WO2016068218A1 - デヒドロゲナーゼ化した変異酵素及びその用途 - Google Patents
デヒドロゲナーゼ化した変異酵素及びその用途 Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- 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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- 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
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03006—Cholesterol oxidase (1.1.3.6)
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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.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
Definitions
- the present invention relates to enzyme mutation technology and use thereof. Specifically, the present invention relates to dehydrogenase-ized cholesterol oxidase and its gene, and a preparation method and use thereof.
- This application claims priority based on Japanese Patent Application No. 2014-219593 filed on Oct. 28, 2014, the entire contents of which are incorporated by reference.
- CHO cholesterol oxidase
- CHDH cholesterol dehydrogenase
- Measures using CHO may be affected by dissolved oxygen in the biosensor measurement sample, and it has been pointed out that dissolved oxygen may affect the measurement results.
- measurement using CHDH has the advantage of not being affected by dissolved oxygen in the biosensor measurement sample.
- NAD NAD
- the measurement using CHDH is not only complicated in operation, but also has a disadvantage in terms of cost, especially in industrial use because NAD is relatively expensive. Therefore, an enzyme that is not affected by dissolved oxygen and does not require the addition of NAD is required.
- an object of the present invention is to provide an enzyme having excellent practicality that can be used for measuring cholesterol.
- a method of bringing CHO closer to CHDH (dehydrogenase conversion) can be considered.
- this method there is a possibility of obtaining an enzyme having the advantages of CHO, that is, having FAD as a coenzyme.
- the enzyme cholesterol can be measured without adding NAD and without being affected by dissolved oxygen.
- Non-patent Document 1 Non-patent Document 1
- Non-patent Document 1 discloses a surprising dehydrogenase conversion occurred far exceeding that already reported.
- a microorganism-derived cholesterol oxidase amino acid sequence comprising an amino acid sequence in which one or more amino acids selected from the group consisting of the following (1) to (8) are substituted with other amino acids, and derived from the microorganism
- mutant enzyme with higher cholesterol dehydrogenase activity CHDH activity / CHO activity
- cholesterol oxidase activity (1) an amino acid corresponding to amino acid 113 in the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid corresponding to amino acid 362 of the amino acid sequence shown in SEQ ID NO: 1; (3) an amino acid corresponding to amino acid 402 of the amino acid sequence shown in SEQ ID NO: 1; (4) an amino acid corresponding to the amino acid at position 412 of the amino acid sequence shown in SEQ ID NO: 1; (5) an amino acid corresponding to the 468th amino acid in the amino acid sequence shown in SEQ ID NO: 1; (6) an amino acid corresponding to amino acid sequence in which one or more amino acids selected from the group consisting of the following (1) to (8) are
- the mutant enzyme according to [1] or [2], wherein the amino acid to be substituted is the amino acid of (2) and the amino acid after substitution is proline.
- the amino acid to be substituted is a combination of the amino acid of (2) and the amino acid of (4), the combination of the amino acid of (2) and the amino acid of (6), the combination of the amino acid of (2) and the amino acid of (7)
- the mutant enzyme according to [1] or [2] which is a combination of the amino acid (2) and the amino acid (8).
- [5] The amino acid after substitution is proline for the amino acid (2), tyrosine for the amino acid (4), methionine or tryptophan for the amino acid (6), and amino acid (7) Is glycine, leucine, threonine or alanine, and the amino acid (8) is cysteine, isoleucine, serine or threonine according to [4].
- [6] The mutant enzyme according to [1], comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 18.
- [7] A gene encoding the mutant enzyme according to any one of [1] to [6].
- [8] The gene according to [7], comprising any one of the nucleotide sequences of SEQ ID NOS: 20 to 36.
- a recombinant DNA comprising the gene according to [7] or [8].
- a method for measuring cholesterol comprising measuring cholesterol in a sample using the mutant enzyme according to any one of [1] to [6].
- a cholesterol measurement reagent comprising the mutant enzyme according to any one of [1] to [6].
- a cholesterol measurement kit comprising the cholesterol measurement reagent according to [12].
- An enzyme agent comprising the mutant enzyme according to any one of [1] to [6].
- a method for preparing a mutant enzyme comprising the following steps (I) to (III): (I) providing a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 18; (II) expressing the nucleic acid; and (III) recovering the expression product.
- Streptomyces sp.-derived CHO (CHO in the figure) L113, M362, L412, D468, and Y483 are highly conserved.
- the homology of the amino acid sequence of each CHO to the amino acid sequence of CHO derived from Streptomyces sp. (SEQ ID NO: 1) is as follows.
- CHO (SEQ ID NO: 37) derived from Streptomyces albulus: 81% CHO derived from Streptomyces virginiae (SEQ ID NO: 38): 85% CHO derived from Streptomyces lavendulae (Streptomyces lavendulae) SEQ ID NO: 39): 84% CHO (SEQ ID NO: 40) derived from Streptomyces natalensis: 82% CHO (SEQ ID NO: 41) derived from Streptomyces 41 natalensis: 82% Streptomyces CHO derived from Streptomyces avermitilis (SEQ ID NO: 42): 82% CHO derived from Streptomyces griseus (SEQ ID NO: 43): 81% CHO derived from Streptomyces hygrospinosus (Streptomyces hygrospinosus) SEQ ID NO: 44): 81% streptoma CHO derived from Streptomyces)
- Mutation M362P and another mutation (mutations around the substrate binding site L113E, L113T, L113V, L113M, L113L, L412K, L412Y, L412P, L412T, L412G, M402D, M402N, M402G, M402W, M402T, D468P, D468Q, D468H , D468Y and D468F) were examined for effects. Evaluation of double mutants. The effect of combining mutation M362P with another mutation (saturation mutation of Y483, mutation of S518, saturation mutation of V519, which is a mutation around the active center) was examined. Comparison of activity ratio (CHDH activity / CHO activity).
- mutant enzyme is an enzyme obtained by mutating or modifying an “underlying enzyme” by the technique disclosed in this specification. “Mutant enzyme”, “mutant enzyme” and “modified enzyme” are used interchangeably.
- the underlying enzyme is typically a wild type enzyme. However, this does not preclude the application of an enzyme that has already been subjected to artificial manipulation to the present invention as a “base enzyme”.
- base enzyme is also referred to as “mutation target enzyme” in the present specification.
- Modify one enzyme (referred to as A enzyme for convenience) to be similar to another enzyme (referred to as enzyme B for convenience), that is, to make one or more properties of enzyme A closer to the corresponding properties of enzyme B
- This is referred to as “converting enzyme A into enzyme B”.
- cholesterol oxidase (CHO) is brought close to cholesterol dehydrogenase (CHDH), that is, cholesterol dehydrogenase activity is imparted or increased while reducing the oxidase activity of CHO.
- CHDH cholesterol dehydrogenase
- dehydrogenase mutant enzyme of the present invention has a higher ratio of CHDH activity to CHO activity (CHDH activity / CHO activity) than the original enzyme (CHO before dehydrogenase).
- amino acid substitution is performed as mutation or modification. Therefore, when the mutant enzyme is compared with the enzyme to be mutated, some amino acid residues are different.
- each amino acid is represented by one letter as follows.
- amino acid residue at the mutation point is expressed by a combination of the above-mentioned one letter representing the type of amino acid and a number representing the amino acid position. For example, if the 362rd methionine is a mutation point, it is expressed as “M362”.
- the first aspect of the present invention relates to an enzyme obtained by mutating a microorganism-derived cholesterol oxidase (CHO) (hereinafter also referred to as “mutant CHO”).
- CHO microorganism-derived cholesterol oxidase
- one or two or more amino acids selected from the group consisting of the following (1) to (8) are substituted with other amino acids in the amino acid sequence of a microorganism-derived CHO (mutation target enzyme). Have the amino acid sequence.
- Amino acid corresponding to amino acid 113 in the amino acid sequence shown in SEQ ID NO: 1 (2) Amino acid corresponding to amino acid 362 in the amino acid sequence shown in SEQ ID NO: 1 (3) Amino acid in position 402 of the amino acid sequence shown in SEQ ID NO: 1 (4) amino acid corresponding to amino acid position 412 of the amino acid sequence shown in SEQ ID NO: 1 (5) amino acid corresponding to amino acid position 468 of the amino acid sequence shown in SEQ ID NO: 1 (6) amino acid sequence shown in SEQ ID NO: 1 (7) amino acid corresponding to amino acid position 518 of the amino acid sequence shown in SEQ ID NO: 1 (8) amino acid corresponding to amino acid position 519 of the amino acid sequence shown in SEQ ID NO: 1
- the 113th amino acid, the 362nd amino acid, the 402th amino acid, the 412th amino acid and the 468th amino acid of the amino acid sequence shown in SEQ ID NO: 1 are around the substrate binding site of Streptomyces sp.CHO. It is thought to play an important role in the interaction with the substrate.
