JP2006217811A - Modified product of pqq (pyrroloquinoline quinone)-dependent glucose dehydrogenase having excellent substrate specificity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a PQQGDH (pyrroloquinoline quinone-dependent glucose dehydrogenase) having improved substrate specificity. <P>SOLUTION: A modified type PQQGDH has a reduced activity for disaccharides as compared with that of a wild type PQQGDH. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a modified glucose dehydrogenase with improved substrate specificity (hereinafter, glucose dehydrogenase is also referred to as GDH), and specifically, pyrroloquinoline quinone (hereinafter, pyrroloquinoline quinone is also referred to as PQQ) as a coenzyme. The present invention relates to a modified PQQ-dependent glucose dehydrogenase (hereinafter, pyrroloquinoline quinone-dependent glucose dehydrogenase is also referred to as PQQGDH), a production method thereof, and a glucose sensor. The modified PQQGDH of the present invention is useful for the determination of glucose in clinical examinations and food analysis.

  PQQGDH is a glucose dehydrogenase using pyrroloquinoline quinone (PQQ) as a coenzyme. Since it catalyzes the reaction of oxidizing glucose to produce gluconolactone, it can be used for blood glucose measurement. Blood glucose concentration is an extremely important index for clinical diagnosis as an important marker of diabetes. Currently, blood glucose concentration is measured mainly by a method using a biosensor using glucose oxidase. However, since the reaction is affected by the dissolved oxygen concentration, there may be an error in the measured value. It was. PQQ-dependent glucose dehydrogenase has attracted attention as a new enzyme that replaces this glucose oxidase.

Our group found that Acinetobacter baumanni NCIMB11517 produces PQQ-dependent glucose dehydrogenase, and constructed a gene cloning and high expression system (see, for example, Patent Document 1). However, PQQ-dependent glucose dehydrogenase has a problem in its substrate specificity compared with glucose oxidase.
Japanese Patent Laid-Open No. 11-243949

  The present invention has been made against the background of the problems of the prior art, and relates to the improvement of the substrate specificity of PQQGDH.

As a result of diligent research to solve the above-mentioned problems, the present inventors can replace the amino acid in a specific region of PQQGDH and / or improve the substrate specificity by mutation by inserting the amino acid. Thus, the present invention has been completed.
In particular, the present invention has been completed by focusing on “amino acid insertion”, which has not been practically practically studied so far.
That is, the present invention
[Claim 1]
Amino acid substitutions, the amino acid sequence of the Acinetobacter genus PQQGDH, (T224A + A236T), (Q168A + T224A + A236T + M342I), (Q168A + 171A + T224A + A236T + M342I), (Q168A + 171S + T224A + A236T + M342I), (M342I + 430P), (E245D + M342I + 430P), (Q168A + L169P + A170M + E245D + M342I + 430P), (Q168A + 169F + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + 430P), ( Q168A + 169Y + L169P + A170L + E245D + M342I + 429D + 430P), (Q168A + 169Y + L169P + A170M + E245D + M342I + N429E + 430P), or modified PQQG with reduced activity on disaccharides compared to wild-type PQQGDH having amino acid mutations at positions equivalent to those in other species .
[Section 2]
Modified PQQGDH, in which stability is reduced by substituting a charged amino acid before and after P in modified PQQGDH whose stability is reduced by substituting a certain amino acid for P or by inserting P at a certain site.
[Section 3]
The modified PQQGDH according to claim 2, wherein substitution or insertion of P is around position 430.
[Claim 4]
The modified PQQGDH according to claim 2 or 3, wherein the charged amino acid is D or E.
[Section 5]
The modified PQQGDH according to claim 1, wherein the disaccharide is maltose.
[Claim 6]
A gene encoding the modified PQQGDH according to any one of claims 1 to 5.
[Claim 7]
A vector comprising the gene according to claim 6.
[Section 8]
A transformant transformed with the vector according to claim 7.
[Claim 9]
A method for producing modified PQQGDH, wherein the transformant according to claim 8 is cultured.
[Section 10]
The composition for glucose measurement containing the modified PQQGDH in any one of Claims 1-5.
[Section 11]
A glucose assay kit comprising the modified PQQGDH according to any one of claims 1 to 5.
[Claim 12]
A glucose sensor comprising the modified PQQGDH according to any one of claims 1 to 5.
[Claim 13]
A glucose measurement method comprising the modified PQQGDH according to any one of claims 1 to 5.
[Section 14]
A method for reducing the activity of PQQGDH on disaccharides, wherein the amino acid mutation according to any one of claims 1 to 5 is performed on PQQGDH.
[Section 15]
A glucose measurement system using PQQGDH, comprising the PQQGLD in which the amino acid mutation according to any one of claims 1 to 5 is performed, and a method for improving measurement accuracy in the glucose measurement system.
[Section 16]
A glucose measurement composition using PQQGDH, comprising the PQQGLD subjected to the amino acid mutation according to any one of claims 1 to 5, wherein the composition for glucose measurement with improved measurement accuracy is produced. Method.

  The modified PQQGDH according to the present invention is an enzyme having a reduced activity on disaccharides compared to wild-type PQQGDH. By using the modified PQQGDH according to the present invention in a glucose assay kit and a glucose sensor, it is possible to perform analysis with higher accuracy than that using wild-type PQQGDH, and a glucose assay kit and glucose sensor with higher stability. Can be provided.

  Hereinafter, the present invention will be described in detail.

  The modified PQQGDH of the present invention has a lower activity against disaccharides than the wild-type PQQGDH.

  The action with respect to a disaccharide means the effect | action which dehydrogenates a disaccharide. Examples of the disaccharide include maltose, sucrose, lactose, cellobiose, and particularly maltose. In the present invention, the decrease in the activity on disaccharides is also expressed as an improvement in substrate specificity.

