriately used for the production of a kit containing the amadoriase of the present invention and a surfactant. That is, this invention provides the manufacturing method of the kit containing amadoriase and surfactant including the step which prepares suitable amadoriase and surfactant. At this time, the amadoriase and the surfactant can be prepared as the same or separate components. If amadoriase and surfactant are provided as separate components in the kit, they can be mixed just prior to the ⁇ FVH measurement.
- the amadoriase contained in the kit of the present invention preferably has a residual activity (%) after 5 minutes after adding the surfactant prepared in the kit to a final concentration, compared to the case where it is not added, Preferably 13% or more, more preferably 15% or more, most preferably 19% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more.
- the remaining activity will be described below.
- the amadoriase contained in the kit of the present invention preferably has a final concentration at the time of measurement of 110 ⁇ g / ml or less, for example, 100 ⁇ g / ml or less, 70 ⁇ g / ml or less, or 50 ⁇ g per 0.01% (w / v) surfactant. / Ml or less.
- the surfactant contained in the kit has a final concentration at the time of measurement of 0.01% (w / v) or more, for example, 0.02% (w / v) or more, 0.04% (w / v).
- the final concentration at the time of measurement refers to the concentration at which the component is finally diluted and glycated hemoglobin is measured. Accordingly, in the kit, a stock solution having a concentration higher than the final concentration at the time of measurement may be used.
- the amadoriase contained in the kit of the present invention may be an amadoriase having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 37 or a variant produced based on this and having improved surfactant resistance.
- the mutant is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 99% or more of sequence identity with SEQ ID NO: 1 or SEQ ID NO: 37
- an amino acid sequence in which one to several amino acids are modified or mutated, deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 37 Can have.
- the amadoriase contained in the kit of the present invention includes the genera Upicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Ugenus, Espergillus, Achaetium genus, Thielavia genus, Chaetomium genus, Gelasinospora genus, Microascus genus, Leptosphaeria genus, Ophioborus genus, Pleospora genus, Coniochaetidium genus, Pichia genus Pichia ium genus Agrobacterium genus may be amadoriase or variants thereof of natural such as from Arthrobacter sp.
- such variants include asparagine at position 262 of the amino acid sequence shown in SEQ ID NO: 1 or 3, valine at position 257, glutamic acid at position 253, glutamine at position 337, glutamic acid at position 340, glutamic acid at position 133, and glutamic acid at position 44.
- an amino acid selected from the group consisting of glutamic acid, glycine at position 256, glutamic acid at position 81, aspartic acid at position 129, aspartic acid at position 132, glutamic acid at position 231, aspartic acid at position 232, and glutamic acid at position 249 There may be one or more amino acid substitutions at the position.
- a person skilled in the art can easily check whether a certain amadoriase or a variant thereof can be used in the kit of the present invention, that is, has a desired surfactant resistance, for example, by the test method described later or the evaluation method of Example 7. be able to.
- a buffering agent the term includes one or more buffering agents unless otherwise specified.
- the buffer solution refers to a solution having a buffering action (buffering ability) that keeps the pH of the solution in a certain range, and the buffering agent refers to a substance that imparts a buffering action to the solution.
- the buffering agent is composed of a weak acid and a salt thereof.
- the salt is called a conjugated salt.
- the buffer is composed of phosphoric acid and its potassium salt
- the base compound is phosphoric acid
- this may be referred to as a phosphate buffer for convenience.
- the concentration for a certain buffering agent refers to the concentration for the base compound obtained by adding together the single form of the compound serving as the base of the buffering agent and the form of its conjugate salt.
- concentration of phosphoric acid and its conjugate salt for example, potassium phosphate
- buffers buffer solutions
- those that maintain the residual activity of amadoriase in the presence of a surfactant or reduce the decrease in the residual activity are particularly preferable.
- such a preferable buffer may be particularly referred to as a buffer having an amadoriase stabilizing action or the buffer of the present invention.
- a buffer having an amadoriase stabilizing action or the buffer of the present invention.
- HEPES does not correspond to the buffering agent having an amadoriase stabilizing action of the present invention.
- the buffering agent having an amadoriase stabilizing action of the present invention does not only keep the pH of the solution constant, but also has an action of stabilizing the amadoriase when buffering the pH.
- the amadoriase stabilizing action referred to with respect to the buffer of the present invention means an action of maintaining the residual activity of amadoriase in the presence of a surfactant or reducing a decrease in the residual activity.
- Such an amadoriase stabilizing action includes a residual amadoriase activity of a solution in which a buffer is not added or a solution using a buffer that does not have the amadoriase stabilizing action of the present invention in the presence of a surfactant, and the buffering agent of the present invention. It can be evaluated by comparing the residual amadoriase activity of the solution using.
- buffer examples include, for example, a boric acid buffer containing boric acid and / or a salt thereof, a tris hydrochloric acid buffer, phosphoric acid and / or a salt thereof.
- a potassium phosphate buffer or sodium phosphate buffer an organic acid buffer containing an organic acid buffer and / or a salt thereof
- Buffers such as citrate buffers containing citric acid and / or salts thereof, monocarboxylic acid buffers containing monocarboxylic acid buffers and / or salts thereof, eg acetate buffers containing acetate buffer and / or salts thereof
- buffer examples include ACES (N- (2-acetamido) -2-aminoethanesulfonic acid), BES (N, N-bis (2-hydroxyethyl) -2. -Aminoethanesulfonic acid), Bicin (N, N-bis (2-hydroxyethyl) glycine), Bis-Tris (bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane), CHES (N-cyclohexyl-2- Aminoethanesulfonic acid), EPPS (4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), HEPPSO (N- (hydroxyethyl) Piperazine-N'-2-hydroxypropanesulfonic acid), MES (2- (N-morpholino) ethanesulfonic acid),
- n may be 0, 1, 2 or 3 and R 10 may independently be H, OH, —CH 2 OH or —COOH] and / or a salt thereof
- buffering agents comprising Further, a phthalic acid buffer containing phthalic acid and / or its salt, a maleic acid buffer containing maleic acid and / or its salt, a fumaric acid buffer containing fumaric acid and / or its salt, glutaric acid and / or its Glutarate buffer containing salt, citraconic acid buffer containing citraconic acid and / or salt thereof, mesaconic acid buffer containing mesaconic acid and / or salt thereof, malonic acid buffer containing malonic acid and / or salt thereof, Tartrate buffer containing tartaric acid and / or salt thereof, succinate buffer containing succinic acid and / or salt thereof, adipate buffer containing adipic acid and / or salt thereof, apple containing malic acid and / or
- buffers that have the amadoriase stabilizing action of the present invention.
- Preferred buffers having an amadoriase stabilizing action of the present invention include phosphate buffer, ACES buffer, citrate buffer, malate buffer, acetate buffer, maleate buffer, citraconic acid buffer, malonate buffer Agents, glutaric acid buffers, tartaric acid buffers, and buffers of formula (IV) such as, but not limited to, MES buffers, MOPS buffers, and MOPSO buffers. Of these, only one type may be applied, or two or more types may be used.
- action of this invention in combination with substances (for example, buffer agent which does not have an amadoriase stabilization effect
- the salt include, but are not limited to, sodium salts, potassium salts, magnesium salts, calcium salts, and ammonium salts of the base compound.
- the buffer of the present invention can be used in the kit or composition of the present invention at an appropriate concentration.
- the amount of the buffer of the present invention added to the kit or composition of the present invention can be calculated based on the final concentration in the measurement solution.
- the final concentration of the buffer of the present invention in the measurement solution is preferably sufficient to buffer pH changes that may occur in the measurement solution.
- the final concentration of the buffer of the present invention in the measurement solution is such that the residual activity of the amadoriase in the solution containing the surfactant is 20% or more, preferably 40% or more, preferably 60% or more, preferably 80 The concentration is at least%.
- the final concentration of the buffer of the present invention is, for example, 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, such as 50 mM or more, 1 M or less, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, 100 mM or less, such as 1 mM to 1 M, It can be 5 mM to 500 mM, 10 mM to 300 mM, for example 50 mM to 100 mM.
- a phosphate buffer When a phosphate buffer is used as the buffer having an amadoriase stabilizing action of the present invention, its concentration can be 50 mM to 500 mM, such as 50 mM to 300 mM, preferably 100 mM to 300 mM.
- a citrate buffer, malic acid buffer, maleic acid, citraconic acid buffer, malonic acid buffer, glutaric acid buffer, or tartaric acid buffer is used as the buffer of the present invention, the concentration is 5 mM to 500 mM, preferably Can be 10 mM to 200 mM, such as 10 mM to 100 mM.
- the concentration is 10 mM to 500 mM, such as 100 mM to 500 mM, such as 150 mM to 300 mM. It can be.
- an ACES buffer is used as the buffer of the present invention, its concentration can be 200 mM to 1 M, such as 200 mM to 500 mM.
- a plurality of buffering agents may be combined as the buffering agent of the present invention. It should be noted that the amount of the buffer of the present invention used in the composition may vary depending on the amount of the stabilizer when a stabilizer is also added to the composition.
- a stabilizer for maintaining the residual activity of amadoriase in the presence of a surfactant or reducing the decrease in the residual activity may be appropriately added to the kit or composition of the present invention.
- a stabilizer refers to a substance that maintains the residual activity of amadoriase in the presence of a surfactant or reduces the decrease in residual activity.
- this expression when referring to a stabilizer, this expression includes one or more stabilizers unless otherwise specified.
- Examples of the stabilizer (stabilizer) included in the kit or composition of the present invention include phosphoric acid, tricarboxylic acid (for example, citric acid), and dicarboxylic acid (for example, malic acid, maleic acid, citraconic acid, malonic acid). , Glutaric acid, tartaric acid), monocarboxylic acids (for example, acetic acid), compounds represented by formula (IV) (for example, MES, MOPS, MOPSO), ammonium sulfate, salts thereof, and any combinations thereof.
- phosphoric acid for example, citric acid
- dicarboxylic acid for example, malic acid, maleic acid, citraconic acid, malonic acid
- monocarboxylic acids for example, acetic acid
- compounds represented by formula (IV) for example, MES, MOPS, MOPSO
- ammonium sulfate for example, MES, MOPS, MOPSO
- the stabilizer of the present invention can be used in the kit or composition of the present invention at an appropriate concentration.
- the amount of the stabilizer added to the kit or composition of the present invention is calculated based on the final concentration in the measurement solution.
- the amount of stabilizer added is 35% or more, 37.5% or more, preferably 40% or more, 45% or more, 50% or more, 55% of the residual activity of amadoriase in a solution containing a surfactant.
- the amount is preferably 60% or more, 65% or more, 70% or more, 75% or more, preferably 80% or more, 85% or more, 90% or more, or 95% or more.
- the stabilizer of the present invention is, for example, a kit such that the final concentration in the measurement solution is 0.1 mM to 100 mM, 0.2 mM to 100 mM, 0.5 mM to 50 mM, 1 mM to 30 mM, 2 mM to 30 mM, 5 mM to 20 mM, or 10 mM to 20 mM. Or it may be added to the composition.
- the amount of stabilizer can vary depending on the amount of the buffer. For example, when a stabilizer is added, in order to prevent a change in pH, the type and amount of a buffering agent to be added may be appropriately selected and adjusted, or the pH of the stabilizer solution may be appropriately adjusted. .
- buffering agents of the present invention particularly for phosphate buffering agents, citrate buffering agents, and MES buffering agents, a concentration that exhibits a buffering ability to keep the pH of the solution constant, that is, for example, 100 mM for phosphate buffering agents, When citrate buffer was used at 50 mM, for example, and MES buffer was used at 150 mM, for example, an amadoriase stabilizing effect was observed.
- This stabilizing effect is lower than the concentration at which phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or its sodium salt effectively exert a buffering effect, specifically Even 5 mM was observed for phosphoric acid, 0.5 mM for citric acid, and 20 mM for MES. From this, phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or its sodium salt, in addition to the amadoriase stabilizing action as the buffer of the present invention, are separately amadoriase. It was confirmed that there is a stabilizing action that leaves the activity.
- amadoriase stabilizing action of the buffer of the present invention for convenience, such an action may be referred to as the amadoriase stabilizing action of the stabilizer of the present invention. Therefore, phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or its sodium salt have the amadoriase stabilizing action of the buffer of the present invention, and the stabilizer of the present invention Has an amadoriase stabilizing effect. In other words, phosphoric acid and / or its potassium salt, citric acid and / or its sodium salt, and MES and / or its sodium salt fall under the buffer of the present invention, while the stabilizer of the present invention. This also applies.
- maleic acid, citraconic acid, malonic acid, glutaric acid, tartaric acid, MOPS, and MOPSO which have an amadoriase stabilizing action
- kit or composition of the present invention contains amadoriase, a surfactant, a stabilizer and / or a buffer
- these can be added to the kit or composition in any order.
- a stabilizer and / or a buffering agent if included is added first, and a surfactant is added afterwards to reduce the residual activity of amadoriase. Can be reduced.
- amadoriase of the present invention obtained by the means as described above is characterized in that the resistance to a surfactant is improved compared to that before modification as a result of mutation in the amino acid sequence due to genetic modification or the like.
- the modified amadoriase is a predetermined surfactant under the reaction conditions described in the activity measurement method and surfactant resistance evaluation method described herein.
- Treatment for example, 0.01% (w / v) hexadecyltrimethylammonium chloride (hereinafter referred to as “CTAC”) is added, and the remaining activity (%) after 5 minutes at 30 ° C. is improved. It is characterized by.
- the residual activity (%) is a percentage (%) of the ratio of the activity after the surfactant treatment when the activity before the surfactant treatment is 100. In the present specification, when the concentration of the surfactant is described as a percentage, this means% (w / v) unless otherwise specified.
