WO2011068050A1 - 変異酵素及びその用途 - Google Patents
変異酵素及びその用途 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
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- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Definitions
- the present invention relates to a mutant enzyme and a method for modifying the enzyme, and provides a dehydrogenase (dehydrogenase) -ized glucose oxidase, a method for preparing the same, and the like.
- dehydrogenase dehydrogenase
- This application claims priority based on Japanese Patent Application No. 2009-277096 filed on Dec. 5, 2009, the entire contents of which are incorporated by reference.
- the biosensor uses glucose oxidase (hereinafter abbreviated as “GO”) and glucose dehydrogenase (hereinafter abbreviated as “GDH”), which are enzymes using glucose as a substrate.
- GO glucose oxidase
- GDH glucose dehydrogenase
- GO has the advantage of high specificity for glucose and excellent thermal stability, the measurement using it is easily affected by dissolved oxygen in the measurement sample, and dissolved oxygen affects the measurement results. The problem of being affected is pointed out.
- PQQ-GDH GDH using pyrroloquinoline (PQQ) as a coenzyme
- PQQ-GDH GDH using pyrroloquinoline (PQQ) as a coenzyme
- PQQ-GDH GDH using pyrroloquinoline (PQQ) as a coenzyme
- PQQ-GDH GDH using pyrroloquinoline (PQQ) as a coenzyme
- GDH uses flavin adenine dinucleotide as a coenzyme as an enzyme that is not affected by dissolved oxygen and acts on glucose in the absence of NAD (P) (hereinafter abbreviated as “FAD-GDH”) It has been known. So far, FAD-GDH has been obtained from Aspergillus oryzae (Non-Patent Documents 1 to 4, Patent Document 4) and Aspergillus tereus (Patent Document 5), respectively.
- the reactivity to xylose is relatively high (for example, in the case of FAD-GDH disclosed in Patent Document 5, the reactivity to xylose is about 10% of the activity to glucose) and the optimum. It is known that the temperature is high (for example, the optimum temperature of FAD-GDH disclosed in Patent Document 4 is about 60 ° C.).
- change of an enzyme is tried energetically for the purpose of improving practicality. Literatures that report modifications of FAD-GDH are shown below (Patent Literatures 6 to 9).
- FAD-GDH has advantages over PQQ-GDH.
- FAD-GDH since FAD-GDH has a relatively high reactivity to xylose, it has a problem that it is difficult to detect an accurate measurement value when measuring blood glucose of a person undergoing a xylose tolerance test.
- the optimum temperature is high, sufficient activity cannot be exhibited when measuring in a low temperature environment such as measurement in a cold region. Under such measurement conditions, temperature correction is necessary and measurement errors are likely to occur.
- the existing FAD-GDH has points to be improved, and further improvement of practicality is desired.
- One of the problems of the present invention is to meet such a demand.
- Another object of the present invention is to provide a novel technique for enzyme modification.
- GO derived from Aspergillus niger was selected, and its amino acid sequence was subjected to multiple alignment with a plurality of known amino acid sequences of FAD-GDH. And by using the alignment results and the three-dimensional structure data of GO, among amino acids near the active center of GO, it is conserved between FAD-GDH (highly common), but GO and FAD-GDH Amino acids differing between were searched. As a result, 13 amino acid positions were identified. Next, mutant enzymes with mutations introduced into these amino acid positions were prepared and their characteristics were examined. As a result, mutant enzymes with an increased ratio of GDH activity to GO activity (GDH activity / GO activity) were observed. Therefore, it was decided to analyze the sequence of the mutant enzyme.
- the inventors succeeded in identifying four amino acid positions effective for increasing GDH activity, that is, for GDH conversion.
- This result suggests that, of the 13 sites identified first, other than the 4 sites are not effective in increasing GDH activity.
- other characteristics such as substrate characteristics, coenzyme specificity, and temperature stability are also observed in the remaining nine positions. It has the potential to be useful for improving and improving.
- GDH activity and other characteristics may be improved by using two or more in combination. Thus, the remaining nine amino acid positions are also valuable and expected to be used.
- the four mutation target positions that have been successfully identified are not limited to single positions, and combinations thereof are effective for GDH conversion.
- the present inventors succeeded in converting GO to GDH, and created a mutant GO having high GDH activity. At the same time, we have succeeded in identifying amino acid positions effective for GO mutation. Some of the obtained mutant GO did not show reactivity to xylose. That is, the present inventors succeeded in obtaining GDH that surpasses existing FAD-GDH in that it does not show reactivity to xylose.
- the two approaches with high structural similarity are based on the modification of the enzyme (particularly, the target of modification). It is proved that the above-mentioned enzyme is effective in imparting the characteristics of the other enzyme or the characteristics of the other enzyme in a more preferable state.
- a mutant enzyme comprising an amino acid sequence in which one or more amino acids selected from the group consisting of the following (1) to (13) are substituted with other amino acids in the amino acid sequence of microorganism-derived glucose oxidase: (1) an amino acid corresponding to amino acid 115 of the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid corresponding to the 131st amino acid in the amino acid sequence shown in SEQ ID NO: 1; (3) an amino acid corresponding to amino acid 132 of the amino acid sequence shown in SEQ ID NO: 1; (4) an amino acid corresponding to amino acid 193 of the amino acid sequence shown in SEQ ID NO: 1; (5) an amino acid corresponding to the 353rd amino acid in the amino acid sequence shown in SEQ ID NO: 1; (6) an amino acid corresponding to the 436th amino acid in the amino acid sequence shown in SEQ ID NO: 1; (7) an amino acid corresponding to the 446th amino acid in the amino acid sequence shown in SEQ
- the amino acid to be substituted is an amino acid of (3), an amino acid of (5), an amino acid of (7) or an amino acid of (12), or a combination of two or more amino acids selected from these The mutant enzyme according to [1] or [2].
- the amino acid after substitution is alanine for the amino acid of (3), alanine for the amino acid of (5), histidine for the amino acid of (7), and serine for the amino acid of (12).
- the amino acid to be substituted is the amino acid (3), the amino acid (7) or the amino acid (12), or a combination of two or more amino acids selected from these amino acids [1] or [2 ]
- Mutant enzyme as described in. [6] The amino acid after substitution is alanine for the amino acid of (3), histidine for the amino acid of (7), and serine, arginine, leucine or proline for the amino acid of (12).
- Mutant enzyme as described in. [7] The mutant enzyme according to [1] or [2], wherein the substituted amino acid is the amino acid (7) and the amino acid (12).
- mutant enzyme according to [7] wherein the amino acid after substitution is histidine for the amino acid of (7) and serine, arginine, leucine or proline for the amino acid of (12).
- mutant enzyme according to [1] comprising the amino acid sequence of any one of SEQ ID NOs: 7 to 21 and 59 to 61.
- the gene according to [10] comprising any one of the nucleotide sequences of SEQ ID NOs: 22 to 36 and 62 to 64.
- a recombinant DNA comprising the gene according to [10] or [11].
- a method for measuring glucose comprising measuring glucose in a sample using the mutant enzyme according to any one of [1] to [9].
- a glucose measurement reagent comprising the mutant enzyme according to any one of [1] to [9].
- a glucose measurement kit comprising the glucose measurement reagent according to [15].
- a method comprising reducing the amount of glucose in an industrial product or a raw material thereof using the mutant enzyme according to any one of [1] to [9].
- An enzyme agent comprising the mutant enzyme according to any one of [1] to [9].
