JP2009225801A - Modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a more practically advantageous enzyme usable as a reagent for measuring blood glucose than the known enzymes used as blood glucose sensors. <P>SOLUTION: Provided is a modified flavin-adenine-dinucleotide-dependent glucose dehydrogenase (FADGDH) with more improved heat stability than wild type FADGDH, the modified FADGDH being derived from preferably a eukaryote, more preferably a filamentous fungus, furthermore preferably a genus Aspergillus fungus, and having a primary structure with at least one amino acid substituted, deleted, inserted or added to FADGDH, for example, having a specific amino acid sequence. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  TECHNICAL FIELD The present invention relates to a modified glucose dehydrogenase (GDH) with improved thermostability, a modified FADGDH-dependent glucose dehydrogenase (FADGDH) having flavin adenine dinucleotide (FAD) as a coenzyme, a method for producing the same, and a glucose sensor It is about.

  Blood glucose self-measurement is important for diabetics to grasp their normal blood glucose level and use it for treatment. An enzyme using glucose as a substrate is used for a sensor used for blood glucose self-measurement. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has been used as an enzyme for blood glucose sensors for a long time because it has the advantage of high specificity for glucose and excellent thermal stability, and its first announcement dates back to about 40 years ago. . In a blood glucose sensor using glucose oxidase, measurement is performed by passing electrons generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone to the electrode through a mediator. There is a problem that dissolved oxygen affects the measured value because the protons generated in the above are easily passed to oxygen.

  In order to avoid such problems, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47) or pyrroloquinoline quinone (herein, pyrroloquinoline quinone is also referred to as PQQ) -dependent glucose dehydrogenase. (EC 1.1.5.2 (formerly EC 1.1.9.17) has been used as an enzyme for blood glucose sensors. These are advantageous in that they are not affected by dissolved oxygen, but the former NAD (P ) -Dependent glucose dehydrogenase (NAD (P) -dependent glucose dehydrogenase is also referred to as NADGDH in this document) is not stable and requires the addition of a coenzyme, while the latter PQQ-dependent glucose dehydrogenase ( In this book, PQQ-dependent glucose dehydrogenase is also referred to as PQQGDH To.) Have poor substrate specificity, there is a drawback of impairing the accuracy of the measurements to act in sugars other than glucose such as maltose and lactose.

  Patent Document 1 discloses an Aspergillus-derived flavin-binding glucose dehydrogenase (herein, flavin-binding glucose dehydrogenase is also referred to as FADGDH). Since this enzyme has an action on xylose of about 10% of the action on glucose, when measuring blood glucose of a person undergoing a xylose tolerance test, the accuracy of the measurement value may be impaired. About thermal stability, it is said that the activity remaining rate is about 89% after treatment at 50 ° C. for 15 minutes and the stability is excellent. Patent Document 2 reports the gene sequence and amino acid sequence thereof.

WO2004 / 058958 WO2006 / 101239

Karlin et al. , Proc. Natl. Acad. Sci. USA (1993) Vol. 90 p5873-5877 Needleman et al. , J. et al. Mol. Biol. (1970) Vol. 48 p444-453 Myers and Miller, CABIOS Vol. 4 p11-17 Pearson et al. , Proc. Natl. Acad. Sci. USA (1988) Vol. 85 p2444-2448

  An object of the present invention is to provide an enzyme that can be used as a reagent for measuring blood glucose level, which is more practically advantageous than the above-mentioned known enzymes for blood glucose sensor.

  Patent Document 2 discloses that flavin-binding glucose dehydrogenase was obtained by liquid culture or wheat bran culture of Aspergillus terreus wild strain, and a gene encoding flavin-binding glucose dehydrogenase derived from Aspergillus tereus genus. In addition, it is described that enzymes obtained by expression in recombinant Escherichia coli, recombinant mold (Aspergillus oryzae), and recombinant yeast (genus Candida) were purified.

  Patent Document 2 discloses several examples of property tests of these enzymes and comparison of characteristics when applied to sensors.

  However, the inventors of the present application considered that the enzyme described in Patent Document 2 is not sufficiently described in terms of industrial requirements and may not satisfy the requirements. For example, an enzyme expressed in Escherichia coli that seems to be most suitable for mass production, which is one of the important requirements of the industry, is not described in the same very important requirement, temperature stability. Is mentioned. Therefore, the present inventor reviewed the prior art from the viewpoint of stable supply of the enzyme with a view to producing the enzyme by genetic recombination. Another object of the present invention is to provide an enzyme that can be used as a reagent for measuring blood glucose level that is more practically advantageous by reducing the activity of xylose by appropriately modifying the amino acid sequence of FADGDH derived from the Aspergillus oryzae strain. Repeated examination.

  As a result, the FADGDH recombinant (rFADGDH) obtained by expression in Escherichia coli, which seems to be most suitable for mass production, is an enzyme obtained by culturing and purifying from a wild strain. It turned out to be greatly inferior compared with.