- the degree of improvement in the residual activity (%) of the modified amadoriase of the present invention is not limited, for example, the residual activity (%) after performing the surfactant treatment on the amadoriase before and after introducing the mutation of the present invention is preferable. Is 13% or more, more preferably 15% or more, most preferably 19% or more, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% %, 95% or 99% or more of the modified amadoriase is included in the present invention.
- 2% or more, preferably 9% or more, and most preferably 19% or more in the numerical comparison between the residual activities (%) after the surfactant treatment of the amadoriase before and after introducing the mutation of the present invention 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more or 99% or more Amadoriase is encompassed by the present invention.
- the amadoriase before introducing the mutation of the present invention when the amadoriase before introducing the mutation of the present invention is treated with a surfactant, all the activity may be lost.
- a standard amadoriase whose activity is not lost even by treatment with a surfactant is used.
- the residual activity after the surfactant treatment may be compared with the residual activity of the amadoriase after the mutation introduction after the surfactant treatment.
- the relative evaluation results differ depending on the degree of surfactant resistance of the amadoriase before mutagenesis. It is difficult to evaluate the absolute surfactant resistance of each mutant by comparing only However, by following the conditions of the examples in the present invention, it is possible to absolutely evaluate the surfactant resistance of each variant.
- the amadoriase of the present invention produced by the Escherichia coli JM109 (pKK223-3-CFP-T7 / 253K) strain included in the present invention is mixed with 0.01% CTAC and subjected to 30 ° C. for 5 minutes.
- the residual activity of CFP-T7 which is an amadoriase before introducing the mutation of the present invention, is 69.9%, whereas the residual activity of the amadoriase after the mutation of the present invention exceeds 72%.
- coli JM109 (pKK223-3-CFP-D7) is mixed with 0.04% CTAC and subjected to 5 minutes at 30 ° C., the mutation of the present invention is introduced.
- the residual activity of CFP-D which is an amadoriase before the introduction, is 12.7%, whereas the residual activity of the amadoriase after mutagenesis of the present invention exceeds 15%.
- the amadoriase with improved surfactant resistance significantly improves the storage stability in the enzyme-containing product and the like, and also uses a strong surfactant to improve the protease degradation efficiency of HbA1c and increase the measurement sensitivity. Since it is stable in some cases, it is very advantageous in industry.
- Method for measuring amadoriase activity Various methods can be used as a method for measuring the activity of amadoriase. As an example, a method for measuring amadoriase activity used in the present invention will be described below.
- Examples of the method for measuring the enzyme activity of amadoriase in the present invention include a method for measuring the amount of hydrogen peroxide produced by the enzyme reaction and a method for measuring the amount of oxygen consumed by the enzyme reaction.
- a method for measuring the amount of hydrogen peroxide will be described.
- fructosyl valine is used as a substrate for the measurement of amadoriase activity in the present invention unless otherwise specified.
- the enzyme titer is defined as 1 U for the amount of enzyme that produces 1 ⁇ mol of hydrogen peroxide per minute when measured using fructosyl valine as a substrate.
- Glycated amino acids such as fructosyl valine and glycated peptides such as fructosyl valyl histidine can be synthesized and purified based on the method of Sakagami et al. (See JP 2001-95598 A).
- Reagent 1 POD-4-AA solution 4.0 kU peroxidase (Kikkoman), 100 mg 4-aminoantipyrine (Tokyo Chemical Industry) 0.1M potassium phosphate buffer Dissolve in (pH 7.0) and make up to 1 L.
- Reagent 2 TOOS solution 500 mg of TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium, manufactured by Dojin Chemical Co., Ltd.) is dissolved in ion-exchanged water and fixed to 100 ml. Yes.
- Reagent 3 Substrate solution (150 mM; final concentration 5 mM) Dissolve 417 mg of fructosyl valine in ion-exchanged water to a constant volume of 10 ml.
- Activity (U / ml) ⁇ ( ⁇ As ⁇ A0) ⁇ 3.0 ⁇ df ⁇ ⁇ (39.2 ⁇ 0.5 ⁇ 0.1) ⁇ As: change in absorbance per minute of reaction solution ⁇ A 0 : change in absorbance per minute of control solution 39.2: millimolar extinction coefficient of quinoneimine dye produced by reaction (mM ⁇ 1 ⁇ cm ⁇ 1 ) 0.5: mol number of quinoneimine dye produced by 1 mol of hydrogen peroxide df: dilution factor
- the crude Amadoriase enzyme solution or the purified Amadoriase preparation is diluted with 30 mM MES / 21 mM Tris buffer (pH 6.5) so as to be about 1.0 U / ml, and CTAC (for example, manufactured by Tokyo Chemical Industry Co., Ltd.) is terminated. After adding to a concentration of 0.01% (w / v) or 0.04%, warm at 30 ° C. for 5 minutes. After warming, the solution was diluted 2-fold with 10 mM phosphate buffer (pH 7.0) containing 0.15% BSA.
- the enzyme activity of the sample before the surfactant treatment and after the surfactant treatment was measured using the above method, and the ratio of the activity after the surfactant treatment when the activity before the surfactant treatment was taken as 100, that is, Surfactant resistance is evaluated by determining the residual activity (%).
- the residual activity of amadoriase is measured using various buffers instead of the 30 mM MES / 21 mM Tris buffer, thereby evaluating the contribution of the buffer to the residual amadoriase activity. be able to.
- 30 mM MES / 21 mM Tris buffer pH 6.5
- phosphate buffer pH 7.0
- citrate buffer pH 6.0
- HEPES buffer pH 7.0
- ACES buffer pH 7.0
- the effect of the stabilizer can be evaluated by further adding various stabilizers and measuring the residual activity of amadoriase.
- the stabilizer to be evaluated is a compound that also has a buffering action
- the amadoriase stabilizing action that does not depend on the contribution of the buffering action of the compound to the residual amadoriase activity, it has an amadoriase stabilizing action.
- Use a buffer that is not used at a concentration sufficient to confer buffering capacity on the solution eg, use HEPES (pH 7.0) at 500 mM
- HEPES pH 7.0
- the concentration sufficient to impart buffering capacity to the solution means that the pH is kept within a certain range (for example, pH 5 to 10, pH 6 to 8) without causing pH fluctuation due to other reagents added to the solution. Refers to the concentration.
- the concentration that is not sufficient to impart buffering capacity to a solution refers to a concentration at which pH changes when a reagent is added to the solution and the pH deviates from a certain range. These concentrations vary depending on the type and amount of other reagents added to the solution, but those skilled in the art can appropriately determine the concentrations by conventional methods. Other conditions and procedures may be the same as those in the surfactant resistance measurement method described above.
- the stabilizer and the buffer are appropriately concentrated in a solution containing the amadoriase of the present invention and a surfactant. It can be added while adjusting, and the residual activity of amadoriase can be measured. Other conditions and procedures may be the same as those in the surfactant resistance measurement method described above.
- Example 1 (Surfactant resistance improvement type mutation) (1) Preparation of recombinant plasmid pKK223-3-CFP-T7 DNA Escherichia coli JM109 (pKK223-3-CFP-T7) strain (International Publication 2007) having a recombinant plasmid containing the CFP-T7 gene (SEQ ID NO: 2) No. 1/25779) to LB-amp medium [1% (W / V) bactotryptone, 0.5% (W / V) peptone, 0.5% (W / V) NaCl, 50 ⁇ g / ml Ampicillin] Inoculated into 2.5 ml and cultured with shaking at 37 ° C. for 20 hours to obtain a culture.
- the culture was collected by centrifugation at 7,000 rpm for 5 minutes to obtain bacterial cells.
- the recombinant plasmid pKK223-3-CFP-T7 was extracted from the cells using QIAGEN tip-100 (Qiagen) and purified, and the recombinant plasmid pKK223-3-CFP-T7 DNA2. 5 ⁇ g was obtained.
- a part of the reaction solution was electrophoresed on a 1.0% agarose gel, and it was confirmed that about 6,000 bp of DNA was specifically amplified.
- the DNA thus obtained was treated with the restriction enzyme DpnI (NEW ENGLAND, manufactured by BIOLABS) to cleave the remaining template DNA, and then transformed into E. coli JM109 and developed on an LB-amp agar medium.
- the grown colonies were inoculated into LB-amp medium and cultured with shaking, and plasmid DNA was isolated by the same method as in (1) above.
- the base sequence of DNA encoding amadoriase in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl genetic analyzer (manufactured by Life Technologies), and as a result, position 262 of the amino acid sequence described in SEQ ID NO: 1 was determined.
- a recombinant plasmid (pKK223-3-CFP-T7-262H) encoding a modified amadoriase in which asparagine was replaced with histidine was obtained.
- the synthetic plasmid pKK223-3-CFP-T7 DNA was used as a template, and the synthetic oligonucleotides KOD, -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-257C) encoding a modified amadoriase in which the valine at position 257 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with cysteine was obtained.
- a recombinant plasmid (pKK223-3-CFP-T7-257S) encoding a modified amadoriase in which the valine at position 257 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with serine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, and the synthetic oligonucleotides SEQ ID NO: 8, 10 -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-257T) encoding a modified amadoriase in which the valine at position 257 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with threonine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides SEQ ID NO: 11, 12 and KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-253K) encoding a modified amadoriase in which glutamic acid at position 253 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- a synthetic plasmid of SEQ ID NOS: 12 and 13, KOD using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-253R) encoding a modified amadoriase in which glutamic acid at position 253 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with arginine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides SEQ ID NO: 14 and 15, KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-337K) encoding a modified amadoriase in which glutamine at position 337 of the amino acid sequence shown in SEQ ID NO: 1 was substituted with lysine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, and the synthetic oligonucleotides SEQ ID NO: 16, 17 and KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-340P) encoding a modified amadoriase in which glutamic acid at position 340 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with proline was obtained.
- the synthetic plasmids of SEQ ID NOS: 18 and 19, KOD using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-133A) encoding a modified amadoriase in which glutamic acid at position 133 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with alanine was obtained.
- the synthetic plasmids of SEQ ID NOS: 19 and 20, KOD using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-133M) encoding a modified amadoriase in which glutamic acid at position 133 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with methionine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides SEQ ID NO: 19, 21 and KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-133K) encoding a modified amadoriase in which glutamic acid at position 133 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides of SEQ ID NOS: 22 and 23, KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-44P) encoding a modified amadoriase in which glutamic acid at position 44 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with proline was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, and the synthetic oligonucleotides KOD, SEQ ID NO: 24, -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-256K) encoding a modified amadoriase in which glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- the synthetic plasmids of SEQ ID NOS: 8 and 25, KOD using the recombinant plasmid pKK223-3-CFP-T7 DNA as a template -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-256R) encoding a modified amadoriase in which glycine at position 256 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with arginine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides SEQ ID NO: 26 and 27, KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-81K) encoding a modified amadoriase in which glutamic acid at position 81 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- a recombinant plasmid (pKK223-3-CFP-T7-129K) encoding a modified amadoriase in which the aspartic acid at position 129 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with lysine was obtained.
- a recombinant plasmid (pKK223-3-CFP-T7-132K) encoding a modified amadoriase in which the aspartic acid at position 132 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with lysine was obtained.
- a recombinant plasmid (pKK223-3-CFP-T7-231K) encoding a modified amadoriase in which glutamic acid at position 231 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- a recombinant plasmid (pKK223-3-CFP-T7-232K) encoding a modified amadoriase in which the aspartic acid at position 232 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- the recombinant plasmid pKK223-3-CFP-T7 DNA was used as a template, the synthetic oligonucleotides SEQ ID NO: 52 and 53, KOD -Plus- (manufactured by Toyobo Co., Ltd.) was used for PCR reaction, transformation of E. coli JM109, and determination of the DNA sequence encoding amadoriase in the plasmid DNA retained by the growing colonies under the same conditions as described above.
- a recombinant plasmid (pKK223-3-CFP-T7-249K) encoding a modified amadoriase in which glutamic acid at position 249 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine was obtained.
- Arginine is glutamine at position 337
- glutamine at position 340 is proline
- glutamine at position 44 is proline
- glutamate at position 133 is alanine
- methionine lysine
- glycine at position 256 is lysine
- Glutamic acid at position 81 is lysine
- Aspartic acid at position 129 is lysine
- Aspartic acid at position 132 is lysine
- Glutamic acid at position 231 is lysine
- 232 Aspartic acid is lysine
- Glutamic acid at position 249 is lysine
- Residual activity is improved both over 72%, and improved over 79% be significant, was improved over 89% but more pronounced. Therefore, it was confirmed that each of these mutation points is a mutation point that improves the surfactant resistance of amadoriase.
- SEQ ID NO: 37 is the amino acid sequence of Curvularia clavata-derived ketoamine oxidase (hereinafter referred to as CcFX) (International Publication No. 2004/104203).
- the gene encoding the amino acid sequence of SEQ ID NO: 37 was obtained by total synthesis of cDNA by the total synthesis of PCR using a conventional gene fragment (including the stop codon TAA). At this time, an EcoRI site and a HindIII site were added to the 5 ′ end and 3 ′ end of SEQ ID NO: 55, respectively.
- the total amino acid sequence predicted from the cloned gene sequence was identical to the sequence of CcFX in FIG.
- the following procedure was performed. First, the gene synthesized above is treated with two types of restriction enzymes, EcoRI site and HindIII site (Takara Bio), and inserted into the EcoRI-HindIII site of pKK-223-3 Vector (GE Healthcare).
- CcFX a surfactant resistance improving mutation
- 129 of CcFX which is a position corresponding to positions 129, 132, 133, 231, 232, 249, 253, 256, and 337 in the genus Coniochaeta (CFP-T7) Mutations were introduced at positions 132, 133, 229, 230, 247, 251, 254, and 335.
