JP2007000020A - Modified pyrroloquinoline quinone (pqq)-dependent glucose dehydrogenase having excellent substrate specificity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a modified PQQGDH (pyrroloquinoline quinone-dependent glucose dehydrogenase) improved in substrate specificity. <P>SOLUTION: The modified PQQGDH has a reduced activity to disaccharides (maltose) as compared with that of a wild type PQQGDH through modifying genes of a PQQGDH (pyrroloquinoline quinone-dependent glucose dehydrogenase) derived from Acinetobacter. The PQQGDH is expected to be utilized to accurate measurement of blood glucose. <P>COPYRIGHT: (C)2007,JPO&INPIT

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 intensive studies to solve the above problems, the present inventors have been able to 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
Item 1.
Modified PQQGDH, which has a pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQGDH), which has at least one of the following mutations and is less active against disaccharides than wild-type PQQGDH.
(1) 125, 128, 142, 168, 169, 170, 224, 230, 236, 345, 351, 416, and 428 in the amino acid sequence of PQQGDH derived from Acinetobacter (2) Aminobacterium-derived PQQGDH amino acid sequence at position 74, 75, 76, 126, 127, 129, at least one position, or at an equivalent position in other species as described above, Item 1. It has an amino acid insertion at at least one position at positions 130, 131, 132, 168, 169, 170, 171 and 344, or equivalent positions in other species.
Item 2. The modified PQQGDH according to Item 1, wherein the disaccharide is maltose.
Item 3.
Item 3. A gene encoding the modified PQQGDH according to Item 1 or 2.
Item 4.
A vector comprising the gene according to Item 3.
Item 5.
Item 5. A transformant transformed with the vector according to Item 4.
Item 6.
A method for producing a modified PQQGDH, wherein the transformant according to Item 5 is cultured.
Item 7.
Item 3. A glucose measurement composition comprising the modified PQQGDH according to Item 1 or 2.
Item 8.
Item 3. A glucose assay kit comprising the modified PQQGDH according to item 1 or 2.
Item 9.
Item 3. A glucose sensor comprising the modified PQQGDH of item 1 or 2.
Item 10.
Item 3. A glucose measurement method comprising the modified PQQGDH according to Item 1 or 2.
Item 11.
3. A method for reducing the activity of PQQGDH on disaccharides, wherein the amino acid mutation of Item 1 or 2 is performed on PQQGDH.
Item 12.
A method for producing PQQGDH, wherein the action of PQQGDH on disaccharides is reduced, wherein the amino acid mutation of Item 1 or 2 is performed on PQQGDH.
Item 13.
A method for improving the accuracy of measurement in a glucose measurement system, characterized in that the glucose measurement system using PQQGDH contains PQQGLD in which the amino acid mutation of Item 1 or 2 has been performed.
Item 14.
A method for producing a glucose measurement composition with improved measurement accuracy, characterized by containing PQQGLD in which the amino acid mutation according to Item 1 or 2 is contained in a glucose measurement system using PQQGDH.

  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 for a glucose assay kit and a glucose sensor, it becomes possible to perform a more accurate analysis than that using a wild type PQQGDH, or a glucose assay kit and a 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. .

  By using a mediator different from our previous invention, the present invention has found that there are mutations that are effective in improving substrate specificity even in combinations of mutations different from the previous invention. Invented. In other words, this is the first example where it has been found that the substrate characteristics of PQQGDH may change depending on the mediator used in activity measurement.

  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.