- amino acids 483, 518 and 519 of the amino acid sequence shown in SEQ ID NO: 1 are located around the active center of Streptomyces sp. CHO and are considered to play an important role in enzyme activity.
- dehydrogenase is achieved by mutating amino acids corresponding to these amino acids, which are considered to play an important role in the properties of CHO.
- the mutant enzyme of the present invention has a feature that the ratio of cholesterol dehydrogenase activity to cholesterol oxidase activity (CHDH activity / CHO activity) is higher than that of the enzyme before mutation (that is, microorganism-derived cholesterol oxidase which is the enzyme to be mutated). Show.
- the “CHDH activity / CHO activity” (calculated by specific activity) of the mutant enzyme is, for example, 5 or more, preferably 10 or more, more preferably 20 or more, and even more preferably 30 or more.
- the mutated enzyme is, for example, 1.0 ⁇ 10 6 times or more, preferably 1.5 ⁇ 10 6 times or more, more preferably 2.0 ⁇ 10 6 times or more, and even more preferably 1.0 ⁇ 10 7 times or more “CHDH” Activity / CHO activity "(compared with specific activity).
- the term “corresponding” when used for amino acid residues in the present specification means that the protein (enzyme) to be compared makes an equivalent contribution to the performance of its function.
- an amino acid sequence to be compared with a reference amino acid sequence that is, the amino acid sequence of SEQ ID NO: 1 is arranged so that an optimal comparison can be made while considering partial homology of the primary structure (amino acid sequence).
- the amino acid at the position corresponding to a specific amino acid in the reference amino acid sequence is identified as a “corresponding amino acid”.
- the “corresponding amino acid” can also be specified by comparing three-dimensional structures (three-dimensional structures). A highly reliable comparison result can be obtained by using the three-dimensional structure information.
- a method of performing alignment while comparing atomic coordinates of the three-dimensional structures of a plurality of enzymes can be employed.
- the three-dimensional structure information of the enzyme to be mutated can be obtained from, for example, Protein Data Bank (http://www.pdbj.org/index_j.html).
- Crystallize the protein is indispensable for determining the three-dimensional structure, but it also has industrial utility as a high purity protein purification method and a high density and stable storage method. In this case, it is preferable to crystallize a protein bound with a substrate or an analog compound thereof as a ligand.
- Diffraction data is collected by irradiating the produced crystal with X-rays. In many cases, protein crystals are damaged by X-ray irradiation and the diffraction ability deteriorates. In that case, a cryogenic measurement technique in which the crystal is rapidly cooled to about ⁇ 173 ° C.
- the heavy atom isomorphous substitution method is a method of obtaining phase information by introducing a metal atom having a large atomic number such as mercury or platinum into a crystal and utilizing the contribution of the metal atom to the X-ray diffraction data of the large X-ray scattering ability. .
- the determined phase can be improved by smoothing the electron density of the solvent region in the crystal. Since water molecules in the solvent region have large fluctuations, almost no electron density is observed, so by approximating the electron density in this region to 0, it is possible to approach the true electron density and thus the phase is improved. . Further, when a plurality of molecules are contained in the asymmetric unit, the phase is further improved by averaging the electron density of these molecules. The protein model is fit to the electron density map calculated using the improved phase in this way. This process is performed on a computer graphic using a program such as QUANTA from MSI (USA). Thereafter, the structure is refined using a program such as X-PLOR of MSI, and the structural analysis is completed.
- crystal structure of a similar protein When the crystal structure of a similar protein is known with respect to the target protein, it can be determined by a molecular replacement method using the atomic coordinates of the known protein. Molecular replacement and structural refinement can be performed using programs such as CNS_SOLVE ver.11.
- a CHO derived from a microorganism that is an enzyme to be mutated a CHO derived from Streptomyces sp. (SEQ ID NO: 1 shows an amino acid sequence (an example)) or a CHO derived from Streptomyces sp. preferable.
- a specific example of the latter is CHO having an amino acid sequence showing 65% or more, preferably 80% or more, more preferably 90% or more homology with the amino acid sequence of SEQ ID NO: 1.
- Examples of such highly homologous CHO include Streptomyces albras-derived CHO (SEQ ID NO: 37), Streptomyces virginie-derived CHO (SEQ ID NO: 38 shows an amino acid sequence (example)), and Streptomyces lavendulae CHO (SEQ ID NO: 39 shows amino acid sequence (example)), Streptomyces chatanoogensis-derived CHO (SEQ ID NO: 40 shows amino acid sequence (example)), Streptomyces natalensis-derived CHO (SEQ ID NO: 41) Shows an amino acid sequence (an example)), Streptomyces emitris-derived CHO (SEQ ID NO: 42 shows an amino acid sequence (an example)), Streptomyces griseus-derived CHO (SEQ ID NO: 43 shows an amino acid sequence (an example)) ), CHO derived from Streptomyces hygrospinosus (SEQ ID NO: 44 amino acid sequence) Streptomyces limosus
- SEQ ID NOs: 1 and 37 to 53 An alignment comparison of SEQ ID NOs: 1 and 37 to 53 is shown in FIGS.
- CHO derived from microorganisms CHO derived from Brevibacterium sterolicam (an amino acid sequence (an example) is shown in SEQ ID NO: 54), CHO derived from Nocardia sp. (Amino acid in SEQ ID NO: 55) And a CHO derived from Burkholderia cepacia (an amino acid sequence (example) is shown in SEQ ID NO: 56)).
- a “corresponding amino acid” may be specified by comparing three-dimensional structures (three-dimensional structures).
- the amino acid of (1) above becomes the 113th amino acid of SEQ ID NO: 1
- the amino acid of (2) above is of SEQ ID NO: 1.
- the amino acid at position 362 is the amino acid at position 402 of SEQ ID NO: 1
- the amino acid at position (4) is the amino acid at position 412 of SEQ ID NO: 1
- the amino acid at position (5) is position 468 of SEQ ID NO: 1.
- the amino acid of (6) is the 483 position amino acid of SEQ ID NO: 1
- the amino acid of (7) is the 518th amino acid of SEQ ID NO: 1
- the amino acid of (8) is the 519th amino acid of SEQ ID NO: 1.
- the amino acid of the above (1) is The amino acid of (76) of SEQ ID NO: 54
- the amino acid of (2) above is the amino acid of position 325 of SEQ ID NO: 54
- the amino acid of (3) is the amino acid of position 365 of SEQ ID NO: 54
- the amino acid of (4) is SEQ ID NO:
- the amino acid of (5) is the amino acid of position 431 of SEQ ID NO: 54
- the amino acid of (6) is the amino acid of position 446 of SEQ ID NO: 54
- the amino acid of (7) is the amino acid of SEQ ID NO: 54
- the amino acid at position 481 is the amino acid at position 482 in SEQ ID NO: 54.
- the amino acid to be substituted is preferably the amino acid (1) or the amino acid (2). These are amino acids that have been confirmed to be extremely effective for dehydrogenase alone, as shown in the Examples described later. Mutant CHO in which these amino acids are substituted exhibits a much higher activity ratio (CHDH activity / CHO activity) than the enzyme before mutation.
- amino acids after substitution are glutamic acid, threonine, valine, methionine, and tryptophan for the amino acid of (1), and proline, glutamic acid, arginine, alanine, and tryptophan for the amino acid of (2).
- Amino acids are aspartic acid, asparagine, glycine, tryptophan, threonine, (4) amino acids are lysine, tyrosine, proline, threonine, glycine, and (5) amino acids are proline, glutamine, histidine, Tyrosine, phenylalanine, methionine, tryptophan for amino acid (6), glycine, leucine, threonine, alanine for amino acid (7), cysteine, isoleucine, serine, threonine for amino acid (8). It is.
- mutant enzyme L113E SEQ ID NO: 2: A mutation of (1), and the amino acid after substitution is glutamic acid.
- Mutant enzyme M362P SEQ ID NO: 3: a mutation of (2), the amino acid after substitution is proline.
