WO2016031611A1

WO2016031611A1 - Mutant enzyme and application thereof

Info

Publication number: WO2016031611A1
Application number: PCT/JP2015/073060
Authority: WO
Inventors: 享一西尾
Original assignee: 天野エンザイム株式会社
Priority date: 2014-08-29
Filing date: 2015-08-17
Publication date: 2016-03-03
Also published as: JPWO2016031611A1; JP6695802B2

Abstract

　The purpose of the present invention is to provide a glucose dehydrogenase-converted enzyme having excellent affinity for glucose. Provided is a mutant enzyme comprising an amino acid sequence in which the amino acid corresponding to amino acid 444 of an amino acid sequence shown by SEQ ID NO: 1 has been substituted by another amino acid in the amino acid sequence of microbial glucose oxidase.

Description

Mutant enzyme and its use

The present invention relates to a mutant enzyme and a method for modifying the enzyme. A dehydrogenase (dehydrogenase) glucose oxidase and a method for preparing the same are provided. This application is based on the priority based on Japanese Patent Application No. 2014-176042 filed on August 29, 2014, and on the basis of Japanese Patent Application No. 2014-233291 filed on November 18, 2014 Priority is claimed and the entire contents of these patent applications are incorporated by reference.

Simple self blood glucose measuring devices using electrochemical biosensors are widely used. The biosensor uses glucose oxidase (hereinafter abbreviated as “GO”) and glucose dehydrogenase (hereinafter abbreviated as “GDH”), which are enzymes using glucose as a substrate. While 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.

On the other hand, GDH using pyrroloquinoline quinone (PQQ) as a coenzyme (hereinafter abbreviated as “PQQ-GDH”) is known as an enzyme that acts on glucose in the absence of NAD (P) without being affected by dissolved oxygen. (See, for example, Patent Documents 1 to 3). However, PQQ-GDH is extracted and isolated because (1) PQQ is easily dissociated from the enzyme, (2) its selectivity for glucose is low, and (3) it is generally present in the membrane fraction. There are problems such as difficulty in operation.

In addition to PQQ-GDH, 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. These FAD-GDHs are reactive to xylose. Is relatively high, there is room for improvement in terms of accuracy when measuring blood sugar in those undergoing a xylose tolerance test. In addition, the modification | 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 14).

On the other hand, FAD-GDH derived from the genus Mucor, which has a relatively low activity on xylose, has been reported (Patent Documents 15 to 20, Non-Patent Document 5), but there is still room for improvement in terms of practicality. .

JP 2000-350588 A JP 2001-197888 A Japanese Patent Laid-Open No. 2001-346587 International Publication No. 2007/139013 Pamphlet International Publication No. 2004/058958 Pamphlet JP 2009-225801 A JP 2009-225800 A JP 2009-159964 A JP 2008-237210 A International Publication No. 2009/084616 Pamphlet International Publication No. 2010/053161 Pamphlet JP 2013-055917 A JP2011-152129A JP 2011-115156 A Japanese Patent No. 4648993 JP 2013-176364 A JP 2013-176363 A JP 2013-116102 A JP 2013-081399 A Japanese Patent No. 5435180 International Publication No. 2011/0668050 Pamphlet

∙ Enzymes for sensor applications are required to be given “accurate measurement values” which are important indicators for sensor performance evaluation. The accuracy of the measurement depends on both the substrate specificity and the substrate affinity of the enzyme used. If an enzyme having excellent substrate specificity and substrate affinity can be used, an accurate blood glucose level can be measured, and the amount of enzyme used can be reduced. That is, accurate measurement can be realized with a small amount of enzyme. Therefore, an object of the present invention is to provide a highly practical mutant enzyme having improved substrate affinity as an enzyme for a biosensor used for blood glucose measurement or the like. Recently, the structure of FAD-GDH was analyzed using the structure of GO, and it was reported that glutamic acid at position 414 (E414) and arginine at position 502 (R502) are important for substrate recognition (non-patented). References 6 and 7) and a modified GDH (Patent Document 21) having the advantages of GO and FAD-GDH have been developed, but the substrate affinity has not been improved.