  For example, aFADGDH obtained from Aspergillus oryzae by the inventors by the method described below maintained about 77% activity in a treatment at 50 ° C. for 15 minutes, but the FADGDH recombinant obtained by expression in E. coli ( raFADGDH) had a thermal stability of about 13% after treatment at 50 ° C. for 15 minutes. The thermal stability of Aspergillus tereus FADGDH recombinant (rtFADGDH) was about 28% after treatment at 50 ° C. for 15 minutes.

  Similarly, for the enzyme whose structure and production method are described in Patent Document 2, the enzyme obtained by expression in Escherichia coli is more stable than the enzyme obtained by culturing and purifying from a wild strain. It is considered that there is a high possibility of being inferior.

  This was thought to be because the enzyme produced by genetic recombination was not added with polysaccharides on the surface and its thermal stability was reduced.

  In the manufacturing process of a blood glucose sensor chip, heat drying may be performed. When a recombinant is used, there is a risk of significant heat inactivation, and it is necessary to improve thermal stability. It was.

  Therefore, we conducted further studies with the aim of providing an enzyme that can be used as a reagent for measuring blood glucose level, which has sufficient thermal stability even if produced by genetic recombination with E. coli, and is more advantageous in practical use. .

  As a result, we overcame the drawbacks related to the thermal stability of known enzymes for blood glucose sensors as described above by appropriately modifying the amino acid sequence of FADGDH derived from Aspergillus oryzae strain or Aspergillus tereus strain, It was possible to provide an enzyme that can be used as a blood glucose level measurement reagent that is more practically advantageous.