The modified PQQGDH of the present invention, which has reduced activity on disaccharides compared to wild-type PQQGDH, includes, for example, amino acid substitutions in the amino acid sequence of the genus Acinetobacter genus PQQGDH, T125R, T125V, T125L, T125G, T125C, T125P, T125H, T125S, T125K, T125A, T125E, F128C, F128E, F128G, F128H, F128I, F128K, F128L, F128P, F128S, F128T, F128V, F128W, F128Y, F128Q, F128M, F128N, F128D, K142Q, K142N, K142H, 142 K142E, K142T, K142L, K142S, K142V, K142P, K142F, K142D, K14 Y, K142I, K142R, K142M, Q168M, Q168K, Q168S, Q168T, Q168C, Q168G, L169A, L169V, L169F, L169M, L169K, L169R, L169S, L169T, L169H, L169C, L169L, L169C, L169H, L169C, L169H A170F, A170P, A170I, A170L, A170E, A170K, A170R, A170S, A170T, A170Y, A170H, A170C, A170N, A170W, A170G, T224A, T224C, T224S, T224A, P230H, P230I, P230M, P230S, P230M, P230P A236C, A236E, A236S, A236V, A236T, I345Y, 345E, I345I, A351T, A351S, E416A, E416A, E416D, E416F, E416G, E416H, E416I, E416L, E416M, E416N, E416P, E416Q, E416R, E416T, E416V, E416T, E416V, E416T Insertion is 74A, 74D, 74E, 74G, 74L, 74N, 74Q, 74R, 74S, 74T, 74V, 74W, 74C, 74I, 74M, 74K, 74Y, 75F, 75L, 75S, 75T, 75V, 75R, 75W 75C, 75G, 75A, 75M, 75N, 75Q, 75D, 75K, 75P, 76G, 76H, 76I, 76L, 76S, 76V, 76W, 76Y, 76Q, 76E, 76D, 76C, 7 6K, 76T, 76A, 76F, 76L, 76P, 126V, 126S, 126L, 126F, 126G, 126P, 126Q, 126C, 126R, 126S, 126D, 126A, 126I, 127Y, 127G, 127V, 127E, 127I, 127N, 127P, 127A, 127C, 127S, 127M, 127D, 127T, 127H, 127K, 127Q, 129N, 129Y, 129E, 129V, 129H, 129T, 129F, 129R, 129L, 129I, 129S, 129W, 129K, 129D, 130F, 130T, 130D, 130R, 130V, 130S, 130I, 130C, 130A, 130P, 130Y, 130N, 130G, 130E, 130K, 130L, 131D, 131A, 131V, 31C, 131P, 131H, 131S, 131F, 131G, 131W, 131R, 131M, 131T, 131I, 132V, 132P, 132L, 132I, 132K, 132E, 168A, 168V, 168M, 168I, 168L, 168T, 168Y, 168H, 168C, 168Q, 168W, 168G, 169A, 169C, 169E, 169G, 169I, 169K, 169L, 169M, 169N, 169R, 169S, 169T, 169V, 169W, 170A, 170C, 170D, 170E, 170F, 170G, 170H, 170I, 170L, 170N, 170P, 170Q, 170R, 170S, 170T, 170V, 170W, 170Y, 171A, 171C, 171D, 171E, 171F, 171 , 171I, 171K, 171L, 171M, 171N, 171P, 171Q, 171R, 171S, 171T, 171V, 171Y, 344P, 344V, 344Q, 344W, 344L, 344F, 344S, 344I, 344H, 344T, 344A, 344N, 344K , and those applied alone 344Y, as these substitutions, insertions multiple mutant mutations are combined, Q168A + L169P + E245D + M342I + A351T, Q168A + L169P + E245D + M342I + A351T + 430P, Q168A + L169P + A170M + E245D + M342I + 345Y, Q168A + L169P + A170M + E245D + M342I + 345W, Q168A + L169P + A170M + E245D + M342I + A351T, Q168A + L169P + A170M + E245D + M342I + A351S, Q168A + L169P + A170M + E245D + M342I + 429G, Q168A + L169P + A170M + E245D + M342I + G428V, Q168A + L169P + A170M + E245D + M342I + A351T + 430P, Q168A + L169P + A170M + E245D + M342I + K142T + 344T, Q168A + L169P + A170M + E245D + M342I + 74X 1 (X 1 = A, D, E, G, L, N, Q, R, S, T, V, W, C, I, M, K, Y), Q168A + L169P + A170M + E245D + M342I + 75X 2 (X 2 = F, L, S, T, V, R, W, C, G, A, M, N Q, D, K, P) , Q168A + L169P + A170M + E245D + M342I + 76X 3 (X 3 = G, H, I, L, S, V, W, Y, Q, E, D, C, K, T, A, F, L, P _{), Q168A + L169P + A170M +} E245D + M342I + T125X 4 (X 4 = R, V, L, G, C, P, H, S, K, A, E), Q168A + L169P + A170M + E245D + M342I + 126X 5 (X 5 = V, S, L, F, G, P, Q , C, R, S, D , A, I), Q168A + L169P + A170M + E245D + M342I + 127X 6 (X 6 = Y, G, V, E, I, N, P, A, C, S, M, D, T, H, K, Q), Q168A + L169P + A170M + E245D + M342I + F128X 7 (X 7 = C E, G, H, I, K, L, P, S, T, V, W, Y, Q, M, N, D), Q168A + L169P + A170M + E245D + M342I + 129X 8 (X 8 = N, Y, E, V, H, T , F, R, L, I, S, W, K, D), Q168A + L169P + A170M + E245D + M342I + 130X ₉ (X ₉ = F, T, D, R, V, S, I, C, A, P, Y, N, G, _{E, K, L), Q168A} + L169P + A170M + E245D + M342I + 131X 10 (X 10 = D, A, V, C, P, H, S, F, G, W, R, M, T, I), Q168A + L169P + A170M + E245D + M342I + 132X 11 (X 11 = V, P, L, I, K, E), Q168A + L169P + A170M + E245D + M342I + _{142X 12 (X 12 = Q,} N, H, W, E, T, L, S, V, P, F, D, Y, I, R, M), Q168A + L169P + A170M + E245D + M342I + T224X 13 (X 13 = A, C, S _{), Q168A + L169P + A170M +} E245D + M342I + P230X 14 (X 14 = H, I, M, S, T, V), Q168A + L169P + A170M + E245D + M342I + A236X 15 (X 15 = C, E, S, V, T), Q168A + L169P + A170M + E245D + M342I + 344X 16 (X 16 = P, V, Q, W, L, F, S, I, H, T, A, N, K, Y), Q168A + L169P + A170M + E245D + M342I + I345X 17 (X 17 = Y, E, I), Q168 _{+ L169P + A170M + E245D + M342I} + E416X 18 (X 18 = A, C, D, F, G, H, I, L, M, N, P, Q, R, T, V, S, Y, K, W), L169P + A170M + E245D + M342I + Q168X 19 (X 19 _{= M, K, S, T} , C, G), Q168A + A170M + E245D + M342I + L169X 20 (X 20 = A, V, F, M, K, R, S, T, H, C, N, Q, W, G), Q168A + L169P + E245D + M342I + A170X ₂₁ (X ₂₁ = V, F, P, I, L, E, K, R, S, T, Y, H, C, N, W, G), Q168A + L169P + A170M + E245D + M342I + 168X ₂₂ (X ₂₂ = A, V, M, I, L, T, Y, H, C, Q, W, G), Q _{68A + L169P + A170M + E245D +} M342I + 169X 23 (X 23 = A, C, E, G, I, K, L, M, N, R, S, T, V, W), Q168A + L169P + A170M + E245D + M342I + 170X 24 (X 24 = A, C, D, E, F , G, H, I, L , N, P, Q, R, S, T, V, W, Y), Q168A + L169P + A170M + E245D + M342I + 171X 25 (X 25 = A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, Y), or any of the wild type having any of the amino acid mutations at equivalent positions in other species Examples include modified PQQGDH, which has a lower activity on disaccharides than PQQGDH.