- a recombinant plasmid containing the CcFX gene (SEQ ID NO: 55) is used as a starting plasmid, and the Escherichia coli JM109 (pKK223-3-CcFX) strain having the same is prepared in the same manner as described in (1) and (2) above. Various mutants were prepared. The sequences of the primers used for mutagenesis are as shown in SEQ ID NOs: 56 to 74. Next, modified amadoriase was produced by the procedure described in (3) above.
- amadoriase was diluted with 20 mM potassium phosphate buffer (pH 7.0), and 0.01% CTAC The surfactant resistance of various modified amadoriases was evaluated. The results are shown in Table 1-2. In addition, even if the activity measurement was performed again 30 minutes after diluting the heated sample twice with the BSA solution, it was confirmed that there was no change in the activity value.
- the residual activity of CcFX was 27.4% under the conditions of this example.
- the residual activity is improved to 34% or more, markedly improved to 56% or more, and more remarkable 64%. It improved to more than%.
- Coniochaeta genus amadoriase and Curvularia clavata ketoamine oxidase have about 80% amino acid sequence identity. Therefore, surfactant resistance is also obtained by introducing mutations at positions corresponding to the above positions of Amadoriase derived from Coniochaeta genus or other species having 80% or more amino acid sequence identity with Curvularia clavata-derived ketoamine oxidase. It is thought that it can be improved.
- surfactant resistance was improved by substitution with lysine or arginine at positions 251 and 335 of CcFX. From this, CcFX's 81-position, 129-position, 132-position, 229-position, 230-position, 247-position, 251-254, and 335-position are interfaced by substitution with lysine or arginine, which is a basic amino acid residue. It is thought that the active agent resistance is improved. The same applies to other various amadoriases.
- surfactant resistance was improved by substitution with alanine or methionine at position 133 of CFP-T7 and substitution with alanine at position 133 of CcFX.
- substitution of CFP-T7 at position 133 and CcFX at position 133 with hydrophobic amino acid residues such as alanine, methionine, valine, isoleucine, leucine, phenylalanine, tryptophan, and proline improves surfactant resistance. It is done. The same applies to other various amadoriases.
- the mechanism by which the mutant amadoriase of the present invention becomes resistant to a surfactant is considered to be as follows, for example.
- replacing the acidic amino acid of amadoriase with a hydrophobic amino acid or basic amino acid reduces the affinity between the amadoriase and the cationic surfactant and protects the amadoriase from the modifying action of the surfactant. It is done.
- lysine or arginine which is a basic amino acid residue
- the basic amino acid residue is repelled from the cationic surfactant, so that the amadoriase is further protected from the modifying action of the surfactant. .
- Example 2 (Accumulation of surfactant-resistant mutations) Based on the knowledge of the surfactant resistance-enhancing mutation found in Example 1, by combining these and accumulating mutations, the aim is to obtain an amadoriase with further enhanced surfactant resistance. An attempt was made to accumulate surfactant resistance-enhancing mutations by producing (double mutant, triple mutant, quadruple mutant, five-fold mutant, six-fold mutant, and seven-fold mutant).
- SEQ ID NO: 3 is the amino acid sequence of the Amadoriase derived from the genus Coniochaeta (hereinafter referred to as “Aminodriase”) into which a substrate specificity-improving mutation (E98A) and a thermal stability-improving mutation (F43Y, G184D, deletion of three amino acid residues at the carboxyl terminus) have been introduced.
- Aminodriase a substrate specificity-improving mutation
- F43Y, G184D thermal stability-improving mutation
- Surfactant resistance-enhancing mutations were accumulated using the plasmid DNA with the CFP-D gene inserted into the pKK223-3 vector as a template.
- Synthetic oligonucleotide (SEQ ID NO: 5, 6, 7, 16, 17, 18, 19, 23, 28, 29, 30, 31, 32, 33, 46, 47, 50, 51, 52, 54), KOD-Plus -Using Toyobo Co., Ltd., PCR reaction, transformation of Escherichia coli JM109 strain, and DNA sequencing of amadoriase in plasmid DNA held by growing colonies were performed under the same conditions as in (2) above. .
- pKK223-3-CFP-D1 is a mutant in which glutamic acid at position 44 is replaced with proline; double mutant in which glutamic acid at position 44 is replaced with proline and glutamic acid at position 340 is replaced with proline.
- PKK223-3-CFP-D2; pKK2233-3 which is a triple mutant in which glutamic acid at position 44 is replaced with proline, asparagine at position 262 is replaced with histidine, and glutamic acid at position 340 is replaced with proline.
- CFP-D3 a quadruple mutant in which glutamic acid at position 44 is replaced with proline, valine at position 257 is replaced with cysteine, asparagine at position 262 is replaced with histidine, and glutamic acid at position 340 is replaced with proline.
- pKK223-3-CFP-D5 / 129K the glutamic acid at position 44 is replaced with proline, the glutamic acid at position 133 is replaced with alanine, the glutamic acid at position 253 is replaced with lysine, and the valine at position 257 is replaced with cysteine;
- Asparagine at position 262 is replaced with histidine, PKK223-3-CFP-D6, which is a six-fold mutant in which glutamic acid at position 340 is replaced with proline;
- E. coli JM109 strain was transformed under the same conditions as described above, and E. coli JM109 (pKK223-3-CFP-D) strain, E. coli JM109 (pKK223-3-CFP-D1) strain, E. coli JM109 (pKK223-3-CFP) were transformed.
- -D2 strain E. coli JM109 (pKK223-3-CFP-D3) strain
- E. coli JM109 (pKK223-3-CFP-D4) strain E. coli JM109 (pKK223-3-CFP-D4 / 232K) strain
- E. coli JM109 (pKK223) -3-CFP-D4 / 249K) E.
- E. coli JM109 (pKK223-3-CFP-D5), E. coli JM109 (pKK223-3-CFP-D5 / 129K), E. coli JM109 (pKK223-3-CFP-D6) Strain E. coli JM109 (pKK223-3-C To obtain a P-D7) strain.
- E. coli JM109 (pKK223-3-CFP-T7) strain, E. coli JM109 (pKK223-3-CFP-D) strain, E. coli JM109 (pKK223-3-CFP-) having the ability to produce modified amadoriase obtained as described above.
- E. coli JM109 (pKK223-3-CFP-D4 / 249K), E. coli JM109 (pKK223-3-CFP-D5), E. coli JM109 (pKK223-3-CFP-D5 / 129K) Escherichia coli JM109 (pKK223-3-CF D6) strain, E. coli JM109 (pKK223-3-CFP-D7) strain was cultured by the above method to prepare a crude enzyme solution 1.5ml of various modified amadoriase.
- the various multiple mutants prepared by combining the various single mutations confirmed in Example 1, all the residual activities were remarkably improved. Specifically, the residual activity in the double mutant in which the glutamic acid at position 44 was replaced with proline and the glutamic acid at position 340 was replaced with proline was 37.1%, which was improved compared to CFP-T7. . Furthermore, the residual activity in the triple mutant in which asparagine at position 262 was replaced with histidine in this mutation was 51.4%, which was further improved as compared with CFP-T7. Furthermore, the residual activity in the quadruple mutant in which valine at position 257 was replaced with cysteine in this mutation was 60.7%, which was significantly improved as compared with CFP-T7.
- the residual activity in the quintuple mutant in which glutamic acid at position 253 was substituted with lysine for this mutation was 95.6%.
- the sixfold mutant in which glutamic acid at position 133 was substituted with alanine for this mutation was 99.2%, and the residual activity in the sevenfold mutant in which glutamine at position 337 was replaced with lysine was 100% in this mutation, which was significantly improved compared to CFP-T7. There was almost no inactivation of amadoriase.
- the residual activity in the quintuple mutant in which the aspartic acid at position 232 of the quadruple mutant CFP-D4 is further substituted with lysine is 66.2%, and the glutamic acid at position 249 in the quadruple mutant CFP-D4 is as follows.
- the remaining activity in the quintuple mutant further substituted with lysine is 91.0%, and the remaining activity in the six-fold mutant in which the aspartic acid at position 129 of the quintuple mutant CFP-D5 is further substituted with lysine was 98.1%, which was markedly improved compared to CFP-T7, and there was almost no inactivation of amadoriase by CTAC.
- the surfactant resistance-enhancing mutation was accumulated.
- the procedure was the same as above except that the CcFX gene was used instead of the CFP-D gene.
- Plasmid DNA retained by PCR reaction, transformation of Escherichia coli JM109 strain and growing colonies using synthetic oligonucleotides (SEQ ID NO: 72, 73) and KOD-Plus- (Toyobo Co., Ltd.) under the same conditions as in (2) above.
- the nucleotide sequence of the DNA encoding the amadoriase was determined.
- pKK223-3-CcFX / 132K / 335K in which aspartic acid at position 132 is substituted with lysine and threonine at position 335 is substituted with lysine; glutamic acid at position 133 is substituted with alanine, and position 335 PKK223-3-CcFX / 133A / 335K in which threonine is replaced with lysine; pKK2233-3 in which glutamic acid at position 229 is replaced with lysine and threonine at position 335 is replaced with lysine CcFX / 229K / 335K; and pKK223-3-CcFX / 251K / 335K, a mutant in which glutamic acid at position 251 was replaced with lysine and threonine at position 335 was replaced with lysine.
- Escherichia coli JM109 strain was transformed under the same conditions as described above, and the transformed strain was cultured by the above-mentioned method to prepare 1.5 ml of crude enzyme solutions of various modified amadoriases.
- the surfactant resistance measurement method described in (4) above was used. Diluted and mixed with 0.01% CTAC, the surfactant resistance of various modified amadoriases was evaluated. The results are shown in Table 2-2. In addition, even if the activity measurement was performed again 30 minutes after diluting the heated sample twice with the BSA solution, it was confirmed that there was no change in the activity value.
- the residual activity of CcFX was 27.4% under the conditions of this example, while various single mutations confirmed in Example 1 were combined.
- the residual activity was significantly improved.
- the CcFX double mutants have improved surfactant resistance, and the effects of mutations accumulate regardless of the type of amadoriase enzyme. It was confirmed.
- Example 3-1 (Evaluation for surfactant TTAC) Instead of the surfactant CTAC used in Example 2, the stability of CFP-D was evaluated using tetradecyltrimethylammonium chloride (hereinafter referred to as “TTAC”).
- TTAC tetradecyltrimethylammonium chloride
- the residual activity in the various multiple mutants produced in Example 2 was significantly improved. Specifically, the residual activity in the double mutant in which glutamic acid at position 44 was replaced with proline and glutamic acid at position 340 was replaced with proline was 69.9%, which was improved compared to CFP-D. . Furthermore, the residual activity in the triple mutant in which asparagine at position 262 was replaced with histidine in this mutation was 85.3%, which was further improved as compared with CFP-D. Furthermore, the residual activity in the quadruple mutant in which valine at position 257 was replaced with cysteine in this mutation was 91.1%, which was significantly improved as compared with CFP-D.
- the residual activity in the quintuple mutant in which glutamic acid at position 253 was substituted with lysine for this mutation was 94.9%, and in the six-fold mutant in which glutamic acid at position 133 was substituted with alanine for this mutation.
- the residual activity was 96.4%, and furthermore, the residual activity in the sevenfold mutant in which glutamine at position 337 was replaced with lysine in this mutation was 100%, which was significantly improved compared to CFP-D, and TTAC There was almost no inactivation of amadoriase.
- Example 3-2 Purification of CFP-T7, CFP-D2, CFP-D7 Using the crude enzymes CFP-T7, CFP-D2 and CFP-D7 obtained in Examples 1 and 2, the prepared crude enzyme solution was equilibrated with 20 mM potassium phosphate buffer (pH 8.0). Adsorbed on Sepharose Fast Flow resin (manufactured by GE Healthcare), then washed with 80 ml of the same buffer, and then adsorbed with 20 mM potassium phosphate buffer (pH 8.0) containing 100 mM NaCl. The eluted protein was eluted, and the fraction showing amadoriase activity was collected.
- 20 mM potassium phosphate buffer pH 8.0
- the obtained fraction showing amadoriase activity was concentrated using Amicon Ultra-15, 30K NMWL (Millipore). Thereafter, it is applied to HiLoad 26/60 Superdex 200 pg (manufactured by GE Healthcare) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing 150 mM NaCl, eluted with the same buffer, and shows the amadoriase activity.
- the fraction was collected to obtain purified samples of wild type and modified amadoriase.
- the obtained purified sample was confirmed to be purified to a single band by analysis by SDS-PAGE.
- the activity of CFP-T7 before mutagenesis was greatly reduced by most of the surfactants except when OTAB and OTAC were used as the surfactants.
- the double mutant CFP-D2 showed better surfactant resistance than CFP-T7 for all types of surfactants tested.
- the seven-fold variant CFP-D7 also showed better surfactant resistance than CFP-T7 for all types of surfactants tested, and in most cases the surfactant more than the double variant CFP-D2 Resistance was improved.
- both D2 and D7 showed surfactant resistance regardless of whether the counter ion (Z) was a chloride ion or a bromide ion.
- both D2 and D7 showed resistance not only to ammonium ion surfactants but also to pyridinium ion surfactants and phosphonium ion surfactants. Was shown to be for various cationic surfactants.
- the surfactant-resistant amadoriase of the present invention has a wide range of surfactants regardless of the type of counterion of the surfactant, regardless of the chain length, and regardless of the basic structure of the cationic surfactant. It has been demonstrated to have a resistance spectrum.
- Example 4 Evaluation for surfactant SDS
- SDS sodium dodecyl sulfate
- the residual activity in the various multiple mutants produced in Example 2 was significantly improved. Specifically, the residual activity in the double mutant in which the glutamic acid at position 44 was replaced with proline and the glutamic acid at position 340 was replaced with proline was 19.2%, which was improved compared to CFP-T7. . Furthermore, the residual activity in the triple mutant in which asparagine at position 262 was replaced with histidine in this mutation was 11.3%, which was improved compared to CFP-T7. Furthermore, the residual activity in the quadruple mutant in which valine at position 257 was replaced with cysteine in this mutation was 17.1%, which was improved as compared with CFP-T7.