That is, the present invention is as follows.
[Claim 1]
Modified FADGDH whose thermal stability is improved by modification.
[Section 2]
Item 2. The modified FADGDH according to Item 1, which is derived from a eukaryote.
[Section 3]
Item 2. FADGDH according to item 1, which is derived from a filamentous fungus.
[Claim 4]
Item 2. FADGDH according to Item 1, which is derived from Aspergillus.
[Section 5]
Item 5. The modified FADGDH according to Items 1 to 4, wherein the thermal stability is improved as compared with wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH).
[Claim 6]
Item 5. The modified FADGDH according to Items 1 to 4, wherein the activity remaining rate after heat treatment at 50 ° C. for 15 minutes is 20% or more in a liquid state.
[Claim 7]
Item 5. The modified FADGDH according to Items 1 to 4, wherein the residual activity ratio after heat treatment at 50 ° C. for 15 minutes is 35% or more in a liquid state.
[Section 8]
Item 5. The modified FADGDH according to Items 1 to 4, wherein the residual activity rate after heat treatment at 50 ° C. for 15 minutes is 40% or more in a liquid state.
[Claim 9]
Item 5. The modified FADGDH according to Items 1 to 4, wherein an activity remaining ratio after heat treatment at 50 ° C for 15 minutes is 70% or more.
[Section 10]
Item 5. The modified FADGDH according to Items 1 to 4, wherein an activity remaining ratio after heat treatment at 50 ° C for 15 minutes is 80% or more.
[Section 11]
A modified FADGDH having a primary structure in which at least one amino acid is substituted, deleted, inserted or added in FADGDH having the amino acid sequence set forth in SEQ ID NO: 2 or 46 in the Sequence Listing.
[Claim 12]
In SEQ ID NO: 2 in the sequence listing, positions 120, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 180, 329, 331, 369, 471, and 551, at least one position in the group consisting of positions 316, 159, 161, 164, 166, 167, 175 in SEQ ID NO: 46 in the sequence listing Modified FADGDH having improved amino acid substitution at positions 325, 327, 365, 547 or equivalent positions in other species as described above.
[Claim 13]
In SEQ ID NO: 2 in the sequence listing, at least the amino acid substitution is K120E, G160E, G160I, G160P, G160S, G160Q, S162A, S162C, S162D, S162E, S162F, S162H, S162L, S162P, G163D, G163K, G163S, G163R, G163R, G163R , S164T, S164Y, L165A, L165I, L165N, L165P, L165V, A166C, A166I, A166K, A166L, A166M, A166P, A166S, 167A, S167P, S167R, S167V, N169P, L169P, L169P , S171K, S171M, S171Q, S171V, V172A, V172C, V172E, V 72I, V172M, V172S, V172W, V172Y, A180G, V329Q, A331C, A331D, A331I, A331K, A331L, A331M, Q331V, K369R, K471R, V551A, V551G, V551T, V551G, 1601 ), (G160S + S167P), (G160Q + S167P), (S162A + S167P), (S162C + S167P), (S162D + S167P), (S162D + S167P), (S162E + S167P), (S162E + S167P), (S162F + S167P), S16F + S167P) (S164T S167P), (S164Y + S167P), (L165A + S167P), (L165I + S167P), (L165P + S171K),, (L165P + V551C) (L165V + V551C), (A166C + S167P), (A166I + S167P), (A166K + S167P), (A166K + S167P), (A166M + S167P), (A166P + S167P) , (A166S + S167P), (S167P + N169K), (S167P + N169P), (S167P + N169Y), (S167P + N169W), (S167P + L170C), (S167P + L170F), 17S17P + S171P, S17P + S171P 67P + S171V), (S167P + V172A), (S167P + V172C), (S167P + V172E), (S167P + V172I), (S167P + V172M), (S167P + V172S), (S167P + V172T), (S167P + V172W), (S167P + V172Y), (S167P + V329Q), (S167P + A331C), (S167P + A331D) , (S167P + A331I), (S167P + A331K), (S167P + A331L), (S167P + A331M), (S167P + A331V), (G163K + V551C), (G163R + V551C), or at least SEQ ID NO: 116 in SEQ ID NO: 46. K116G, K116L K116F, K116Q, Q159A, Q159K, Q159N, Q159P, Q159V, Q159L, E161C, N164Y, N164V, N164C, T166F, T166Y, T166W, T167L, T167V, T167S, G175K, S325S, G175K, S3S A modified FADGDH having an amino acid substitution at any of S325V, S325Y, S327E, Q365R, V547S, V547C, V547A, and V547Q, or an amino acid substitution at the same position in other species as described above.
[Section 14]
Item 15. The modified FADGDH according to Item 1-14, wherein the pH stability is improved by modification.
[Section 15]
Item 16. The modified FAD-GDH according to item 1-15, wherein the residual activity after treatment at 25 ° C. for 16 hours at pH 4.5 to pH 6.5 is 80% or more.
[Section 16]
Item 16. The modified FAD-GDH according to item 1-15, wherein the residual activity after treatment at 25 ° C. for 16 hours at pH 4.5 to pH 6.5 is 90% or more.
[Section 17]
A variant FADGDH having improved amino acid stability having an amino acid substitution at at least one position in the group consisting of positions 163, 167, and 551 in SEQ ID NO: 2, or a position equivalent to the above in another species.
[Section 18]
In at least SEQ ID NO: 2 in the sequence listing, the amino acid substitution is at least one of S167P, V551C, (G163K + V551C), (G163R + V551C), or pH stability having an amino acid substitution at a position in the same arrangement in other species as described above Improved modified FADGDH.
[Section 19]
A gene encoding the modified FADGDH according to any one of claims 1 to 18.
[Section 20]
A vector comprising the gene according to claim 19.
[Claim 21]
A transformant transformed with the vector according to claim 20.
[Item 22]
A method for producing a modified FADGDH, wherein the transformant according to claim 21 is cultured.
[Section 23]
A glucose assay kit comprising the modified FADGDH according to any one of claims 1 to 18.
[Claim 24]
A glucose sensor comprising the modified FADGDH according to any one of claims 1 to 18.
[Claim 25]
A glucose measurement method comprising the modified FADGDH according to any one of claims 1 to 18.
[Claim 26]
A modified FADGDH having improved substrate specificity over a flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) derived from wild-type Aspergillus oryzae, the 53rd position in the amino acid sequence of SEQ ID NO: 2 or a position equivalent thereto A modified FADGDH having an amino acid substitution and improved thermal stability.
[Section 27]
Item 27. The modified FADGDH according to Item 26, wherein the activity on xylose is 5.0% or less of the activity on glucose.
[Claim 28]
Item 27. The modified FADGDH according to Item 26, which has an amino acid substitution at any position in the group consisting of G53H, G53N, G53K, G53M, G53T, G53V and G53C in SEQ ID NO: 2, or an equivalent position thereof.
[Item 29]
Item 27. The modified FADGDH according to Item 26, which has an amino acid substitution at one or more positions in the group consisting of positions 163, 167, and 551 in the amino acid sequence of SEQ ID NO: 2, or a position equivalent thereto.
[Section 30]
Item 32. The modified FADGDH according to Item 29, which has an amino acid substitution at a position equivalent to or any one of the group consisting of (G53H + S167P), (G53N + S167P), (G53H + S167P) and (G53N + G163R + V551C) in the amino acid sequence of SEQ ID NO: 2. .
[Claim 31]
Item 32. A gene encoding the modified FADGDH according to any one of Items 26 to 30, a vector containing the gene, a transformant transformed with the vector, and the transformation A method for producing modified FADGDH, comprising culturing a body.
[Section 32]
Item 31. A glucose assay kit comprising the modified FADGDH of any one of Items 26 to 30. [Section 33]
Item 31. A glucose measurement method comprising the modified FADGDH of any one of Items 26 to 30.

  The modified FADGDH according to Item 26 has a lower effect on pentose than the wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH). An example of the pentose is xylose. The modified FADGDH according to Item 26 has an effect on xylose of 5.0% or less of the activity on glucose as compared with the wild type FADGDH. In addition, the effect | action with respect to xylose means the relative ratio% (glucose is set to 1) of the reaction rate when glucose is used as a substrate and xylose is used as a substrate.

  The modified FADGDH according to Item 26 preferably has improved thermal stability as compared to wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH). In the modified FADGDH according to Item 26, the residual activity rate after heat treatment at 50 ° C. for 15 minutes is 20% or more, preferably 40% or more, and more preferably 70% or more. If such stability can be maintained, it is possible to heat and dry during formulation.