Judgment whether the action with respect to a disaccharide has fallen is performed as follows.
In the activity measurement method described in Test Example 1 described later, PQQGDH activity value (a) when D-glucose was used as a substrate solution using wild-type PQQGDH, and the disaccharide was used instead of D-glucose as a substrate solution. The PQQGDH activity value (b) is measured, and the relative value ((b) / (a) × 100) with respect to the measured value when glucose is used as the substrate is set to 100. Next, the same operation is performed using the modified PQQGDH, and the values are compared and determined.

  The modified PQQGDH of the present invention is included in the modified PQQGDH of the present invention, regardless of whether the activity on glucose is increased, unchanged or decreased, as long as the activity on the disaccharide is lower than that on the wild type PQQGDH. .

  The modified PQQGDH of the present invention includes those in which the action on disaccharides in the measurement of glucose concentration is lower than when wild-type PQQGDH is used. Preferably, the action property to maltose is reduced. The effect on maltose is preferably 90% or less, more preferably 70% or less, still more preferably 40% or less, particularly 20% or less of wild-type PQQGDH.

  The modified PQQGDH of the present invention preferably has an action on maltose of 90% or less of the action on glucose. More preferably, it is 70% or less, more preferably 40% or less, particularly 20% or less.

  Examples of the modified PQQGDH of the present invention having a reduced activity on disaccharides compared to wild-type PQQGDH include, for example, (T224A + I234A + A236A + A2361A), (Q168A + T224A + A236A + 71. ), (E245D + M342I + 430P), (Q168A + L169P + A170M + E245D + M342I + 430P), (Q168A + 169F + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169E + D43 P), (Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P), (Q168A + 169Y + L169P + A170M + E245D + M342I + N429E + 430P) which is the same as the above, or Q which is equivalent to the above. The modified PQQGDH is exemplified.

  Here, for example, “T224A” means that T (Thr) at position 224 is replaced with A (Ala). “171A” means that A (Ala) is inserted at a position after the 171st position. It means that A was inserted after the amino acid at position 171. Multiple mutants are represented by hyphens that are represented by the same principle.

  In addition, for example, PQQGDH multiple mutants “Q168A + 169F + L169P + A170L + E245D + M342I + N429D + 430P” have Q at position 168 as A, L at position 169 as P, 170 at position A as L, E at position 245 as D, M at position 342 Is substituted with I at position 429 with D, F is inserted after position 168 replaced with A, and P is inserted after position 429 replaced with D. For convenience, the numbers dealt with here correspond to the positions on the amino acid sequence of the wild type PQQGDH of Acinetobacter baumannii NCIMB11517 strain, and the amino acid position is moved backward by insertion of the front amino acid. Even if it slips, the number representing the position is not corrected.

  P that has been mutated to reduce its activity on disaccharides may be a factor that reduces the stability of the enzyme, and modified PQQGDH, which has improved stability by placing charged amino acids before and after that, Illustrated.

  In this example, 430P is exemplified as an example of P that is mutagenized to reduce its activity on disaccharides, but it is also effective for modified PQQGDH whose stability has been reduced by N429P mutagenesis. .

  Preferred forms of the charged amino acids are acidic amino acids D and E, which are arranged to stabilize the P chain measurement, and can be either before or after P.

  Multiple mutants with any combination of substitutions or insertions shown in the next paragraph contribute to an increase in the substrate specificity of PQQGDH.

(T224A + A236T), (Q168A + T224A + A236T + M342I), (Q168A + 171A + T224A + A236T + M342I), (Q168A + 171S + T224A + A236T + M342I), (M342I + 430P), (E245D + M342I + 430P), (Q168A + L169P + A170M + E245D + M342I + 430P), (Q168A + 169F + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P), (Q168A + 169Y + L169P + A170M + E245D + 342I + N429E + 430P)

  The present invention is also a method for reducing the activity of PQQGDH on disaccharides, characterized in that the amino acid insertion described above is performed in PQQGDH.

  The present invention also relates to a glucose measurement system using PQQGDH, which comprises the PQQGLD into which the amino acid insertion according to any one of claims 1 to 6 has been performed, and the measurement accuracy in the glucose measurement system. It is a way to improve.

  The invention of the present application also includes a composition for measuring glucose with improved measurement accuracy characterized by containing PQQGLD in which the amino acid insertion according to any one of claims 1 to 6 is included in a glucose measurement system using PQQGDH This is a method for manufacturing a product.

  The amino acid sequence of PQQGDH derived from the genus Acinetobacter is preferably the amino acid sequence of PQQGDH derived from Acinetobacter calcoaceticus or Acinetobacter baumannii. Among them, SEQ ID NO: 1 is preferable. The wild-type PQQGDH protein represented by SEQ ID NO: 1 and its base sequence represented by SEQ ID NO: 2 originate from Acinetobacter baumanni NCIMB11517 strain, and are disclosed in JP-A-11-243949 Yes. In the above and SEQ ID NO: 1, the amino acid notation is numbered with 1 aspartic acid from which the signal sequence has been removed.

  Acinetobacter baumannii NCIMB11517 strain was previously classified as Acinetobacter calcaceticus.

  The modified PQQGDH of the present invention has a portion of other amino acid residues deleted or substituted as long as it has glucose dehydrogenase activity, and preferably has no substantial adverse effect on the action on disaccharides. Other amino acid residues may be added or substituted.

By the way, at the time of filing this application, the results of X-ray crystal structure analysis of an enzyme derived from Acinetobacter calcoaceticus LMD79.41 were reported, and the higher-order structure of the enzyme including the active center was clarified. (See Non-Patent Documents 1, 2, 3, and 4.)
J. et al. Mol. Biol. , 289, 319-333 (1999) PNAS, 96 (21), 11787-11791 (1999) The EMBO Journal, 18 (19), 5187-5194 (1999) Protein Science, 9, 1265-1273 (2000)

Although research on the correlation between the structure and function of the enzyme has been carried out based on the knowledge about the higher-order structure, it cannot be said that it has been clarified yet. For example, glucose is introduced by introducing a mutation into a specific site of a structural gene of an amino acid residue in a loop region (W6BC) connecting the B strand and the C strand of the sixth W-motif of water-soluble glucose dehydrogenase. However, it has been considered that the selectivity to can be improved (see, for example, Patent Document 2). However, only the effects disclosed in the examples have been demonstrated.
JP 2001-197888 A

  Here, based on the results of the present invention, the knowledge about these higher-order structures is reviewed. For modification of the action on disaccharides, amino acids involved in PQQ binding and / or surrounding amino acids, glucose binding There may be a possibility that at least one of amino acids involved in and / or surrounding amino acids, amino acids involved in calcium ion binding and / or surrounding amino acids is involved.