- Example 5 Measurement of fructosyl peptide sample under surfactant mixing
- CFP-T7 and CFP-D7 purified enzymes obtained in Example 3-2 the following samples were measured.
- the activity values of CFP-T7 and CFP-D7 were determined according to the method described in the method for measuring amadoriase activity using a final concentration of 5 mM ⁇ FVH as a substrate and adjusting to pH 7.0 (reagent 1).
- the amount of change in absorbance of CFP-T7 was 0.150 under 0% CTAC mixing, and CFP ⁇ under 0.005% CTAC mixing.
- the absorbance change amount of T7 was 0.111.
- the change in absorbance of CFP-T7 is 0.077 under 0.01% CTAC mixing, and the change in absorbance of CFP-T7 is 0.031 under 0.02% CTAC mixing.
- CFP-D7 the change in absorbance of CFP-D7 is 0.140 under 0.02% CTAC mixing, and that of CFP-D7 under 0.2% CTAC mixing.
- the amount of change in absorbance was 0.069. That is, the amount of change in absorbance decreased as CFP-T7 mixed with CTAC, whereas the decrease in amount of change after absorption was suppressed in CFP-D7, and the absorbance was further increased in the presence of 0.1% or more of CTAC. The amount of change became constant. Further, although the amount of decrease in absorbance change was large up to the critical micelle concentration of CTAC of 1.3 mM (0.04%), the change in influence on amadoriase was small at concentrations higher than the critical micelle concentration. Therefore, it was found that CFP-D7 exists stably even under a high CTAC concentration mixture, and ⁇ FVH can be measured.
- Example 6 Quantitative determination of fructosyl peptide sample with surfactant
- CFP-T7 and CFP-D7 the linearity of ⁇ FVH measured values was compared in the range of 0.5 to 3 ⁇ M under CTAC mixing.
- CFP-T7 is mixed with 0.01% or 0.02% CTAC, and is further 4.2 ⁇ M, 8.4 ⁇ M, 13 ⁇ M, 17 ⁇ M, 21 ⁇ M or 25 ⁇ M ⁇ FVH, ie 0.5 ⁇ M at the final concentration.
- Fig. 4 shows the results of measuring ⁇ FVH.
- Fig. 4 shows the results of measuring ⁇ FVH using CFP-D7 under a mixture of 0.02% CTAC.
- Fig. 5 shows the results of mixing with 0.2% CTAC. The results of measurement of ⁇ FVH using CFP-D7.
- the correlation coefficient of ⁇ FVH of 0.5 ⁇ M to 3.0 ⁇ M with CFP-T7 was as high as 0.924 under 0.01% CTAC mixing. However, the correlation coefficient was as low as 0.625 under 0.02% CTAC mixing. On the other hand, when CFP-D7 was used, the correlation coefficient of 0.5 ⁇ M to 3.0 ⁇ M ⁇ FVH showed a high linearity of 0.965 even under 0.2% CTAC mixing.
- Sunk HbA1c manufactured by Arkray
- HbA1c measurement kit a whole blood sample is reacted with amadoriase in a 416-fold diluted state.
- the HbA1c value in the NGSP value is 6.5%
- the total Hb concentration is 141 to 150 g / l
- the blood sample is measured by dilution 416 times
- the concentration of ⁇ FVH cut out by the protease is 0.50 to 0.53 ⁇ M.
- the boundary value of the HbA1c value for actual diabetes diagnosis is NGSP value of 6.5%. Therefore, it can be said that CFP-D7 can be fully utilized in actual HbA1c measurement, and that measurement sensitivity can be increased by using a strong surfactant, for example, CTAC.
- Example 7 Evaluation of surfactant resistance of various amadoriases
- a surfactant preferably an ionic surfactant
- the surfactant derived from the genus Coniochaeta is modified as described above. Improved resistance.
- amadoriase other than Coniochaeta genus amadoriase can measure HbA1c in the presence of an ionic surfactant.
- ⁇ FVH was measured by combining fructosyl peptide oxidase derived from Phaeosphaeria nodorum, ketoamine oxidase derived from Neocosmospora vasinfecta, and an ionic surfactant.
- SEQ ID NO: 40 is the amino acid sequence of Phaeosphaeria nodorum-derived fructosyl peptide oxidase (hereinafter referred to as PnFX) (see Biotechnology and Bioengineering, 106, 358-366, 2058-366).
- PnFX Phaeosphaeria nodorum-derived fructosyl peptide oxidase
- the gene encoding the amino acid sequence of SEQ ID NO: 40 was obtained by total synthesis of cDNA by total synthesis by PCR of gene fragments, which is a standard method.
- the recombinant plasmid pET22b-PnFX was obtained, and the E. coli BL21 (DE3) strain was transformed under the same conditions as above to obtain the E. coli BL21 (DE3) (pET22b-PnFX) strain.
- the Escherichia coli BL21 (DE3) (pET22b-PnFX) strain having the ability to produce PnFX obtained as described above was inoculated into an LB-amp medium supplemented with IPTG so as to have a final concentration of 0.1 mM. For 16 hours. Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 8.0), suspended in the same buffer solution, subjected to ultrasonic disruption, and centrifuged at 20,000 ⁇ g for 10 minutes. Separated to prepare a crude enzyme solution.
- the prepared crude enzyme solution of PnFX was purified according to the method described in the aforementioned non-patent document (Biotechnology and Bioengineering, 106, 358-366, 2010). That is, the crude enzyme solution was fractionated with ammonium sulfate, dialyzed with 10 mM potassium phosphate buffer (pH 8.0), purified by anion exchange chromatography (in this example, using Q Sepharose Fast Flow), and gel Purification was performed by filtration chromatography (HiLoad 26/600 Suprdex 200 was used in this example). The obtained fraction was analyzed by SDS-PAGE to confirm that it had been purified to a purity free from other contaminating proteins, and was used as a purified PnFX preparation.
- Neocosmospora vasinfecta SEQ ID NO: 38 is the amino acid sequence of Neocosmospora vasinfecta-derived ketoamine oxidase (NvFX), and is a recombinant in which a gene encoding the amino acid sequence of SEQ ID NO: 38 (SEQ ID NO: 45) is inserted.
- NvFX Neocosmospora vasinfecta-derived ketoamine oxidase
- the Escherichia coli BL21 (DE3) strain was transformed in the same manner as in Example 1, and the resulting Escherichia coli BL21 (DE3) (pET22b-NvFX) strain was cultured by the above method to prepare a crude enzyme solution of NvFX. .
- the prepared crude enzyme solution was adsorbed on Q Sepharose Fast Flow resin (manufactured by GE Healthcare) equilibrated with 10 mM potassium phosphate buffer (pH 8.0), and then 10 mM potassium phosphate buffer (containing 20 mM NaCl) ( The resin was washed with pH 8.0), and then NvFX adsorbed on the resin was eluted and recovered with 10 mM potassium phosphate buffer (pH 8.0) containing 300 mM NaCl.
- NvFX-containing crude enzyme solution was applied to a HiLoad 26/600 Superdex 200 column equilibrated with 20 mM MES-NaOH buffer (pH 7.0) containing 150 mM NaCl, and NvFX was eluted with the same buffer. Fractions showing fructosyl amino acid oxidase activity (amadoriase activity) were collected. The obtained fraction was analyzed by SDS-PAGE to confirm that it had been purified to a purity free from other contaminating proteins, and was used as a purified sample of NvFX.
- the absorbance change amount of PnFX is 0.173 under 0% CTAC mixture, and the absorbance change amount is 0.0173 under 0.01% CTAC mixture. It was 0.115.
- the change in absorbance of PnFX is 0.084 under 0.02% CTAC mixing, whereas the change in absorbance of CFP-T7 is 0.031 under 0.02% CTAC mixing.
- the change in absorbance of PnFX was 0.026, whereas the change in absorbance of CFP-T7 was 0.005. That is, it can be said that PnFX can measure ⁇ FVH as a substrate in a mixture of CTAC at a high concentration of 0.02% or more.
- Example 8 Evaluation of surfactant resistance of amadoriase in the presence of various buffers
- a surfactant resistance of amadoriase was improved when a buffer other than 30 mM MES / 21 mM Tris buffer (pH 6.5) was used was examined.
- CFP-T7 purified as described above was used as a sample, the final concentration of CTAC was 0.01%, and various buffers, specifically phosphoric acid, were used instead of 30 mM MES / 21 mM Tris buffer (pH 6.5).
- Example 9 Evaluation of surfactant resistance of amadoriase when each stabilizer is added
- Stabilizers include phosphoric acid, tricarboxylic acid (eg citric acid), dicarboxylic acid (eg malic acid, maleic acid, citraconic acid, malonic acid, glutaric acid, tartaric acid), monocarboxylic acid (eg acetic acid), MES , MOPS, MOPSO, and ammonium sulfate were used.
- CHES was used as a comparative example.
- formula (IV) such as ammonium sulfate
- amadoriase CFP-T7 into which the mutation of the present invention was not introduced and the amadoriase CFP-D2 into which the mutation of the present invention was introduced were improved in surfactant resistance.
- the amadoriase stabilizing action of the stabilizer of the present invention is different from the amadoriase stabilizing action of the buffer of the present invention.
- Table 8 when MES is used as the buffer of the present invention, the residual activity of CFP-T7 amadoriase is 14.3% at a concentration of 50 mM, and the residual activity is 17.6% at a concentration of 100 mM. It was confirmed that the residual activity was 62.9% at a concentration of 150 mM.
- amadoriase stabilizing action of the stabilizer of the present invention is a stabilizing action different from the amadoriase stabilizing action of the buffer of the present invention.
- dicarboxylic acids, MOPS, and MOPSO that have shown a stabilizing action are considered to be the same because they can be used as buffering agents.
- the surfactant resistance was also added to PnFX in the same manner as CFP-T7 when phosphoric acid, citric acid, malic acid, acetic acid, MES, and ammonium sulfate were added.
- the improvement effect was shown. Therefore, it can be said that phosphoric acid, tricarboxylic acid, dicarboxylic acid, monocarboxylic acid, MES, and ammonium sulfate are useful as stabilizers for improving the surfactant resistance of a wide variety of amadoriases. Since the amino acid sequence identity between CFP-T7 and PnFX is 75%, it can be said that Amadoriase having 75% amino acid sequence identity with CFP-T7 has the above-mentioned effects.