- Mutant enzyme M362E SEQ ID NO: 4: mutation of (2), the amino acid after substitution is glutamic acid.
- Mutant enzyme M362R SEQ ID NO: 5 (2) mutation, the amino acid after substitution is arginine.
- Mutant enzyme M362A SEQ ID NO: 6 (2) mutation, the amino acid after substitution is alanine.
- Mutant enzyme M362W SEQ ID NO: 7 (2) mutation, the substituted amino acid is tryptophan.
- amino acids (1) to (8) two or more amino acids may be substituted. Preferred combinations of amino acids to be substituted are listed below. (2) and (4) combination (2) and (6) combination (2) and (7) combination (2) and (8) combination
- mutant enzymes obtained by applying the above combinations are shown below.
- the combination of (2) and (6), the combination of (2) and (7), and ( A combination of 2) and (8) is preferred.
- a particularly preferred combination is the combination of (2) and (6).
- the mutant to which the combination of (2) and (6) was applied (SEQ ID NO: 10) showed a very high activity ratio (CHDH activity / CHO activity).
- the protein after the mutation may have the same function as the protein before the mutation. That is, the amino acid sequence mutation does not substantially affect the protein function, and the protein function may be maintained before and after the mutation.
- amino acids when compared with a mutant CHO consisting of an amino acid sequence in which one or more amino acids selected from the group consisting of (1) to (8) above are substituted with other amino acids, amino acids Although slight differences in sequence are observed (however, differences in amino acid sequence occur at positions other than the positions where the amino acid substitution is performed), but no substantial difference in characteristics is observed It can be regarded as an enzyme substantially identical to CHO.
- “Slight difference in amino acid sequence” as used herein typically means deletion of one to several amino acids (upper limit is 3, 5, 7, 10) constituting an amino acid sequence, It means that a mutation (change) has occurred in the amino acid sequence by substitution or addition, insertion, or a combination of 1 to several amino acids (the upper limit is 3, 5, 7, 10).
- the identity (%) between the amino acid sequence of “substantially identical enzyme” and the amino acid sequence of the above-mentioned mutant CHO as a reference is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more, and most preferably 99% or more.
- the difference in amino acid sequence may occur at a plurality of positions. “Slight differences in amino acid sequence” are preferably caused by conservative amino acid substitutions.
- the second aspect of the present invention provides a nucleic acid related to the mutant CHO of the present invention. That is, a gene encoding a mutant CHO, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a mutant CHO, and a nucleic acid that can be used as a primer for amplifying or mutating a nucleic acid encoding a mutant CHO Is provided.
- Genes encoding mutant CHO are typically used for the preparation of mutant CHO. According to a genetic engineering preparation method using a gene encoding a mutant CHO, it is possible to obtain a mutant CHO in a more homogeneous state. This method can also be said to be a suitable method when preparing a large amount of mutant CHO.
- the use of the gene encoding the mutant CHO is not limited to the preparation of the mutant CHO.
- the nucleic acid can also be used as an experimental tool for elucidating the mechanism of action of mutant CHO or as a tool for designing or creating a further mutant of an enzyme.
- the “gene encoding a mutant CHO” refers to a nucleic acid from which the mutant CHO is obtained when it is expressed, and of course a nucleic acid having a base sequence corresponding to the amino acid sequence of the mutant CHO.
- a nucleic acid obtained by adding a sequence that does not encode an amino acid sequence to such a nucleic acid is also included. Codon degeneracy is also considered.
- mutant CHO examples include SEQ ID NOs: 20 to 36. These sequences are genes encoding a mutant CHO in which a specific amino acid substitution has been made to CHO derived from Streptomyces sp. The amino acid substitution in each sequence is as follows.
- the nucleic acid of the present invention is isolated by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. Can be prepared.
- a nucleic acid (hereinafter referred to as “the base sequence of a protein encoding the mutant CHO of the present invention that is equivalent in function but having a different base sequence when compared to the base sequence of the gene encoding the mutant CHO of the present invention”). Also referred to as “homologous nucleic acid.”
- a base sequence defining a homologous nucleic acid is also referred to as “homologous base sequence”).
- An example of a homologous nucleic acid consists of a base sequence containing one or more base substitutions, deletions, insertions, additions, or inversions based on the base sequence of the nucleic acid encoding the mutant CHO of the present invention.
- a DNA encoding a protein having a typical enzymatic activity that is, CHDH activity.
- Base substitution or deletion may occur at a plurality of sites.
- the term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it depends on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the nucleic acid It is.
- homologous nucleic acids include, for example, restriction enzyme treatment, treatment with exonuclease, DNA ligase, etc., site-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) It can be obtained by introducing mutations by mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York). Homologous nucleic acids can also be obtained by other methods such as ultraviolet irradiation.
- Another aspect of the present invention relates to a nucleic acid having a base sequence complementary to the base sequence of the gene encoding the mutant CHO of the present invention. Still another embodiment of the present invention is at least about 60%, 70%, 80%, 90%, 95%, 99% of the base sequence of the gene encoding the mutant CHO of the present invention or a base sequence complementary thereto. %, 99.9% nucleic acid having the same base sequence is provided.
- Yet another aspect of the present invention relates to a nucleic acid having a base sequence that hybridizes under stringent conditions to a base sequence of a gene encoding the mutant CHO of the present invention or a base sequence complementary to the homologous base sequence thereof.
- the “stringent conditions” here are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) Can be set with reference to.
- hybridization solution 50% formamide, 10 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml denaturation
- 5 ⁇ Denhardt solution 1% SDS
- 10% dextran sulfate 10 ⁇ g / ml denaturation
- incubation at about 42 ° C to about 50 ° C using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), followed by washing at about 65 ° C to about 70 ° C using 0.1 x SSC, 0.1% SDS can be mentioned.
- Further preferable stringent conditions include, for example, 50% formamide, 5 ⁇ SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).
- nucleic acid having a base sequence of a gene encoding the mutant CHO of the present invention or a part of a base sequence complementary thereto.
- a nucleic acid fragment can be used for detecting, identifying, and / or amplifying a nucleic acid having a base sequence of a gene encoding the mutant CHO of the present invention.
- the nucleic acid fragment is, for example, a nucleotide portion continuous in the base sequence of the gene encoding the mutant CHO of the present invention (eg, about 10 to about 100 bases in length, preferably about 20 to about 100 bases in length, more preferably about 30 to about 100 in length).
- Base length is designed to include at least a portion that hybridizes.
- a nucleic acid fragment can be labeled.
- fluorescent substances, enzymes, and radioisotopes can be used.
- Still another aspect of the present invention relates to a recombinant DNA containing the gene of the present invention (gene encoding mutant CHO).
- the recombinant DNA of the present invention is provided, for example, in the form of a vector.
- vector refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.
- An appropriate vector is selected according to the purpose of use (cloning, protein expression) and in consideration of the type of host cell.
- Examples of vectors using insect cells as hosts include pAc and pVL, and examples of vectors using mammalian cells as hosts include pCDM8 and pMT2PC.
- the vector of the present invention is preferably an expression vector.
- “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell.
- Expression vectors usually contain a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like.
- An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence / absence (and extent) of introduction of the expression vector can be confirmed using a selection marker.
- Insertion of the nucleic acid of the present invention into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. are performed using standard recombinant DNA techniques (for example, Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press and New York, which can be referred to, are known methods using restriction enzymes and DNA ligases).
- E. coli Escherichia coli
- budding yeast Sacharomyces cerevisiae
- E. coli E. coli BL21 (DE3) pLysS when T7 promoter is used, and E. coli JM109 otherwise.
- budding yeast include budding yeast SHY2, budding yeast AH22, or budding yeast INVSc1 (Invitrogen).
- microorganism that is, a transformant
- the microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention.
- calcium chloride method Frnal of Molecular Biology (J. Mol. Biol.), Volume 53, pp. 159 (1970)
- Hanahan Method Journal of Molecular Biology, Volume 166, 557) (1983)
- SEM Gene, 96, 23 (1990)
- Chung et al. Proceedings of the National Academy of Sciences of the USA, 86 Vol., P.
- microorganism of the present invention is used for producing the mutant CHO of the present invention. (Method for preparing mutant enzyme described below) Refer to the column).
- the third aspect of the present invention relates to the use of mutant CHO.
- a method for measuring cholesterol using mutant CHO is provided.
- the amount of cholesterol in a sample is measured using the redox reaction by this enzyme.