- a method for designing a mutant enzyme comprising the following steps (i) and (ii): (i) one or more amino acids selected from the group consisting of the following (1) to (13) in the amino acid sequence of the enzyme to be mutated, which is a microorganism-derived glucose oxidase or a microorganism-derived flavin adenine dinucleotide-dependent glucose dehydrogenase Steps to identify: (1) an amino acid corresponding to amino acid 115 of the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid corresponding to the 131st amino acid in the amino acid sequence shown in SEQ ID NO: 1; (3) an amino acid corresponding to amino acid 132 of the amino acid sequence shown in SEQ ID NO: 1; (4) an amino acid corresponding to amino acid 193 of the amino acid sequence shown in SEQ ID NO: 1; (5) an amino acid corresponding to the 353rd amino acid in the amino acid sequence shown in SEQ ID NO: 1; (6) an amino acid corresponding to the 436
- the enzyme to be mutated is a microorganism-derived glucose oxidase, and the amino acid substituted in step (i) is the amino acid of (3), the amino acid of (5), the amino acid of (7) or the amino acid of (12), or The design method according to [19], which is a combination of two or more amino acids selected from these.
- the enzyme to be mutated is a microorganism-derived glucose oxidase, and the amino acid to be substituted in step (i) is selected from (3) amino acid, (7) amino acid or (12) amino acid, or from these The design method according to [19], which is a combination of two or more amino acids.
- a method for preparing a mutant enzyme comprising the following steps (I) to (III): (I) A nucleic acid encoding an amino acid sequence of any one of SEQ ID NOs: 7 to 21, 59 to 61, or an amino acid sequence constructed by the design method according to any one of [19] to [26] is prepared. Step; (II) expressing the nucleic acid; and (III) recovering the expression product.
- amino acids near the active center of GO it is conserved between FAD-GDH (highly common), but the amino that is different between GO and FAD-GDH is underlined, from the N-terminal side.
- the numbers are assigned in order.
- the arrow is the amino acid at the active center of GO.
- a HindIII site (boxed) and Kozak sequence (underlined) are added to the 5 ′ end, and an XhoI site (boxed) is added to the 3 ′ end.
- the second amino acid is changed from glutamine to serine by adding the Kozak sequence.
- the gene sequence of Aspergillus niger GO is shown in SEQ ID NO: 38.
- Plate assay results A library (Saccharomyces cerevisiae) containing the plasmid into which the mutation was introduced was prepared, and after growing colonies were replicated on an expression plate, GO activity and GDH activity were examined by a plate assay. The table
- pYES-GO is an unmutated transformant
- pYES2 is a transformant transformed with a plasmid prior to gene insertion
- GO is “Amano” 2 (Amano Enzyme).
- FAD-GDH represents GDH “Amano” 8 (Amano Enzyme), respectively.
- the graph which shows the result of the activity confirmation in a liquid culture. GOH activity and GO activity of each mutant enzyme transformant are indicated by bars, and GDH activity / GO activity is indicated by broken lines.
- pYES-GO represents an unmutated transformant.
- pYES-GO is an unmutated transformant
- pYES2 is a transformant transformed with a plasmid prior to gene insertion
- GO is “Amano” 2 (Amano Enzyme).
- FAD-GDH represents GDH “Amano” 8 (Amano Enzyme), respectively.
- surface which shows the variation
- the reactivity to each substrate was calculated as a relative value to the reactivity to glucose.
- pYES-GO represents an unmutated transformant.
- amino acid corresponding to amino acid position 446 in the amino acid sequence shown in SEQ ID NO: 1 amino acid equivalent to position 472 in the amino acid sequence shown in SEQ ID NO: 1.
- GDH / GO activity ratios of various enzymes with different amino acids after substitution were compared. *: GO activity is below detection limit.
- Substrate specificity of D446H and V582P multiple mutant enzymes. The reactivity to maltose, xylose, fructose, galactose, mannose and lactose was compared with FAD-GDH (GDH “Amano” 8 (Amano Enzyme)).
- 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”. In this specification, the “base enzyme” is also referred to as “mutation target enzyme” or “target enzyme”.
- 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”.
- characteristics are enzyme activity (eg glucose oxidase activity when enzyme A is glucose oxidase), substrate specificity, temperature characteristics (optimum temperature, temperature stability, etc.), pH characteristics (optimum pH) PH stability), coenzyme specificity, and reactivity with mediators.
- Enzyme mutated glucose oxidase 1st aspect of this invention is related with the enzyme (henceforth "mutant GO") which mutated glucose oxidase (GO) derived from microorganisms.
- mutant GO mutated glucose oxidase
- one or more amino acids selected from the group consisting of the following (1) to (13) are substituted with other amino acids in the amino acid sequence of microorganism-derived GO (mutation target enzyme). Have the amino acid sequence.
- Amino acid corresponding to amino acid position 115 of the amino acid sequence shown in SEQ ID NO: 1 (2) Amino acid corresponding to amino acid position 131 of the amino acid sequence shown in SEQ ID NO: 1 (3) Amino acid position 132 of the amino acid sequence shown in SEQ ID NO: 1 (4) amino acid corresponding to amino acid 193 of the amino acid sequence shown in SEQ ID NO: 1 (5) amino acid corresponding to amino acid 353 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 446 of the amino acid sequence shown in SEQ ID NO: 1 (8) amino acid corresponding to amino acid position 472 of the amino acid sequence shown in SEQ ID NO: 1 (9) SEQ ID NO: 1 (10) amino acid corresponding to amino acid 535 of the amino acid sequence shown in SEQ ID NO: 1 (11) amino acid corresponding to amino acid 511 of the amino acid sequence shown in SEQ ID NO: 1 Amino acids corresponding to Amino Acids (12)
- amino acids at position 582, amino acid 582, and 583 are amino acids found by comparing Aspergillus niger GO with multiple types of FAD-GDH, and are near the active center of GO and are characteristic of GO .
- the properties of enzymes are improved and improved by mutating amino acids corresponding to these amino acids, which are considered to play an important role in the properties of GO.
- 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.
- Examples of GO derived from microorganisms that are enzymes to be mutated are GO derived from Aspergillus niger and GO derived from Penicillium amagasakiens.
- the amino acid sequence of Aspergillus niger-derived GO and the amino acid sequence of Penicillium amagasakiens-derived GO registered in public databases are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
- SEQ ID NO: 1 and SEQ ID NO: 2 amino acid sequence of Penicillium amagasakiens
- the amino acid of (1) above is the 115th amino acid of SEQ ID NO: 1
- the amino acid of (2) is the 131st amino acid of SEQ ID NO: 1.
- the amino acid of (3) above is amino acid 132 of SEQ ID NO. 1
- the amino acid of (4) above is amino acid 193 of SEQ ID NO. 1
- the amino acid of (5) above is amino acid 353 of SEQ ID NO.
- the amino acid of (6) is amino acid 436 of SEQ ID NO: 1
- the amino acid of (7) is amino acid 446 of SEQ ID NO: 1
- the amino acid of (8) is amino acid 472 of SEQ ID NO: 1
- the amino acid of (9) is the 511th amino acid of SEQ ID NO: 1
- the amino acid of (10) is the 535th amino acid of SEQ ID NO: 1
- the amino acid of (11) is the 537th amino acid of SEQ ID NO: 1
- (12 ) Amino acid is SEQ ID NO: Becomes the position 582 amino acids, amino acid of the above (13) is 583 amino acid positions of SEQ ID NO: 1.