The modified FADGDH according to Item 26 preferably has improved pH stability over the wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH). The modified FADGDH according to Item 26 has a residual activity of 80% or more after treatment at 25 ° C. for 16 hours at pH 4.5 to pH 7.0, or 25 ° C. at pH 4.5 to pH 6.5. The residual activity after 16 hours of treatment is 80% or more, preferably 90% or more.
[Section 34]
Modified FADGDH with improved thermal stability over wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), preferably from eukaryotes, more preferably from filamentous fungi, more preferably from Aspergillus spp. FADGDH. Here, FADGDH preferably has an activity remaining ratio after heat treatment at 50 ° C. for 15 minutes of 20% or more, more preferably 40% or more, and further preferably 80% or more.
[Claim 35]
In FADGDH having the amino acid sequence described in SEQ ID NO: 2, it is a modified FADGDH having a primary structure in which at least one amino acid is substituted, deleted, inserted or added. For example, in SEQ ID NO: 2, position 120, 160 , 162, 163, 164, 165, 166, 167, 170, 171, 172, 180, 329, 331, 369, 369, 471 and 551 A modified FADGDH with improved thermal stability having an amino acid substitution at least at one of the positions, or at a position equivalent to the above in another species. Here, modified FADGDH having an amino acid substitution at at least one of positions 162, 163, 167, and 551 can be exemplified as a more preferable example.
[Claim 36]
Modified FADGDH having improved thermal stability over flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) derived from wild-type Aspergillus oryzae, at positions 163 and / or 551 in the amino acid sequence of SEQ ID NO: 2, or A modified FADGDH having an amino acid substitution at an equivalent position and having improved thermal stability.
[Section 37]
G163D, G163K, G163L, G163R, V551A, V551C, V551T, V551Q, V551S, V551Y, (G163D + S167P), (L165P + V551C), (L165V + V551K), (155) Or a modified FADGDH as described in the previous paragraph, having an equivalent amino acid substitution at any or the equivalent position.
[Section 38]
In addition to positions 163 and / or 551 in the amino acid sequence of SEQ ID NO: 2, in addition, positions 120, 160, 162, 164, 165, 166, 167, 170, 170, 171 and 172, A modified FADGDH having an amino acid substitution at any one or more positions in the group consisting of positions 180, 329, 331, 369, and 471, or an equivalent position.
[Section 39]
Modified FADGDH with improved pH stability over wild-type flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), preferably from eukaryotes, more preferably from filamentous fungi, more preferably from Aspergillus spp. FADGDH
[Claim 40].
In addition to positions 163 and / or 551 in the amino acid sequence of SEQ ID NO: 2, it is a modified FADGDH having an improved pH stability, further having an amino acid substitution at position 167 or equivalent position.
[Section 41]
The modified FADGDH described in the previous paragraph, which has an equivalent amino acid substitution at any position within the group consisting of S167P, V551C, (G163K + V551C) and (G163R + V551C) in the amino acid sequence of SEQ ID NO: 2, or an equivalent position thereto.
[Section 42]
48. The FADGDH of any one of Items 34 to 41, wherein the residual activity after treatment at 25 ° C. for 16 hours at pH 4.5 to pH 6.5 is 80% or more, preferably 90% or more.
[Section 43]
A variant FADGDH having improved amino acid stability having an amino acid substitution at at least one position in the group consisting of positions 163, 167, and 551 in SEQ ID NO: 2, or a position equivalent to the above in another species.
[Item 44]
In SEQ ID NO: 2, the amino acid substitution is at least one of S167P, V551C, (G163K + V551C), (G163R + V551C), or a modification with improved pH stability having an amino acid substitution at a position in the same arrangement in other species Type FADGDH.
[Section 45]
Item 44. The gene encoding the modified FADGDH according to any one of Items 34 to 44, a vector containing the gene, a transformant transformed with the vector, and the modified FADGDH characterized by culturing the transformant Manufacturing method.
[Claim 46]
45. A glucose assay kit or glucose sensor comprising the modified FADGDH of any one of Items 34 to 44.
[Section 47]
Item 45. A glucose measurement method comprising the modified FADGDH according to any one of Items 34 to 44.

  In the modified FADGDH of the present invention, in particular, G163K, G163L, G163R, S167P, V551A, V551C, V551Q, V551S, V551Y, (G160I + S167P), (S162F + S167P), (S167P + N169Y), S17P + N16Y, S17P + N16Y ), (S167P + V172I), (S167P + V172W), (G163K + V551C) (G163R + V551C) amino acid substitution contributes to the improvement of the thermal stability of the modified FADGDH.

  Here, “K120E” means that K (Lys) at position 120 is replaced with E (Glu). Also, “(G160E + S167P)” is a substitution of G at position 160 with E and S at position 167 with P, and + is a multiple (double in this example) mutant having both substitutions. Means.