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Abstract
Description
(i)配列番号1、3、または37に示すアミノ酸配列に1または数個のアミノ酸の欠失、挿入、付加、および/または置換がなされたアミノ酸配列を有し、かつ/又は
(ii)配列番号1、3、または37に示すアミノ酸配列と少なくとも70%の同一性を有するアミノ酸配列を有する、
前記アマドリアーゼ。
(i)262位のアスパラギン、
(ii)257位のバリン、
(iii)249位のグルタミン酸
(iv)253位のグルタミン酸、
(v)337位のグルタミン、
(vi)340位のグルタミン酸、
(vii)232位のアスパラギン酸、
(viii)129位のアスパラギン酸、
(ix)132位のアスパラギン酸、
(x)133位のグルタミン酸、
(xi)44位のグルタミン酸、
(xii)256位のグリシン、
(xiii)231位のグルタミン酸、及び
(xiv)81位のグルタミン酸、
よりなる群から選択されるアミノ酸に対応する位置で1つまたはそれ以上のアミノ酸残基の置換を有する、[1]又は[2]記載のアマドリアーゼ。
以下の(i)から(xiv)の置換:
(i)262位のアスパラギンがヒスチジンに置換されている;
(ii)257位のバリンがシステイン、セリン、トレオニンに置換されている;
(iii)249位のグルタミン酸がリジン、アルギニンに置換されている;
(iv)253位のグルタミン酸がリジン、アルギニンに置換されている;
(v)337位のグルタミンがリジン、アルギニンに置換されている;
(vi)340位のグルタミン酸がプロリンに置換されている;
(vii)232位のアスパラギン酸がリジン、アルギニンに置換されている;
(viii)129位のアスパラギン酸がリジン、アルギニンに置換されている;
(ix)132位のアスパラギン酸がリジン、アルギニンに置換されている;
(x)133位のグルタミン酸がアラニン、メチオニン、リジン、アルギニンに置換されている;
(xi)44位のグルタミン酸がプロリンに置換されている;
(xii)256位のグリシンがリジン、アルギニンに置換されている;
(xiii)231位のグルタミン酸がリジン、アルギニンに置換されている;及び
(xiv)81位のグルタミン酸がリジン、アルギニンに置換されている;
のいずれか1以上を有する、[1]~[3]のいずれかに記載のアマドリアーゼ。
(i)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(ii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(iii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(iv)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および232位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換;
(v)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および249位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換;
(vi)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(vii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および129位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換;
(viii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;並びに
(ix)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、337位のグルタミンに対応する位置のアミノ酸のリジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換
よりなる群から選択されるアミノ酸残基の置換を有する、[1]~[4]のいずれかに記載のアマドリアーゼ。
以下の(i)から(ix)の置換:
(i)247位のグルタミン酸がリジン、アルギニンに置換されている;
(ii)251位のグルタミン酸がリジン、アルギニンに置換されている;
(iii)335位のトレオニンがリジン、アルギニンに置換されている;
(iv)230位のアスパラギン酸がリジン、アルギニンに置換されている;
(v)129位のアスパラギン酸がリジン、アルギニンに置換されている;
(vi)132位のアスパラギン酸がリジン、アルギニンに置換されている;
(vii)133位のグルタミン酸がアラニン、メチオニン、リジン、アルギニンに置換されている;
(viii)254位のアスパラギンがリジン、アルギニンに置換されている;及び
(ix)229位のグルタミン酸がリジン、アルギニンに置換されている;
のいずれか1以上を有する、[1]~[3]のいずれかに記載のアマドリアーゼ。
(i)251位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンの置換;
(ii)132位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;
(iii)133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;並びに
(iv)229位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;
よりなる群から選択されるアミノ酸残基の置換を有する、[1]~[3]および[6]のいずれかに記載のアマドリアーゼ。
(i)[10]記載の宿主細胞を培養する工程;
(ii)宿主細胞に含まれるアマドリアーゼ遺伝子を発現させる工程;及び
(iii)培養物からアマドリアーゼを単離する工程を含む、アマドリアーゼを製造する方法。
(i)界面活性剤を添加してから5分後の残存活性(%)が、添加しない場合と比較して、15%以上残存し、かつ/又は
(ii)終濃度0.04%の界面活性剤の存在下において、発色基質N-(カルボキシメチルアミノカルボニル)-4,4′-ビス(ジメチルアミノ)ジフェニルアミンナトリウム(DA-64)を添加し5分間反応させた後の751nmにおける吸光度と、糖化アミノ酸溶液または糖化ペプチド溶液の代わりにイオン交換水を用いた対照液添加から5分後の751nmにおける吸光度との差が0.006以上となるアマドリアーゼである、
[13]に記載の組成物。
以下の一般式(II)で表されるピリジニウム塩、
以下の一般式(III)で表されるホスホニウム塩、
並びにドデシル硫酸ナトリウムからなる群より選択される1以上のイオン性界面活性剤である、[13]~[16]のいずれかに記載の組成物。
1-デシルピリジニウムクロリド、1-デシルピリジニウムブロミド、1-ドデシルピリジニウムクロリド、1-ドデシルピリジニウムブロミド、1-テトラデシルピリジニウムクロリド、1-テトラデシルピリジニウムブロミド、1-ヘキサデシルピリジニウムクロリド、1-ヘキサデシルピリジニウムブロミド、N-セチル-2-メチルピリジニウムクロリド、N-セチル-2-メチルピリジニウムブロミド、N-セチル-3-メチルピリジニウムクロリド、N-セチル-3-メチルピリジニウムブロミド、N-セチル-4-メチルピリジニウムクロリド、N-セチル-4-メチルピリジニウムブロミド、1-オクタデシルピリジニウムクロリド、1-オクタデシルピリジニウムブロミド、1-エイコシルピリジニウムクロリド及び1-エイコシルピリジニウムブロミド、
テトラエチルホスホニウムクロリド、テトラエチルホスホニウムブロミド、トリブチルメチルホスホニウムクロリド、トリブチルメチルホスホニウムブロミド、トリブチルメチルホスホニウムヨージド、テトラブチルホスホニウムクロリド、テトラブチルホスホニウムブロミド、テトラ-n-オクチルホスホニウムクロリド、テトラ-n-オクチルホスホニウムブロミド、トリブチルドデシルホスホニウムクロリド、トリブチルドデシルホスホニウムブロミド、トリブチルヘキサデシルホスホニウムクロリド、トリブチルヘキサデシルホスホニウムブロミド、メチルトリフェニルホスホニウムクロリド、メチルトリフェニルホスホニウムブロミド、メチルトリフェニルホスホニウムヨージド、テトラフェニルホスホニウムクロリド、並びにテトラフェニルホスホニウムブロミド、
からなる群より選択される1以上のイオン性界面活性剤である、[17]に記載の組成物。
硫酸アンモニウム及びこれらの組合せからなる群より選択される1以上の安定化剤をさらに含む、[13]に記載の糖化ヘモグロビン測定用組成物。
アマドリアーゼは、ケトアミンオキシダーゼ、フルクトシルアミノ酸オキシダーゼ、フルクトシルペプチドオキシダーゼ、フルクトシルアミンオキシダーゼ等とも称され、酸素の存在下で、イミノ2酢酸またはその誘導体(アマドリ化合物)を酸化して、グリオキシル酸またはα-ケトアルデヒド、アミノ酸またはペプチドおよび過酸化水素を生成する反応を触媒する酵素のことをいう。アマドリアーゼは、自然界に広く分布しており、微生物や、動物または植物起源の酵素を探索することにより、得ることができる。微生物においては、例えば、糸状菌、酵母または細菌等から得ることができる。
(2)257位のバリンの置換、例えば、システイン、セリン、トレオニンへの置換。
(3)249位のグルタミン酸の置換、例えば、リジン、アルギニンへの置換。
(4)253位のグルタミン酸の置換、例えば、リジン、アルギニンへの置換。
(5)337位のグルタミンの置換、例えば、リジン、アルギニンへの置換。
(6)340位のグルタミン酸の置換、例えば、プロリンへの置換。
(7)232位のアスパラギン酸の置換、例えば、リジン、アルギニンへの置換。
(8)129位のアスパラギン酸の置換、例えば、リジン、アルギニンへの置換。
(9)132位のアスパラギン酸の置換、例えば、リジン、アルギニンへの置換。
(10)133位のグルタミン酸の置換、例えば、アラニン、メチオニン、リジン、アルギニンへの置換。
(11)44位のグルタミン酸の置換、例えば、プロリンへの置換。
(12)256位のグリシンの置換、例えば、リジン、アルギニンへの置換。
(13)231位のグルタミン酸の置換、例えば、リジン、アルギニンへの置換。
(14)81位のグルタミン酸の置換、例えば、リジン、アルギニンへの置換。
(11)-(6) 44位のグルタミン酸の置換および340位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換を有する変異体。
(11)-(1)-(6) 44位のグルタミン酸の置換、262位のアスパラギンの置換および340位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換を有する変異体。
(11)-(2)-(1)-(6) 44位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換および340位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換を有する変異体。
(11)-(7)-(2)-(1)-(6) 44位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換、340位のグルタミン酸の置換、および232位のアスパラギン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、および232位のアスパラギン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換を有する変異体。
(11)-(3)-(2)-(1)-(6) 44位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換、340位のグルタミン酸の置換、および249位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、および249位のグルタミン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換を有する変異体。
(11)-(8)-(4)-(2)-(1)-(6) 44位のグルタミン酸の置換、253位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換、340位のグルタミン酸の置換、および129位のアスパラギン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、および129位のアスパラギン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換を有する変異体。
(11)-(10)-(4)-(2)-(1)-(6) 44位のグルタミン酸の置換、133位のグルタミン酸の置換、253位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換および340位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換を有する変異体。
(11)-(10)-(4)-(2)-(1)-(5)-(6) 44位のグルタミン酸の置換、133位のグルタミン酸の置換、253位のグルタミン酸の置換、257位のバリンの置換、262位のアスパラギンの置換、337位のグルタミンのリジンへの置換および340位のグルタミン酸の置換、例えば、44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンまたはアルギニンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、337位のグルタミンに対応する位置のアミノ酸のリジンまたはアルギニンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換を有する変異体。
(i)247位のグルタミン酸の置換、例えばリジン、アルギニンへの置換;
(ii)251位のグルタミン酸の置換、例えばリジン、アルギニンへの置換;
(iii)335位のトレオニンの置換、例えばリジン、アルギニンへの置換;
(iv)230位のアスパラギン酸の置換、例えばリジン、アルギニンへの置換;
(v)129位のアスパラギン酸の置換、例えばリジン、アルギニンへの置換;
(vi)132位のアスパラギン酸の置換、例えばリジン、アルギニンへの置換;
(vii)133位のグルタミン酸の置換、例えばアラニン、メチオニン、リジン、アルギニンへの置換;
(viii)254位のアスパラギンの置換、例えばリジン、アルギニンへの置換;及び
(ix)229位のグルタミン酸の置換、例えばリジン、アルギニンへの置換。
配列番号37に示すアミノ酸配列において、以下の(i)から(iv):
(i)251位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンの置換を有する変異体;
(ii)132位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換を有する変異体;
(iii)133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換を有する変異体;並びに
(iv)229位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換を有する変異体。
これらのアマドリアーゼをコードする本発明の遺伝子(以下、単に「アマドリアーゼ遺伝子」ともいう。)を得るには、通常一般的に用いられている遺伝子のクローニング方法が用いられる。例えば、アマドリアーゼ生産能を有する微生物菌体や種々の細胞から常法、例えば、Current Protocols in Molecular Biology(WILEY Interscience,1989)記載の方法により、染色体DNAまたはmRNAを抽出することができる。さらにmRNAを鋳型としてcDNAを合成することができる。このようにして得られた染色体DNAまたはcDNAを用いて、染色体DNAまたはcDNAのライブラリーを作製することができる。