The modified PQQGDH of the present invention is a PQQ-dependent glucose dehydrogenase described in SEQ ID NO: 1, amino acids involved in PQQ binding and / or surrounding amino acids, and / or amino acids involved in glucose binding and / or Or the surrounding amino acid is substituted.
On the other hand, Non-Patent Documents 3 and 4 include Y344, W346, R228, N229, K377, R406, R408, D424 as amino acids that bind to PQQ, and Q76, D143, H144, D163, as amino acids that bind to glucose. Q168, L169, etc. are described.

The modified PQQGDH of the present invention includes the PQQ-dependent glucose dehydrogenase described in SEQ ID NO: 1 in which amino acids involved in calcium ion binding and / or surrounding amino acids are substituted.
On the other hand, Non-Patent Document 1 describes P248, G247, Q246, D252, T348, and the like as amino acids that bind to the active center calcium ion.

  The modified PQQGDH of the present invention includes those obtained by mutating amino acids located within a radius of 15 mm, preferably within a radius of 10 mm, from the active center in the active three-dimensional structure of the wild-type enzyme.

  The modified PQQGDH of the present invention includes those obtained by mutating amino acids located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme. In particular, when the substrate is glucose, those obtained by mutating amino acids located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme are preferable.

  The modified PQQGDH of the present invention includes those obtained by mutating amino acids located within a radius of 10 mm from the OH group that binds to the 1st carbon of the substrate in the active three-dimensional structure of the wild-type enzyme . In particular, when the substrate is glucose, those obtained by mutating amino acids located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme are preferable.

  The modified PQQGDH of the present invention includes those obtained by mutating amino acids located within a radius of 10 mm from the OH group that binds to the carbon at the 2-position of the substrate in the active three-dimensional structure of the wild-type enzyme. . In particular, when the substrate is glucose, those obtained by mutating amino acids located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme are preferable.

  In accordance with the above teachings, those skilled in the art will refer to the wild-type PQQGDH protein represented by SEQ ID NO: 1 originating from the Acinetobacter baumannii NCIMB11517 strain and its base sequence represented by SEQ ID NO: 2 Derived from other sources (whether natural, modified, or artificially synthesized) with high homology (preferably having a homology of 80% or higher, more preferably 90% or higher) As for the modified PQQGDH, a position corresponding to SEQ ID NO: 1 (equivalent position) is found, and an amino acid residue is substituted in the region, so that the disaccharide is more than the wild type PQQGDH without excessive trial and error. It is possible to obtain a modified PQQGDH having a reduced activity on.

  For example, when the amino acid sequence of SEQ ID NO: 1 is compared with the amino acid sequence of the enzyme derived from Acinetobacter calcoaceticus LMD79.41, the difference is slight and the homology is 92.3% (including the signal sequence). ) And are very similar, it can be easily recognized which amino acid residue of an enzyme of another origin corresponds to a certain residue in SEQ ID NO: 1. Then, according to the present invention, the amino acid residue is substituted with another amino acid residue and / or another amino acid is inserted at one or more of such sites, so that the action on disaccharides is greater than that of wild-type PQQGDH. Modified PQQGDH having reduced properties can be obtained. These modified PQQGDH glucose dehydrogenases are also included within the scope of the present invention.

  The gene encoding the modified PQQGDH of the present invention may be obtained by modifying a DNA fragment containing a gene encoding wild-type PQQGDH obtained from various sources such as microorganisms. Specifically, for example, Acinetobacter calcoaceticus, Acinetobacter baumannii, Pseudomonas aeruginosa, Pseudomonas fluseus, Pseudomonas plutida, Pseudomonas plutida Examples include oxidative bacteria such as dance, and enterobacteria such as Agrobacterium radiobacter, Escherichia coli, and Klebsiella aerogenes. However, it is difficult to modify a membrane enzyme present in Escherichia coli to make it soluble, and the origin is Acinetobacter, more preferably Acinetobacter calcoaceticus or Acinetobacter baumannii with high homology. It is preferable to select any of the soluble PQQGDH.

  As a method for modifying the gene encoding wild-type PQQGDH, a commonly performed technique for modifying genetic information is used. That is, a DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. As a specific method for converting bases in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, QuickChange SiteDirected MutagenStained MutaseNeste, etc.) Use of the chain reaction method (PCR) is mentioned.

The prepared DNA having genetic information of the modified protein is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified protein.
When using a plasmid as a vector, for example, pBluescript, pUC18, etc. can be used when Escherichia coli is used as a host microorganism. Examples of host microorganisms that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method for transferring the recombinant DNA in the presence of calcium ions can be employed. An electroporation method may be used. Furthermore, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

  Such genes may be extracted from these strains or chemically synthesized. Furthermore, it is possible to obtain a DNA fragment containing the PQQGDH gene by using the PCR method.

In the present invention, a method for obtaining a gene encoding PQQGDH includes the following methods. For example, after separating and purifying the chromosome of Acinetobacter calcoaceticus NCIB11517, DNA was cleaved using sonication, restriction enzyme treatment, etc., linear expression vector and DNA at the blunt or sticky ends of both DNAs The recombinant vector is constructed by ligation and closing with ligase or the like. After transferring the recombinant vector into a replicable host microorganism, screening is performed using the expression of the vector marker and enzyme activity as an index, and a recombinant vector containing a gene encoding GDH having PQQ as a prosthetic group is retained. To obtain microorganisms.
Acinetobacter calcoaceticus NCIB11517 can be obtained from NCIMB or UKNCC.