- the present invention is used, for example, for measuring blood cholesterol level, measuring food cholesterol level, and the like.
- the present invention also provides a reagent for measuring cholesterol containing the present enzyme.
- the reagent is used in the above-described method for measuring cholesterol according to the present invention.
- the present invention further provides a kit (cholesterol measurement kit) for carrying out the cholesterol measurement method of the present invention.
- the kit of the present invention includes a reagent for measuring cholesterol containing the present enzyme, a reagent for reaction, a buffer solution, a cholesterol standard solution and the like as optional elements.
- an instruction manual is usually attached to the cholesterol measurement kit of the present invention.
- a further aspect of the present invention relates to a method for preparing a mutant enzyme.
- a mutant CHO successfully obtained by the present inventors is prepared by a genetic engineering technique.
- a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 18 is prepared (step (I)).
- the “nucleic acid encoding a specific amino acid sequence” is a nucleic acid from which a polypeptide having the amino acid sequence is obtained when it is expressed, not to mention a nucleic acid comprising a base sequence corresponding to the amino acid sequence.
- nucleic acid encoding any amino acid sequence of SEQ ID NOs: 2 to 18 refers to the sequence information disclosed in this specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, It can be prepared in an isolated state by using a biochemical method or the like.
- amino acid sequences of SEQ ID NOs: 2 to 18 are the amino acid sequences of CHO derived from Streptomyces sp.
- nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 18 is also obtained by adding a necessary mutation to the gene encoding CHO derived from Streptomyces sp. (SEQ ID NO: 19).
- Many methods for position-specific base sequence substitution are known in the art (see, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York), and an appropriate method is selected from them. Can be used.
- a position-specific mutation introducing method a position-specific amino acid saturation mutation method can be employed.
- the position-specific amino acid saturation mutation method is a “Semi-rational, semi-random” technique in which amino acid saturation mutation is introduced by estimating the position where the desired function is involved based on the three-dimensional structure of the protein (J. Mol. Biol. 331, 585-592 (2003)).
- a site-specific amino acid saturation mutation can be introduced by using a kit such as Quick change (Stratagene) and overlap extention PCR (Nucleic Acid Res. 16,7351-7367 (1988)).
- a DNA polymerase used for PCR Taq polymerase or the like can be used.
- it is preferable to use a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo), Pfu turbo (Stratagene).
- step (II) the prepared nucleic acid is expressed (step (II)).
- an expression vector into which the nucleic acid is inserted is prepared, and a host cell is transformed using the expression vector.
- “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell.
- Expression vectors usually contain a promoter sequence necessary for the expression of the inserted nucleic acid, an enhancer sequence that promotes expression, and the like.
- An expression vector containing a selectable marker can also be used. When such an expression vector is used, the presence / absence (and extent) of introduction of the expression vector can be confirmed using a selection marker.
- the transformant is cultured under conditions where a mutant enzyme as an expression product is produced.
- the transformant may be cultured according to a conventional method.
- the carbon source used in the medium may be any assimitable carbon compound.
- glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used.
- the nitrogen source may be any nitrogen compound that can be used.
- peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used.
- phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.
- the culture temperature can be set within the range of 30 ° C to 40 ° C (preferably around 37 ° C).
- the culture time can be set in consideration of the growth characteristics of the transformant to be cultured and the production characteristics of the mutant enzyme.
- the pH of the medium is adjusted so that the transformant grows and the enzyme is produced.
- the pH of the medium is about 6.0 to 9.0 (preferably around pH 7.0).
- the expression product (mutant enzyme) is recovered (step (III)).
- the culture solution containing the cultured microbial cells can be used as it is or after concentration, removal of impurities, etc., it can be used as an enzyme solution.
- the expression product is once recovered from the culture solution or microbial cells. If the expression product is a secreted protein, it can be recovered from the culture solution, and if not, it can be recovered from the fungus body.
- the culture supernatant is filtered and centrifuged to remove insoluble matters, followed by concentration under reduced pressure, membrane concentration, salting out using ammonium sulfate or sodium sulfate, methanol, ethanol, acetone, etc.
- chromatographic methods such as fractional precipitation, dialysis, heat treatment, isoelectric point treatment, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography (eg, Sephadex gel (GE Healthcare Bioscience)) Separation using a combination of gel filtration, DEAE Sepharose CL-6B (GE Healthcare Bioscience), Octyl Sepharose CL-6B (GE Healthcare Bioscience), CM Sepharose CL-6B (GE Healthcare Bioscience) Purify and obtain the purified product of mutant enzyme Door can be.
- DEAE Sepharose CL-6B GE Healthcare Bioscience
- Octyl Sepharose CL-6B GE Healthcare Bioscience
- CM Sepharose CL-6B GE Healthcare Bioscience
- the microbial cells are collected by filtering, centrifuging, etc., and then the microbial cells are subjected to mechanical methods such as pressure treatment, ultrasonic treatment, or enzymatic methods such as lysozyme. After destruction by the method, a purified product of the mutant enzyme can be obtained by separation and purification in the same manner as described above.
- the purified enzyme obtained as described above by pulverizing it by, for example, freeze drying, vacuum drying or spray drying.
- the purified enzyme may be dissolved in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer in advance.
- a phosphate buffer or a triethanolamine buffer can be used.
- PIPES, MES, or MOPS is mentioned as a GOOD buffer here.
- cell-free synthesis system (cell-free transcription system, cell-free transcription / translation system) refers to a ribosome derived from a live cell (or obtained by a genetic engineering technique), not a live cell. This refers to the in vitro synthesis of mRNA and protein encoded by a template nucleic acid (DNA or mRNA) using transcription / translation factors.
- a cell extract obtained by purifying a cell disruption solution as needed is generally used.
- Cell extracts generally contain ribosomes necessary for protein synthesis, various factors such as initiation factors, and various enzymes such as tRNA.
- ribosomes necessary for protein synthesis
- various factors such as initiation factors
- various enzymes such as tRNA.
- other substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate are added to the cell extract.
- a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.
- cell-free transcription / translation system is used interchangeably with a cell-free protein synthesis system, in-vitro translation system or in-vitro transcription / translation system.
- RNA is used as a template to synthesize proteins.
- total RNA, mRNA, in vitro transcript and the like are used.
- the other in vitro transcription / translation system uses DNA as a template.
- the template DNA should contain a ribosome binding region and preferably contain an appropriate terminator sequence.
- conditions to which factors necessary for each reaction are added are set so that the transcription reaction and the translation reaction proceed continuously.
- coli DH5 ⁇ was transformed. 5. SOC (100 ⁇ L / tube) was added and re-cultured (37 ° C., 1 h). 6. The whole amount was applied to an LB + Amp (100 ⁇ g / mL) plate and cultured (37 ° C., O / N). 7. Plasmid was extracted using QIA Prep (Qiagen). 8. The extracted plasmid (4 ⁇ L) was used to transform E. coli BL21 (pGKJE-8). 9. SOC (1 mL / tube) was added and re-cultured (37 ° C., 1 h). 10.
- ⁇ CHO activity measurement method 0.02 sample diluted from 1 to 1,000 times with enzyme extracted from bacterial cells (or enzyme extracted from bacterial cells and purified) with dilution buffer (50 mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH 7.0)) mL and reaction solution (0.1 M phosphate buffer (pH 7.0) 25.5 mL, substrate solution (5.3% cholesterol solution (w / v)) 2 mL, 1.76 g / dL 4-AA solution 0.5 mL, 5 g / dL Mix 1 mL of phenol solution and 1 mL of 250 U / mL PO-3 (PO “Amano” 3) solution).
- dilution buffer 50 mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH 7.0)
- reaction solution 0.1 M phosphate buffer (pH 7.0) 25.5 mL
- substrate solution 5.3% cholesterol solution (w / v)
- the enzyme activity (U) is calculated with 1 U as the amount of enzyme that produces 1 ⁇ mol of H 2 O 2 per minute under the measurement conditions.
- ⁇ CHDH activity measurement method 0.02 sample diluted from 1 to 1,000 times with enzyme extracted from bacterial cells (or enzyme extracted from bacterial cells and purified) with dilution buffer (50 mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH 7.0)) mL and reaction solution (0.1 M phosphate buffer (pH 7.0) 25 mL, substrate solution (5.3% cholesterol solution (w / v)) 2 mL, 3 mmol / L PMS solution 2 mL, 6.6 mmol / L NTB solution 1 After mixing 0.2 mL, react at 37 ° C. for 0.5 hour, and measure the absorbance at 570 nm. The enzyme activity (U) is calculated with 1 U as the amount of enzyme that produces 0.5 ⁇ mol of diformazan dye per minute under the measurement conditions.