  The heat-dryable level is a state in which the residual activity after treatment at 50 ° C. for 15 minutes is 20% or more, preferably 40% or more, and more preferably 60% or more. This is a state in which there is residual activity.

  The improvement in the stability of FADGDH according to the present invention reduces the heat inactivation of the enzyme during preparation of the glucose measurement reagent, glucose assay kit, and glucose sensor, thereby reducing the amount of the enzyme used and improving the measurement accuracy.

Fig. 3 shows the pH stability of AST (wild-type), modified FADGDH purified preparations derived from Asperolize. pH 3.5 to 6.3: acetate buffer, pH 6.3 to 7.3, PIPES buffer, pH 7.3 to 8.8: Tris-HCl buffer, pH 6.0 to 7.7: phosphate buffer were used, respectively.

  The present invention includes modified FADGDH with improved thermal stability over recombinant FADGDH.

  Wild-type Aspergillus oryzae-derived FADGDH represented by SEQ ID NO: 2 was obtained by the following method.

  The present inventors estimated and obtained a glucose dehydrogenase gene derived from Aspergillus oryzae using a database of National Center for Biotechnology Information (hereinafter referred to as NCBI), and used the gene to obtain glucose derived from Aspergillus oryzae from E. coli. It has been found that dehydrogenase can be obtained.

  In order to obtain the Aspergillus oryzae-derived GDH gene, purification of GDH was attempted using various chromatographies from the culture supernatant of its own Aspergillus oryzae TI strain, but it was difficult to obtain high-purity GDH. There has been no choice but to give up cloning using a partial amino acid sequence, which is one of the common methods of gene acquisition. However, we found that Penicillium lilacinoechinatum NBRC6231 strain produced GDH and succeeded in determining the partial amino acid sequence using purified enzyme. Then, based on the determined amino acid sequence, P.P. A part of lilacinoechinatum NBRC6231-derived GDH gene was obtained and the nucleotide sequence was determined (1356 bp). Finally, the Aspergillus oryzae GDH gene was estimated and obtained based on this base sequence. The outline is shown in <Experimental example 1> <Experimental example 2> below.

<Experimental example 1>
[Estimation of Aspergillus oryzae-derived glucose dehydrogenase (hereinafter also referred to as AOGDH) gene]
[1] Acquisition of GDH derived from Aspergillus oryzae The L dry strain of Aspergillus oryzae TI strain was inoculated into potato dextrose agar medium (manufactured by Difco) and recovered by incubating at 25 ° C. The restored mycelium on the plate was collected together with the agar and suspended in filter sterilized water. 6 L of production medium (1% malt extract, 1.5% soybean peptide, 0.1% MgSO4 · 7 hydrate, 2% glucose, pH 6.5) was prepared in two 10 L jar fermenters, After autoclaving at 15 ° C. for 15 minutes and allowing to cool, the above mycelial suspension was inoculated and cultured at 30 ° C. with aeration and stirring. After 64 hours from the start of the culture, the culture was stopped, the mycelium was removed by filtration, and the filtrate containing GDH activity was collected. Low molecular weight substances were removed from the collected supernatant by ultrafiltration membrane (molecular weight 10,000 cut). Next, ammonium sulfate was added and dissolved to 60% saturation, ammonium sulfate fractionation was performed, and the supernatant fraction containing GDH was collected by a centrifuge, and then adsorbed on an Octyl-Sepharose column, and ammonium sulfate saturation 60%. Fractions with GDH activity were collected by gradient elution at ˜0%. The obtained GDH solution was desalted using a G-25-Sepharose column, and then 60% saturated ammonium sulfate was added and dissolved, adsorbed on the Phenyl-Sepharose column, and ammonium sulfate saturated 60%. Fractions with GDH activity were collected by gradient elution at ˜0%. Further, this was heated at 50 ° C. for 45 minutes, and then centrifuged to obtain a supernatant. The solution obtained through the above steps was used as a purified GDH preparation (AOGDH). In the purification process, 20 mM potassium phosphate buffer (pH 6.5) was used as the buffer. Furthermore, in order to determine the partial amino acid sequence of AOGDH, although purification was attempted by various means such as ion exchange chromatography and gel filtration chromatography, a purified sample with high purity that can be used for partial amino acid sequencing is obtained. I couldn't.