  Subsequently, the microorganism holding the recombinant vector can be cultured, the recombinant vector can be isolated and purified from the cells of the cultured microorganism, and the gene encoding GDH can be collected from the expression vector. For example, the chromosomal DNA of Acinetobacter calcoaceticus NCIB11517, which is a gene donor, is specifically collected as follows.

  A culture solution obtained by stirring and culturing the gene-donating microorganism for 1 to 3 days, for example, is collected by centrifugation, and then lysed to prepare a GDH gene-containing lysate containing PQQ as a prosthetic group can do. As a method for lysis, for example, treatment is performed with a lytic enzyme such as lysozyme, and a protease, other enzyme, or a surfactant such as sodium lauryl sulfate (SDS) is used in combination as necessary. Further, it may be combined with a physical crushing method such as freeze-thawing or French press treatment.

  In order to separate and purify DNA from the lysate obtained as described above, a conventional method is used, for example, by appropriately combining methods such as deproteinization treatment by phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment. be able to.

  A method of cleaving DNA separated and purified from microorganisms can be performed by, for example, ultrasonic treatment, restriction enzyme treatment, or the like. Type II restriction enzymes that act on specific nucleotide sequences are suitable.

  As a vector for cloning, a vector constructed for gene recombination from a phage or plasmid that can autonomously grow in a host microorganism is suitable. Examples of the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as a host microorganism. Examples of plasmids include pBR322, pUC19, and pBluescript when Escherichia coli is used as a host microorganism.

  At the time of cloning, a vector fragment can be obtained by cleaving the above-described vector with the restriction enzyme used to cleave the microbial DNA that is the gene donor encoding GDH described above. It is not necessary to use the same restriction enzyme as that used in the above. The method for binding the microbial DNA fragment and the vector DNA fragment may be a method using a known DNA ligase. For example, after annealing of the sticky end of the microbial DNA fragment and the sticky end of the vector fragment, an appropriate DNA ligase is used. A recombinant vector of a microbial DNA fragment and a vector DNA fragment is prepared by use. If necessary, after annealing, it can be transferred to a host microorganism and a recombinant vector can be prepared using in vivo DNA ligase.

  The host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, can autonomously propagate, and can express a foreign gene. Generally, Escherichia coli W3110, Escherichia coli C600, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli DH5α, and the like can be used.

  As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method or an electroporation method using calcium treatment can be used.

  The microorganism which is a transformant obtained as described above can stably produce a large amount of GDH by being cultured in a nutrient medium. The selection of whether or not the target recombinant vector is transferred to the host microorganism may be performed by searching for a microorganism that simultaneously expresses GDH activity by adding a drug resistance marker of the vector holding the target DNA and PQQ. For example, a microorganism that grows in a selective medium based on a drug resistance marker and generates GDH may be selected.

The base sequence of the GDH gene having PQQ as a prosthetic group obtained by the above method was decoded by the dideoxy method described in Science, Vol. 214, 1205 (1981). The amino acid sequence of GDH was estimated from the base sequence determined as described above.

  As described above, transfer from a recombinant vector having a GDH gene having PQQ selected once as a prosthetic group to a recombinant vector capable of replicating in a microorganism capable of producing PQQ retains the GDH gene. It can be easily carried out by collecting DNA, which is a GDH gene, from a recombinant vector by a restriction enzyme or PCR method and binding it to other vector fragments. Moreover, the transformation of the microorganism which has PQQ production ability by these vectors can use the competent cell method by electrolysis, the electroporation method, etc.

  Examples of microorganisms capable of producing PQQ include methanol-utilizing bacteria such as the genus Methylobacterium, acetic acid bacteria belonging to the genus Acetobacter and Gluconobacter, and the genus Flavobacterium And bacteria such as Pseudomonas and Acinetobacter. Among them, a host-vector system that can use Pseudomonas bacteria and Acinetobacter bacteria is established and is preferable because it is easy to use.

  For Pseudomonas bacteria, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas petitida and the like can be used. As Acinetobacter bacteria, Acinetobacter calcoaceticus, Acinetobacter baumannii and the like can be used.

  As a recombinant vector that can be replicated in the above-mentioned microorganism, a vector derived from RSF1010 or a vector having a similar replicon can be used for Pseudomonas bacteria. For example, pKT240, pMMB24, etc. (MM Bagdasarian et al., Gene, 26, 273 (1983)), pCN40, pCN60, etc. (CC Nito et al., Gene, 87, 145 (1990)), pTS1137, etc. be able to. Further, pME290 and the like (Y. Itoh et al., Gene, 36, 27 (1985)), pNI111, pNI20C (N. Itoh et al., J. Biochem., 110, 614 (1991)) can also be used.

  In bacteria belonging to the genus Acinetobacter, pWM43 and the like (W. Minas et al., Appl. Environ. Microbiol., 59, 2807 (1993)), pKT230, pWH1266 and the like (M. Hunger et al., Gene, 87, 45 (1990)) are used as vectors. Is available.

  Microorganisms which are transformants thus obtained can stably produce a large amount of modified protein by being cultured in a nutrient medium. The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional physiological properties of the host, and in many cases, the culture is performed in liquid culture. Industrially, aeration and agitation culture is advantageous.

  As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. 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 appropriately changed within the range in which the fungus grows and produces modified PQQGDH. However, in the case of the microorganism having the PQQ production ability as described above, it is preferably about 20 to 42 ° C. Although the culture time varies somewhat depending on conditions, the culture may be completed at an appropriate time in consideration of the time when the modified PQQGDH reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed as long as the bacteria grow and produce modified PQQGDH, but is preferably in the range of about pH 6.0 to 9.0.