There are two main approaches to obtaining highly practical GDH: (1) a method for modifying existing GDH (FAD-GDH or PQQ-GDH) and (2) a method for screening microorganisms. These approaches already have many attempts (for example,

Patent Documents

6 and 7 listed above) and are unlikely to lead to the creation of more effective enzymes in the future. In view of this situation, the present inventors' research group noted that glucose oxidase (GO) does not have the above-mentioned problems of FAD-GDH, as reported in the above-mentioned Patent Document 21, and Focusing on the fact that FAD-GDH has a relatively high homology in amino acid sequence, a new approach was adopted in which GDH activity was imparted to GO, that is, GO was converted to GDH. As a result, GO was successfully converted to GDH, a mutant GO with high GDH activity was created, and at the same time, an amino acid position effective for GO mutation was also successfully identified (Patent Document 21).

However, when the substrate affinity of the enzyme obtained in the above report was examined, the substrate affinity was lower than that of GO before mutation. In view of this fact, it was considered that improving the affinity of the substrate was important for improving the performance of the sensor, and the study was advanced. As a result of intensive studies, the inventors succeeded in obtaining a mutant enzyme having improved substrate affinity and succeeded in identifying a mutation position effective for improving substrate specificity.

By the way, in view of the common technical knowledge that the same kind of enzyme has a high structure (primary structure, three-dimensional structure) similarity, and the possibility that the same mutation produces the same effect, the Aspergillus niger shown in the examples described later. Mutation methods found by the present inventors can be applied to GO of Penicillium ガ amagasakiense and other microbially derived GO, which have a very high structural similarity with other GOs. I can say that.

The following invention is based on the above results and considerations.
[1] Mutant enzyme comprising an amino acid sequence in which the amino acid of (1) below is substituted with another amino acid in the amino acid sequence of glucose oxidase derived from a microorganism:
(1) An amino acid corresponding to the 444th amino acid in the amino acid sequence shown in SEQ ID NO: 1.
[2] The mutant enzyme according to [1], wherein the amino acid after substitution is arginine, glutamic acid, glutamine, leucine, methionine, threonine, tryptophan, cysteine, valine, or isoleucine.
[3] The mutant enzyme according to [1], wherein the amino acid after substitution is arginine, glutamic acid or glutamine.
[4] The mutant enzyme according to [1], wherein the amino acid after substitution is arginine.
[5] In the amino acid sequence of the glucose oxidase derived from microorganisms, in addition to the amino acid of (1), the amino acid sequence of (1) to [4] is composed of an amino acid sequence in which the amino acid of (2) below is substituted with another amino acid: The mutant enzyme according to any one of the above:
(2) An amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1.
[6] The mutant enzyme according to [5], wherein the amino acid after substitution for the amino acid of (2) is glycine, cysteine, proline, serine, glutamine, asparagine, or glutamic acid.
[7] The mutant enzyme according to any one of [1] to [6], wherein the amino acid sequence of the microorganism-derived glucose oxidase is the amino acid sequence of SEQ ID NO: 1, 2, or 15.
[8] The mutant enzyme according to [1], comprising the amino acid sequence of SEQ ID NO: 3 or 4.
[9] A gene encoding the mutant enzyme according to any one of [1] to [8].
[10] The gene according to [9], comprising the nucleotide sequence of SEQ ID NO: 5 or 6.
[11] A recombinant DNA comprising the gene according to [9] or [10].
[12] A recombinant vector comprising the gene according to [9] or [10].
[13] A microorganism having the recombinant DNA according to [11].
[14] A glucose measuring method, wherein glucose in a sample is measured using the mutant enzyme according to any one of [1] to [8].
[15] A glucose measurement reagent comprising the mutant enzyme according to any one of [1] to [8].
[16] A glucose measurement kit comprising the glucose measurement reagent according to [15].
[17] 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 [8].
[18] An enzyme agent comprising the mutant enzyme according to any one of [1] to [8].
[19] A method for designing a mutant enzyme comprising the following steps (i) and (ii):
(i) A step of specifying the following amino acid (1) in the amino acid sequence of the enzyme to be mutated, which is a microorganism-derived glucose oxidase:
(1) an amino acid corresponding to the 444th amino acid of the amino acid sequence shown in SEQ ID NO: 1;
(ii) A step of constructing an amino acid sequence in which the amino acid sequence specified in step (i) is substituted with another amino acid based on the amino acid sequence of the enzyme to be mutated.
[20] The design method according to [19], wherein in step (i), the following amino acid (2) is specified in addition to the amino acid (1):
(2) An amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1.
[21] The design method according to [19] or [20], wherein the microorganism-derived glucose oxidase is glucose oxidase of Aspergillus niger or Penicillium amagasakiens.
[22] The design method according to [21], wherein the amino acid sequence of glucose oxidase is the amino acid sequence of SEQ ID NO: 1 or 2.
[23] A method for preparing a mutant enzyme comprising the following steps (I) to (III):
(I) a step of preparing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3 or 4, or the amino acid sequence constructed by the design method according to any one of [19] to [22];
(II) expressing the nucleic acid; and (III) recovering the expression product.