本発明において用いることのできるベクターとしては、上記プラスミドに限定されることなくそれ以外の、例えば、バクテリオファージ、コスミド等の当業者に公知の任意のベクターを用いることができる。具体的には、例えば、pBluescriptII SK+(STRATAGENE社製)等が好ましい。
アマドリアーゼ遺伝子の変異処理は、企図する変異形態に応じた、公知の任意の方法で行うことができる。すなわち、アマドリアーゼ遺伝子あるいは当該遺伝子の組み込まれた組換え体DNAと変異原となる薬剤とを接触・作用させる方法;紫外線照射法;遺伝子工学的手法;またはタンパク質工学的手法を駆使する方法等を広く用いることができる。
上述の如くして得られたアマドリアーゼ遺伝子を、常法により、バクテリオファージ、コスミド、または原核細胞もしくは真核細胞の形質転換に用いられるプラスミド等のベクターに組み込み、各々のベクターに対応する宿主を常法により、形質転換または形質導入をすることができる。例えば、得られた組換え体DNAを用いて、任意の宿主、例えば、エッシェリシア属に属する微生物、具体例としては大腸菌K-12株、好ましくは大腸菌JM109株、大腸菌DH5α株(ともにタカラバイオ社製)や大腸菌B株、好ましくは大腸菌BL21株(ニッポンジーン社製)等を形質転換またはそれらに形質導入してそれぞれの菌株を得ることができる。
アミノ酸配列の同一性は、GENETYX Ver.11(ゼネティックス社製)のマキシマムマッチングやサーチホモロジー等のプログラムまたはDNASIS Pro(日立ソフト社製)のマキシマムマッチングやマルチプルアライメント等のプログラムにより計算することができる。
「アミノ酸に対応する位置」とは、配列番号1に示すConiochaeta属由来のアマドリアーゼのアミノ酸配列の特定の位置のアミノ酸に対応する他の生物種由来のアマドリアーゼのアミノ酸配列における位置をいう。
なお、本発明において、「配列番号1記載のアミノ酸配列の44位のグルタミン酸に対応する位置」とは、確定したアマドリアーゼのアミノ酸配列を、配列番号1に示されるConiochaeta属由来のアマドリアーゼのアミノ酸配列と比較した場合に、配列番号1のアマドリアーゼの44位のグルタミン酸に対応するアミノ酸を意味するものである。これにより、上記の「相当する位置のアミノ酸残基」を特定する方法でアミノ酸配列を整列させた図1-1により特定することができる。
上記のようにして得られた界面活性剤耐性の優れたアマドリアーゼの生産能を有する菌株を用いて、当該アマドリアーゼを生産するには、この菌株を通常の固体培養法で培養してもよいが、可能な限り液体培養法を採用して培養するのが好ましい。
本発明における界面活性剤としては、本発明のHbA1cの測定方法を可能とする界面活性剤であれば特に制限は無く、非イオン性界面活性剤やイオン性の界面活性剤、例えば、カチオン性界面活性剤、アニオン性界面活性剤、両性界面活性剤等が挙げられるが、特にカチオン性界面活性剤、アニオン性界面活性剤が好ましい。本明細書において界面活性剤というとき、特に断らない限り、この表現は1以上の界面活性剤を包含するものとする。
本発明はアマドリアーゼ及び界面活性剤を含む糖化ヘモグロビン測定用キットを提供する。界面活性剤は非イオン性又はイオン性界面活性剤とすることができる。また、アマドリアーゼと界面活性剤とは、キット中に同一又は別個の成分として含まれ得る。一般に、同一の成分としてアマドリアーゼと界面活性剤とがキット中に含まれる場合、界面活性剤はアマドリアーゼを失活させない濃度で含まれるのが好ましい。別個の成分としてアマドリアーゼと界面活性剤とがキット中に含まれる場合、界面活性剤は測定時における終濃度よりも高濃度のストック溶液を用いてもよい。このストック溶液を適宜希釈して、測定に用いる溶液を調製する。
本発明のキット又は組成物には、アマドリアーゼの活性が失活しない範囲であるpH5.0~pH10.0、好ましくはpH6.0~pH8.0の範囲で緩衝能を有する緩衝剤または緩衝液を適宜加えて良い。本明細書において緩衝剤というとき、特に断らない限り、この用語は1以上の緩衝剤を包含するものとする。緩衝液とは溶液のpHを一定範囲に保つ緩衝作用(緩衝能)のある溶液のことをいい、緩衝剤とは溶液に緩衝作用を付与する物質をいう。緩衝剤は、弱酸を例にとると、弱酸とその塩から構成され、この場合、当該塩を共役塩と呼ぶ。例えば緩衝剤がリン酸とそのカリウム塩から構成される場合、本明細書ではベースとなる化合物がリン酸であることからこれを便宜上、リン酸緩衝剤と呼ぶことがある。またある緩衝剤についての濃度は、当該緩衝剤のベースとなる化合物の単独形態とその共役塩の形態とを合計したベース化合物についての濃度をいう。例えば100mMのリン酸緩衝剤というとき、これは終濃度として溶液に含まれるリン酸及びその共役塩(例えばリン酸カリウム塩)を合計したリン酸濃度が100mMであることをいう。
本発明のキット又は組成物には、界面活性剤存在下でのアマドリアーゼの残存活性を維持する又は残存活性の低下を軽減する安定化剤を適宜加えてもよい。本明細書において安定化剤とは、界面活性剤存在下でのアマドリアーゼの残存活性を維持する又は残存活性の低下を軽減する物質を言う。本明細書において安定化剤というとき、特に断らない限り、この表現は1以上の安定化剤を包含するものとする。本発明のキット又は組成物に含まれる安定化剤(安定剤)としては、例えば、リン酸、トリカルボン酸(例えば、クエン酸)、ジカルボン酸(例えば、リンゴ酸、マレイン酸、シトラコン酸、マロン酸、グルタル酸、酒石酸)、モノカルボン酸(例えば、酢酸)、式(IV)で表される化合物(例えばMES、MOPS、MOPSO)、硫酸アンモニウム、これらの塩及びこれらの任意の組合せが挙げられる。
上記のような手段で得られる本発明のアマドリアーゼは、遺伝子改変等により、そのアミノ酸配列に変異を生じた結果、改変前のものと比較して界面活性剤耐性が向上していることを特徴とする。具体的には、改変前のアマドリアーゼの活性と比較して、改変アマドリアーゼは、本明細書中に記載の活性測定方法および界面活性剤耐性評価方法に記載した反応条件下で、所定の界面活性剤処理、例えば、0.01%(w/v)のヘキサデシルトリメチルアンモニウムクロリド(以下、「CTAC」と表す)を添加してから、30℃、5分後の残存活性(%)が、向上していることを特徴とする。ここで、残存活性(%)とは、界面活性剤処理前の活性を100とした場合の界面活性剤処理後の活性の割合をパーセンテージ(%)で表したものである。なお、本明細書において界面活性剤の濃度をパーセンテージで記載する場合、特に断らない限りこれは%(w/v)を意味するものとする。
アマドリアーゼの活性の測定方法としては、種々の方法を用いることができるが、一例として、以下に、本発明で用いるアマドリアーゼ活性の測定方法について説明する。
本発明におけるアマドリアーゼの酵素活性の測定方法としては、酵素の反応により生成する過酸化水素量を測定する方法や酵素反応により消費する酸素量を測定する方法などが主な測定方法として挙げられる。以下に、一例として、過酸化水素量を測定する方法について示す。
(1)試薬1:POD-4-AA溶液
4.0kUのパーオキシダーゼ(キッコーマン社製)、100mgの4-アミノアンチピリン(東京化成工業社製)を0.1Mのリン酸カリウム緩衝液(pH7.0)に溶解し、1Lに定容する。
500mgのTOOS(N-エチル-N-(2-ヒドロキシ-3-スルホプロピル)-m-トルイジンナトリウム、同仁化学社製)をイオン交換水に溶解し、100mlに定容する。
フルクトシルバリン417mgをイオン交換水に溶解して10mlに定容する。
2.7mlの試薬1,100μlの試薬2、および100μlの試薬3を混和し、37℃で5分間予備加温する。その後、酵素液を100μl加えてよく混ぜた後、分光光度計(U-3010、日立ハイテクノロジーズ社製)により、555nmにおける吸光度を測定する。測定値は、555nmにおける1分後から3分後の1分間あたりの吸光度変化とする。なお対照液は、100μlの試薬3の代わりに100μlのイオン交換水を加える以外は前記と同様に調製する。37℃、1分当たりに生成される過酸化水素のマイクロモル数を酵素液中の活性単位(U)とし、下記の式に従って算出する。
活性(U/ml)= {(ΔAs-ΔA0)×3.0×df}÷(39.2×0.5×0.1)
ΔAs : 反応液の1分間あたりの吸光度変化
ΔA0 : 対照液の1分間あたりの吸光度変化
39.2: 反応により生成されるキノンイミン色素の
ミリモル吸光係数(mM-1・cm-1)
0.5 : 1molの過酸化水素による生成されるキノンイミン色素のmol数
df : 希釈係数
アマドリアーゼ粗酵素液、またはアマドリアーゼ精製標品を約1.0U/mlとなるように、30mM MES/21mM Tris緩衝液(pH6.5)で希釈し、CTAC(例えば、東京化成工業社製)を終濃度0.01%(w/v)または0.04%となるように添加後、30℃にて5分間加温する。加温後、0.15%のBSAを含む10mM リン酸緩衝液(pH7.0)で2倍希釈し、上述のB.の方法を用いて界面活性剤処理前と界面活性剤処理後のサンプルの酵素活性を測定し、界面活性剤処理前の活性を100とした場合の界面活性剤処理後の活性の割合、すなわち、残存活性(%)を求めることにより、界面活性剤耐性を評価する。
上記の界面活性剤耐性測定方法において、30mM MES/21mM Tris緩衝液の代わりに種々の緩衝剤を用いてアマドリアーゼの残存活性の測定を行うことで、緩衝剤によるアマドリアーゼ活性残存への寄与を評価することができる。例えば30mM MES/21mM Tris緩衝液(pH 6.5)の代わりに、リン酸緩衝液(pH 7.0)、クエン酸緩衝液(pH 6.0)、HEPES緩衝液(pH 7.0)、ACES緩衝液(pH 7.0)等を用いることができる。他の条件及び手順は上記の界面活性剤耐性測定方法と同様としうる。
上記の界面活性剤耐性測定方法において、種々の安定化剤をさらに添加してアマドリアーゼの残存活性の測定を行うことで、当該安定化剤の作用を評価することができる。評価対象である安定化剤が緩衝作用をも有する化合物である場合、当該化合物の緩衝作用によるアマドリアーゼ活性残存への寄与によらないアマドリアーゼ安定化作用を評価するためには、アマドリアーゼ安定化作用を有しない緩衝剤を溶液に緩衝能を付与するのに十分な濃度で使用しつつ(例えばHEPES(pH 7.0)を500mMにて使用)、安定化剤を低濃度で、すなわち溶液に緩衝能を付与するには十分でない低濃度で用いることができる。溶液に緩衝能を付与するのに十分な濃度とは、当該溶液に添加する他の試薬に起因するpH変動が生じずpHが一定の範囲(例えばpH5~10、pH6~8)に保たれる濃度をいう。溶液に緩衝能を付与するには十分でない濃度とは、当該溶液に他の試薬を添加するとpH変動が生じpHが一定範囲から逸脱する濃度をいう。これらの濃度は溶液に添加される他の試薬の種類及び量に応じて変化するが、当業者であれば慣用法により該濃度を適宜決定することができる。他の条件及び手順は上記の界面活性剤耐性測定方法と同様としうる。
本発明の緩衝剤と、本発明の安定化剤とを併用した場合のアマドリアーゼ安定作用を評価するために、本発明のアマドリアーゼ及び界面活性剤を含む溶液に、安定化剤及び緩衝剤を適宜濃度調整しつつ添加し、アマドリアーゼの残存活性を測定することもできる。他の条件及び手順は上記の界面活性剤耐性測定方法と同様としうる。
(界面活性剤耐性向上型変異について)
(1)組換え体プラスミドpKK223-3-CFP-T7 DNAの調製
CFP-T7遺伝子(配列番号2)を含む組換え体プラスミドを有する大腸菌JM109(pKK223-3-CFP-T7)株(国際公開2007/125779号参照)を、LB-amp培地[1%(W/V) バクトトリプトン、0.5%(W/V) ペプトン、0.5%(W/V) NaCl、50μg/ml Ampicilin]2.5mlに接種して、37℃で20時間振とう培養し、培養物を得た。
得られた組換え体プラスミドpKK223-3-CFP-T7 DNAを鋳型として、配列番号5、6の合成オリゴヌクレオチド、KOD-Plus-(東洋紡績社製)を用い、以下の条件でPCR反応を行った。
上記の手順により得られた上記組換え体プラスミドを保持するそれぞれの大腸菌JM109株を、0.1mMのIPTGを添加したLB-amp培地3mlにおいて、30℃で16時間培養した。その後、各菌体をpH7.0の0.01Mリン酸緩衝液で洗浄、超音波破砕、15,000rpmで10分間遠心分離し、各粗酵素液1.5mlを調製した。
このようにして調製した各粗酵素液をサンプルとし、CTACの終濃度を0.01%として、上記の界面活性剤耐性測定法に従って、各種改変型アマドリアーゼの界面活性剤耐性評価を行った。結果を表1-1に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。表1-1において、CFP-T7は、大腸菌JM109(pKK223-3-CFP-T7)株由来のアマドリアーゼを示す。なお、本実施例では大腸菌JM109(pKK223-3-CFP-T7)株由来のアマドリアーゼであるCFP-T7を変異元酵素としたため、表中に記載の「アミノ酸変異」の記載には、CFP-T7に既に導入済みの各種変異点は含めていない。
配列番号37はCurvularia clavata由来ケトアミンオキシダーゼ(以降CcFXと称する)のアミノ酸配列である(国際公開第2004/104203号)。配列番号37のアミノ酸配列をコードする遺伝子(配列番号55)を定法である遺伝子断片のPCRによる全合成によりcDNAを全合成することで取得した(終止コドンTAAを含む)。このとき、配列番号55の5´末端、3´末端にはそれぞれEcoRIサイトとHindIIIサイトを付加した。また、クローニングした遺伝子配列から予想されるアミノ酸配列全長は図1のCcFXの配列と一致していることを確認した。続いて、取得した配列番号55の遺伝子を大腸菌で発現させるために、以下の手順を行った。まず、上記で全合成した遺伝子をEcoRIサイトとHindIIIサイト(タカラバイオ社製)の2種類の制限酵素で処理し、pKK-223-3 Vector(GEヘルスケア社製)のEcoRI-HindIIIサイトに挿入することで、組換え体プラスミドpKK223-3-CcFXを取得し、上記と同様の条件で大腸菌JM109株を形質転換し、大腸菌JM109(pKK223-3-CcFX)株を得た。
(界面活性剤耐性向上型変異の蓄積)
実施例1で見出された界面活性剤耐性向上変異の知見に基づき、これらを組み合わせて変異を蓄積させることにより、さらに界面活性剤耐性を高めたアマドリアーゼを取得することを目的として、多重変異体(2重変異体、3重変異体、4重変異体、5重変異体、6重変異体、7重変異体)の作製による界面活性剤耐性向上型変異の蓄積を試みた。
(界面活性剤TTACに対する評価)
実施例2で用いた界面活性剤CTACに変わり、テトラデシルトリメチルアンモニウムクロリド(以下、「TTAC」と表記。)を用いて、CFP-Dの安定性を評価した。実施例1に準じた界面活性剤耐性測定方法に従って、ただし、界面活性剤処理条件に関しては、アマドリアーゼを20mM リン酸カリウム緩衝液(pH7.0)で希釈し、0.04%のTTACの混合を行うことで、各種改変型アマドリアーゼの界面活性剤耐性評価を行った。結果を表3-1に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
(CFP-T7、CFP-D2、CFP-D7の精製)
実施例1,2で得たCFP―T7、CFP-D2およびCFP-D7の粗酵素を用いて、調製した粗酵素液を20mM リン酸カリウム緩衝液(pH8.0)で平衡化した4mlのQ Sepharose Fast Flow樹脂(GEヘルスケア社製)に吸着させ、次に80mlの同緩衝液で樹脂を洗浄し、続いて100mM NaClを含む20mM リン酸カリウム緩衝液(pH8.0)で樹脂に吸着していた蛋白質を溶出させ、アマドリアーゼ活性を示す画分を回収した。