- the amino acid of (1) is the 115th amino acid of SEQ ID NO: 2
- the amino acid of (2) is SEQ ID NO: 2
- the amino acid of (3) is the amino acid of position 132 of SEQ ID NO. 2
- the amino acid of (4) is the amino acid of position 193 of SEQ ID NO: 2
- the amino acid of (5) is the amino acid of SEQ ID NO: 2.
- the amino acid at position 353 is the amino acid at position 436 in SEQ ID NO: 2
- the amino acid at position 7 is amino acid at position 446 in SEQ ID NO: 2
- the amino acid at position 8 is position 472 in SEQ ID NO: 2.
- the amino acid of (9) above becomes amino acid 511 of SEQ ID NO. 2
- the amino acid of above (10) becomes amino acid 535 of SEQ ID NO. 2
- the amino acid of above (11) becomes amino acid 537 of SEQ ID NO.
- the above (12) Acid becomes 582 of the amino acid of SEQ ID NO: 2
- amino acids of the above (13) is 583 amino acid positions of SEQ ID NO: 2.
- the amino acid to be substituted is preferably the amino acid (3), the amino acid (5), the amino acid (7) or the amino acid (12). These are amino acids that have been confirmed to be effective in improving GDH activity, as shown in the Examples below. Mutant GO in which at least one of these amino acids is substituted can exhibit high GDH activity compared to the enzyme before mutation.
- a specific example of the mutant GO in which the amino acid of (3) is substituted is an enzyme having the amino acid sequence of SEQ ID NO: 7.
- mutant GO in which the amino acid of (5) is substituted is an enzyme consisting of the amino acid sequence of SEQ ID NO: 8
- a specific example of mutant GO in which the amino acid of (7) is substituted is SEQ ID NO: 9
- a specific example of the mutant GO in which the amino acid (12) is substituted is an enzyme consisting of the amino acid sequence of SEQ ID NO: 10. All of these mutant GOs show high GDH activity compared to the Aspergillus niger-derived GO which is an enzyme before mutation.
- amino acid after substitution is not particularly limited, but the amino acid after substitution is preferably selected so as not to fall under the so-called “conservative amino acid substitution”.
- conservative amino acid substitution refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties.
- a basic side chain eg lysine, arginine, histidine
- an acidic side chain eg aspartic acid, glutamic acid
- an uncharged polar side chain eg glycine, asparagine, glutamine, serine, threonine, tyrosine
- Cysteine eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- ⁇ -branched side chains eg threonine, valine, isoleucine
- aromatic side chains eg tyrosine, phenylalanine, Like tryptophan and histidine.
- Conservative amino acid substitutions are typically substitutions between amino acid residues within the same family.
- amino acids after substitution are alanine for amino acid (3), alanine for amino acid (5), histidine for amino acid (7), serine for amino acid (12) Arginine, leucine and proline.
- amino acids (1) to (13) two or more amino acids may be substituted.
- Examples of amino acid combinations to be substituted are listed below. (3) and (5) combination (3) and (7) combination (3) and (12) combination (5) and (7) combination (5) and (12) combination (7) and ( Combination of (12) Combination of (3), (5) and (7) Combination of (3), (5) and (12) Combination of (3), (7) and (12) (5) and (7) Combination of (12) and (12) Combination of (3), (5), (7) and (12)
- SEQ ID NOs: 11 to 21 Examples of amino acid sequences of mutant enzymes obtained by applying the above combinations are shown in SEQ ID NOs: 11 to 21, respectively. These sequences are amino acid sequences of mutant GO obtained by applying the above combination to GO of Aspergillus niger.
- the correspondence between the combinations of SEQ ID NOs and mutations is as follows.
- SEQ ID NO: 11 Combination of (3) and (5)
- SEQ ID NO: 12 Combination of (3) and (7)
- SEQ ID NO: 13 Combination of (3) and (12)
- SEQ ID NO: 14 (5) and (7)
- SEQ ID NO: 15 Combination of (5) and (12)
- SEQ ID NO: 16 Combination of (7) and (12)
- SEQ ID NO: 17 Combination of (3), (5) and (7)
- SEQ ID NO: 19 Combination of (3), (7) and (12)
- SEQ ID NO: 20 Combination of (5), (7) and (12)
- SEQ ID NO: 21 Combination of (3), (5), (7) and (12)
- SEQ ID NO: 59 Combination of (7) and (12)
- SEQ ID NO: 60 Combination of (7) and (12)
- (7) and (12) is preferred.
- a particularly preferred combination is the combination of (7) and (12) (specific examples of the amino acid sequence of the mutant enzyme of this combination are SEQ ID NOs: 16, 59 to 61 as described above).
- the amino acid after substitution is preferably histidine for (7), and serine (SEQ ID NO: 16), arginine (SEQ ID NO: 59), leucine (SEQ ID NO: 60) or proline (SEQ ID NO: 61) for (12).
- the amino acid after substitution is proline.
- 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 GO consisting of an amino acid sequence in which one or two or more amino acids selected from the group consisting of (1) to (13) 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 that is substantially identical to GO.
- “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 GO 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 GO of the present invention. Namely, a gene encoding a mutant GO, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a mutant GO, and a nucleic acid that can be used as a primer for amplifying or mutating a nucleic acid encoding a mutant GO Is provided.
- the gene encoding mutant GO is typically used for the preparation of mutant GO. According to a genetic engineering preparation method using a gene encoding a mutant GO, it is possible to obtain a mutant GO in a more homogeneous state. In addition, this method can be said to be a suitable method even when a large amount of mutant GO is prepared. In addition, the use of the gene encoding the mutant GO is not limited to the preparation of the mutant GO.
- the nucleic acid can also be used as an experimental tool for elucidating the mechanism of action of mutant GO, or as a tool for designing or creating a further mutant of an enzyme.
- the “gene encoding a mutant GO” refers to a nucleic acid from which the mutant GO 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 GO.
- 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.
- sequence of the gene encoding the mutant GO are shown in SEQ ID NOs: 22-36 and 62-64. These sequences are genes encoding a mutant GO in which a specific amino acid substitution has been made in GO of Aspergillus niger. 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 gene encoding the mutant GO of the present invention, which is equivalent in function to the protein encoded by the same but has a different base sequence”). Also referred to as “homologous nucleic acid.”
- a base sequence defining a homologous nucleic acid is also referred to as “homologous base sequence”).
- a homologous nucleic acid it 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 GO of the present invention.
- a DNA encoding a protein having a typical enzyme activity ie, GDH 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 varies depending 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 GO 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 GO of the present invention or a base sequence complementary thereto. %, 99.9% nucleic acid having the same base sequence is provided.
- Still another embodiment 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 GO of the present invention or a base sequence complementary to the base sequence homologous thereto.
- 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 GO 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 GO 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 GO of the present invention (for example, 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 (a gene encoding a mutant GO).
- the recombinant DNA of the present invention is provided, for example, in the form of a vector.
- the term “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.
- M13 phage or a modified version thereof, ⁇ phage or a modified version thereof, pBR322 or a modified version thereof (pB325, pAT153, pUC8, etc.) such as E. coli host vector, pYepSec1, pMFa, pYES2
- 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).
- the host cell is preferably a microorganism such as Escherichia coli (Escherichia coli) or budding yeast (Saccharomyces cerevisiae) from the viewpoint of ease of handling, but the recombinant DNA can be replicated and the mutant GO gene can be used. Any host cell capable of expression can be used.
- E. coli include 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 GO of the present invention. (See below for how to prepare mutant enzymes) Column).
- the third aspect of the present invention relates to the use of mutant GO.