Comparison of the amino acid sequence of GO derived from Aspergillus niger and GO derived from Penicillium amagasakiens. The amino acids to be mutated are underlined. “*” Represents identity, “:” represents conserved substitutions, and “.” Represents semi-conserved substitutions. GDH activity and GO activity of an enzyme introduced with a mutation at amino acid 444 (S444) of GO derived from Aspergillus niger. GDH activity and GO activity of an enzyme introduced with a mutation at amino acid 582 (V582) of GO derived from Aspergillus niger. Affinity to glucose of multiple mutant enzyme GOM6 (S444R, V582P) (measurement result of Km value). Substrate specificity of multiple mutant enzyme GOM6 (S444R, V582P). The results are shown in comparison with the multiple mutant GOM2 (D446H, V582P) and the enzyme before mutation (GO derived from Aspergillus niger). Comparison of amino acid sequences of various Aspergillus niger derived GOs. Gi 110294440 (ABG66642.1) (SEQ ID NO: 8), gi 238801174 (ACR56326.1) (SEQ ID NO: 9), gi 470268262 (AGI04246.1) (SEQ ID NO: 10) gi 55975635 (AAV68194.1) (sequence) No. 11), gi 310687275 (ADP03053.1) (SEQ ID NO: 12), gi 393716500 (AFN20671.1) (SEQ ID NO: 13), gi 170676331 (ACB30370.1) (SEQ ID NO: 14), gi 121529 (sp P13006.1 , GOX_ASPNG) (SEQ ID NO: 15), gi 13236685 (AAF59929.2, AF234246_1) (SEQ ID NO: 16), gi 656365428 (AID16306.1) (SEQ ID NO: 17), GO-1 (SEQ ID NO: 1). The same amino acid is shown in dark shading. It can be seen that the identity between the sequences is high. Continuation of FIG. Continuation of FIG. Continuation of FIG. GDH activity and GO activity of various mutated enzymes. Aspergillus niger-derived GO (SEQ ID NO: 1) multiple mutation enzyme GOM6 (S444R, V582P), multiple mutation enzyme GOM7 (S444Q, V582P), multiple mutation enzyme GOM8 (S444E, V582P), another Aspergillus niger-derived GO The multiple mutation enzyme 1cf3M6 (S444R, V582P) of (SEQ ID NO: 15) and the multiple mutation enzyme 1pgeM6 (N444R, V582P) of GO derived from Penicillium amagasakiens (SEQ ID NO: 2) were compared. Affinity of various multiple mutant enzymes for glucose (measurement result of Km value). Aspergillus niger-derived GO (SEQ ID NO: 1) multiple mutation enzyme GOM6 (S444R, V582P), multiple mutation enzyme GOM7 (S444Q, V582P), multiple mutation enzyme GOM8 (S444E, V582P), another Aspergillus niger-derived GO The multiple mutation enzyme 1cf3M6 (S444R, V582P) of (SEQ ID NO: 15) and the multiple mutation enzyme 1pgeM6 (N444R, V582P) of GO derived from Penicillium amagasakiens (SEQ ID NO: 2) were compared. Substrate specificity of various mutated enzymes. Aspergillus niger-derived GO (SEQ ID NO: 1) multiple mutation enzyme GOM6 (S444R, V582P), multiple mutation enzyme GOM7 (S444Q, V582P), multiple mutation enzyme GOM8 (S444E, V582P), another Aspergillus niger-derived GO The multiple mutation enzyme 1cf3M6 (S444R, V582P) of (SEQ ID NO: 15) and the multiple mutation enzyme 1pgeM6 (N444R, V582P) of GO derived from Penicillium amagasakiens (SEQ ID NO: 2) were compared.