[2] Acquisition of Penicillium filamentous fungus-derived GDH Using Penicillium lilacinoechinatum NBRC6231 as a GDH-producing fungus derived from Penicillium filamentous fungus, culturing and purifying according to the same procedure as the above Aspergillus oryzae strain TI, by SDS electrophoresis An almost uniform purified sample was obtained.
[Production of cDNA]
Penicillium lilacinoechinatum NBRC6231 was cultured according to the above method (however, the culture time in the jar fermenter was 24 hours), and the mycelium was collected by filtration with filter paper. The obtained mycelia were immediately frozen in liquid nitrogen, and the mycelium was pulverized using a cool mill (Toyobo Co., Ltd.). Total RNA was extracted from the pulverized cells using Sepakol RNA I (Nacalai Tesque) according to the protocol of this kit. From the total RNA obtained, mRNA was purified using Origotex-dt30 (Daiichi Chemicals Co., Ltd.), and RT-PCR was performed using RiverTra-Plus-TM (Toyobo Co., Ltd.) using this as a template. The obtained product was subjected to agarose electrophoresis, and a portion corresponding to a chain length of 0.5 to 4.0 kb was cut out. CDNA was extracted and purified from the excised gel fragment using MagExtractor-PCR & Gel Clean Up- (manufactured by Toyobo Co., Ltd.) to obtain a cDNA sample.
[Determination of GDH gene partial sequence]
The above-purified NBRC6231-derived GDH is dissolved in Tris-HCl buffer (pH 6.8) containing 0.1% SDS and 10% glycerol, so that the Glu-specific V8 endoprotease has a final concentration of 10 μg / ml. Partial degradation was performed by adding and incubating at 37 ° C. for 16 hours. This sample was electrophoresed on a gel with an acrylamide concentration of 16% to separate the peptides. Peptide molecules present in this gel were transferred to a PVDF membrane by a semi-dry method using a blotting buffer (1.4% glycine, 0.3% Tris, 20% ethanol). The peptide transcribed on the PVDF membrane was stained using a CBB staining kit (Gel Code Blue Stain Reagent manufactured by PIERCE), and two bands of the visualized peptide fragment were cut out and the internal amino acid sequence was analyzed by a peptide sequencer. . The resulting amino acid sequences were IGGVVDTSLKVYGT (SEQ ID NO: 37) and WGGGTKQTVRAKGALGTGT (SEQ ID NO: 38). Based on this sequence, a degenerate primer containing a mixed base was prepared, and PCR was performed using NBRC6231-derived cDNA as a template. An amplified product was obtained and confirmed by agarose gel electrophoresis. A single band of about 1.4 kb was obtained. Met. This band was cut out, extracted and purified using Toyobo's MagExtractor-PCR & Gel Clean Up-. The purified DNA fragment was TA-cloned with TARGET Clone-Plus- (Toyobo), and Escherichia coli JM109 competent cell (Toyobo) was transformed with the resulting vector by heat shock. From the transformed clones, colonies in which insert insertion was confirmed by blue-white determination were extracted and purified using a MagExtractor-Plasmid- (manufactured by Toyobo), and the nucleotide sequence of the insert was determined using plasmid sequence-specific primers. Determined (1356 bp).
[Estimation of AOGDH gene]
Based on the determined base sequence, a homology search was performed from the homepage of “NCBI BLAST” (http://www.ncbi.nlm.nih.gov/BLAST/) to estimate the AOGDH gene. AOGDH and P.E. The homology at the amino acid level with the GDH partial sequence derived from lilacinoechinatum NBRC6231 was 49%.

<Experimental example 2>
[Acquisition of Aspergillus oryzae-derived glucose dehydrogenase gene and introduction into Escherichia coli]
In order to obtain the AOGDH gene, mRNA was prepared from the cells of Aspergillus oryzae TI strain, and cDNA was synthesized. Two types of oligo DNAs shown in SEQ ID NOs: 39 and 40 were synthesized, and AOGDH gene was amplified using KOD Plus DNA polymerase (manufactured by Toyobo Co., Ltd.) using the prepared cDNA as a template. The DNA fragment was treated with restriction enzymes NdeI and BamHI, pBluescript (in which the NdeI site was introduced in the form of matching the atg of the NdeI recognition sequence in accordance with the translation initiation codon atg of LacZ), the recombinant plasmid was inserted into the NdeI-BamHI site. It was constructed. Escherichia coli DH5α (Toyobo Co., Ltd.) was transformed with this recombinant plasmid. A plasmid was extracted from the transformant according to a conventional method, and the base sequence of the AOGDH gene was determined (SEQ ID NO: 41). As a result, it was revealed that the amino acid residue deduced from the cDNA sequence consists of 593 amino acids (SEQ ID NO: 42). The database predicted GDH was 588 amino acids, suggesting that the number of amino acid residues is different from that of TI strain GDH. In addition, about this gene, the arrangement | sequence was confirmed using TI strain | stump | stock genomic DNA, and the gene adjacent region was also confirmed using the RACE method. In addition, a recombinant plasmid having a DNA sequence based on the database was constructed using the PCR method, and a transformant was obtained in the same manner. These transformants were cultured with shaking at 30 ° C. for 16 hours in 200 ml of a liquid medium (Terrific broth) containing 100 μg / ml ampicillin. When the GDH activity was confirmed for the cell disruption solution, the GDH activity could not be confirmed in the transformant having the GDH sequence estimated from the database. However, the transformant having the TI strain-derived GDH sequence was not found in the cell body. 8.0 U of GDH activity was obtained per 1 ml of culture solution. The GDH activity of the culture supernatant of Aspergillus oryzae TI strain carried out in Example 1 was 0.2 U / ml.