  Although the culture solution containing the cells producing the modified PQQGDH in the culture can be collected and used as it is, in general, when the modified PQQGDH is present in the culture solution, it is filtered and centrifuged. It is used after separating the modified PQQGDH-containing solution and microbial cells by separation or the like. When the modified PQQGDH is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial cells are collected by a mechanical method or an enzymatic method such as lysozyme. In addition, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize GDH, and it is separated and collected as an aqueous solution.

  The GDH-containing solution obtained as described above is precipitated by, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractional precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. You just have to let them know. Heat treatment and isoelectric point treatment are also effective purification means. Thereafter, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

  For example, separation and purification by gel filtration using Sephadex gel (Pharmacia Biotech), column chromatography such as DEAE Sepharose CL-6B (Pharmacia Biotech), octyl Sepharose CL-6B (Pharmacia Biotech), etc. Goods can be obtained. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

  The purified enzyme obtained as described above can be pulverized and distributed, for example, by freeze drying, vacuum drying, spray drying, or the like. At this time, the purified enzyme may be one dissolved in a phosphate buffer, a Tris-HCl buffer, or a GOOD buffer. Preferred are GOOD buffers, with PIPES, MES or MOPS buffers being particularly preferred. Further, GDH can be further stabilized by adding calcium ions or salts thereof, amino acids such as glutamic acid, glutamine, and lysine, and serum albumin.

  Although the manufacturing method of the modified protein of this invention is not specifically limited, It is possible to manufacture in the procedure as shown below. As a method for modifying the amino acid sequence constituting the protein, a commonly used technique for modifying genetic information is used. That is, a DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. As a specific method for converting bases in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, QuickChange SiteDirected MutagenStained MutaseNeste, etc.) Use of the chain reaction method (PCR) is mentioned.

  In the present invention, 74, 75, 76, 142, 168, 169, 170, 171, 224, 230, 230, 236, 244, 245 of PQQGDH shown in SEQ ID NO: 1 Focusing on positions 258, 342, 344, 345, 416, 429, and 430, a library in which mutations were randomly introduced into these amino acid sites using the above mutation introduction kit was prepared, and the substrate When the change in specificity was used as an indicator, a PQQGDH variant with improved substrate specificity could be obtained. With regard to substrate specificity, (T224A + A236T), (Q168A + T224A + A236T + M342I), (Q168A + 171A + T224A + A236T + M342I), (Q168A + 171S + T224A + A236T + M342I), (M342I + 430P), (E245D + M342I + 430P), (Q168A + L169P + A170M + E245D + M342I + 430P), (Q168A + 169F + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P), (Q168A + 169Y + L169P + A 70M + E245D + M342I + N429E + 430P) multiple mutant are particularly preferred.

The modified PQQGDH protein of the present invention can take various forms such as liquid (aqueous solution, suspension, etc.), powder, and lyophilized. The lyophilization method is not particularly limited and may be performed according to a conventional method. The composition containing the enzyme of the present invention is not limited to a lyophilized product, and may be in a solution state in which the lyophilized product is redissolved.
The modified PQQGDH protein of the present invention can be used as a glucose measurement composition. The modified PQQGDH protein or glucose measurement composition of the present invention can be used as a glucose assay kit or a glucose sensor when measuring glucose. The glucose measurement composition of the present invention may be in a purified state or may contain various additives depending on the form and method of use.
For example, the purified modified protein can be stabilized by the following method.

  (1) One or more compounds selected from the group consisting of (1) aspartic acid, glutamic acid, α-ketoglutaric acid, malic acid, α-ketogluconic acid, α-cyclodextrin, and salts thereof And (2) By coexisting albumin, the modified protein can be further stabilized.

  In the lyophilized composition, the PQQGDH content varies depending on the origin of the enzyme, but is usually suitably used in the range of about 5 to 50% (weight ratio). When converted into enzyme activity, it is preferably used in the range of 100 to 2000 U / mg.

  Examples of salts of aspartic acid, glutamic acid, α-ketoglutaric acid, malic acid, and α-ketogluconic acid include salts of sodium, potassium, ammonium, calcium, magnesium, and the like, but are not particularly limited. The addition amount of the above compound, its salt and α-cyclodextrin is preferably in the range of 1 to 90% (weight ratio). These substances may be used alone or in combination.

  The buffer solution to be contained is not particularly limited, and examples thereof include Tris buffer solution, phosphate buffer solution, borate buffer solution, and GOOD buffer solution. The pH of the buffer is adjusted in the range of about 5.0 to 9.0 according to the purpose of use. The content of the buffering agent in the lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.1 to 30% (weight ratio). used.

  Examples of albumin that can be used include bovine serum albumin (BSA) and ovalbumin (OVA). BSA is particularly preferable. The content of the albumin is preferably 1 to 80% (weight ratio), more preferably 5 to 70% (weight ratio).

  Further, other stabilizers and the like may be added to the composition within a range that does not particularly adversely affect the reaction of PQQGDH. The blending method of the stabilizer of the present invention is not particularly limited. For example, a method of blending a stabilizer in a buffer solution containing PQQGDH, a method of blending PQQGDH in a buffer solution containing a stabilizer, or a method of blending PQQGDH and a stabilizer in a buffer solution at the same time can be mentioned.

Moreover, the stabilization effect is acquired even if calcium ion is added. That is, the modified protein can be stabilized by containing calcium ions or calcium salts. Examples of calcium salts include calcium chloride, inorganic acid such as calcium acetate or calcium citrate, or calcium salt of organic acid. In the aqueous composition, the content of calcium ions is preferably 1 × 10 ⁻⁴ to 1 × 10 ⁻² M.

  The stabilizing effect by containing calcium ions or calcium salts is further improved by containing an amino acid selected from the group consisting of glutamic acid, glutamine and lysine.

  One or more amino acids selected from the group consisting of glutamic acid, glutamine and lysine may be used. In the aqueous composition, the content of an amino acid selected from the group consisting of glutamic acid, glutamine and lysine is preferably 0.01 to 0.2% by weight.