For convenience of explanation, some terms used in connection with the present invention are defined below.
(the term)
The term “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”. Examples of "characteristics" here 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. The mutant GO of the present invention has an amino acid sequence in which the following amino acid (1) is substituted with another amino acid in the amino acid sequence of GO (mutation target enzyme) derived from a microorganism.
(1) Amino acid corresponding to the 444th amino acid of the amino acid sequence shown in SEQ ID NO: 1

As shown in Examples described later, the amino acid at position 444 is important for the affinity for glucose when GO is converted to glucose dehydrogenase (GDH). In the present invention, the affinity for glucose is improved by mutating an amino acid corresponding to the amino acid.

Here, 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. For example, 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”. Can do. Instead of, or in addition to, the comparison of primary structures, 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. In this case, 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).

An example of a method for determining a protein tertiary structure by X-ray crystal structure analysis is shown below.
(1) Crystallize the protein. Crystallization 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.
(2) 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. and diffraction data is collected in that state has recently become widespread. Finally, in order to collect high resolution data used for structure determination, synchrotron radiation having high luminance is used.
(3) In order to perform crystal structure analysis, phase information is required in addition to diffraction data. When the crystal structure of a protein related to the target protein is unknown, it is impossible to determine the structure by the molecular replacement method, and the phase problem must be solved by the heavy atom isomorphous replacement method. 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. 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 GO derived from Aspergillus niger and the amino acid sequence of GO derived from Penicillium amagasakiens are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In addition, an alignment comparison of these two amino acid sequences is shown in FIG.

When Aspergillus niger-derived GO having the amino acid sequence of SEQ ID NO: 1 is used as the mutation target enzyme, the amino acid of the above (1) is the 444th amino acid of SEQ ID NO: 1. On the other hand, when Penicillium amagasakiens-derived GO having the amino acid sequence of SEQ ID NO: 2 is used as the enzyme to be mutated, the amino acid of the above (1) is the 444th amino acid of SEQ ID NO: 2.

Preferably, in addition to the above amino acid (1), the following amino acid (2) is also substituted. That is, as a preferred embodiment of the present invention, a mutant GO in which two amino acids are substituted is provided.
(2) Amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1

The amino acid (2) above is important for GDH activity of GO. In the present invention, a GDHase having high affinity for glucose is obtained by substituting the amino acid of (2) in addition to the amino acid of (1).

The type of amino acid after substitution is not particularly limited. As examples of the amino acid after substitution, the amino acid (1) is arginine, glutamic acid, glutamine, leucine, methionine, threonine, tryptophan, cysteine, valine or isoleucine. Arginine, glutamic acid or glutamine is preferable, and arginine is particularly preferable. On the other hand, for the amino acid (2), the amino acid after substitution is, for example, glycine, cysteine, proline, serine, glutamine, asparagine, or glutamic acid. Preferred is cysteine or proline, and particularly preferred is proline.

Specific examples of the mutant enzyme of the present invention include an enzyme having the amino acid sequence of SEQ ID NO: 3 and an enzyme having the amino acid sequence of SEQ ID NO: 4. The former is an enzyme in which the amino acid of (1) above is substituted with arginine for Aspergillus niger-derived GO, and the latter is an enzyme in which the amino acid of (1) above is substituted with arginine for Aspergillus niger-derived GO. ) Is an enzyme in which the amino acid is replaced with proline.

By the way, in general, when a part of the amino acid sequence of a certain protein is mutated, 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. In consideration of this common general knowledge, a mutant GO consisting of an amino acid sequence in which the amino acid in (1) above is replaced with another amino acid, or an amino acid in which the amino acids in (1) and (2) above are each replaced with another amino acid. A slight difference in the amino acid sequence is observed when compared with the mutant GO consisting of the sequence (however, the difference in the amino acid sequence occurs at a position other than the position where the amino acid substitution is performed), but the characteristics are substantially Those in which no difference is observed can be regarded as substantially the same enzyme as the mutant 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.

(Nucleic acid encoding mutant GO)
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. For example, 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.

In the present specification, 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. In addition, 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.

Examples of gene sequences encoding mutant GO are shown in SEQ ID NOs: 5 and 6. The sequence of SEQ ID NO: 5 is a gene encoding a mutant GO in which the amino acid substitution of (1) above (substitution S444R for arginine) has been performed on GO of Aspergillus niger. Similarly, the sequence of SEQ ID NO: 6 was subjected to the above amino acid substitution (substitution S444R for arginine) and amino acid substitution (substitution for proline V582P) of (2) above to Aspergillus niger GO. It is a gene encoding mutant GO.