種々の界面活性剤を用い、上記のようにして得た精製酵素CFP-T7、CFP-D2およびCFP-D7の安定性を評価した。実施例1に準じた界面活性剤耐性測定方法に従って、ただし、界面活性剤処理条件に関しては、アマドリアーゼを20mM リン酸カリウム緩衝液(pH7.0)で希釈し、各種濃度の界面活性剤の混合を行うことで、各種改変型アマドリアーゼの界面活性剤耐性評価を行った。結果を表3-2に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
(界面活性剤SDSに対する評価)
実施例2で用いた界面活性剤CTACに変わり、ドデシル硫酸ナトリウム(以下、「SDS」と表記。)を用いて、CFP-Dの安定性を評価した。実施例1に準じた界面活性剤耐性測定方法に従って、ただし、界面活性剤処理条件に関しては、アマドリアーゼを30mM MES/21mM Tris緩衝液(pH6.5)で希釈し、0.04%のSDSの混合を行うことで、各種改変型アマドリアーゼの界面活性剤耐性評価を行った。結果を表4に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
実施例3-2で得たCFP-T7、およびCFP-D7の精製酵素を用いて、下記に示した試料の測定を実施した。なお、CFP-T7、およびCFP-D7の活性値はアマドリアーゼ活性の測定方法に記載の方法に従い、終濃度5mMのαFVHを基質として、pH7.0に調整した(試薬1)を用いて決定した。
(試薬4)αFVH 125mgをイオン交換水に溶解し、10mlに定容することにより、基質溶液30mMを得た。さらに、CTAC溶液で714倍希釈することにより、42μMのαFVH/0%~0.2%のCTAC溶液を得た。
(試薬5)
0.21mM DA-64(N-(カルボキシメチルアミノカルボニル)-4,4′-ビス(ジメチルアミノ)ジフェニルアミンナトリウム、和光純薬工業社製)
20mM リン酸カリウム緩衝液(pH7.0)
(試薬6)
6.7U/ml CFP-T7、あるいはCFP-D7
19U/ml パーオキシダーゼ(キッコーマン社製)
20mM リン酸カリウム緩衝液(pH7.0)
予め37℃で5分間加温しておいた135μlの(試薬5)と上記(11)で調製した25μlの試料の混合溶液に対し、50μlの(試薬6)を添加して反応を開始し、37℃で5分間反応後の波長751nmにおける吸光度を自動分析装置Bio Majesty JCA‐BM1650(日本電子)で測定した。基質溶液の代わりにイオン交換水を用いて作製した(試薬4)に対しても、同様に操作して測定した波長751nmにおける吸光度(試薬ブランク)を対照として、下記の式により、それぞれの試料を測定した場合の吸光度変化量(差)を算出した。発色基質DA-64の終濃度は0.15mMであり、基質ありの場合はαFVHの終濃度は5μMであり、吸光度測定に用いたセルの長さ(光路)は1cmであった。
吸光度変化量=ΔAes-Ae0
ΔAes:反応開始から5分経過後の吸光度
ΔAe0:対照液を添加した場合の反応開始から5分経過後の吸光度
(界面活性剤混合下におけるフルクトシルペプチド試料の定量)
CFP-T7、およびCFP-D7の精製酵素を用いて、CTAC混合下において、0.5~3μMの範囲でαFVH測定値の直線性を比較した。CFP-T7は、0.01%または0.02%のCTAC混合をし、さらに4.2μM、8.4μM、13μM、17μM、21μMまたは25μMのαFVH、つまり終濃度では、0.5μM、1.0μM、1.5μM、2.0μM、2.5μMまたは3.0μMのαFVHを用いる条件で、実施例5と同様に吸光度変化量を測定し、相関係数を算出した。同様に、CFP-D7は、0.02%または0.2%のCTAC混合をし、上記と同様のαFVHを用いる条件で、実施例5と同様に吸光度変化量を測定し、相関係数を算出した。その結果を表6に示し、各相関データを図2、3、4、5に示す。図2は、0.01%のCTACの混合下でCFP-T7を用いて、αFVHを測定した結果であり、図3は、0.02%のCTACの混合下でCFP-T7を用いて、αFVHを測定した結果であり、図4は、0.02%のCTACの混合下でCFP-D7を用いて、αFVHを測定した結果であり、図5は、0.2%のCTACの混合下でCFP-D7を用いて、αFVHを測定した結果である。
(各種アマドリアーゼの界面活性剤耐性の評価)
界面活性剤、好ましくはイオン性界面活性剤の存在下においても活性が残存するアマドリアーゼを含む糖化ヘモグロビン測定用組成物を提供するために、上述のようにConiochaeta属由来アマドリアーゼを改変して界面活性剤耐性を向上させた。また、Coniochaeta属由来アマドリアーゼ以外のアマドリアーゼにおいては、イオン性界面活性剤存在下においてのHbA1c測定が可能か知られていない。そこで、Phaeosphaeria nodorum由来のフルクトシルペプチドオキシダーゼおよびNeocosmospora vasinfecta由来のケトアミンオキシダーゼとイオン性界面活性剤を組み合わせて、αFVHの測定を試みた。
配列番号40はPhaeosphaeria nodorum由来フルクトシルペプチドオキシダーゼ(以降PnFXと称する)のアミノ酸配列である(Biotechnology and Bioengineering, 106, 358-366, 2010参照)。配列番号40のアミノ酸配列をコードする遺伝子(配列番号44)を定法である遺伝子断片のPCRによる全合成によりcDNAを全合成することで取得した。このとき、配列番号40の5´末端、3´末端にはそれぞれNdeIサイトとBamHIサイトを付加した。また、クローニングした遺伝子配列から予想されるアミノ酸配列全長は図1のPnFXの配列と一致していることを確認した。続いて、取得した配列番号44の遺伝子を大腸菌で発現させるために、以下の手順を行った。まず、上記で全合成した遺伝子をNdeIサイトとBamHI(タカラバイオ社製)の2種類の制限酵素で処理し、pET-22b(+) Vector(ノバジェン社製)のNdeI-BamHIサイトに挿入することで、組換え体プラスミドpET22b-PnFXを取得し、上記と同様の条件で大腸菌BL21(DE3)株を形質転換し、大腸菌BL21(DE3)(pET22b-PnFX)株を得た。
配列番号38はNeocosmospora vasinfecta由来ケトアミンオキシダーゼ(NvFX)のアミノ酸配列であり、配列番号38のアミノ酸配列をコードする遺伝子(配列番号45)を挿入した組換え体プラスミドpET22b―NvFXを大腸菌で発現させることにより、NvFXの活性が確認されている(国際公開第2012/18094号参照)。実施例1と同様に大腸菌BL21(DE3)株を形質転換し、得られた大腸菌BL21(DE3)(pET22b―NvFX)株を用いて上記の方法で培養して、NvFXの粗酵素液を調製した。
(各種緩衝剤存在下におけるアマドリアーゼの界面活性剤耐性評価)
30mM MES/21mM Tris緩衝剤(pH6.5)以外の緩衝剤を用いた場合に、アマドリアーゼの界面活性剤耐性が向上するか検討した。上記のように精製したCFP-T7をサンプルとし、CTACの終濃度を0.01%として、30mM MES/21mM Tris緩衝剤(pH6.5)の代わりに各種緩衝剤、具体的にはリン酸とリン酸カリウムとを含むリン酸緩衝剤(pH7.0)、クエン酸とクエン酸ナトリウムとを含むクエン酸緩衝剤(pH6.0)、MESとそのナトリウム塩とを含むMES緩衝剤(pH7.0)、HEPESとそのナトリウム塩とを含むHEPES緩衝剤(pH7.0)、ACESとそのナトリウム塩とを含むACES緩衝剤(pH7.0)の存在下において、実施例1の界面活性剤耐性測定法に従って、CFP-T7の界面活性剤耐性評価を行った。結果を表8に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
(各安定化剤添加時におけるアマドリアーゼの界面活性剤耐性評価)
種々の安定化剤を添加した際に、アマドリアーゼの界面活性剤耐性が向上するか検討した。安定化剤として、リン酸、トリカルボン酸(例えば、クエン酸)、ジカルボン酸(例えば、リンゴ酸、マレイン酸、シトラコン酸、マロン酸、グルタル酸、酒石酸)、モノカルボン酸(例えば、酢酸)、MES、MOPS、MOPSO、硫酸アンモニウムを用いた。また比較例としてCHESを用いた。安定化剤を添加した際に、pHの変化を防ぐようにするために、緩衝液は500mM HEPES(pH7.0)を用い、上記のように精製したCFP-T7をサンプルとし、CTACの終濃度を0.01%として、さらに、種々の安定化剤を添加し、実施例1の界面活性剤耐性測定法に従って、CFP-T7の界面活性剤耐性評価を行った。結果を表9-1、9-2に示す。また、500mM HEPES(pH7.0)緩衝剤を用い、上記のように精製したCFP-D2をサンプルとし、さらに、種々の安定化剤を添加し、実施例1の界面活性剤耐性測定法に従って、ただし、界面活性剤処理条件に関しては、CTACの終濃度を0.08%、処理温度を37℃とより過酷な条件を設定し、CFP-D2の界面活性剤耐性評価を行った。結果を表9-3に示す。実際に、安定化剤を添加した際に、pHは実際に7.0を示すことを確認した。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
に包含されるMES(n=1, R10はH)、MOPS(n=2, R10はいずれもH)、MOPSO(n=2, R10はOHおよびH)はいずれもアマドリアーゼ安定化作用を示した。以上のことから、リン酸、トリカルボン酸、ジカルボン酸、モノカルボン酸、MES、MOPS、MOPSO等の式(IV)で表される化合物、硫酸アンモニウムには、アマドリアーゼの界面活性剤耐性を向上させる効果があることを示している。また、本発明の変異を導入していないアマドリアーゼCFP-T7についても、本発明の変異を導入したアマドリアーゼCFP-D2についても界面活性剤耐性の向上が観察された。
Coniochaeta属由来アマドリアーゼ以外のアマドリアーゼとして、例えば、PnFXに対し、上記の安定化剤は界面活性剤耐性向上効果があるか検討した。安定化剤は上記と同様のものを用い、安定化剤を添加した際に、pHの変化を防ぐようにするために、緩衝液は300mM HEPES(pH7.0)を用い、上記のように精製したPnFXをサンプルとし、CTACの終濃度を0.04%として、上記と同様に、PnFXの界面活性剤耐性評価を行った。実際に、安定化剤を添加した際に、pHは実際に7.0を示すことを確認した。結果を表10に示す。なお加温後のサンプルをBSA溶液で2倍希釈してから30分後に再度活性測定を行っても、活性値に変化がないことを確認した。
配列番号2. CFP-T7の遺伝子配列
配列番号3. CFP-Dのアミノ酸配列
配列番号4. CFP-Dの遺伝子配列
配列番号5. N262H導入プライマーFw
配列番号6. N262X導入プライマーRv
配列番号7. V257C導入プライマーFw
配列番号8. V257X導入プライマーRv
配列番号9. V257S導入プライマーFw
配列番号10. V257T導入プライマーFw
配列番号11. E253K導入プライマーFw
配列番号12. E253X導入プライマーRv
配列番号13. E253R導入プライマーFw
配列番号14. Q337K導入プライマーFw
配列番号15. Q337X導入プライマーRv
配列番号16. E340P導入プライマーFw
配列番号17. E340X導入プライマーRv
配列番号18. E133A導入プライマーFw
配列番号19. E133X導入プライマーRv
配列番号20. E133M導入プライマーFw
配列番号21. E133K導入プライマーFw
配列番号22. E44P導入プライマーFw
配列番号23. E44X導入プライマーRv
配列番号24. G256K導入プライマーFw
配列番号25. G256R導入プライマーFw
配列番号26. E81K導入プライマーFw
配列番号27. E81X導入プライマーRv
配列番号28. F43Y/E44P導入プライマーFw
配列番号29. V257X/N262H導入プライマーRv
配列番号30. E253K/V257C導入プライマーFw
配列番号31. E253X/V257C/N262H導入プライマーRv
配列番号32. Q337K/E340P導入プライマーFw
配列番号33. Q337X/E340P導入プライマーRv
配列番号34. Eupenicillium terrenum由来のアマドリアーゼ
配列番号35. Pyrenochaeta sp.由来のケトアミンオキシダーゼ
配列番号36. Arthrinium sp.由来のケトアミンオキシダーゼ
配列番号37. Curvularia clavata由来のケトアミンオキシダーゼ
配列番号38. Neocosmospora vasinfecta由来のケトアミンオキシダーゼ
配列番号39. Cryptococcus neoformans由来のフルクトシルアミノ酸オキシダーゼ
配列番号40. Phaeosphaeria nodorum由来のフルクトシルペプチドオキシダーゼ
配列番号41. Aspergillus nidulans由来のフルクトシルアミノ酸オキシダーゼ
配列番号42. Ulocladium sp.由来のフルクトシルアミノ酸オキシダーゼ
配列番号43. Penicillium crysogenum由来のフルクトシルアミノ酸オキシダーゼ
配列番号44. Phaeosphaeria nodorum由来のフルクトシルペプチドオキシダーゼの遺伝子
配列番号45. Neocosmospora vasinfecta由来のケトアミンオキシダーゼの遺伝子
配列番号46. D129K導入プライマーFw
配列番号47. D129K導入プライマーRv
配列番号48. D132K導入プライマーFw
配列番号49. E231K導入プライマーFw
配列番号50. E231X導入プライマーRv
配列番号51. D232K導入プライマーFw
配列番号52. E249K導入プライマーFw
配列番号53. E249K導入プライマーRv
配列番号54. E249K/V257C導入プライマーRv
配列番号55. Curvularia clavata由来のケトアミンオキシダーゼの遺伝子
配列番号56. CcFXのD129K導入プライマーFw
配列番号57. CcFXのD129K導入プライマーRv
配列番号58. CcFXのD132K導入プライマーFw
配列番号59. CcFXのE133K導入プライマーFw
配列番号60. CcFXのE133A導入プライマーFw
配列番号61. CcFXのE133X導入プライマーRv
配列番号62. CcFXのE229K導入プライマーFw
配列番号63. CcFXのD230K導入プライマーFw
配列番号64. CcFXのD230X導入プライマーRv
配列番号65. CcFXのE247K導入プライマーFw
配列番号66. CcFXのE247K導入プライマーRv
配列番号67. CcFXのE251K導入プライマーFw
配列番号68. CcFXのE251X導入プライマーRv
配列番号69. CcFXのE251R導入プライマーFw
配列番号70. CcFXのN254K導入プライマーFw
配列番号71. CcFXのN254K導入プライマーRv
配列番号72. CcFXのT335K導入プライマーFw
配列番号73. CcFXのT335X導入プライマーRv
配列番号74. CcFXのT335R導入プライマーFw
Claims (26)
- 界面活性剤を添加してから5分後の残存活性(%)が、配列番号1、3、または37に示すアミノ酸配列を有するアマドリアーゼと比較して向上しているアマドリアーゼであって、
(i)配列番号1、3、または37に示すアミノ酸配列に1または数個のアミノ酸の欠失、挿入、付加、および/または置換がなされたアミノ酸配列を有し、かつ/又は
(ii)配列番号1、3、または37に示すアミノ酸配列と少なくとも70%の同一性を有するアミノ酸配列を有する、
前記アマドリアーゼ。 - 界面活性剤がイオン性界面活性剤である、請求項1記載のアマドリアーゼ。