  Further, serum albumin may be contained. When serum albumin is added to the aqueous composition, the content is preferably 0.05 to 0.5% by weight.

  As a buffering agent, a normal thing is used, and what makes pH of a composition 5-10 normally is preferable. Specifically, Tris-HCl, boric acid, and Good's buffer are used, but any buffer that does not form an insoluble salt with calcium can be used.

  If necessary, other components such as surfactants, stabilizers and excipients may be added to the aqueous composition.

  The composition for measuring glucose of the present invention may be configured so as not to contain a protein component other than the host-derived protein component (for example, a biological substance such as BSA).

  In this case, the composition consists essentially of modified PQQGDH and calcium or a calcium salt, and a buffer. Moreover, after containing these, an amino acid or an organic acid may be further added. Moreover, if it contains these, an aqueous composition and a lyophilized material will not ask | require.

  Examples of the supply form of calcium include calcium salts of inorganic acids or organic acids such as calcium chloride, calcium acetate, and calcium citrate. In the powder composition, the calcium content (W / W) is preferably 0.05% to 5%.

Any buffering agent that is generally used may be used, and usually the buffering agent having a pH of 5 to 10 is preferable. However, those that form an insoluble salt with calcium such as a phosphate buffer are not preferred. Those having an amino group end such as trisaminomethane are not preferable because the PQQ bond of PQQ-dependent glucose dehydrogenase is unstable. For this reason, more preferably as a buffering agent, a buffering agent such as boric acid or acetic acid, BES, Bicine, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TES Good buffer such as Tricine.
In the powder composition, the content (W / W) of the buffering agent is preferably 1.0% to 50%.

In the present invention, glucose can be measured by the following various methods.
Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified PQQGDH according to the present invention. The glucose assay kit of the present invention comprises a modified PQQGDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified PQQGDH of the present invention, buffers necessary for the assay, mediators, a glucose standard solution for creating a calibration curve, and directions for use. The modified PQQGDH according to the present invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution. Preferably, the modified PQQGDH of the present invention is provided in the form of a holo, but can be provided in the form of an apoenzyme and can be holoed at the time of use.

Glucose Sensor The present invention also features a glucose sensor that uses a modified PQQGDH according to the present invention. As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination thereof may be used. Preferably, the modified PQQGDH of the present invention is immobilized on the electrode in a holo form, but may be immobilized in the form of an apoenzyme and the PQQ provided as a separate layer or in solution. Typically, the modified PQQGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde.

The glucose concentration can be measured as follows. The buffer is placed in a thermostatic cell, and PQQ and CaCl ₂ and mediator are added to maintain a constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. As the working electrode, an electrode on which the modified PQQGDH of the present invention is immobilized is used, and a counter electrode (for example, platinum electrode) and a reference electrode (for example, Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

Hereinafter, the present invention will be described in more detail based on examples.
Example 1: Construction of PQQ-dependent glucose dehydrogenase gene expression plasmid The wild-type PQQ-dependent glucose dehydrogenase expression plasmid pNPG5 was transformed into the multiple cloning site of the vector pBluescript SK (-) at Acinetobacter baumannii. A structural gene encoding a PQQ-dependent glucose dehydrogenase derived from NCIMB11517 strain is inserted. The base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of PQQ-dependent glucose dehydrogenase deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.

Example 2: Preparation of mutant PQQ-dependent glucose dehydrogenase A recombinant plasmid pNPG5 containing a wild-type PQQ-dependent glucose dehydrogenase gene and a synthetic oligo of about 40 mer containing a triplet encoding the amino acid at the mutation introduction site in the center Based on the nucleotide, mutation treatment operation was performed according to the protocol using QuickChange ^™ Site-Directed Mutagenesis Kit (manufactured by STRATAGENE), and 74, 75, 76, 142, 168, 169, 170, Create a multiple mutation library with mutations randomly introduced at positions 171, 224, 230, 236, 244, 245, 258, 342, 344, 345, 416, 429 and 430 did. Then, the nucleotide sequence of the candidate strain obtained by screening using the change in substrate specificity as an index is determined, and the 224th threonine of the amino acid sequence described in SEQ ID NO: 1 is replaced with alanine and the 236th alanine is replaced with threonine. A recombinant plasmid (pNPG5-T224A + A236T) encoding the mutant PQQ-dependent glucose dehydrogenase was obtained.
Next, based on pNPG5-T224A + A236T, using synthetic oligonucleotides of other non-mutated sites, library preparation and screening using substrate specificity as an index are performed in the same manner as described above. Recombinant plasmids encoding mutant PQQ-dependent glucose dehydrogenases with improved substrate specificity (pNPG5-Q168A + T224A + A236T + M342I, pNPG5-Q168A + 171A + T224A + A236T + M342I, pNPG5-Q168A + 171S + T236M + I236T were obtained).
Others As described above, library preparation and screening were performed, and recombinant plasmids (pNPG5-M342I + 430P, pNPG5-E245D + M342I + 430P, pNPG5- Q168A + L169P + A170M + E245D + M342I + 430P, pNPG5-Q168A + 169F + L169P + A170L + E245D + M342I + 430P, pNPG5-Q168A + 169Y + L169P + A170L + E245D + M342I + 430P, pNPG5-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P, pNPG5-Q168A + 169Y + L169P + A170M + E 45D + M342I + N429E + 430P) acquires, Escherichia coli competent cells each recombinant plasmid (Escherichia coli JM109; manufactured by Toyobo) was transformed, were obtained transformant, respectively.