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.

In another embodiment of the present invention, 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.” In addition, a base sequence defining a homologous nucleic acid is also referred to as “homologous base sequence”). As an example of 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. And 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 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.

Such 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. As stringent conditions, for example, 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 μg / ml denaturation Conditions of 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 μg / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

Still another embodiment of the present invention provides a nucleic acid (nucleic acid fragment) having a base sequence of a gene encoding the mutant GO of the present invention or a part of a base sequence complementary thereto. Such 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. When used as a probe, a nucleic acid fragment can be labeled. For labeling, for example, 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. As used herein, 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 form thereof, λ phage or a modified form thereof, pBR322 or a modified form thereof (pB325, pAT153, pUC8, etc.), etc., as a vector using E. coli as a host, 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. Examples of E. coli include E. coli BL21 (DE3) pLysS when T7 promoter is used, and E. coli JM109 otherwise. Examples of budding yeast include budding yeast SHY2, budding yeast AH22, or budding yeast INVSc1 (Invitrogen).

Another aspect of the present invention relates to a microorganism (that is, a transformant) having the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention. For example, calcium chloride method (Journal 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. 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. USA 81, 7161-7165 (1984)), lipofection (Felgner, PL et al., Proc. Natl. Acad. Sci. USA 84,7413-7417 (1984)) etc. The 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).

(Use of Mutation GO)
The third aspect of the present invention relates to the use of mutant GO. In this aspect, first, a glucose measurement method using a mutant GO is provided. In the glucose measuring method of the present invention, 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. Moreover, you may utilize this invention in order to investigate a fermentation degree in the manufacturing process of fermented foods (for example, vinegar) or fermented drinks (for example, beer and liquor).

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. In addition, an instruction manual is usually attached to the glucose measurement kit of the present invention.

As a further application 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. Provides a method for reducing the glucose content and an enzyme agent used in the application. For example, when the mutant GO of the present invention is applied to food, 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). As the excipient, 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. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

(Method for designing mutant enzymes)
Another aspect of the present invention relates to a method for designing a mutant enzyme. In the design method of the present invention, the following steps (i) and (ii) are performed.
Step (i): The following amino acid (1) is specified in the amino acid sequence of the enzyme to be mutated, which is a microorganism-derived glucose oxidase (microorganism-derived GO).
(1) Amino acid corresponding to the 444th amino acid of the amino acid sequence shown in SEQ ID NO: 1

In the present invention, by replacing the amino acid of (1) above, the affinity for glucose when GO is converted to GDH is improved.

The enzyme to be mutated in the design method of the present invention is a microorganism-derived GO. The enzyme to be mutated is typically a wild-type enzyme (an enzyme found in nature). However, this does not preclude that an enzyme that has already undergone some mutation or modification is used as the enzyme to be mutated. Examples of microorganism-derived GO are Aspergillus niger GO and Penicillium amagasakiens GO. The amino acid sequence (one example) of the enzyme exemplified here is shown below. In a preferred embodiment, an enzyme consisting of any one of these amino acid sequences is an enzyme to be mutated.
GO of Aspergillus niger: amino acid sequence of SEQ ID NO: 1 GO of Penicillium amagasakiense: amino acid sequence of SEQ ID NO: 2

For Aspergillus niger GO, several sequences are known in addition to the above sequence (SEQ ID NO: 1). 6 to 9 show alignment comparisons of amino acid sequences of various Aspergillus niger-derived GOs.

Preferably, in step (i), in addition to the amino acid of (1) above, the following amino acid of (2) is specified.
(2) Amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1

In the present invention, the following step (ii) is performed after step (i).
Step (ii): Based on the amino acid sequence of the enzyme to be mutated, an amino acid sequence in which the amino acid sequence specified in step (i) is substituted with another amino acid is constructed.