  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 170th position. 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 the sake of convenience, the numbers dealt with here correspond to positions on the amino acid sequence of 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.

  Multiple mutants with any combination of substitutions or insertions shown in the next paragraph contribute to improving the substrate specificity of PQQGDH. The combinations of multiple mutants obtained in the present invention are shown below.

Q168A + L169P + E245D + M342I + A351T, Q168A + L169P + E245D + M342I + A351T + 430P, Q168A + L169P + A170M + E245D + M342I + 345Y, Q168A + L169P + A170M + E245D + M342I + 345W, Q168A + L169P + A170M + E245D + M342I + A351T, Q168A + L169P + A170M + E245D + M342I + A351S, Q168A + L169P + A170M + E245D + M342I + 429G, Q168A + L169P + A170M + E245D + M342I + G428V, Q168A + L169P + A170M + E245D + M342I + A351T + 430P, Q168A + L169P + A1 _{0M + E245D + M342I + K142T +} 344T, Q168A + L169P + A170M + E245D + M342I + 74X 1 (X 1 = A, D, E, G, L, N, Q, R, S, T, V, W, C, I, M, K, Y), Q168A + L169P + A170M + E245D + M342I + 75X 2 (X 2 = F , L, S, T, V, R, W, C, G, A, M, N, Q, D, K, P), Q168A + L169P + A170M + E245D + M342I + 76X ₃ (X ₃ = G, H, I, L, S, V, W, Y, Q, E, D, C, K, T, A, F, L, P), Q168A + L169P + A170M + E245D + M342I + T125X ₄ (X ₄ = R, V, L, G, C, P, H, S, K, A E), Q168A + L169P + A170M + E _{45D + M342I + 126X 5 (X} 5 = V, S, L, F, G, P, Q, C, R, S, D, A, I), Q168A + L169P + A170M + E245D + M342I + 127X 6 (X 6 = Y, G, V, E, I, N , P, A, C, S, M, D, T, H, K, Q), Q168A + L169P + A170M + E245D + M342I + F128X ₇ (X ₇ = C, E, G, H, I, K, L, P, S, T, V, _{W, Y, Q, M,} N, D), Q168A + L169P + A170M + E245D + M342I + 129X 8 (X 8 = N, Y, E, V, H, T, F, R, L, I, S, W, K, D), Q168A + L169P + A170M + E245D + M342I + 130X 9 (X ₉ = F, T, D, R, V, S, I, C, A, P, Y, N, G, _{E, K, L), Q168A} + L169P + A170M + E245D + M342I + 131X 10 (X 10 = D, A, V, C, P, H, S, F, G, W, R, M, T, I), Q168A + L169P + A170M + E245D + M342I + 132X 11 (X 11 = V, P, L, I, K, E), Q168A + L169P + A170M + E245D + M342I + K142X 12 (X 12 = Q, N, H, W, E, T, L, S, V, P, F, D, Y, I, R, M), _{Q168A + L169P + A170M + E245D +} M342I + T224X 13 (X 13 = A, C, S), Q168A + L169P + A170M + E245D + M342I + P230X 14 (X 14 = H, I, M, S, T, V), Q168A + L169P + A170 _{+ E245D + M342I + A236X 15 (} X 15 = C, E, S, V, T), Q168A + L169P + A170M + E245D + M342I + 344X 16 (X 16 = P, V, Q, W, L, F, S, I, H, T, A, N, K, Y _{), Q168A + L169P + A170M +} E245D + M342I + I345X 17 (X 17 = Y, E, I), Q168A + L169P + A170M + E245D + M342I + E416X 18 (X 18 = A, C, D, F, G, H, I, L, M, N, P, Q, R, T, V _{, S, Y, K, W} ), L169P + A170M + E245D + M342I + Q168X 19 (X 19 = M, K, S, T, C, G), Q168A + A170M + E245D + M342I + L169X 20 (X 20 = A, V, F, M, K, R, S, T, H, C, N, Q, W, G), Q168A + L169P + E245D + M342I + A170X ₂₁ (X ₂₁ = V, F, P, I, L, E, K, R, S, T _{, Y, H, C, N} , W, G), Q168A + L169P + A170M + E245D + M342I + 168X 22 (X 22 = A, V, M, I, L, T, Y, H, C, Q, W, G), Q168A + L169P + A170M + E245D + M342I + 169X 23 (X 23 = A, C, E, G , I, K, L, M, N, R, S, T, V, W), Q168A + L169P + A170M + E245D + M342I + 170X 24 (X 24 = A, C, D, E, F, G, H, I, L, N, P, Q, R, S, T, V, W, Y), Q168A + L169P + A170M + E2 _{45D + M342I + 171X 25 (X} 25 = A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, Y)

  The present invention is also a method for reducing the activity of PQQGDH on disaccharides, characterized by performing the above-mentioned amino acid mutation in PQQGDH.

  The present invention also relates to a glucose measurement system using PQQGDH, which comprises the PQQGLD in which the amino acid mutation according to any one of claims 1 to 5 is performed. It is a way to improve.

  The invention of the present application also comprises a composition for measuring glucose with improved measurement accuracy, characterized by containing PQQGLD having the amino acid mutation according to any one of claims 1 to 5 in a glucose measuring 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. In this specification, Acinetobacter baumannii NCIMB 11517 and Acinetobacter calcoaceticus NCIMB 11517 are synonymous.

  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 or inserted.
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 an amino acid involved in calcium ion binding and / or an amino acid around it is substituted or inserted.
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 in the vicinity of an amino acid 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, one obtained by introducing a mutation in the vicinity of an amino acid located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme is 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, one obtained by introducing a mutation in the vicinity of an amino acid located within a radius of 10 mm from the substrate in the active three-dimensional structure of the wild-type enzyme is 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 produced 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 the host microorganism that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, Escherichia coli DH5α, and the like. 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 baumannii 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.