<Experimental example 3>
[Introduction of Aspergillus oryzae-derived glucose dehydrogenase (hereinafter referred to as AOGDH) gene into Escherichia coli]
When FAD-GDH after cleavage of the signal peptide is mFAD-GDH, only M is added to the N-terminus of mFAD-GDH, and the N-terminus of mFAD-GDH has a form of 1 amino acid, expressed as S2. .

  In S2, PCR is carried out using the oligonucleotide of SEQ ID NO: 43 as an N-terminal primer and a combination with the primer of SEQ ID NO: 44, and a recombinant plasmid having a DNA sequence (SEQ ID NO: 1) encoding S2 in the same procedure And a transformant was obtained in the same manner.

  It was confirmed that the plasmid having the modified FAD-GDH DNA sequence had no sequence errors in DNA sequencing.

  This transformant was subjected to liquid culture for 1 to 2 days in a TB medium using a 10 L-jar fermenter. After collecting the cells in each culture phase, the cells were ultrasonically disrupted to confirm the GDH activity. By deleting the amino acid sequence that appears to be a signal peptide, its GDH productivity increased.

  The wild type Aspergillus terreus-derived FADGDH represented by SEQ ID NO: 46 was obtained by the following method.

<Experimental example 4>
Preparation of cDNA Aspergillus tereus NBRC33026 (purchased from National Institute of Technology and Evaluation) L dry specimen was inoculated into potato dextrose agar medium (manufactured by Difco) and restored by incubating at 25 ° C. Prepare 50 ml of 1.5% soybean peptide, 2% glucose, 1% malto extract pH 6.5 in a 500 ml Sakaguchi flask, collect the mycelium on the restored plate together with the agar, inoculate it, and shake at 30 ° C for 24 hours. Culture was performed and the cells were collected. The obtained bacterial cells were immediately frozen in liquid nitrogen and pulverized using a Toyobo cool mill. From the crushed cells, total RNA was extracted according to the protocol of this kit using Sepasol RNA I manufactured by Nacalai Tesque, and cDNA was prepared by performing RT-PCR using Toyobo's RiverTra-Plus-TM as a template.

<Experimental example 5>
Sequencing of GDH gene We have succeeded in cloning GDH genes derived from Aspergillus oryzae, Penicillium lilacinochinulatum, and Penicillium italicum, and have obtained base sequence information. In order to clone the GDH gene from Aspergillus terreus, the deduced amino acid sequences of the above three types of GDH were aligned, and degenerate primers were prepared based on the sequence of the highly homologous region. When PCR was performed using the genomic DNA prepared in <Experimental Example 4> as a template, amplification products were observed. The amplified product was subcloned and the nucleotide sequence was determined. Based on the determined GDH partial sequence, 5′-side and 3′-side adjacent regions of the sequence portion were determined by the RACE method. The sequence from the start codon to the end codon of the determined gene region is shown in SEQ ID NO: 45, and the amino acid sequence deduced from this sequence is shown in SEQ ID NO: 46. The Aspergillus terreus FERM BP-08578-derived coenzyme-linked glucose dehydrogenase shown in Patent Document 1 has a very high homology of about 98.5% at the amino acid level and is essentially equivalent. It is thought that. “Homology” refers to an optimal alignment when two amino acid sequences are aligned using mathematical algorithms known in the art (preferably the algorithm uses one or the other of the sequences for optimal alignment). The percentage of the same amino acid residue with respect to all overlapping amino acids in the case of introduction of gaps in both). Examples of such an algorithm include those described in Non-Patent Documents 1 to 4, but are not limited thereto.

<Experimental example 6>
Production of GDH Recombinant Plasmid and Recombinant A signal peptide was predicted for the amino acid sequence encoded by the DNA sequence of SEQ ID NO: 45 using SignalP 3.0 Server. Based on this result, in order to remove the signal peptide, the N-terminal sequence 25 codon was deleted, and PCR primers were prepared so as to amplify the sequence amplification with the addition of the start codon (ATG) (SEQ ID NOs: 47 and 48). . Using these primers, gene amplification was performed using NBRC33026 cDNA as a template with KOD Plus DNA polymerase (Toyobo Co., Ltd.). The amplified fragment is treated with restriction enzymes NdeI and BamHI, inserted between the NdeI-BamHI sites of pBluescript (in which the NdeI site is introduced in a form that matches the ATG of the NdeI recognition sequence in accordance with the translation start codon ATG of LacZ) A plasmid was constructed (pAtGDH-s2-7). Using this recombinant plasmid, Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) was transformed to obtain an Aspergillus terreus-derived GDH recombinant. The transformant was cultured with shaking at 30 ° C. for 16 hours in 200 ml of a liquid medium (Terrific broth) containing 100 μg / ml ampicillin. When the GDH activity was confirmed for the microbial cell disruption solution, 1.0 U GDH activity per 1 ml of the culture solution was obtained in the microbial cell.

The modified FADGDH of the present invention can be obtained by performing amino acid substitution at any position shown above in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 46.