(Method for preparing mutant enzyme)
A further aspect of the present invention relates to a method for preparing a mutant enzyme. In one embodiment of the method for preparing a mutant enzyme of the present invention, a mutant GO successfully obtained by the present inventors is prepared by a genetic engineering technique. In this embodiment, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3 or 4 is prepared (Step (I)). Here, 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. In addition, an extra sequence (either a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence) may be added to such a nucleic acid. Codon degeneracy is also considered. "Nucleic acid encoding the amino acid sequence of SEQ ID NO: 3 or 4" refers to the sequence information disclosed in this specification or the attached sequence listing, and uses standard genetic engineering techniques, molecular biological techniques, biochemical By using a technique or the like, it can be prepared in an isolated state. Here, all of the amino acid sequences of SEQ ID NO: 3 or 4 are obtained by mutating the amino acid sequence of Aspergillus niger-derived GO. Therefore, a nucleic acid (gene) encoding the amino acid sequence of SEQ ID NO: 3 or 4 can also be obtained by adding a necessary mutation to the gene encoding Aspergillus niger-derived GO (SEQ ID NO: 7). 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. As 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)). For example, 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)). As a DNA polymerase used for PCR, Taq polymerase or the like can be used. However, it is preferable to use a highly accurate DNA polymerase such as KOD-PLUS- (Toyobo), Pfu turbo (Stratagene).

In another embodiment of the present invention, a mutant enzyme is prepared based on the amino acid sequence designed by the design method of the present invention. In this embodiment, in step (I), a nucleic acid encoding an amino acid sequence constructed by the designing method of the present invention is prepared. For example, based on the amino acid sequence constructed by the design method of the present invention, a necessary mutation (that is, substitution of an amino acid at a specific position in a protein as an expression product) is added to a gene encoding an enzyme to be mutated. A nucleic acid (gene) encoding the mutant enzyme is obtained.

Following step (I), the prepared nucleic acid is expressed (step (II)). For example, first, 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.

Next, 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. For example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. The nitrogen source may be any nitrogen compound that can be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, 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 in consideration of the growth characteristics of the transformant to be cultured and the production characteristics of the mutant enzyme. Preferably, it can be set within the range of 30 ° C. to 40 ° C. (more 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. Preferably, the pH of the medium is about 6.0 to 9.0 (preferably around pH 7.0).

Subsequently, the expression product (mutant enzyme) is recovered (step (III)). Although 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. In general, 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. When recovering from the culture solution, for example, 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. Various 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. On the other hand, when recovering from the microbial cells, 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.

It is also possible to provide the purified enzyme obtained as described above by pulverizing it by, for example, freeze drying, vacuum drying or spray drying. At that time, the purified enzyme may be dissolved in a phosphate buffer, triethanolamine buffer, Tris-HCl buffer or GOOD buffer in advance. Preferably, a phosphate buffer or a triethanolamine buffer can be used. In addition, PIPES, MES, or MOPS is mentioned as a GOOD buffer here.

Usually, gene expression and expression product (mutant enzyme) are collected using an appropriate host-vector system as described above, but a cell-free synthesis system may be used. 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. In a cell-free synthesis system, 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. When protein is synthesized, 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. Of course, a ribosome, various factors, and / or various enzymes prepared separately may be supplemented as necessary during protein synthesis.

Development of a transcription / translation system that reconstitutes each molecule (factor) necessary for protein synthesis has also been reported (Shimizu, Y. et al .: Nature Biotech., 19, 751-755, 2001). In this synthesis system, three types of initiation factors constituting bacterial protein synthesis system, three types of elongation factors, four types of factors involved in termination, 20 types of aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and Genes of 31 kinds of factors consisting of methionyl tRNA formyltransferase are amplified from the Escherichia coli genome, and the protein synthesis system is reconstructed in vitro using these genes. In the present invention, such a reconstructed synthesis system may be used.

The term “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. In an in vitro translation system, RNA is used as a template to synthesize proteins. As the template RNA, 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. In the in vitro transcription / translation system, conditions to which factors necessary for each reaction are added are set so that the transcription reaction and the translation reaction proceed continuously.

According to previous studies, a multiple mutant enzyme (referred to as “GOM2”) in which two mutations (D446H and V582P) are added to Aspergillus niger-derived GO exhibits high GDH activity and excellent substrate specificity. It became clear (patent document 21). When the characteristics of this multiple mutant enzyme (GOM2) were examined in detail, there was room for improvement in terms of substrate affinity for glucose. Therefore, considering that improving the substrate affinity is important for improving the performance of the sensor, the following examination was performed.