- dilution buffer 50 mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH 7.0)
- reaction solution 0.1 M
- ⁇ Result> The results (part of experimental data; data for M402 and L412 are omitted) are shown in FIG. Based on the activity ratio ( ⁇ 570nm / ⁇ 500nm), 5 mutation sites around the substrate binding site (L113, M362, M402, L412, D468) and 3 mutation sites around the active center (Y483, S518, V519) was selected.
- ⁇ 570 is the difference between the OD value of the sample at the end of the 0.5 hour reaction in the CHDH activity measurement method and the OD value of the same blank
- ⁇ 500 is the OD value of the sample at the end of the 0.5 hour reaction in the CHO activity measurement method. It is the difference of the OD value of the same blank.
- E. coli BL21 (pGKJE-8) was transformed with the ligation reaction solution (10 ⁇ L / tube). 5.
- the LB + Amp (100 ⁇ g / mL) + Cm (20 ⁇ g / mL) plate was applied and cultured (37 ° C., O / N). 6.
- Activity was confirmed using a CHO activity measurement method and a CHDH activity measurement method. 9. The activity ratio (CHDH / CHO) was compared.
- L113 Glutamic acid, threonine, valine, methionine, tryptophan M362: Proline, glutamic acid, arginine, alanine, tryptophan M402: Aspartic acid, asparagine, glycine, tryptophan, threonine L412: Lysine, tyrosine, proline, threonine, glycine D468: Proline, glutamine , Histidine, tyrosine, phenylalanine Y483: methionine, tryptophan S518: glycine, leucine, threonine, alanine V519: cysteine, isoleucine, serine, threonine
- Ligation treatment (16 ° C., 2 h) was performed using DpnI treatment solution (2 ⁇ L). 4. E. coli BL21 (pGKJE-8) was transformed with the ligation reaction solution (10 ⁇ L / tube). 5. The LB + Amp (100 ⁇ g / mL) + Cm (20 ⁇ g / mL) plate was applied and cultured (37 ° C., O / N). 6. After pre-culture in LB Broth (invitrogen), main culture was performed in Teriffic Broth (invitrogen). 7. After the main culture, the cells were collected and the enzyme was extracted using B-per (TaKaRa). 8. Activity was confirmed using a CHO activity measurement method and a CHDH activity measurement method. 9. The activity ratio (CHDH / CHO) was compared.
- Combination 1 M362P + L412Y Combination 2: M362P + Y483M Combination 3: M362P + Y483W Combination 4: M362P + S518G Combination 5: M362P + S518L Combination 6: M362P + S518T Combination 7: M362P + S518A Combination 8: M362P + V519C Combination 9: M362P + V519I Combination 10: M362P + V519S Combination 11: M362P + V519T
- the specific activity of the dehydrogenated mutant enzymes was determined by the following procedure. For comparison, the specific activity of the wild-type enzyme and the previously reported mutant enzyme (V228A) was also calculated. 1. For each mutant enzyme, the transformed E. coli was cultured (preculture / main culture). The culture conditions were in accordance with the above experiment. 2.
- Bacteria were collected from the culture and purified (bacteria collection ⁇ bacterial crushing (bead crushing) ⁇ supernatant collection ⁇ aggregation treatment ⁇ column purification (DEAE Sepharose, Butyl-S Sepharose) ⁇ desalting and concentration). 3. Activity measurement (CHO, CHDH) and protein concentration were measured to calculate specific activity. 4. CHDH / CHO (specific activity) was compared.
- the activity ratio (CHDH activity / CHO activity) was about 2 to 20 times that of a single mutation.
- the activity value U / mL in FIG. 8 is calculated from the difference between the OD values of the samples at 3 minutes and 5 minutes during the 0.5 hour reaction in the activity measurement method.
- the mutant CHO of the present invention is useful for detection and quantification of cholesterol in a sample.
- the mutant CHO of the present invention has both the advantages of CHO (no need to add a coenzyme during measurement) and CHDH (not affected by dissolved oxygen), and its utility value is high. Since the mutant CHO of the present invention includes the coenzyme FAD, it is not necessary to add a coenzyme (NAD in the case of CHDH), and simple and low-cost measurement is possible.
Abstract
Description
[1]微生物由来コレステロールオキシダーゼのアミノ酸配列において、以下の(1)~(8)からなる群より選択される一又は二以上のアミノ酸が他のアミノ酸に置換されたアミノ酸配列からなり、前記微生物由来コレステロールオキシダーゼに比較して、コレステロールオキシダーゼ活性に対するコレステロールデヒドロゲナーゼ活性(CHDH活性/CHO活性)が高い、変異酵素:
(1)配列番号1に示すアミノ酸配列の113位アミノ酸に相当するアミノ酸;
(2)配列番号1に示すアミノ酸配列の362位アミノ酸に相当するアミノ酸;
(3)配列番号1に示すアミノ酸配列の402位アミノ酸に相当するアミノ酸;
(4)配列番号1に示すアミノ酸配列の412位アミノ酸に相当するアミノ酸;
(5)配列番号1に示すアミノ酸配列の468位アミノ酸に相当するアミノ酸;
(6)配列番号1に示すアミノ酸配列の483位アミノ酸に相当するアミノ酸;
(7)配列番号1に示すアミノ酸配列の518位アミノ酸に相当するアミノ酸;
(8)配列番号1に示すアミノ酸配列の519位アミノ酸に相当するアミノ酸。
[2]前記微生物由来コレステロールオキシダーゼのアミノ酸配列が、配列番号1のアミノ酸配列と65%以上の同一性を示す配列である、[1]に記載の変異酵素。
[3]置換されるアミノ酸が(2)のアミノ酸であり、置換後のアミノ酸がプロリンである、[1]又は[2]に記載の変異酵素。
[4]置換されるアミノ酸が、(2)のアミノ酸と(4)のアミノ酸の組合せ、(2)のアミノ酸と(6)のアミノ酸の組合せ、(2)のアミノ酸と(7)のアミノ酸の組合せ、又は(2)のアミノ酸と(8)のアミノ酸の組合せである、[1]又は[2]に記載の変異酵素。
[5]置換後のアミノ酸が、(2)のアミノ酸についてはプロリンであり、(4)のアミノ酸についてはチロシンであり、(6)のアミノ酸についてはメチオニン又はトリプトファンであり、(7)のアミノ酸についてはグリシン、ロイシン、トレオニン又はアラニンであり、(8)のアミノ酸についてはシステイン、イソロイシン、セリン又はトレオニンである、[4]に記載の変異酵素。
[6]配列番号2~18のいずれかのアミノ酸配列からなる、[1]に記載の変異酵素。
[7][1]~[6]のいずれか一項に記載の変異酵素をコードする遺伝子。
[8]配列番号20~36のいずれかの塩基配列を含む、[7]に記載の遺伝子。
[9][7]又は[8]に記載の遺伝子を含む組換えDNA。
[10][9]に記載の組換えDNAを保有する微生物。
[11][1]~[6]のいずれか一項に記載の変異酵素を用いて試料中のコレステロールを測定することを特徴とする、コレステロール測定法。
[12][1]~[6]のいずれか一項に記載の変異酵素を含むことを特徴とするコレステロール測定用試薬。
[13][12]に記載のコレステロール測定用試薬を含む、コレステロール測定用キット。
[14][1]~[6]のいずれか一項に記載の変異酵素を含有する酵素剤。
[15]以下のステップ(I)~(III)を含む、変異酵素の調製法:
(I)配列番号2~18のいずれかのアミノ酸配列をコードする核酸を用意するステップ;
(II)前記核酸を発現させるステップ、及び
(III)発現産物を回収するステップ。
(用語)
用語「変異酵素」とは、本明細書が開示する手法によって、「基になる酵素」を変異ないし改変して得られる酵素である。「変異酵素」、「変異型酵素」及び「改変型酵素」は置換可能に用いられる。基になる酵素は典型的には野生型酵素である。但し、既に人為的操作が施されている酵素を「基になる酵素」として本発明に適用することを妨げるものではない。尚、「基になる酵素」のことを本明細書では「変異対象酵素」とも呼ぶ。
メチオニン:M、セリン:S、アラニン:A、トレオニン:T、バリン:V、チロシン:Y、ロイシン:L、アスパラギン:N、イソロイシン:I、グルタミン:Q、プロリン:P、アスパラギン酸:D、フェニルアラニン:F、グルタミン酸:E、トリプトファン:W、リジン:K、システイン:C、アルギニン:R、グリシン:G、ヒスチジン:H
本発明の第1の局面は微生物由来のコレステロールオキシダーゼ(CHO)を変異させた酵素(以下「変異CHO」とも呼ぶ)に関する。本発明の変異CHOは、微生物由来のCHO(変異対象酵素)のアミノ酸配列において、以下の(1)~(8)からなる群より選択される一又は二以上のアミノ酸が他のアミノ酸に置換されたアミノ酸配列を有する。
(1)配列番号1に示すアミノ酸配列の113位アミノ酸に相当するアミノ酸
(2)配列番号1に示すアミノ酸配列の362位アミノ酸に相当するアミノ酸
(3)配列番号1に示すアミノ酸配列の402位アミノ酸に相当するアミノ酸
(4)配列番号1に示すアミノ酸配列の412位アミノ酸に相当するアミノ酸
(5)配列番号1に示すアミノ酸配列の468位アミノ酸に相当するアミノ酸
(6)配列番号1に示すアミノ酸配列の483位アミノ酸に相当するアミノ酸
(7)配列番号1に示すアミノ酸配列の518位アミノ酸に相当するアミノ酸
(8)配列番号1に示すアミノ酸配列の519位アミノ酸に相当するアミノ酸
(1)タンパク質を結晶化する。結晶化は、立体構造決定のためには欠かせないが、それ以外にも、タンパク質の高純度の精製法、高密度で安定な保存法として産業上の有用性もある。この場合、リガンドとして基質もしくはそのアナログ化合物を結合したタンパク質を結晶化すると良い。
(2)作製した結晶にX線を照射して回折データを収集する。なお、タンパク質結晶はX線照射によりダメージを受け回折能が劣化するケースが多々ある。その場合、結晶を急激に-173℃程度に冷却し、その状態で回折データを収集する低温測定技術が最近普及してきた。なお、最終的に、構造決定に利用する高分解能データを収集するために、輝度の高いシンクロトロン放射光が利用される。
(3)結晶構造解析を行うには、回折データに加えて、位相情報が必要になる。目的のタンパク質に対して、類縁のタンパク質の結晶構造が未知の場合、分子置換法で構造決定することは不可能であり、重原子同型置換法により位相問題が解決されなくてはならない。重原子同型置換法は、水銀や白金等原子番号が大きな金属原子を結晶に導入し、金属原子の大きなX線散乱能のX線回折データへの寄与を利用して位相情報を得る方法である。決定された位相は、結晶中の溶媒領域の電子密度を平滑化することにより改善することが可能である。溶媒領域の水分子は揺らぎが大きいために電子密度がほとんど観測されないので、この領域の電子密度を0に近似することにより、真の電子密度に近づくことができ、ひいては位相が改善されるのである。また、非対称単位に複数の分子が含まれている場合、これらの分子の電子密度を平均化することにより位相が更に大幅に改善される。このようにして改善された位相を用いて計算した電子密度図にタンパク質のモデルをフィットさせる。このプロセスは、コンピューターグラフィックス上で、MSI社(アメリカ)のQUANTA等のプログラムを用いて行われる。この後、MSI社のX-PLOR等のプログラムを用いて、構造精密化を行い、構造解析は完了する。目的のタンパク質に対して、類縁のタンパク質の結晶構造が既知の場合は、既知タンパク質の原子座標を用いて分子置換法により決定できる。分子置換と構造精密化はプログラム CNS_SOLVE ver.11などを用いて行うことができる。
変異酵素L113E:配列番号2:(1)の変異であり、置換後のアミノ酸はグルタミン酸。
変異酵素M362P:配列番号3:(2)の変異であり、置換後のアミノ酸はプロリン。
変異酵素M362E:配列番号4:(2)の変異であり、置換後のアミノ酸はグルタミン酸。
変異酵素M362R:配列番号5:(2)の変異であり、置換後のアミノ酸はアルギニン。
変異酵素M362A:配列番号6:(2)の変異であり、置換後のアミノ酸はアラニン。
変異酵素M362W:配列番号7:(2)の変異であり、置換後のアミノ酸はトリプトファン。
(2)と(4)の組合せ
(2)と(6)の組合せ
(2)と(7)の組合せ
(2)と(8)の組合せ
変異酵素(M362P+L412Y):配列番号8:(2)と(4)の組合せであり、(2)の置換後のアミノ酸はプロリン、(4)の置換後のアミノ酸はチロシン。
変異酵素(M362P+Y483M):配列番号9:(2)と(6)の組合せであり、(2)の置換後のアミノ酸はプロリン、(6)の置換後のアミノ酸はメチオニン。
変異酵素(M362P+Y483W):配列番号10:(2)と(6)の組合せあり、(2)の置換後のアミノ酸はプロリン、(6)の置換後のアミノ酸はトリプトファン。
変異酵素(M362P+S518G):配列番号11:(2)と(7)の組合せあり、(2)の置換後のアミノ酸はプロリン、(7)の置換後のアミノ酸はグリシン。
変異酵素(M362P+S518L):配列番号12:(2)と(7)の組合せあり、(2)の置換後のアミノ酸はプロリン、(7)の置換後のアミノ酸はロイシン。
変異酵素(M362P+S518T):配列番号13:(2)と(7)の組合せあり、(2)の置換後のアミノ酸はプロリン、(7)の置換後のアミノ酸はトレオニン。
変異酵素(M362P+S518A):配列番号14:(2)と(7)の組合せあり、(2)の置換後のアミノ酸はプロリン、(7)の置換後のアミノ酸はアラニン。
変異酵素(M362P+V519C):配列番号15:(2)と(8)の組合せあり、(2)の置換後のアミノ酸はプロリン、(8)の置換後のアミノ酸はシステイン。
変異酵素(M362P+V519I):配列番号16:(2)と(8)の組合せあり、(2)の置換後のアミノ酸はプロリン、(8)の置換後のアミノ酸はイソロイシン。
変異酵素(M362P+V519S):配列番号17:(2)と(8)の組合せあり、(2)の置換後のアミノ酸はプロリン、(8)の置換後のアミノ酸はセリン。
変異酵素(M362P+V519T):配列番号18:(2)と(8)の組合せあり、(2)の置換後のアミノ酸はプロリン、(8)の置換後のアミノ酸はトレオニン。