  For example, a culture solution obtained by stirring and culturing the gene-donating microorganism for 1 to 3 days 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-mentioned vector with the restriction enzyme used for cleaving 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 may be 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 grow, 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, positions 125, 128, 142, 168, 169, 170, 224, 230, 236, 345, 351, 416, 428 and PQQGDH shown in SEQ ID NO: 1 Amino acid substitution at at least one of the positions, or 74, 75, 76, 126, 127, 129, 130, 131, 132, 168, 169, 170, 171, Focusing on the insertion of amino acids at at least one position of positions 344 and 344, a library in which mutations are randomly introduced into these amino acid sites using the above-described mutation introduction kit is prepared, and screening is performed using changes in substrate specificity as an index. As a result, a PQQGDH variant with improved substrate specificity could be obtained. The substrate specificity sex Nikanshite teeth, Q168A + L169P + E245D + M342I + A351T, Q168A + L169P + E245D + M342I + A351T + 430P, Q168A + L169P + A170M + E245D + M342I + 345Y, Q168A + L169P + A170M + E245D + M342I + 345W, Q168A + L169P + A170M + E245D + M342I + A351T, Q168A + L169P + A170M + E245D + M342I + A351S, Q168A + L169P + A170M + E245D + M342I + 429G, Q168A + L169P + A170M + E245D + M342I + G428V, Q168A + L169P + A170M + E245D + M342I + A351T + 430P, Q16 _{A + L169P + A170M + E245D +} M342I + K142T + 344T, Q168A + L169P + A170M + E245D + M342I + 74X 1 (X 1 = A, D, E, G, L, N, Q, R, S, T, V, W, C, I, M, K, Y), Q168A + L169P + A170M + E245D + M342I + 75X 2 (X 2 = F , L, S, T, V, R, W, C, G, A, M, N, Q, D, K, P), Q168A + L169P + A170M + E245D + M342I + 76X ₃ (X ₃ = G, H, I, L, S, V, W, Y, Q, E, D, C, K, T, A, F, L, P),
_{Q168A + L169P + A170M + E245D +} M342I + T125X 4 (X 4 = R, V, L, G, C, P, H, S, K, A, E), Q168A + L169P + A170M + E245D + M342I + 126X 5 (X 5 = V, S, L, F, G, P, Q, C , R, S, D, A , I), Q168A + L169P + A170M + E245D + M342I + 127X 6 (X 6 = Y, G, V, E, I, N, P, A, C, S, M, D, T, H, K, Q) _{, Q168A + L169P + A170M + E245D} + M342I + F128X 7 (X 7 = C, E, G, H, I, K, L, P, S, T, V, W, Y, Q, M, N, D), Q168A + L169P + A170M + E245D + M342I + 129X 8 (X 8 = N, Y, E, V, H, T F, R, L, I, S, W, K, D), Q168A + L169P + A170M + E245D + M342I + 130X 9 (X 9 = F, T, D, R, V, S, I, C, A, P, Y, N, G, E _{, K, L), Q168A +} L169P + A170M + E245D + M342I + 131X 10 (X 10 = D, A, V, C, P, H, S, F, G, W, R, M, T, I), Q168A + L169P + A170M + E245D + M342I + 132X 11 (X 11 = V, P _{, L, I, K, E} ), Q168A + L169P + A170M + E245D + M342I + K142X 12 (X 12 = Q, N, H, W, E, T, L, S, V, P, F, D, Y, I, R, M), Q168A + L169P + A170M + E245D + M342I + T224X ₁₃ (X _{13 = A, C, S)} , Q168A + L169P + A170M + E245D + M342I + P230X 14 (X 14 = H, I, M, S, T, V), Q168A + L169P + A170M + E245D + M342I + A236X 15 (X 15 = C, E, S, V, T), Q168A + L169P + A170M + E245D + M342I + 344X 16 (X 16 = P, V, Q, W , L, F, S, I, H, T, A, N, K, Y), Q168A + L169P + A170M + E245D + M342I + I345X 17 (X 17 = Y, E, I), Q168A + L169P + A170M + E245D + M342I + E416X 18 (X 18 = A , C, D, F, G, H, I, L, M, N, P, Q, R, T, V, S, Y, K, W), L169P + A1 _{70M + E245D + M342I + Q168X 19} (X 19 = M, K, S, T, C, G), Q168A + A170M + E245D + M342I + L169X 20 (X 20 = A, V, F, M, K, R, S, T, H, C, N, Q, W _{, G), Q168A + L169P +} E245D + M342I + A170X 21 (X 21 = V, F, P, I, L, E, K, R, S, T, Y, H, C, N, W, G), Q168A + L169P + A170M + E245D + M342I + 168X 22 (X 22 = A , V, M, I, L , T, Y, H, C, Q, W, G), Q168A + L169P + A170M + E245D + M342I + 169X 23 (X 23 = A, C, E, G, I, K, L, M, N, R, S, T, V, W), Q168A + L169P + A17 _{M + E245D + M342I + 170X 24} (X 24 = A, C, D, E, F, G, H, I, L, N, P, Q, R, S, T, V, W, Y), Q168A + L169P + A170M + E245D + M342I + 171X 25 (X 25 = A , C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, Y) 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 such as sodium, potassium, ammonium, calcium, and magnesium, 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, calcium salts of inorganic acids such as calcium acetate and calcium citrate, and organic acids. 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. As immobilization methods, there are 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. It may be fixed or adsorbed and fixed on the electrode, or a combination of these 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 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.