- a glucose measurement method using a mutant GO is provided.
- the amount of glucose in a sample is measured using an oxidation-reduction reaction by this enzyme.
- the present invention is used, for example, for measurement of blood glucose level, measurement of glucose concentration in foods (such as seasonings and beverages), and the like.
- the present invention also provides a glucose measuring reagent containing the present enzyme.
- the reagent is used in the glucose measurement method of the present invention described above.
- the present invention further provides a kit (glucose measurement kit) for carrying out the glucose measurement method of the present invention.
- the kit of the present invention includes a reagent for measuring glucose containing the present enzyme, a reagent for reaction, a buffer solution, a glucose standard solution and the like as optional elements.
- an instruction manual is usually attached to the glucose measurement kit of the present invention.
- the mutant GO of the present invention is allowed to act on industrial products (various processed foods, confectionery, soft drinks, alcoholic beverages, foods such as dietary supplements, and cosmetics) or their raw materials.
- industrial products various processed foods, confectionery, soft drinks, alcoholic beverages, foods such as dietary supplements, and cosmetics
- the Maillard reaction can be suppressed by reducing the glucose content.
- the enzyme agent of the present invention may contain excipients, buffers, suspension agents, stabilizers, preservatives, preservatives, physiological saline and the like in addition to the active ingredient (mutant GO).
- starch dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
- Phosphate, citrate, acetate, etc. can be used as the buffer.
- propylene glycol, ascorbic acid or the like can be used.
- preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
- a microorganism-derived glucose oxidase microorganism-derived GO
- microorganism-derived FDA-GDH microorganism-derived flavin adenine dinucleotide-dependent glucose dehydrogenase
- Amino acid corresponding to amino acid position 115 of the amino acid sequence shown in SEQ ID NO: 1 (2) Amino acid corresponding to amino acid position 131 of the amino acid sequence shown in SEQ ID NO: 1 (3) Amino acid position 132 of the amino acid sequence shown in SEQ ID NO: 1 (4) amino acid corresponding to amino acid 193 of the amino acid sequence shown in SEQ ID NO: 1 (5) amino acid corresponding to amino acid 353 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 446 of the amino acid sequence shown in SEQ ID NO: 1 (8) amino acid corresponding to amino acid position 472 of the amino acid sequence shown in SEQ ID NO: 1 (9) SEQ ID NO: 1 (10) amino acid corresponding to amino acid 535 of the amino acid sequence shown in SEQ ID NO: 1 (11) amino acid corresponding to amino acid 511 of the amino acid sequence shown in SEQ ID NO: 1 Amino acids corresponding to Amino Acids (12)
- the above substitution target amino acids (1) to (13) were found by comparing the microorganism-derived GO with a plurality of types of microorganism-derived FAD-GDH. It is expected that the properties of the enzyme will be changed by substituting these amino acids. Examples of properties that can be changed include GO activity, GDH activity, substrate specificity, temperature characteristics (optimum temperature, temperature stability, etc.), pH characteristics (optimum pH, pH stability), coenzyme specificity, Reactivity with mediators.
- the enzyme to be mutated in the design method of the present invention is microorganism-derived GO or microorganism-derived FAD-GDH.
- the enzyme to be mutated is typically a wild-type enzyme (an enzyme found in nature). However, this does not preclude the use of an enzyme that has already undergone some mutation or modification as the enzyme to be displaced.
- microorganism-derived GO examples are GO of Aspergillus niger and GO of Penicillium amagasakiens
- microorganism-derived FAD-GDH examples are FAD-GDH of Penicillium italicam, FAD-GDH of Penicillium lilacino ethinulanium, Aspergillus ⁇ Oryzae FAD-GDH and Aspergillus tereus FAD-GDH.
- the amino acid sequences of the enzymes exemplified here are listed below in the public database. In a preferred embodiment, an enzyme consisting of any one of these amino acid sequences is an enzyme to be mutated.
- Aspergillus niger GO amino acid sequence of SEQ ID NO: 1
- Penicillium amagasakiense GO amino acid sequence of SEQ ID NO: 2
- FAD-GDH SEQ ID NO: 3
- FAD-GDH SEQ ID NO: 4
- FAD-GDH SEQ ID NO: 5 amino acid sequence Aspergillus terreus
- FAD -GDH Amino acid sequence of SEQ ID NO: 6.
- amino acids corresponding to the amino acids (1) to (13) above are shown together in the table of FIG.
- step (ii) is performed after step (i).
- amino acid after substitution is not particularly limited. Therefore, it may be a conservative amino acid substitution or a non-conservative amino acid substitution.
- conservative amino acid substitution refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties.
- a basic side chain for example, lysine, arginine, histidine
- an acidic side chain for example, aspartic acid, glutamic acid
- an uncharged polar side chain for example, asparagine, glutamine, serine, threonine, tyrosine, cysteine
- Non-polar side chains eg glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- ⁇ -branched side chains eg threonine, valine, isoleucine
- aromatic side chains eg tyrosine, phenylalanine, Like tryptophan
- a conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.
- a further aspect of the present invention relates to a method for preparing a mutant enzyme.
- a mutant GO 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: 7 to 10 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: 7 to 10 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. Here, all of the amino acid sequences of SEQ ID NOs: 7 to 10 are obtained by mutating the amino acid sequence of Aspergillus niger-derived GO.
- nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 7 to 10 can also be obtained by adding necessary mutations to the gene encoding Aspergillus niger-derived GO (SEQ ID NO: 38). It can.
- 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).
- a mutant enzyme is prepared based on the amino acid sequence designed by the design method of the present invention.
- a nucleic acid encoding an amino acid sequence constructed by the designing method of the present invention is prepared.
- a necessary mutation that is, substitution of an amino acid at a specific position in a protein as an expression product
- a nucleic acid (gene) encoding the mutant enzyme is obtained.
- 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 insolubles, 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 a purified product of the 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.
- Alignment comparison of GO and FAD-GDH Alignment comparison of GO derived from Aspergillus niger and FAD-GDH derived from Aspergillus oryzae, Aspergillus tereus, Penicillium italicam, Penicillium lyracino ethinulanium, and amino acid sequences are already known, and already From the three-dimensional structure of Aspergillus niger derived GO, the three-dimensional structure of which is conserved between FAD-GDH among amino acids near the active center of GO (highly common), but GO and FAD Different amino acids were searched for between -GDH (FIGS. 2 and 3).
- ClustalW2 European Molecular Biology Laboratory
- EBI European Bioinformatics Institute
- Genomic DNA was extracted from Aspergillus niger GO-1 bacteria (owned by Amano Enzyme) using Gen Elute Genomic DNA kit (Sigma), and the GO gene was obtained by PCR.
- Gen Elute Genomic DNA kit (Sigma)
- the PCR conditions are shown below.
- composition of reaction solution 10 x LA buffer (Takara Bio Inc.) 5 ⁇ L 2.5mM dNTPs (Takara Bio Inc.) 8 ⁇ L 25 mM MgCl 2 (Takara Bio Inc.) 5 ⁇ L Forward primer (50 ⁇ M) 1 ⁇ L Reverse primer (50 ⁇ M) 1 ⁇ L Template 1 ⁇ L LA Taq (Takara Bio Inc.) 0.5 ⁇ L stH 2 O 28.5 ⁇ L
- reaction conditions After reacting at 94 ° C for 2 minutes, the reaction cycle of 94 ° C for 30 seconds, 52 ° C for 30 seconds and 72 ° C for 2 minutes was repeated 35 times, followed by reaction at 72 ° C for 7 minutes, and finally at 4 ° C. Neglect
- the transformed plasmid was transformed into E. coli DH5 ⁇ , followed by plasmid extraction to prepare a mutation library.