本発明の第2の局面は本発明の変異CHOに関連する核酸を提供する。即ち、変異CHOをコードする遺伝子、変異CHOをコードする核酸を同定するためのプローブとして用いることができる核酸、変異CHOをコードする核酸を増幅又は突然変異等させるためのプライマーとして用いることができる核酸が提供される。
配列番号20:L113E
配列番号21:M362P
配列番号22:M362E
配列番号23:M362R
配列番号24:M362A
配列番号25:M362W
配列番号26:M362P及びL412Y
配列番号27:M362P及びY483M
配列番号28:M362P及びY483W
配列番号29:M362P及びS518G
配列番号30:M362P及びS518L
配列番号31:M362P及びS518T
配列番号32:M362P及びS518A
配列番号33:M362P及びV519C
配列番号34:M362P及びV519I
配列番号35:M362P及びV519S
配列番号36:M362P及びV519T
本発明の第3の局面は変異CHOの用途に関する。この局面では、変異CHOを用いたコレステロール測定法が提供される。本発明のコレステロール測定法では本酵素による酸化還元反応を利用して試料中のコレステロール量を測定する。本発明は例えば血中コレステロール量の測定、食品中コレステロール量の測定などに利用される。
本発明の更なる局面は変異酵素の調製法に関する。本発明の変異酵素調製法の一態様では、本発明者らが取得に成功した変異CHOを遺伝子工学的手法で調製する。この態様の場合、配列番号2~18のいずれかのアミノ酸配列をコードする核酸を用意する(ステップ(I))。ここで、「特定のアミノ酸配列をコードする核酸」は、それを発現させた場合に当該アミノ酸配列を有するポリペプチドが得られる核酸であり、当該アミノ酸配列に対応する塩基配列からなる核酸は勿論のこと、そのような核酸に余分な配列(アミノ酸配列をコードする配列であっても、アミノ酸配列をコードしない配列であってもよい)が付加されていてもよい。また、コドンの縮重も考慮される。「配列番号2~18のいずれかのアミノ酸配列をコードする核酸」は、本明細書又は添付の配列表が開示する配列情報を参考にし、標準的な遺伝子工学的手法、分子生物学的手法、生化学的手法などを用いることによって、単離された状態に調製することができる。ここで、配列番号2~18のアミノ酸配列はいずれも、ストレプトマイセス sp.由来CHOのアミノ酸配列に変異を施したものである。従って、ストレプトマイセス sp.由来CHOをコードする遺伝子(配列番号19)に対して必要な変異を加えることによっても、配列番号2~18のいずれかのアミノ酸配列をコードする核酸(遺伝子)を得ることができる。位置特異的塩基配列置換のための方法は当該技術分野において数多く知られており(例えば、Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New Yorkを参照)、その中から適切な方法を選択して用いることができる。位置特異的変異導入法として、位置特異的アミノ酸飽和変異法を採用することができる。位置特異的アミノ酸飽和変異法は、タンパクの立体構造を基に、求める機能の関与する位置を推定し、アミノ酸飽和変異を導入する「Semi-rational,semi-random」手法である(J.Mol.Biol.331,585-592(2003))。例えば、Quick change(ストラタジーン社)等のキット、Overlap extention PCR(Nucleic Acid Res. 16,7351-7367(1988))を用いて位置特異的アミノ酸飽和変異を導入することが可能である。PCRに用いるDNAポリメラーゼはTaqポリメラーゼ等を用いることができる。但し、KOD-PLUS-(東洋紡社)、Pfu turbo(ストラタジーン社)などの精度の高いDNAポリメラーゼを用いることが好ましい。
既知のコレステロールオキシダーゼ(Streptomyces sp. SA-COO)の立体構造情報(プロテインデータバンク(PDB): 1MXT)をもとに、ストレプトマイセス sp.由来コレステロールオキシダーゼ(CHO)(配列番号1)の立体構造を予測し、変異点を選択した。変異点は、基質結合部位周辺(18箇所)、及び活性中心周辺(9箇所)を選択した。
A.で特定した変異点からデヒドロゲナーゼ化に効果的な変異点を、ランダムライブラリーを用いて選抜することにした。
デヒドロゲナーゼ化に効果的な変異点を以下の方法で選抜した。
1. PCR(反応液:25μL/チューブ)により、ランダム変異を導入した。尚、テンプレートにはpC4-CHOA1 No.1(プラスミド:pColdIV、挿入遺伝子:cho遺伝子(BspHI-HindIII))を使用した。
2. PCR反応液(25μL/チューブ)に、制限酵素DpnI(1.5μL/チューブ)を添加して処理(37℃、1h)した。
3. DpnI処理液(2μL)を用いて、ライゲーション処理(16℃、2h)した。
4. ライゲーション反応液(10μL/チューブ)を用いて、E.coli DH5αに形質転換した。
5. SOC(100μL/チューブ)を添加して、復帰培養(37℃、1h)した。
6. 全量をLB+Amp(100μg/mL)プレートに塗布して、培養(37℃、O/N)した。
7. QIA Prep(Qiagen)を用いてプラスミドを抽出した。
8. 抽出したプラスミド(4μL)を用いて、E.coli BL21(pGKJE-8)を形質転換した。
9. SOC(1mL/チューブ)を添加して、復帰培養(37℃、1h)した。
10. LB+Amp(100μg/mL)+Cm(20μg/mL)プレートに塗布して、培養(37℃、O/N)した。
11. LB Broth(invitrogen)にて前培養後、Teriffic Broth(invitrogen)にて本培養した。
12. 本培養後、菌体を回収して、B-per(TaKaRa)を用いて酵素を抽出した。
13. CHO活性測定法、CHDH活性測定法を用いて、活性を確認した。
14. 活性比(CHDH/CHO)で比較した。
菌体から抽出した酵素(又は菌体から抽出し、精製した酵素)を希釈バッファー(50mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH7.0))で1~1,000倍希釈したサンプル0.02mLと反応液(0.1M りん酸緩衝液 (pH7.0) 25.5mL、基質溶液 (5.3% コレステロール溶液 (w/v)) 2 mL、1.76 g/dL 4-A.A溶液 0.5 mL、5 g/dL フェノール溶液 1 mL、250 U/mL PO-3 (PO "Amano" 3)溶液 1 mLを混合)0.2mLを混合した後、37℃で0.5時間反応させ、吸光度500nmを測定する。本測定条件下で1分間に1μmolのH2O2を生成する酵素量を1Uとして酵素活性(U)を算出する。
菌体から抽出した酵素(又は菌体から抽出し、精製した酵素)を希釈バッファー(50mM PIPES, 0.1% Triton X-100, 0.1% BSA (pH7.0))で1~1,000倍希釈したサンプル0.02mLと反応液(0.1M りん酸緩衝液 (pH7.0) 25mL、基質溶液 (5.3% コレステロール溶液 (w/v)) 2 mL、3 mmol/L PMS溶液 2 mL、6.6 mmol/L NTB溶液 1 mLを混合)0.2mLを混合した後、37℃で0.5時間反応させ、吸光度570nmを測定する。本測定条件下で1分間に0.5μmolのジホルマザン色素を生成する酵素量を1Uとして酵素活性(U)を算出する。
結果(実験データの一部。M402及びL412のデータは省略)を図4に示す。活性比(ΔΔ570nm/Δ500nm)をもとに、基質の結合部位周辺の変異点として5箇所(L113、M362、M402、L412、D468)と、活性中心周辺の変異点として3箇所(Y483、S518、V519)を選抜した。尚、Δ570は、CHDH活性測定法における0.5時間の反応終了時のサンプルのOD値と同ブランクのOD値の差であり、Δ500はCHO活性測定法における0.5時間の反応終了時のサンプルのOD値と同ブランクのOD値の差である。
B.で選抜した変異点について、デヒドロゲナーゼ化に効果のあるアミノ酸を飽和ライブラリーを用いて検討することにした。
デヒドロゲナーゼ化に効果の高いアミノ酸を以下の方法で特定した。尚、反応条件や培養条件等はB.の方法と同様である。
1. PCR(反応液:25μL/チューブ)により、飽和変異を導入した。尚、テンプレートにはpC4-CHOA1 No.1(プラスミド:pColdIV、挿入遺伝子:cho遺伝子(BspHI-HindIII))を使用した。
2. PCR反応液(25μL/チューブ)に、制限酵素DpnI(1.5μL/チューブ)を添加して処理(37℃、1h)した。
3. DpnI処理液(2μL)を用いて、ライゲーション処理(16℃、2h)した。
4. ライゲーション反応液(10μL/チューブ)を用いて、E.coli BL21(pGKJE-8)を形質転換した。
5. LB+Amp(100μg/mL)+Cm(20μg/mL)プレートに塗布して、培養(37℃、O/N)した。
6. LB Broth(invitrogen)にて前培養後、Teriffic Broth(invitrogen)にて本培養した。
7. 本培養後、菌体を回収して、B-per(TaKaRa)を用いて酵素を抽出した。
8. CHO活性測定法、CHDH活性測定法を用いて、活性を確認した。
9. 活性比(CHDH/CHO)で比較した。