1. Identification of mutation position In order to improve substrate affinity, mutations designed to replace multiple amino acids are introduced into the surrounding amino acids at each mutation site of the mutant enzyme GOM2, and GDH is introduced by introducing mutations into S444. It has been found that

2. Evaluation of GDH activity Genomic DNA was extracted from Aspergillus niger GO-1 (owned by Amano Enzyme) using Gen Elute Plant Genomic DNA kit (Sigma), and the GO gene was obtained by PCR. Mutated glucose oxidase designed to substitute multiple amino acids into S444 using the pYES-GO-KP-2 plasmid as a template by inserting the amplified product after PCR into pYES2. A plasmid with The transformed plasmid was transformed into Escherichia coli DH5α, 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 expression in liquid culture was referred to the manual for pYES2.

20 μL of the culture supernatant of mutant glucose oxidase designed to substitute 20 kinds of amino acids into S444 for 200 μL of each reagent was added and reacted at 37 ° C. Absorbance was measured 5 minutes and 10 minutes after the start of the reaction, and GO activity and GDH activity were determined from the difference in absorbance. Aspergillus niger GO-1 derived GO (indicated as GO) and Aspergillus niger GO-1 derived GO multiple mutation enzyme (GOM2) with two mutations (D446H and V582P) added for comparison (Control).
<Reagent for GO assay>
Phenolic phosphate buffer solution 21mL
1mol / L glucose 3mL
25u / mL PO-3 5mL
0.4g / dL 4-AA 1mL
<Reagent for GDH assay>
50 mM PIPES-NaOH (cont.0.1% Triton X-100) pH 7.0 23 mL
1mol / L glucose 3mL
3mmol / L PMS 3mL
6.6mmol / L NTB 1mL

Fig. 2 shows the measurement results for S444. Substituted amino acids that increase GDH activity / GO activity are R, E, Q, L, M, T, W, C, V, and I. Among these, substitution with R, E, or Q is effective for GDH, and substitution with R can be evaluated as being most preferable. On the other hand, the measurement results for V582 are shown in FIG. Substituted amino acids that increase GDH activity / GO activity are N, S, E, Q, P, C, and G. Of these, substitution with P is particularly effective for GDH.

3. Evaluation of substrate affinity Based on the above results, glucose of multiple mutants (S444R, V582P: referred to as “GOM6”) in which the amino acid at position 444 was substituted from serine to arginine and the amino acid at position 582 was replaced from valine to proline The affinity for was evaluated by the following method in comparison with multiple mutants (GOM2).

Liquid culture of GOM2 and GOM6 transformants was performed, and DEAE-Sepharose purification and desalting concentration were performed to obtain a purified enzyme solution. The Km value was calculated by measurement using the purified enzyme solution obtained. The expression in liquid culture was referred to the manual for pYES2.

The Km value for glucose of GOM6 was 22.9 × 10 ⁻³ mol / L (FIG. 4), which was about 1/5 of the Km value of GOM2 (116 × 10 ⁻³ mol / L). That is, it was revealed that GOM6 has a much higher affinity for glucose than GOM2. The Km value for glucose of Aspergillus niger-derived GO was 12.9 × 10 ⁻³ mol / L.

4). Confirmation of substrate specificity The substrate specificity of the multiple mutant (GOM6) was evaluated as follows. That is, 20 μL of the purified enzyme used in the substrate affinity confirmation experiment was added to 200 μL of each reagent and reacted at 37 ° C. Absorbance was measured at 5 and 10 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.
<Substrate specificity confirmation GDH assay reagent>
50 mM PIPES-NaOH (cont.0.1% Triton X-100) pH 7.0 23 mL
1mol / L substrate 3mL
3mmol / L PMS 3mL
6.6mmol / L NTB 1mL

The results are shown in FIG. The multiple mutant (GOM6) showed the same substrate specificity as the pre-mutation enzyme (GO derived from Aspergillus niger) and was confirmed to be highly practical.

5. Preparation and characterization of other GO-derived multiple mutant enzymes In order to verify the versatility of successfully identified mutation sites (S444, V582), multiple mutants were prepared for other GOs. Specifically, multiple mutants 1cf3M6 (S444R, V582P) of GO derived from Aspergillus niger (gi 121529, SEQ ID NO: 15) and GO derived from Penicillium amagasakiens registered in public databases (SEQ ID NO: Multiple mutant 1pgeM6 (N444R, V582P) of 2) was prepared, and GDH activity and GO activity, substrate affinity, and substrate specificity were examined. In addition, the characteristics of the multiple mutants GOM7 (S444Q, V582P) and GOM8 (S444E, V582P) differing in amino acid after substitution with GOM6 were also examined. The preparation method and activity measurement method of each multiple mutant were the same as in the above experiment.