- the obtained library was transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen), and the grown colonies were replicated on an expression plate, and then expression and mutagenesis were confirmed by a plate assay (FIG. 5). Regarding F436, the growth of the mutant enzyme-transformed strain could not be confirmed.
- the experiment operation referred to the manual of pYES2.
- T132 The amino acid sequence of the mutagenesis point was confirmed for the mutant enzyme transformant in which a large change was observed in the GDH / GO activity ratio (FIG. 8). Effective mutations were identified based on the results shown in FIGS. First, for T132, the GDH / GO activity ratio of the mutant enzyme-transformed strain having T132A (substitution of threonine to alanine) is higher than that of the mutant enzyme-transformed strain having T132V (substitution of threonine to valine). Therefore, T132A was made an effective mutation. For T353, two mutant T353A and T353H were found in the mutant enzyme transformed strain (5-1-5), but the mutant enzyme transformed strains (5-1-9, 5-1-44) having T353A alone.
- Mutant enzyme transformants (3-1-26, 3-2-26, 5-1-5, 5-1-9, 5-1-44, 7-1-7, 7-2 for 200 ⁇ L of each reagent -17, 7-2-30, 7-2-42, 12-1-49) was added at 20 ⁇ L, and reacted at 37 ° C. Absorbance was measured 10 minutes and 60 minutes after the start of the reaction, and GDH activity was determined from the difference in absorbance. The GDH activity when each substrate was used was expressed as a ratio to the GDH activity (100%) when glucose was used as the substrate. In addition, a transformant having unmutated GO (indicated as pYES-GO in FIG. 9) was used as a control.
- composition of reaction solution 10 x LA buffer (Takara Bio Inc.) 5 ⁇ L 2.5mM dNTPs (Takara Bio Inc.) 8 ⁇ L 25mM MgCl2 (Takara Bio Inc.) 5 ⁇ L Forward primer (50 ⁇ M) 1 ⁇ L Reverse primer (50 ⁇ M) 1 ⁇ L Template 1 ⁇ L LA Taq (Takara Bio Inc.) 0.5 ⁇ L stH2O 28.5 ⁇ L
- reaction conditions After reacting at 94 ° C for 2 minutes, the reaction cycle of 94 ° C for 30 seconds, 52 ° C for 30 seconds and 72 ° C for 2 minutes was repeated 35 times, followed by reaction at 72 ° C for 7 minutes, and finally at 4 ° C. Neglect
- the amplified product after PCR was inserted into pYES2 to form a pYES-GO-3 plasmid, and the sequence of the insert was confirmed. Since no problems were observed in the sequence, the following synthetic oligonucleotides designed to replace T132A, T353A, D446H, and V582S with the constructed pYES-GO-3 plasmid as a template and synthetic oligonucleotides complementary to them were used. On the basis, QuikChange Site-Directed Mutagensesis Kit (Stratagene) was used to perform a mutation operation according to the attached protocol to construct a plasmid having multiple mutant glucose oxidase.
- QuikChange Site-Directed Mutagensesis Kit (Stratagene) was used to perform a mutation operation according to the attached protocol to construct a plasmid having multiple mutant glucose oxidase.
- GO-T132A-1 CTCGTCAACGGTGGCGCTTGGACTCGCCCCCAC (SEQ ID NO: 55)
- GO-T353A-1 CCTTCAGGACCAGACCGCTTCTACCGTCCGCTCAC (SEQ ID NO: 56)
- GO-D446H-1 GGAGTGGCCAGTTTCCATGTGTGGGATCTTCTGC (SEQ ID NO: 57)
- GO-V582S-1 CGCAAATGTCGTCCCATTCTATGACGGTCTTTTATGCCATGG (SEQ ID NO: 58)
- the transformed plasmid was transformed into E. coli DH5 ⁇ , followed by plasmid extraction to prepare a mutation library.
- the obtained library was transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen), and the grown colonies were subjected to liquid culture to examine GO activity and GDH activity.
- the experiment operation referred to the manual of pYES2.
- a transformant having an unmutated GO with a histidine tag inserted (denoted as pYES-GO-3), a transformant transformed with a plasmid before inserting the GO gene (denoted as pYES-2), GO ” Amano ”2 (shown as GO) and GDH” Amano ”8 (shown as FAD-GDH) were the comparison targets (controls).
- the specific activity of the obtained purified enzyme was about 3 to 8 u / mg (protein).
- the substrate specificity in GDH activity was investigated using the purified enzyme.
- the transformed plasmid was transformed into Escherichia coli DH5 ⁇ , followed by plasmid extraction to prepare a D446 and V582 multiple mutation library.
- the resulting library was transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen), and the obtained transformant was subjected to liquid culture using a 96-well deep well, and D446H and V582S, which were combinations prior to the study, were obtained. As a control, those having improved GDH activity and GDH / GO activity ratio were obtained.
- the experiment operation referred to the manual of pYES2.
- a mutant GO partial purified enzyme (D446H, V582P multiple mutant enzyme) was added to 200 ⁇ L of each reagent and reacted at 37 ° C. Absorbance was measured 10 minutes and 30 minutes after the start of the reaction, and GDH activity was determined from the difference in absorbance. The GDH activity when each substrate was used was expressed as a ratio to the GDH activity (100%) when glucose was used as the substrate. GDH “Amano” 8 (shown as FAD-GDH in FIGS. 6 and 7) was used as a control.
- the mutant GO partially purified enzyme (D446H, V582P multiple mutant enzyme) showed no reactivity to xylose and was much superior in substrate specificity compared to GDH “Amano” 8.
- the mutant GO provided by the present invention is useful for detecting and quantifying the amount of glucose in a sample.
- the design method / preparation method of the present invention is used as a means for obtaining GDH or GO having improved characteristics. In particular, it is expected to be used as a means for obtaining GDH-converted GO or GO-converted GDH.