結果を図5に示す。各変異点について、以下の通り、有効な置換後のアミノ酸が特定された。尚、活性中心周辺の変異点(Y483、S518、V519)については、他の変異点と組合せた場合の効果(図7)も考慮して有効な置換後のアミノ酸を特定した。尚、図5~7における活性比(CHDH/CHO)は、活性測定法における0.5時間の反応中、20分の時点と30分の時点(反応終了時)のサンプルのOD値の差から、CHOとCHDHの各々についてU/mLを算出し、比率で表したものである。
L113:グルタミン酸、トレオニン、バリン、メチオニン、トリプトファン
M362:プロリン、グルタミン酸、アルギニン、アラニン、トリプトファン
M402:アスパラギン酸、アスパラギン、グリシン、トリプトファン、トレオニン
L412:リジン、チロシン、プロリン、トレオニン、グリシン
D468:プロリン、グルタミン、ヒスチジン、チロシン、フェニルアラニン
Y483:メチオニン、トリプトファン
S518:グリシン、ロイシン、トレオニン、アラニン
V519:システイン、イソロイシン、セリン、トレオニン
変異点の組合せの効果について検討した。
変異点を組み合わせた場合の効果を以下の方法で検討した。尚、反応条件や培養条件等はB.の方法と同様である。
1. PCR(反応液:25μL/チューブ)により、飽和変異を導入した。尚、テンプレートにはpC4-CHOA1 No.1 - M362P(プラスミド:pColdIV、挿入遺伝子:M362P変異を含むcho遺伝子(BspHI-HindIII))を使用した。
2. PCR反応液(25μL/チューブ)に、制限酵素DpnI(1.5μL/チューブ)を添加して処理(37℃、1h)した。
3. DpnI処理液(2μL)を用いて、ライゲーション処理(16℃、2h)した。
4. ライゲーション反応液(10μL/チューブ)を用いて、E.coli BL21(pGKJE-8)を形質転換した。
5. LB+Amp(100μg/mL)+Cm(20μg/mL)プレートに塗布して、培養(37℃、O/N)した。
6. LB Broth(invitrogen)にて前培養後、Teriffic Broth(invitrogen)にて本培養した。
7. 本培養後、菌体を回収して、B-per(TaKaRa)を用いて酵素を抽出した。
8. CHO活性測定法、CHDH活性測定法を用いて、活性を確認した。
9. 活性比(CHDH/CHO)にて比較した。
結果を図6及び図7に示す。変異点の組合せにより、デヒドロゲナーゼ化の向上が確認された。M362P(単独)よりも活性比(CHDH/CHO)が高い変異の組合せを以下に示す。これらの組合せの中で、組合せ1(M362P+L412Y)は特に高い活性比を示した。尚、各組合せについて、変異酵素のアミノ酸配列を配列番号8(組合せ1)、配列番号9(組合せ2)、配列番号10(組合せ3)、配列番号11(組合せ4)、配列番号12(組合せ5)、配列番号13(組合せ6)、配列番号14(組合せ7)、配列番号15(組合せ8)、配列番号16(組合せ9)、配列番号17(組合せ10)、配列番号18(組合せ11)に示す。
組合せ1:M362P+L412Y
組合せ2:M362P+Y483M
組合せ3:M362P+Y483W
組合せ4:M362P+S518G
組合せ5:M362P+S518L
組合せ6:M362P+S518T
組合せ7:M362P+S518A
組合せ8:M362P+V519C
組合せ9:M362P+V519I
組合せ10:M362P+V519S
組合せ11:M362P+V519T
デヒドロゲナーゼ化された変異酵素を精製し、比活性の確認をした。
デヒドロゲナーゼ化された変異酵素(M362P、M362P+L412Y、M362P+Y483W、M362P+S518T、M362P+V519C)の比活性を以下の手順で求めた。尚、比較のために、野生型酵素と既報の変異酵素(V228A)の比活性も算出した。
1. 各変異酵素について、形質転換後の大腸菌を培養(前培養・本培養)した。培養条件は上記の実験に準じた。
2. 培養液から菌体を回収して、精製(集菌→菌体破砕(ビーズ破砕)→上清回収→凝集処理→カラム精製(DEAE Sepharose、Buthyl-S Sepharose)→脱塩濃縮)した。
3. 活性測定(CHO、CHDH)、タンパク濃度を測定して、比活性を算出した。
4. CHDH/CHO(比活性)を比較した。
結果を図8に示す。各変異酵素は野生型よりも優位にデヒドロゲナーゼ化していることが確認された。驚くべきことに、活性比(CHDH活性/CHO活性)は、単独の変異(M362P)であっても野生型の約1.9×106倍に向上した。デヒドロゲナーゼ化の程度は、既知の変異(Katsuhiro Kojima et al., Journal of molecular catalysis B: Enzymatic 88(2013) 41-46で報告されたV191A)に対応する変異酵素(V228A:V191に対応するアミノ酸残基V228がアラニンに置換された変異体)を遙かに凌駕する。また、変異を組み合わせることにより、単独の変異よりも活性比(CHDH活性/CHO活性)が約2~20倍となった。尚、図8における活性値U/mLは、活性測定法における0.5時間の反応中、3分の時点と5分の時点のサンプルのOD値の差より算出している。
各変異酵素の調製に使用したストレプトマイセス sp.CHO(配列番号1)とブレビバクテリウム・ステロリカムCHO(配列番号54)の構造を、コンピュータソフトウエア(Molecule Operating Environment(Chemical Computing Group社製))を用い、FADにてスーパーポーズをかけて重ね合わせた。両者の構造は高い同一性を示し、各アミノ酸残基は概ね一致した。ストレプトマイセス sp.CHOにおいてデヒドロゲナーゼ化に関与することが判明したアミノ酸残基(L113、M362、M402、L412、D468、Y483、S518、V519)に対応する、ブレビバクテリウムCHOのアミノ酸残基を図9に示した。これらのアミノ酸残基(L113に対応するP76、M362に対応するM325、M402に対応するL365、L412に対応するL375、D468に対応するD431、Y483に対応するY446、S518に対応するN481、V519に対応するV482)を同様に変異させることにより、ブレビバクテリウム・ステロリカムCHOもデヒドロゲナーゼ化することが当然に予想される。
Claims (15)
- 微生物由来コレステロールオキシダーゼのアミノ酸配列において、以下の(1)~(8)からなる群より選択される一又は二以上のアミノ酸が他のアミノ酸に置換されたアミノ酸配列からなり、前記微生物由来コレステロールオキシダーゼに比較して、コレステロールオキシダーゼ活性に対するコレステロールデヒドロゲナーゼ活性(CHDH活性/CHO活性)が高い、変異酵素:
(1)配列番号1に示すアミノ酸配列の113位アミノ酸に相当するアミノ酸;
(2)配列番号1に示すアミノ酸配列の362位アミノ酸に相当するアミノ酸;
(3)配列番号1に示すアミノ酸配列の402位アミノ酸に相当するアミノ酸;
(4)配列番号1に示すアミノ酸配列の412位アミノ酸に相当するアミノ酸;
(5)配列番号1に示すアミノ酸配列の468位アミノ酸に相当するアミノ酸;
(6)配列番号1に示すアミノ酸配列の483位アミノ酸に相当するアミノ酸;
(7)配列番号1に示すアミノ酸配列の518位アミノ酸に相当するアミノ酸;
(8)配列番号1に示すアミノ酸配列の519位アミノ酸に相当するアミノ酸。 - 前記微生物由来コレステロールオキシダーゼのアミノ酸配列が、配列番号1のアミノ酸配列と65%以上の同一性を示す配列である、請求項1に記載の変異酵素。
- 置換されるアミノ酸が(2)のアミノ酸であり、置換後のアミノ酸がプロリンである、請求項1又は2に記載の変異酵素。
- 置換されるアミノ酸が、(2)のアミノ酸と(4)のアミノ酸の組合せ、(2)のアミノ酸と(6)のアミノ酸の組合せ、(2)のアミノ酸と(7)のアミノ酸の組合せ、又は(2)のアミノ酸と(8)のアミノ酸の組合せである、請求項1又は2に記載の変異酵素。
- 置換後のアミノ酸が、(2)のアミノ酸についてはプロリンであり、(4)のアミノ酸についてはチロシンであり、(6)のアミノ酸についてはメチオニン又はトリプトファンであり、(7)のアミノ酸についてはグリシン、ロイシン、トレオニン又はアラニンであり、(8)のアミノ酸についてはシステイン、イソロイシン、セリン又はトレオニンである、請求項4に記載の変異酵素。
- 配列番号2~18のいずれかのアミノ酸配列からなる、請求項1に記載の変異酵素。
- 請求項1~6のいずれか一項に記載の変異酵素をコードする遺伝子。
- 配列番号20~36のいずれかの塩基配列を含む、請求項7に記載の遺伝子。
- 請求項7又は8に記載の遺伝子を含む組換えDNA。
- 請求項9に記載の組換えDNAを保有する微生物。
- 請求項1~6のいずれか一項に記載の変異酵素を用いて試料中のコレステロールを測定することを特徴とする、コレステロール測定法。
- 請求項1~6のいずれか一項に記載の変異酵素を含むことを特徴とするコレステロール測定用試薬。
- 請求項12に記載のコレステロール測定用試薬を含む、コレステロール測定用キット。
- 請求項1~6のいずれか一項に記載の変異酵素を含有する酵素剤。
- 以下のステップ(I)~(III)を含む、変異酵素の調製法:
(I)配列番号2~18のいずれかのアミノ酸配列をコードする核酸を用意するステップ;
(II)前記核酸を発現させるステップ、及び
(III)発現産物を回収するステップ。
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