The mediator used in the glucose measurement composition, glucose assay kit, glucose sensor, or glucose measurement method of the present invention is not particularly limited, but preferably 2,6-dichlorophenol-indophenol (abbreviated as DCPIP) is used. Excellent effect when it is.
Furthermore, the mutation site of the present invention is not only effective when DCPIP is used as a mediator, but also when ferrocene or a derivative thereof (for example, potassium ferricyanide, phenazine methosulfate, etc.) is used as a mediator. Confirm that you can see. Among them, a favorable effect is seen when potassium ferricyanide (Fe) is used as a mediator.
These mediators can be obtained commercially. DCPIP can be purchased from Merck.

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 positions 125, 128, 142, 168, 169, 170, 224, Amino acid substitution at positions 230, 236, 345, 351, 416 and 428, 74, 75, 76, 126, 127, 129, 130, 131, 132, 168 169th, 170th, 71 position, and the mutant libraries randomly mutated amino acid insertions was produced in 344-position. Then, the base sequence of the candidate strain obtained by screening using the change in substrate specificity as an index is determined, and the amino acid sequence described in SEQ ID NO: 1 is 168th glutamine is alanine, 168th glutamine is alanine, 169th A recombinant plasmid (pNPG5-Q168A + L169P + A170M + E245D + M342I) encoding a mutant PQQ-dependent glucose dehydrogenase in which leucine is replaced with proline, 170th alanine is methionine, 245th glutamic acid is aspartic acid, and 342nd methionine is isoleucine. ).
Next, on the basis of pNPG5-Q168A + L169P + A170M + E245D + M342I, using synthetic oligonucleotides of other non-mutated sites, library preparation and screening with substrate specificity as an index are performed in the same manner as described above. A recombinant plasmid (pNPG5-Q168A + L169P + A170M + E245D + M342I + A351T) encoding a mutant PQQ-dependent glucose dehydrogenase with improved substrate specificity was obtained.
In the same manner as described above, a library was prepared and screened to obtain a recombinant plasmid encoding a multiple mutant PQQ-dependent glucose dehydrogenase with improved recording specificity. Tent cells (Escherichia coli JM109; manufactured by Toyobo Co., Ltd.) were transformed to obtain the transformants.

Example 3: Construction of an expression vector that can be replicated in Pseudomonas bacteria 5 μg of the recombinant plasmid pNPG5-Q168A + L169P + A170M + E245D + M342I + A351T DNA obtained in Example 2 was cleaved with restriction enzymes BamHI and XhoI (manufactured by Toyobo), 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-Q168A + L169P + A170M + E245D + M342I + A351T. For other recombinant plasmids, expression plasmids were obtained in the same manner as described above. .

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-Q168A + L169P + A170M + E245D + M342I + A351T 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.
For each expression plasmid, the transformant was obtained in the same manner as described above.