Abstract
Description
[1]微生物由来グルコースオキシダーゼのアミノ酸配列において、以下の(1)~(13)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る変異酵素:
(1)配列番号1に示すアミノ酸配列の115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸;
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸;
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸;
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸;
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸;
(13)配列番号1に示すアミノ酸配列の583位アミノ酸に相当するアミノ酸。
[2]微生物由来グルコースオキシダーゼのアミノ酸配列が配列番号1又は2のアミノ酸配列である、[1]に記載の変異酵素。
[3]置換されるアミノ酸が、(3)のアミノ酸、(5)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、[1]又は[2]に記載の変異酵素。
[4]置換後のアミノ酸が、(3)のアミノ酸についてはアラニンであり、(5)のアミノ酸についてはアラニンであり、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、[3]に記載の変異酵素。
[5]置換されるアミノ酸が、(3)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、[1]又は[2]に記載の変異酵素。
[6]置換後のアミノ酸が、(3)のアミノ酸についてはアラニンであり、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、[5]に記載の変異酵素。
[7]置換されるアミノ酸が、(7)のアミノ酸及び(12)のアミノ酸である、[1]又は[2]に記載の変異酵素。
[8]置換後のアミノ酸が、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、[7]に記載の変異酵素。
[9]配列番号7~21、59~61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。
[10][1]~[9]のいずれか一項に記載の変異酵素をコードする遺伝子。
[11]配列番号22~36、62~64のいずれかの塩基配列を含む、[10]に記載の遺伝子。
[12][10]又は[11]に記載の遺伝子を含む組換えDNA。
[13][12]に記載の組換えDNAを保有する微生物。
[14][1]~[9]のいずれか一項に記載の変異酵素を用いて試料中のグルコースを測定することを特徴とする、グルコース測定法。
[15][1]~[9]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グルコース測定用試薬。
[16][15]に記載のグルコース測定用試薬を含む、グルコース測定用キット。
[17][1]~[9]のいずれか一項に記載の変異酵素を用いて工業製品又はその原料中のグルコース量を低下させることを特徴とする方法。
[18][1]~[9]のいずれか一項に記載の変異酵素を含有する酵素剤。
[19]以下のステップ(i)及び(ii)を含む、変異酵素の設計法:
(i)微生物由来グルコースオキシダーゼ又は微生物由来フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼである変異対象酵素のアミノ酸配列において、以下の(1)~(13)からなる群より選択される一又は二以上のアミノ酸を特定するステップ:
(1)配列番号1に示すアミノ酸配列の115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸;
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸;
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸;
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸;
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸;
(13)配列番号1に示すアミノ酸配列の583位アミノ酸に相当するアミノ酸;
(ii)変異対象酵素のアミノ酸配列を基にして、ステップ(i)で特定されたアミノ酸配列が他のアミノ酸に置換されたアミノ酸配列を構築するステップ。
[20]変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(3)のアミノ酸、(5)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、[19]に記載の設計法。
[21]変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(3)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、[19]に記載の設計法。
[22]変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(7)のアミノ酸及び(12)のアミノ酸である、[19]に記載の設計法。
[23]微生物由来グルコースオキシダーゼが、アスペルギルス・ニガー又はペニシリウム・アマガサキエンスのグルコースオキシダーゼである、[20]~[22]のいずれか一項に記載の設計法。
[24]グルコースオキシダーゼのアミノ酸配列が配列番号1又は2のアミノ酸配列である、[23]に記載の設計法。
[25]変異対象酵素が、ペニシリウム・イタリカム、ペニシリウム・リラシノエチヌラティウム、アスペルギルス・オリゼ又はアスペルギルス・テレウスのフラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼである、[19]に記載の設計法。
[26]フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼのアミノ酸配列が配列番号3~6のいずれかのアミノ酸配列である、[25]に記載の設計法。
[27]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7~21、59~61のいずれかのアミノ酸配列、又は[19]~[26]のいずれか一項に記載の設計法によって構築された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」又は「標的酵素」とも呼ぶ。
本発明の第1の局面は微生物由来のグルコースオキシダーゼ(GO)を変異させた酵素(以下「変異GO」とも呼ぶ)に関する。本発明の変異GOは、微生物由来のGO(変異対象酵素)のアミノ酸配列において、以下の(1)~(13)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115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸
(13)配列番号1に示すアミノ酸配列の583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。
(3)と(5)の組合せ
(3)と(7)の組合せ
(3)と(12)の組合せ
(5)と(7)の組合せ
(5)と(12)の組合せ
(7)と(12)の組合せ
(3)と(5)と(7)の組合せ
(3)と(5)と(12)の組合せ
(3)と(7)と(12)の組合せ
(5)と(7)と(12)の組合せ
(3)と(5)と(7)と(12)の組合せ
配列番号11: (3)と(5)の組合せ
配列番号12: (3)と(7)の組合せ
配列番号13: (3)と(12)の組合せ
配列番号14: (5)と(7)の組合せ
配列番号15: (5)と(12)の組合せ
配列番号16: (7)と(12)の組合せ
配列番号17: (3)と(5)と(7)の組合せ
配列番号18: (3)と(5)と(12)の組合せ
配列番号19: (3)と(7)と(12)の組合せ
配列番号20: (5)と(7)と(12)の組合せ
配列番号21: (3)と(5)と(7)と(12)の組合せ
配列番号59: (7)と(12)の組合せ
配列番号60: (7)と(12)の組合せ
配列番号61: (7)と(12)の組合せ
本発明の第2の局面は本発明の変異GOに関連する核酸を提供する。即ち、変異GOをコードする遺伝子、変異GO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GO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22: T132A
配列番号23: T353A
配列番号24: D446H
配列番号25: V582S
配列番号26: T132A及びT353A
配列番号27: T132A及びD446H
配列番号28: T132A及びV582S
配列番号29: T353A及びD446H
配列番号30: T353A及びV582S
配列番号31: D446H及びV582S
配列番号32: T132A、T353A及びD446H
配列番号33: T132A、T353A及びV582S
配列番号34: T132A、D446H及びV582S
配列番号35: T353A、D446H及びV582S
配列番号36: T132A、T353A、D446H及びV582S
配列番号62: D446H及びV582R
配列番号63: D446H及びV582L
配列番号64: D446H及びV582P
本発明の第3の局面は変異GOの用途に関する。この局面ではまず、変異GOを用いたグルコース測定法が提供される。本発明のグルコース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グルコース量を測定する。本発明は例えば血糖値の測定、食品(調味料や飲料など)中のグルコース濃度の測定などに利用される。また、発酵食品(例えば食酢)又は発酵飲料(例えばビールや酒)の製造工程において発酵度を調べるために本発明を利用してもよい。
本発明の別の局面は変異酵素の設計法に関する。本発明の設計法では、以下のステップ(i)及び(ii)を実施する。
ステップ(i):微生物由来グルコースオキシダーゼ(微生物由来GO)又は微生物由来フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼ(微生物由来FDA-GDH)である変異対象酵素のアミノ酸配列において、以下の群より選択される一又は二以上のアミノ酸を特定する。
(1)配列番号1に示すアミノ酸配列の115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸
(13)配列番号1に示すアミノ酸配列の583位アミノ酸に相当するアミノ酸
アスペルギルス・ニガー(Aspergillus niger)のGO: 配列番号1のアミノ酸配列