Example 3: Construction of an expression vector capable of replicating in Pseudomonas bacteria 5 μg of the recombinant plasmid pNPG5-T224A + A236T DNA obtained in Example 2 was digested with restriction enzymes BamHI and XhoI (manufactured by Toyobo Co., Ltd.), and mutant PQQ-dependent glucose The structural gene part of dehydrogenase was isolated. The isolated DNA was reacted with pTM33 (1 μg) cleaved with BamHI and XhoI with 1 unit of T4 DNA ligase at 16 ° C. for 16 hours to ligate the DNA. Ligated DNA was transformed with Escherichia coli DH5α competent cells. The obtained expression plasmid was designated as pNPG6-T224A + A236T.
pNPG5-Q168A + T224A + A236T + M342I, pNPG5-Q168A + 171A + T224A + A236T + M342I, pNPG5-Q168A + 171S + T224A + A236T + M342I, pNPG5-M342I + 430P, pNPG5-E245D + M342I + 430P, pNPG5-Q168A + L169P + A170M + E245D + M342I + 430P, pNPG5-Q168A + 169F + L169P + A170L + E245D + M342I + 430P, pNPG5-Q168A + 169Y + L169P + A170L + E245D + M342I + 430P, pNPG5-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P, pNPG5-Q1 8A + 169Y + L169P + A170M + E245D + M342I + N429E + for 430P each recombinant plasmid was also acquire the expression plasmid in the same manner as the above-mentioned method. The resulting expression plasmid, respectively pNPG6-Q168A + T224A + A236T + M342I, pNPG6-Q168A + 171A + T224A + A236T + M342I, pNPG6-Q168A + 171S + T224A + A236T + M342I, pNPG6-M342I + 430P, pNPG6-E245D + M342I + 430P, pNPG6-Q168A + L169P + A170M + E245D + M342I + 430P, pNPG6-Q168A + 169F + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429 + 430P, was named pNPG6-Q168A + 169Y + L169P + A170M + E245D + M342I + N429E + 430P.

Example 4: Preparation of Pseudomonas genus transformant Pseudomonas putida TE3493 (Microtechnology 12298) was cultured in LBG medium (LB medium + 0.3% glycerol) at 30 ° C. for 16 hours and centrifuged (12 The bacterial cells were collected by 2,000 rpm for 10 minutes, and 8 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells to suspend the bacterial cells. The bacterial cells were collected again by centrifugation (12,000 rpm, 10 minutes), and 0.4 ml of 5 mM K-phosphate buffer (pH 7.0) containing 300 mM sucrose ice-cooled was added to the bacterial cells. Suspended.
0.5 μg of the expression plasmid pNPG6-T224A + A236T obtained in Example 3 was added to the suspension and transformed by electroporation. A target transformant was obtained from a colony grown on an LB agar medium containing 100 μg / ml streptomycin.
pNPG6-Q168A + T224A + A236T + M342I, pNPG6-Q168A + 171A + T224A + A236T + M342I, pNPG6-Q168A + 171S + T224A + A236T + M342I, pNPG6-M342I + 430P, pNPG6-E245D + M342I + 430P, pNPG6-Q168A + L169P + A170M + E245D + M342I + 430P, pNPG6-Q168A + 169F + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P, pNPG6-Q1 8A + 169Y + L169P + A170M + E245D + M342I + N429E + 430P as the same way as the above also for each expression plasmid were obtained transformant, respectively.

Test example 1
Method for measuring GDH activity Measurement principle D-glucose + PMS + PQQGDH → D-glucono-1,5-lactone + PMS (red)
The presence of diformazan formed by the reduction of nitrotetrazolium blue (NTB) by 2PMS (red) + NTB → 2PMS + diformazan phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 570 nm.
Definition of Unit One unit refers to the amount of PQQGDH enzyme that forms 0.5 mmol of diformazan per minute under the conditions described below.
(3) Method reagents A. D-glucose solution: 0.5 M (0.9 g D-glucose (molecular weight 180.16) / 10 ml H ₂ O)
B. PIPES-NaOH buffer, pH 6.5: 50 mM (1.51 g of PIPES (molecular weight 302.36) suspended in 60 mL of water is dissolved in 5N NaOH and 2.2 ml of 10% Triton X-100 is added. The pH was adjusted to 6.5 ± 0.05 at 25 ° C. using 5N NaOH, and water was added to make 100 ml.)
C. PMS solution: 3.0 mM (9.19 mg phenazine methosulfate (molecular weight 817.65) / 10 ml H ₂ O)
D. NTB solution: 6.6 mM (53.96 mg of nitrotetrazolium blue (molecular weight 817.65) / 10 ml H ₂ O)
E. Enzyme dilution: 50 mM PIPES-NaOH buffer (pH 6.5) containing 1 mM CaCl ₂ , 0.1% Triton X-100, 0.1% BSA
Procedure Prepare the following reaction mixture in a light-proof bottle and store on ice (prepared at the time of use)
1.8 ml D-glucose solution (A)
24.6 ml PIPES-NaOH buffer (pH 6.5) (B)
2.0ml PMS solution (C)
1.0ml NTB solution (D)

The concentrations in the assay mixture are as follows:
PIPES buffer 42 mM
D-glucose 30 mM
PMS 0.20 mM
NTB 0.22mM

3.0 ml of the reaction mixture was placed in a test tube (made of plastic) and preheated at 37 ° C. for 5 minutes.
0.1 ml enzyme solution was added and mixed by gentle inversion.
The increase in absorbance for water at 570 nm was recorded for 4-5 minutes on a spectrophotometer while maintaining at 37 ° C., and ΔOD per minute from the initial linear portion of the curve was calculated (OD test).
At the same time, the same method was repeated except that the enzyme diluent (E) was added instead of the enzyme solution, and a blank (ΔOD blank) was measured.
Immediately before the assay, the enzyme powder was dissolved in an ice-cold enzyme diluent (E) and diluted to 0.1-0.8 U / ml with the same buffer (use of a plastic tube for adhesion of the enzyme) Is preferred).
Calculation Activity is calculated using the following formula:
U / ml = {ΔOD / min (ΔOD test−ΔOD blank) × Vt × df} / (20.1 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.1 ml)
Vs: Sample volume (1.0 ml)
20.1: 1/2 mmol molecular extinction coefficient of diformazan 1.0: optical path length (cm)
df: Dilution factor C: Enzyme concentration in solution (c mg / ml)