The measurement results of GDH activity and GO activity of each multiple mutant are shown in FIG. It can be seen that the multiple mutants GOM7, GOM8, 1cf3M6 and 1pgeM6 are all highly GDH-ized. On the other hand, the Km value of each multiple mutant is equal to or less than the Km value of GOM6, and shows high substrate affinity (FIG. 11). Each multiple mutant is also excellent in substrate specificity (FIG. 12).

As described above, it was confirmed that each of the multiple mutants exhibited the expected characteristics and the versatility of the mutation positions that were successfully identified was high.

The mutant GO of the present invention is useful for detecting and quantifying the amount of glucose in a sample. The affinity of the mutant GO of the present invention for glucose is high. Therefore, if the mutant GO of the present invention is used for a glucose sensor, improvement in measurement accuracy can be expected.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims

In the amino acid sequence of glucose oxidase derived from microorganisms, a mutant enzyme comprising an amino acid sequence in which the amino acid of (1) below is replaced with another amino acid:
(1) An amino acid corresponding to the 444th amino acid in the amino acid sequence shown in SEQ ID NO: 1.
The mutant enzyme according to claim 1, wherein the amino acid after substitution is arginine, glutamic acid, glutamine, leucine, methionine, threonine, tryptophan, cysteine, valine or isoleucine.
The mutant enzyme according to claim 1, wherein the amino acid after substitution is arginine, glutamic acid or glutamine.
The mutant enzyme according to claim 1, wherein the amino acid after substitution is arginine.
The amino acid sequence of the microorganism-derived glucose oxidase comprises an amino acid sequence in which the following amino acid (2) is substituted with another amino acid in addition to the amino acid (1): The described mutant enzyme:
(2) An amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1.
The mutant enzyme according to claim 5, wherein the amino acid after substitution of the amino acid of (2) is glycine, cysteine, proline, serine, glutamine, asparagine or glutamic acid.
The mutant enzyme according to any one of claims 1 to 6, wherein the amino acid sequence of the microorganism-derived glucose oxidase is the amino acid sequence of SEQ ID NO: 1, 2, or 15.
The mutant enzyme according to claim 1, comprising the amino acid sequence of SEQ ID NO: 3 or 4.
A gene encoding the mutant enzyme according to any one of claims 1 to 8.
The gene according to claim 9, comprising the nucleotide sequence of SEQ ID NO: 5 or 6.
Recombinant DNA containing the gene according to claim 9 or 10.
A recombinant vector comprising the gene according to claim 9 or 10.
A microorganism having the recombinant DNA according to claim 11.
A glucose measuring method, wherein glucose in a sample is measured using the mutant enzyme according to any one of claims 1 to 8.
A glucose measuring reagent comprising the mutant enzyme according to any one of claims 1 to 8.
A glucose measurement kit comprising the glucose measurement reagent according to claim 15.
A method of reducing the amount of glucose in an industrial product or its raw material using the mutant enzyme according to any one of claims 1 to 8.
An enzyme agent comprising the mutant enzyme according to any one of claims 1 to 8.
A method for designing a mutant enzyme comprising the following steps (i) and (ii):
(i) A step of specifying the following amino acid (1) in the amino acid sequence of the enzyme to be mutated, which is a microorganism-derived glucose oxidase:
(1) an amino acid corresponding to the 444th amino acid of the amino acid sequence shown in SEQ ID NO: 1;
(ii) A step of constructing an amino acid sequence in which the amino acid sequence specified in step (i) is substituted with another amino acid based on the amino acid sequence of the enzyme to be mutated.
The design method according to claim 19, wherein, in step (i), in addition to the amino acid of (1), the following amino acid of (2) is specified:
(2) An amino acid corresponding to amino acid 582 of the amino acid sequence shown in SEQ ID NO: 1.
The design method according to claim 19 or 20, wherein the microorganism-derived glucose oxidase is glucose oxidase of Aspergillus niger or Penicillium amagasakiens.
The design method according to claim 21, wherein the amino acid sequence of glucose oxidase is the amino acid sequence of SEQ ID NO: 1 or 2.
A method for preparing a mutant enzyme comprising the following steps (I) to (III):
(I) preparing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3 or 4, or the amino acid sequence constructed by the design method according to any one of claims 19 to 22;
(II) expressing the nucleic acid; and (III) recovering the expression product.