ペニシリウム・アマガサキエンス(Penicillium amagasakiense)のGO: 配列番号2のアミノ酸配列
ペニシリウム・イタリカム(Penicillium italicum)のFAD-GDH: 配列番号3のアミノ酸配列
ペニシリウム・リラシノエチヌラティウム(Penicillium lilacinoechinulatum)のFAD-GDH: 配列番号4のアミノ酸配列
アスペルギルス・オリゼ(Aspergillus oryzae)のFAD-GDH: 配列番号5のアミノ酸配列
アスペルギルス・テレウス(Aspergillus terreus)のFAD-GDH: 配列番号6のアミノ酸配列
尚、以上例示した各酵素について、上記(1)~(13)のアミノ酸に該当するアミノ酸を図10の表にまとめて示す。
ステップ(ii):変異対象酵素のアミノ酸配列を基にして、ステップ(i)で特定されたアミノ酸配列が他のアミノ酸に置換されたアミノ酸配列を構築する。
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GO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7~10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7~10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7~10のアミノ酸配列はいずれも、アスペルギルス・ニガー由来GOのアミノ酸配列に変異を施したものである。従って、アスペルギルス・ニガー由来GOをコードする遺伝子(配列番号38)に対して必要な変異を加えることによっても、配列番号7~10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。
アスペルギルス・ニガー由来GOと現在、アミノ酸配列の分かっているアスペルギルス・オリゼ、アスペルギルス・テレウス、ペニシリウム・イタリカム、ペニシリウム・リラシノエチヌラティウム由来FAD-GDHのアライメント比較、及びすでに立体構造が明らかとなっているアスペルギルス・ニガー由来GOの立体構造から、GOの活性中心付近にあるアミノ酸の中で、FAD-GDH間では保存されている(共通性が高い)が、GOとFAD-GDHの間においては相違するアミノ酸を検索した(図2、3)。尚、アライメント比較にはClustalW2(EMBL(European Molecular Biology Laboratory)-EBI(European Bioinformatics Institute)のホームページ内に専用のサイトが設けられている。http://www.ebi.ac.uk/Tools/clustalw2/index.html)を用いた。
GO遺伝子については、過去、大腸菌で発現させた報告が無いことからpYES2(インビトロジェン社)のHindIII-XhoII部分にGO遺伝子を挿入し、サッカロマイセス・セレビシエ(S.cerevisiae)を宿主として発現させることとした。
10×LAバッファー(タカラバイオ株式会社) 5μL
2.5mM dNTPs(タカラバイオ株式会社) 8μL
25mM MgCl2(タカラバイオ株式会社) 5μL
フォワードプライマー(50μM) 1μL
リバースプライマー(50μM) 1μL
Template 1μL
LA Taq(タカラバイオ株式会社) 0.5μL
stH2O 28.5μL
フォワードプライマー:GATCAGAAGCTTAAAAAAATGTCTACTCTCCTTGTGAGCTCG(配列番号39)
リバースプライマー:GATCAGCTCGAGTCACTGCATGGAAGCATAATC(配列番号40)
94℃で2分反応させた後、94℃で30秒、52℃で30秒、72℃で2分の反応サイクルを35回繰り返した後、72℃で7分反応させ、最後に4℃で放置
GO-L115-変異用プライマー:CCACCAACAATCAGACTGCGNNNATCCGCTCCGGAAATGG(配列番号41)
GO-G131-変異用プライマー:GCTCTACCCTCGTCAACGGTNNNACCTGGACTCGCCCC(配列番号42)
GO-T132-変異用プライマー:CTCGTCAACGGTGGCNNNTGGACTCGCCCCCAC(配列番号43)
GO-V193-変異用プライマー:CATGGTATCAATGGTACTNNNCACGCCGGACCCCGCG(配列番号44)
GO-T353-変異用プライマー:CAACCTTCAGGACCAGACCNNNTCTACCGTCCGCTCAC(配列番号45)
GO-F436-変異用プライマー:GTCGCATACTCGGAACTCNNNCTCGACACGGCCGGAG(配列番号46)
GO-D446-変異用プライマー:GCCGGAGTGGCCAGTTTCNNNGTGTGGGATCTTCTGC(配列番号47)
GO-Y472-変異用プライマー:CATCCTCCGCCATTTCGCANNNGACCCTCAGTACTTTCTCAAC(配列番号48)
GO-I551-変異用プライマー:CTTATTTCGCTGGAGAGACTNNNCCCGGTGACAACCTCGC(配列番号49)
GO-P535-変異用プライマー:CCCGTACAACTTCCGCNNNAACTACCATGGTGTGGGTACTTG(配列番号50)
GO-Y537-変異用プライマー:GTACAACTTCCGCCCTAACNNNCATGGTGTGGGTACTTGCTC(配列番号51)
GO-V582-変異用プライマー:CTACGCAAATGTCGTCCCATNNNATGACGGTCTTTTATGCCATGG(配列番号52)
GO-M583-変異用プライマー:CTACGCAAATGTCGTCCCATGTTNNNACGGTCTTTTATGCCATGG(配列番号53)
各々の発色液を80mmのろ紙に浸した後、プレートの上にのせ発色を確認した。
<GOアッセイ>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 20mL
10% グルコース 5mL
25u/mL PO”Amano”3(天野エンザイム社) 5mL
o-ジアニジン 5mg
<GDHアッセイ>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 23mL
10% グルコース 5mL
3mmol/L 1-メトキシPMS 1mL
6.6mmol/L NTB 1mL
プレートアッセイで確認できた陽性コロニー(GOアッセイで発色せず、GDHアッセイで発色したもの)について液体培養を行い、GO活性及びGDH活性を調べた。尚、実験操作はpYES2のマニュアルを参考にした。
<GOアッセイ用試薬>
フェノール含有リン酸緩衝液 19mL
10% グルコース 5mL
25u/mL PO”Amano”3(天野エンザイム社) 5mL
0.4g/dL 4-アミノアンチピリン 1mL
<GDHアッセイ用試薬>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 21mL
10% グルコース 5mL
3mmol/L PMS 3mL
6.6mmol/L NTB 1mL
T132A:配列番号7:配列番号22
T353A:配列番号8:配列番号23
D446H:配列番号9:配列番号24
V582S:配列番号10:配列番号25
T132A及びT353A:配列番号11:配列番号26
T132A及びD446H:配列番号12:配列番号27
T132A及びV582S:配列番号13:配列番号28
T353A及びD446H:配列番号14:配列番号29
T353A及びV582S:配列番号15:配列番号30
D446H及びV582S:配列番号16:配列番号31
T132A、T353A及びD446H:配列番号17:配列番号32
T132A、T353A及びV582S:配列番号18:配列番号33
T132A、D446H及びV582S:配列番号19:配列番号34
T353A、D446H及びV582S:配列番号20:配列番号35
T132A、T353A、D446H及びV582S:配列番号21:配列番号36
有効な変異を有する酵素(変異型GO)についてGDH活性における基質特異性を調べた。
<GDHアッセイ用試薬>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 21mL
10% 基質 5mL
3mmol/L PMS 1mL
6.6mmol/L NTB 3mL
有効な変異と考えられた遺伝子変異の組み合わせについて、効果を検証した。後の精製を簡略化するため、アスペルギルス・ニガーGO-1号菌由来グルコースオキシダーゼ遺伝子のC末端にヒスチジンタグを付加したGO遺伝子(GO-3)をPCR反応により作製し、pYES2に挿入してpYES-GO-3プラスミドを構築した。
10×LAバッファー(タカラバイオ株式会社) 5μL
2.5mM dNTPs(タカラバイオ株式会社) 8μL
25mM MgCl2(タカラバイオ株式会社) 5μL
フォワードプライマー(50μM) 1μL
リバースプライマー(50μM) 1μL
Template 1μL
LA Taq(タカラバイオ株式会社) 0.5μL
stH2O 28.5μL
フォワードプライマー:GATCAGAAGCTTAAAAAAATGTCTACTCTCCTTGTGAGCTCG(配列番号39)
リバースプライマー:GATCAGCTCGAGTCAATGGTGATGGTGATGATGCTGCATGGAAGCATAATC(配列番号54)
94℃で2分反応させた後、94℃で30秒、52℃で30秒、72℃で2分の反応サイクルを35回繰り返した後、72℃で7分反応させ、最後に4℃で放置
GO-T353A-1:CCTTCAGGACCAGACCGCTTCTACCGTCCGCTCAC(配列番号56)
GO-D446H-1:GGAGTGGCCAGTTTCCATGTGTGGGATCTTCTGC(配列番号57)
GO-V582S-1:CGCAAATGTCGTCCCATTCTATGACGGTCTTTTATGCCATGG(配列番号58)
フェーノール含有リン酸緩衝液 19mL
10% グルコース 5mL
25u/mL PO-3 5mL
0.4g/dL 4-A.A 1mL
<GDHアッセイ用試薬>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 21mL
10% グルコース 5mL
3mmol/L PMS 3mL
6.6mmol/L NTB 1mL
次に、効果の認められたT132A、D446H、V582Sの組み合わせ(T132A及びD446H、T132A及びV582S、D446H及びV582S、T132A、D446H及びV582S)について、形質転換株の液体培養を行い、Ni-Sepharoseを用いて精製後、比活性及び基質特異性を調べた。なお、液体培養での発現はpYES2のマニュアルを参考にした。
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH7.0 21mL
10% グルコース 5mL
3mmol/L PMS 3mL
6.6mmol/L NTB 1mL
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 21mL