Example 5: Preparation of holo-type purified expression enzyme 500 ml of terrific broth was dispensed into a 2 L Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then separately sterile filtered streptomycin to 100 μg / ml. Added. The medium was inoculated with 5 ml of Pseudomonas putida TE3493 (pNPG6-T224A + A236T) previously cultured in PY medium containing 100 μg / ml streptomycin at 30 ° C. for 24 hours, and cultured at 30 ° C. for 40 hours with aeration and agitation. The PQQ-dependent glucose dehydrogenase activity at the end of the culture was about 30 U / ml per 1 ml of the culture solution in the activity measurement.
The cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.0), disrupted by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. . The obtained crude enzyme solution was separated and purified by HiTrap-SP (Amersham-Pharmacia) ion exchange column chromatography. Next, after dialyzing with 10 mM PIPES-NaOH buffer (pH 6.5), calcium chloride was added to a final concentration of 1 mM. Finally, it was separated and purified by HiTrap-DEAE (Amersham-Pharmacia) ion exchange column chromatography to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band by SDS-PAGE.
pNPG6-Q168A + T224A + A236T + M342I, pNPG6-Q168A + 171A + T224A + A236T + M342I, pNPG6-Q168A + 171S + T224A + A236T + M342I, pNPG6-M342I + 430P, pNPG6-E245D + M342I + 430P, pNPG6-Q168A + L169P + A170M + E245D + M342I + 430P, pNPG6-Q168A + 169F + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + 430P, pNPG6-Q168A + 169Y + L169P + A170L + E245D + M342I + N429D + 430P, pNPG6-Q1 8A + 169Y + L169P + A170M + E245D + M342I + N429E + also Pseudomonas putida TE3493 transformants by 430P obtained the purified enzyme preparation in the same manner as the above-mentioned method.
The performance was evaluated using the purified enzyme thus obtained.

Substrate specificity The activity of PQQGDH was measured according to the above activity measurement method. The dehydrogenase activity value when glucose is used as the substrate solution and the dehydrogenase activity value when maltose is used as the substrate solution are measured, and the relative value is obtained when the measured value when glucose is used as the substrate is 100. It was. For dehydrogenase activity when maltose was used as a substrate solution, a 0.5 M maltose solution was prepared and used for activity measurement. The results are shown in Table 1.
In Table 1, “T224A” in the left column indicates that T at position 224 is replaced with A. In addition, “171A” in the left column indicates that A is inserted after 171st place. In “M342I + 430P”, multiple mutants are represented by connecting those represented by the same principle as described above with a + symbol.
The maltose activity is that the activity value of these multiple mutants PQQGDH with glucose as a substrate is 100% and the activity value with respect to maltose is lower as the ratio (%) is lower than the activity value when maltose is used as a substrate. Was evaluated as having decreased.
In addition, the thermal stability of these multiple mutants PQQGDH is determined by measuring the residual activity (%) after treatment of the bacterial cell disruption solution at 45 ° C. for 1 hour, and comparing it with wild type PQQGDH. evaluated.

  In the wild type PQQGDH, the reactivity of glucose and maltose is almost equal, whereas in the modified PQQGDH of the present invention, the reactivity of maltose is lowered.

  According to the present invention, PQQGDH having improved substrate specificity can be obtained. This modified PQQGDH can be used for glucose assay kits and glucose sensors.

Claims (16)

  Amino acid substitutions, the amino acid sequence of the Acinetobacter genus PQQGDH, (T224A + A236T), (Q168A + T224A + A236T + M342I), (Q168A + 171A + T224A + A236T + M342I), (Q168A + 171S + T224A + A236T + M342I), (M342I + 430P), (E245D + M342I + 430P), (Q168A + L169P + A170M + E245D + M342I + 430P), (Q168A + 169F + L169P + A170L + E245D + M342I + 430P), (Q168A + 169Y + L169P + A170L + E245D + M342I + 430P), ( Q168A + 169Y + L169P + A170L + E245D + M342I + 429D + 430P), (Q168A + 169Y + L169P + A170M + E245D + M342I + N429E + 430P), or modified PQQG with reduced activity on disaccharides compared to wild-type PQQGDH having amino acid mutations at positions equivalent to those in other species .   Modified PQQGDH, in which stability is reduced by substituting a charged amino acid before and after P in modified PQQGDH whose stability is reduced by substituting a certain amino acid for P or by inserting P at a certain site.   The modified PQQGDH according to claim 2, wherein substitution or insertion of P is around position 430. The modified PQQGDH according to claim 2 or 3, wherein the charged amino acid is D or E.   The modified PQQGDH according to claim 1, wherein the disaccharide is maltose.   A gene encoding the modified PQQGDH according to any one of claims 1 to 5.   A vector comprising the gene according to claim 6.   A transformant transformed with the vector according to claim 7.   A method for producing a modified PQQGDH, wherein the transformant according to claim 8 is cultured.   A composition for measuring glucose comprising the modified PQQGDH according to any one of claims 1 to 5.   A glucose assay kit comprising the modified PQQGDH according to any one of claims 1 to 5.   A glucose sensor comprising the modified PQQGDH according to any one of claims 1 to 5.   A glucose measurement method comprising the modified PQQGDH according to any one of claims 1 to 5.   A method for reducing the activity of PQQGDH on disaccharides, wherein the amino acid mutation according to any one of claims 1 to 5 is performed on PQQGDH.   A glucose measurement system using PQQGDH, comprising the PQQGLD in which the amino acid mutation according to any one of claims 1 to 5 is performed, and a method for improving measurement accuracy in the glucose measurement system.   A glucose measurement composition using PQQGDH, comprising the PQQGLD subjected to the amino acid mutation according to any one of claims 1 to 5, wherein the composition for glucose measurement with improved measurement accuracy is produced. Method.