- 配列番号1または3に示すアミノ酸配列における以下の(i)から(xiv):
(i)262位のアスパラギン、
(ii)257位のバリン、
(iii)249位のグルタミン酸
(iv)253位のグルタミン酸、
(v)337位のグルタミン、
(vi)340位のグルタミン酸、
(vii)232位のアスパラギン酸、
(viii)129位のアスパラギン酸、
(ix)132位のアスパラギン酸、
(x)133位のグルタミン酸、
(xi)44位のグルタミン酸、
(xii)256位のグリシン、
(xiii)231位のグルタミン酸、及び
(xiv)81位のグルタミン酸、
よりなる群から選択されるアミノ酸に対応する位置で1つまたはそれ以上のアミノ酸残基の置換を有する、請求項1又は2記載のアマドリアーゼ。 - 配列番号1または3に示すアミノ酸配列のアミノ酸が、
以下の(i)から(xiv)の置換:
(i)262位のアスパラギンがヒスチジンに置換されている;
(ii)257位のバリンがシステイン、セリン、トレオニンに置換されている;
(iii)249位のグルタミン酸がリジン、アルギニンに置換されている;
(iv)253位のグルタミン酸がリジン、アルギニンに置換されている;
(v)337位のグルタミンがリジン、アルギニンに置換されている;
(vi)340位のグルタミン酸がプロリンに置換されている;
(vii)232位のアスパラギン酸がリジン、アルギニンに置換されている;
(viii)129位のアスパラギン酸がリジン、アルギニンに置換されている;
(ix)132位のアスパラギン酸がリジン、アルギニンに置換されている;
(x)133位のグルタミン酸がアラニン、メチオニン、リジン、アルギニンに置換されている;
(xi)44位のグルタミン酸がプロリンに置換されている;
(xii)256位のグリシンがリジン、アルギニンに置換されている;
(xiii)231位のグルタミン酸がリジン、アルギニンに置換されている;及び
(xiv)81位のグルタミン酸がリジン、アルギニンに置換されている;
のいずれか1以上を有する、請求項1~3のいずれか1項に記載のアマドリアーゼ。 - 配列番号1または3に示すアミノ酸配列において、以下の(i)から(ix):
(i)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(ii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(iii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(iv)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および232位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換;
(v)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および249位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換;
(vi)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;
(vii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換および129位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換;
(viii)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換;並びに
(ix)44位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換、133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換、253位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換、257位のバリンに対応する位置のアミノ酸のシステインへの置換、262位のアスパラギンに対応する位置のアミノ酸のヒスチジンへの置換、337位のグルタミンに対応する位置のアミノ酸のリジンへの置換および340位のグルタミン酸に対応する位置のアミノ酸のプロリンへの置換
よりなる群から選択されるアミノ酸残基の置換を有する、請求項1~4のいずれか1項に記載のアマドリアーゼ。 - 配列番号37に示すアミノ酸配列のアミノ酸が、
以下の(i)から(ix)の置換:
(i)247位のグルタミン酸がリジン、アルギニンに置換されている;
(ii)251位のグルタミン酸がリジン、アルギニンに置換されている;
(iii)335位のトレオニンがリジン、アルギニンに置換されている;
(iv)230位のアスパラギン酸がリジン、アルギニンに置換されている;
(v)129位のアスパラギン酸がリジン、アルギニンに置換されている;
(vi)132位のアスパラギン酸がリジン、アルギニンに置換されている;
(vii)133位のグルタミン酸がアラニン、メチオニン、リジン、アルギニンに置換されている;
(viii)254位のアスパラギンがリジン、アルギニンに置換されている;及び
(ix)229位のグルタミン酸がリジン、アルギニンに置換されている;
のいずれか1以上を有する、請求項1~3のいずれか1項に記載のアマドリアーゼ。 - 配列番号37に示すアミノ酸配列において、以下の(i)から(iv):
(i)251位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンの置換;
(ii)132位のアスパラギン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;
(iii)133位のグルタミン酸に対応する位置のアミノ酸のアラニンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;並びに
(iv)229位のグルタミン酸に対応する位置のアミノ酸のリジンへの置換および335位のトレオニンに対応する位置のアミノ酸のリジンへの置換;
よりなる群から選択されるアミノ酸残基の置換を有する、請求項1~3および6のいずれか1項に記載のアマドリアーゼ。 - 請求項1~7のいずれか1項に記載のアミノ酸配列をコードするアマドリアーゼ遺伝子。
- 請求項8記載のアマドリアーゼ遺伝子を含む組換えベクター。
- 請求項9記載の組換えベクターを含む宿主細胞。
- 以下の工程:
(i)請求項10記載の宿主細胞を培養する工程;
(ii)宿主細胞に含まれるアマドリアーゼ遺伝子を発現させる工程;及び
(iii)培養物からアマドリアーゼを単離する工程を含む、アマドリアーゼを製造する方法。 - 請求項1~7のいずれかに記載のアマドリアーゼを含む、糖化ヘモグロビンの測定に用いるための組成物。
- 1以上の界面活性剤及びアマドリアーゼを含む、糖化ヘモグロビン測定用組成物。
- アマドリアーゼが、
(i)界面活性剤を添加してから5分後の残存活性(%)が、添加しない場合と比較して、15%以上残存し、かつ/又は
(ii)終濃度0.04%の界面活性剤の存在下において、発色基質N-(カルボキシメチルアミノカルボニル)-4,4′-ビス(ジメチルアミノ)ジフェニルアミンナトリウム(DA-64)を添加し5分間反応させた後の751nmにおける吸光度と、糖化アミノ酸溶液または糖化ペプチド溶液の代わりにイオン交換水を用いた対照液添加から5分後の751nmにおける吸光度との差が0.006以上となるアマドリアーゼである、
請求項13に記載の組成物。 - アマドリアーゼが配列番号1、3、37または40に示すアミノ酸配列と少なくとも70%の同一性を有するアミノ酸配列を有するアマドリアーゼである、請求項13又は14に記載の組成物。
- 界面活性剤が、70mM以下の臨界ミセル濃度を有するものである、請求項13~15のいずれか1項に記載の組成物。
- 界面活性剤が、以下の一般式(I)で表される第四級アンモニウム塩、
以下の一般式(II)で表されるピリジニウム塩、
以下の一般式(III)で表されるホスホニウム塩、
並びにドデシル硫酸ナトリウムからなる群より選択される1以上のイオン性界面活性剤である、請求項13~16のいずれか1項に記載の組成物。 - 界面活性剤が、オクチルトリメチルアンモニウムクロリド、オクチルトリメチルアンモニウムブロミド、ジオクチルジメチルアンモニウムクロリド、ジオクチルジメチルアンモニウムブロミド、デシルトリメチルアンモニウムクロリド、デシルトリメチルアンモニウムブロミド、ドデシルトリメチルアンモニウムクロリド、ドデシルトリメチルアンモニウムブロミド、テトラデシルトリメチルアンモニウムクロリド、テトラデシルトリメチルアンモニウムブロミド、ヘキサデシルトリメチルアンモニウムクロリド、ヘキサデシルトリメチルアンモニウムブロミド、オクタデシルトリメチルアンモニウムクロリド、オクタデシルトリメチルアンモニウムブロミド、エイコシルトリメチルアンモニウムクロリド及びエイコシルトリメチルアンモニウムブロミド、ベンジルドデシルジメチルアンモニウムクロリド、ベンジルドデシルジメチルアンモニウムブロミド、ベンジルテトラデシルジメチルアンモニウムクロリド、ベンジルテトラデシルジメチルアンモニウムブロミド、ベンジルセチルジメチルアンモニウムクロリド、およびベンジルセチルジメチルアンモニウムブロミド、
1-デシルピリジニウムクロリド、1-デシルピリジニウムブロミド、1-ドデシルピリジニウムクロリド、1-ドデシルピリジニウムブロミド、1-テトラデシルピリジニウムクロリド、1-テトラデシルピリジニウムブロミド、1-ヘキサデシルピリジニウムクロリド、1-ヘキサデシルピリジニウムブロミド、N-セチル-2-メチルピリジニウムクロリド、N-セチル-2-メチルピリジニウムブロミド、N-セチル-3-メチルピリジニウムクロリド、N-セチル-3-メチルピリジニウムブロミド、N-セチル-4-メチルピリジニウムクロリド、N-セチル-4-メチルピリジニウムブロミド、1-オクタデシルピリジニウムクロリド、1-オクタデシルピリジニウムブロミド、1-エイコシルピリジニウムクロリド及び1-エイコシルピリジニウムブロミド、
テトラエチルホスホニウムクロリド、テトラエチルホスホニウムブロミド、トリブチルメチルホスホニウムクロリド、トリブチルメチルホスホニウムブロミド、トリブチルメチルホスホニウムヨージド、テトラブチルホスホニウムクロリド、テトラブチルホスホニウムブロミド、テトラ-n-オクチルホスホニウムクロリド、テトラ-n-オクチルホスホニウムブロミド、トリブチルドデシルホスホニウムクロリド、トリブチルドデシルホスホニウムブロミド、トリブチルヘキサデシルホスホニウムクロリド、トリブチルヘキサデシルホスホニウムブロミド、メチルトリフェニルホスホニウムクロリド、メチルトリフェニルホスホニウムブロミド、メチルトリフェニルホスホニウムヨージド、テトラフェニルホスホニウムクロリド、並びにテトラフェニルホスホニウムブロミド、
からなる群より選択される1以上のイオン性界面活性剤である、請求項17に記載の組成物。 - 含まれる界面活性剤が、測定時における終濃度として、0.01%(w/v)以上である、請求項13~18のいずれか1項に記載の組成物。
- ホウ酸緩衝剤、トリス塩酸緩衝剤、リン酸緩衝剤、クエン酸緩衝剤、フマル酸緩衝剤、グルタル酸緩衝剤、シトラコン酸緩衝剤、メサコン酸緩衝剤、マロン酸緩衝剤、酒石酸緩衝剤、コハク酸緩衝剤、アジピン酸緩衝剤、ACES(N-(2-アセトアミド)-2-アミノエタンスルホン酸)緩衝剤、BES(N,N-ビス(2-ヒドロキシエチル)-2-アミノエタンスルホン酸)緩衝剤、Bicin(N,N-ビス(2-ヒドロキシエチル)グリシン)緩衝剤、Bis-Tris(ビス(2-ヒドロキシエチル)イミノトリス(ヒドロキシメチル)メタン)緩衝剤、EPPS(4-(2-ヒドロキシエチル)-1-ピペラジンプロパンスルホン酸)緩衝剤、HEPPSO(N-(ヒドロキシエチル)ピペラジン-N'-2-ヒドロキシプロパンスルホン酸)緩衝剤、MES(2-(N-モルホリノ)エタンスルホン酸)緩衝剤、MOPS(3-(N-モルホリノ)プロパンスルホン酸)緩衝剤、MOPSO(2-ヒドロキシ-3-モルホリノプロパンスルホン酸)緩衝剤、PIPES(ピペラジン-N,N'-ビス(2-エタンスルホン酸))緩衝剤、POPSO(ピペラジン-1,4-ビス(2-ヒドロキシプロパンスルホン酸))緩衝剤、TAPS(N-トリス(ヒドロキシメチル)メチル-3-アミノプロパンスルホン酸)緩衝剤、TAPSO(3-[N-トリス(ヒドロキシメチル)メチルアミノ]-2-ヒドロキシプロパンスルホン酸)緩衝剤、TES(N-トリス(ヒドロキシメチル)メチル-2-アミノエタンスルホン酸)緩衝剤、トリシン(N-トリス(ヒドロキシメチル)メチルグリシン)緩衝剤及びこれらの組合せからなる群より選択される1以上の緩衝剤をさらに含む、請求項13に記載の糖化ヘモグロビン測定用組成物。
- 測定溶液における終濃度が100mM以上となるリン酸緩衝剤、測定溶液における終濃度が10mM以上となるクエン酸緩衝剤、測定溶液における終濃度が150mM以上となるMES(2-(N-モルホリノ)エタンスルホン酸)緩衝剤、測定溶液における終濃度が100mM以上となるMOPS(3-(N-モルホリノ)プロパンスルホン酸)緩衝剤、測定溶液における終濃度が100mM以上となるMOPSO(2-ヒドロキシ-3-モルホリノプロパンスルホン酸)緩衝剤、及び測定溶液における終濃度が200mM以上となるACES(N-(2-アセトアミド)-2-アミノエタンスルホン酸)緩衝剤からなる群より選択される1以上の緩衝剤を含む、請求項20に記載の組成物。
- トリカルボン酸がクエン酸であるか、または、ジカルボン酸がフマル酸、グルタル酸、シトラコン酸、メサコン酸、マロン酸、酒石酸、コハク酸、アジピン酸、マレイン酸、リンゴ酸及びこれらの組合せからなる群より選択されるものであるか、モノカルボン酸が酢酸であるか、または、式(IV)の化合物がMES、MOPS、MOPSO及びこれらの組合せからなる群より選択されるものである、請求項22に記載の組成物。
- 前記安定化剤が、測定溶液における終濃度が2mM以上となるリン酸、測定溶液における終濃度が0.2mM以上となるクエン酸、測定溶液における終濃度が2mM以上となるリンゴ酸、測定溶液における終濃度が2mM以上となるマレイン酸、測定溶液における終濃度が2mM以上となるシトラコン酸、測定溶液における終濃度が2mM以上となるマロン酸、測定溶液における終濃度が2mM以上となるグルタル酸、測定溶液における終濃度が2mM以上となる酒石酸、測定溶液における終濃度が10mM以上となる酢酸、測定溶液における終濃度が10mM以上となるMES(2-(N-モルホリノ)エタンスルホン酸)、測定溶液における終濃度が10mM以上となるMOPS(3-(N-モルホリノ)プロパンスルホン酸)、測定溶液における終濃度が10mM以上となるMOPSO(2-ヒドロキシ-3-モルホリノプロパンスルホン酸)、測定溶液における終濃度が2mM以上となる硫酸アンモニウム、及びこれらの組合せからなる群より選択される1以上の安定化剤である、請求項22または23に記載の組成物。
- 請求項20又は21に記載の緩衝剤、及び、請求項22、23又は24に記載の安定化剤を含む糖化ヘモグロビン測定用組成物。
- アマドリアーゼが、配列番号1、配列番号37もしくは配列番号40のアミノ酸配列を有するアマドリアーゼ、または請求項1~7のいずれか1項に記載のアマドリアーゼである、請求項20~25のいずれか1項に記載の組成物。
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WO2016063984A1 (ja) * | 2014-10-24 | 2016-04-28 | キッコーマン株式会社 | デヒドロゲナーゼ活性の向上したアマドリアーゼ |
WO2016072520A1 (ja) * | 2014-11-07 | 2016-05-12 | キッコーマン株式会社 | アニオン性界面活性剤耐性が向上したアマドリアーゼ |
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