10% 基質 5mL
3mmol/L PMS 3mL
6.6mmol/L NTB 1mL
D446及びV582の変異組み合わせについて、各々のアミノ酸が最適なアミノ酸の組み合わせになるように、pYES-GO-K-P-2プラスミドを鋳型として、配列番号47及び配列番号52の合成オリゴヌクレオチドとそれに相補的な合成オリゴヌクレオチドを基に、QuikChange Site-Directed Mutagensesis Kit(ストラタジーン社)を用い、添付のプロトコールに従って変異操作を行い、変異グルコースオキシダーゼを有するプラスミドを構築した。変異導入後のプラスミドを大腸菌DH5αに形質転換後、プラスミド抽出を行い、D446及びV582多重変異ライブラリーを作製した。得られたライブラリーをサッカロマイセス・セレビシエINVSc1(インビトロジェン社)に形質転換し、得られた形質転換体について、96穴ディープウェルを用いて液体培養を行い、検討実施前の組み合わせであるD446H及びV582Sをコントロールとして、GDH活性及びGDH/GO活性比が向上したものを取得した。尚、実験操作はpYES2のマニュアルを参考にした。
(1)培養及び精製
液体培養での発現はpYES2のマニュアルを参考にし、保存プレートより500mL坂口フラスコに調製した100mLの0.67% アミノ酸不含酵母ニトロゲンベース(日本ベクトン・ディッキンソン株式会社製)、2% グルコースを含有する培地(pH5.4)に接種し、30℃、140回転/分、20時間前培養を行った。
変異型GO部分精製酵素(D446H,V582P多重変異酵素)について、GDH活性における基質特異性を調べた。
<GDHアッセイ用試薬>
50mM PIPES-NaOH(cont. 0.1% Triton X-100) pH 7.0 21mL
10% 基質 5mL
3mmol/L PMS 3mL
6.6mmol/L NTB 1mL
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
配列番号41~53、55~58:人工配列の説明:変異導入用プライマー
Claims (27)
- 微生物由来グルコースオキシダーゼのアミノ酸配列において、以下の(1)~(13)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る変異酵素:
(1)配列番号1に示すアミノ酸配列の115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸;
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸;
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸;
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸;
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸;
(13)配列番号1に示すアミノ酸配列の583位アミノ酸に相当するアミノ酸。 - 微生物由来グルコースオキシダーゼのアミノ酸配列が配列番号1又は2のアミノ酸配列である、請求項1に記載の変異酵素。
- 置換されるアミノ酸が、(3)のアミノ酸、(5)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上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、(3)のアミノ酸についてはアラニンであり、(5)のアミノ酸についてはアラニンであり、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、請求項3に記載の変異酵素。
- 置換されるアミノ酸が、(3)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上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、(3)のアミノ酸についてはアラニンであり、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、請求項5に記載の変異酵素。
- 置換されるアミノ酸が、(7)のアミノ酸及び(12)のアミノ酸である、請求項1又は2に記載の変異酵素。
- 置換後のアミノ酸が、(7)のアミノ酸についてはヒスチジンであり、(12)のアミノ酸についてはセリン、アルギニン、ロイシン又はプロリンである、請求項7に記載の変異酵素。
- 配列番号7~21、59~61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1~9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22~36、62~64のいずれかの塩基配列を含む、請求項10に記載の遺伝子。
- 請求項10又は11に記載の遺伝子を含む組換えDNA。
- 請求項12に記載の組換えDNAを保有する微生物。
- 請求項1~9のいずれか一項に記載の変異酵素を用いて試料中のグルコースを測定することを特徴とする、グルコース測定法。
- 請求項1~9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グルコース測定用試薬。
- 請求項15に記載のグルコース測定用試薬を含む、グルコース測定用キット。
- 請求項1~9のいずれか一項に記載の変異酵素を用いて工業製品又はその原料中のグルコース量を低下させることを特徴とする方法。
- 請求項1~9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)及び(ii)を含む、変異酵素の設計法:
(i)微生物由来グルコースオキシダーゼ又は微生物由来フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼである変異対象酵素のアミノ酸配列において、以下の(1)~(13)からなる群より選択される一又は二以上のアミノ酸を特定するステップ:
(1)配列番号1に示すアミノ酸配列の115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131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132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193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353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436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446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472位アミノ酸に相当するアミノ酸;
(9)配列番号1に示すアミノ酸配列の511位アミノ酸に相当するアミノ酸;
(10)配列番号1に示すアミノ酸配列の535位アミノ酸に相当するアミノ酸;
(11)配列番号1に示すアミノ酸配列の537位アミノ酸に相当するアミノ酸;
(12)配列番号1に示すアミノ酸配列の582位アミノ酸に相当するアミノ酸;
(13)配列番号1に示すアミノ酸配列の583位アミノ酸に相当するアミノ酸;
(ii)変異対象酵素のアミノ酸配列を基にして、ステップ(i)で特定されたアミノ酸配列が他のアミノ酸に置換されたアミノ酸配列を構築するステップ。 - 変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(3)のアミノ酸、(5)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、請求項19に記載の設計法。
- 変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(3)のアミノ酸、(7)のアミノ酸又は(12)のアミノ酸、或いはこれらの中から選択される二以上のアミノ酸の組合せである、請求項19に記載の設計法。
- 変異対象酵素が微生物由来グルコースオキシダーゼであり、ステップ(i)において置換されるアミノ酸が、(7)のアミノ酸及び(12)のアミノ酸である、請求項19に記載の設計法。
- 微生物由来グルコースオキシダーゼが、アスペルギルス・ニガー又はペニシリウム・アマガサキエンスのグルコースオキシダーゼである、請求項20~22のいずれか一項に記載の設計法。
- グルコースオキシダーゼのアミノ酸配列が配列番号1又は2のアミノ酸配列である、請求項23に記載の設計法。
- 変異対象酵素が、ペニシリウム・イタリカム、ペニシリウム・リラシノエチヌラティウム、アスペルギルス・オリゼ又はアスペルギルス・テレウスのフラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼである、請求項19に記載の設計法。
- フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼのアミノ酸配列が配列番号3~6のいずれかのアミノ酸配列である、請求項25に記載の設計法。
- 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7~21、59~61のいずれかのアミノ酸配列、又は請求項19~26のいずれか一項に記載の設計法によって構築された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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US9458434B2 (en) | 2016-10-04 |
JPWO2011068050A1 (ja) | 2013-04-18 |
US8883434B2 (en) | 2014-11-11 |
JP6059784B2 (ja) | 2017-01-11 |
JP5846687B2 (ja) | 2016-01-20 |
EP2508600B1 (en) | 2015-08-12 |
KR20120099043A (ko) | 2012-09-06 |
CN102770536B (zh) | 2014-04-30 |
CN102770536A (zh) | 2012-11-07 |
EP2508600A4 (en) | 2013-06-12 |
KR101814588B1 (ko) | 2018-01-04 |
EP2508600A1 (en) | 2012-10-10 |
US20150024461A1 (en) | 2015-01-22 |
CN103981158A (zh) | 2014-08-13 |
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US20120244565A1 (en) | 2012-09-27 |
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