Test example 1
Method for measuring GDH activity Measurement principle D-glucose + PMS + PQQGDH → D-glucono-1,5-lactone + PMS (red)
PMS (red) + DCPIP → PMS + DCPIP (red)
The presence of DCPIP (red) formed by the reduction of 2,6-dichlorophenol-indophenol (DCPIP) with phenazine methosulfate (PMS) (red) was measured spectrophotometrically at 600 nm.
Definition of Unit One unit refers to the amount of PQQGDH enzyme that forms 1.0 mmol of DCPIP (red) per minute under the conditions described below.
(3) Method reagents A. D- glucose solution: 1.0 M (1.8 g D- glucose (molecular weight 180.16) / 10ml 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: 24 mM (73.52 mg phenazine methosulfate (molecular weight 817.65) / 10 ml H ₂ O)
D. DCPIP solution: 2.0 mM (6.5 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. 4.5 ml D-glucose solution (A)
21.9 ml PIPES-NaOH buffer (pH 6.5) (B)
2.0ml PMS solution (C)
1.0 ml DCPIP solution (D)

The concentrations in the assay mixture are as follows:
PIPES buffer 36 mM
D-glucose 148 mM
PMS 1.58mM
NTB 0.066 mM

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 decrease in absorbance for water at 600 nm was recorded on a spectrophotometer while maintaining at 37 ° C. for 4-5 minutes, and the 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 ice-cooled enzyme diluent (E) and diluted to 0.05-0.10 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} / (16.8 × 1.0 × Vs)
U / mg = (U / ml) × 1 / C
Vt: Total volume (3.1 ml)
Vs: Sample volume (0.1 ml)
16.8: Millimolecular extinction coefficient of DCPIP under the above measurement conditions (cm ² / micromol)
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. This medium was inoculated with 5 ml of Pseudomonas putida TE3493 (pNPG6-Q168A + L169P + A170M + E245D + M342I + A351T) previously cultured in PY medium containing 100 μg / ml streptomycin at 30 ° C. for 24 hours, and cultured with aeration at 30 ° C. for 40 hours. The PQQ-dependent glucose dehydrogenase activity at the end of the culture was about 5 U / ml per ml of the culture medium 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.
For other Pseudomonas putida TE3493 transformants using other Pseudomonas expression plasmids, purified enzyme preparations were obtained in the same manner as described above.
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 Tables 1 to 5.
In Table 1, “T125R” in the left column indicates that T at position 125 is replaced with R. Further, “74A” in the left column indicates that A is inserted after the 73rd place. In “Q168A + L169P + E245D + M342I + A351T”, multiple mutants are represented by connecting those represented by the same principle as described above with a + symbol.

  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 (14)

Modified PQQGDH, which has a pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQGDH), which has at least one of the following mutations and is less active against disaccharides than wild-type PQQGDH.
(1) 125, 128, 142, 168, 169, 170, 224, 230, 236, 345, 351, 416, and 428 in the amino acid sequence of PQQGDH derived from Acinetobacter (2) Aminobacterium-derived PQQGDH amino acid sequence at position 74, 75, 76, 126, 127, 129, at least one position, or at an equivalent position in other species as described above, Has amino acid insertions at at least one of positions 130, 131, 132, 168, 169, 170, 171 and 344, or equivalent positions in other species   The modified PQQGDH according to claim 1, wherein the disaccharide is maltose.   A gene encoding the modified PQQGDH according to claim 1 or 2.   A vector comprising the gene according to claim 3.   A transformant transformed with the vector according to claim 4.   A method for producing a modified PQQGDH, wherein the transformant according to claim 5 is cultured.   A composition for measuring glucose comprising the modified PQQGDH according to claim 1 or 2.   A glucose assay kit comprising the modified PQQGDH according to claim 1 or 2.   A glucose sensor comprising the modified PQQGDH according to claim 1 or 2.   A glucose measurement method comprising the modified PQQGDH according to claim 1 or 2.   A method for reducing the activity of PQQGDH on disaccharides, wherein the amino acid mutation according to claim 1 or 2 is performed on PQQGDH.   A method for producing PQQGDH in which the action of PQQGDH on disaccharides is reduced, wherein the amino acid mutation according to claim 1 or 2 is performed on PQQGDH.   A glucose measurement system using PQQGDH, comprising the PQQGLD subjected to the amino acid mutation according to claim 1 or 2, and improving the measurement accuracy in the glucose measurement system.   A glucose measurement system using PQQGDH, comprising the PQQGLD subjected to the amino acid mutation according to claim 1 or 2, and a method for producing a glucose measurement composition with improved measurement accuracy.