  For example, the “position equivalent to the amino acid sequence of SEQ ID NO: 2” means that the amino acid sequence of SEQ ID NO: 2 is homologous to SEQ ID NO: 2 (preferably 60% or more, more preferably 80% or more, more preferably 90% or more. ) Means the same position in the alignment when aligned with another GDH having an amino acid sequence.

The present invention with improved substrate specificity and / or thermal stability has amino acid substitutions in at least one of positions 53, 163, 167, 551 and 551 in the amino acid sequence of SEQ ID NO: 2. A modified FADGDH is exemplified.
A modified FADGDH selected from the group consisting of G53H, G53N, G53K, G53M, G53T, G53V, G53C, G163R, S167P, and V551C in the amino acid sequence of SEQ ID NO: 2.

  Here, “G53H” means that G (Gly) at position 53 is replaced with (His). In particular, the amino acid substitution of G53H, G53N, G53K, G53M, G53T, G53V, G53C contributes to the improvement of the substrate specificity of modified FADGDH, and the amino acid substitution of G53H + S167P, G53N + S167P, G53N + G163R + V551C is the substrate specificity of modified FADGDH And / or contributes to improvement of stability.

  In the amino acid sequence of SEQ ID NO: 2, the present invention with improved thermal stability comprises the following positions: 120, 160, 162, 163, 164, 165, 166, 167, 169, 169, 170, 171 Examples include modified FADGDH having an amino acid substitution at at least one of positions 172, 180, 329, 331, 369, 471 and 551. Among the above, modified FADGDH having an amino acid substitution at at least one of positions 162, 163, 167, 551 and 551.

  In the amino acid sequence of SEQ ID NO: 2, the amino acid substitution is K120E, G160E, G160I, G160P, G160S, G160Q, S162A, S162C, S162D, S162E, S162F, S162H, S162L, S162P, G163D, G163K, G163L, T163F, S163F , S164Y, L165A, L165I, L165N, L165P, L165V, A166C, A166I, A166K, A166L, A166M, A166P, A166S, 167A, S167P, S167R, S167V, N169K, N169K, N169K, N169P , S171M, S171Q, S171V, V172A, V172C, V172E, V172 , V172M, V172S, V172W, V172Y, A180G, V329Q, A331C, A331D, A331I, A331K, A331L, A331M, Q331V, K369R, K471R, V551A, V551C, V551T, V551T, and V551T FADGDH.

  Here, “K120E” means that K (Lys) at position 120 is replaced with E (Glu).

  In particular, G163K, G163L, G163R, S167P, V551A, V551C, V551Q, V551S, V551Y, (G160I + S167P), (S162F + S167P), (S167P + N169Y), (S167P + L171I), S17P + L171I), S17P + L171I) ), (G163K + V551C) (G163R + V551C) amino acid substitution contributes to the improvement of the thermal stability of the modified FADGDH.

  Further, in the amino acid sequence of SEQ ID NO: 46, amino acid substitution at at least one of positions 116, 159, 161, 164, 166, 167, 175, 325, 327, 365, and 547 The modified FADGDH having

  Preferably, in the amino acid sequence of SEQ ID NO: 46, the amino acid substitution is K116D, K116G, K116L, K116F, K116Q, Q159A, Q159K, Q159N, Q159P, Q159V, Q159L, E161C, N164Y, N164V, N164C, T166F, T166T, T166L, , T167V, T167S, G175K, S325A, S325G, S325K, S325Q, S325R, S325T, S325V, S325Y, S327E, Q365R, V547S, V547C, V547A, V547Q.

  Here, “K116D” means that K (Lys) at position 116 is replaced with D (Asp).

  Method for producing modified FADGDH obtained by modifying FADGDH derived from wild-type Aspergillus oryzae represented by SEQ ID NO: 2 or method for producing modified FADGDH obtained by modifying FADGDH derived from wild-type Aspergillus tereus represented by SEQ ID NO: 46 Is not particularly limited, but can be manufactured by the following procedure. As a method for modifying the amino acid sequence constituting FADGDH, a commonly performed technique for modifying genetic information is used. That is, 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 a base sequence in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, Quick Change Site Directed; Alternatively, the polymerase chain reaction (PCR) can be used.

  The prepared DNA having the genetic information of the modified FADGDH is transferred to a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified FADGDH. As the plasmid at this time, for example, Escherichia coli JM109, Escherichia coli DH5, Escherichia coli W3110, Escherichia coli C600, 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 a microorganism belonging to Escherichia coli, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. A poration method may be used. Furthermore, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

  The microorganism, which is the transformant thus obtained, can stably produce a large amount of modified FADGDH 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. Usually, the culture is performed in liquid culture, but industrially, aeration and agitation culture is performed. Is advantageous. As a nutrient source of the medium, those commonly used for culturing microorganisms are widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. Any nitrogen compound that can be used may be used for reducing nitrogen, such as peptone, meat extract, yeast extract, casein hydrolyzed monster, soybean cake alkaline decomposition product, and the like. 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 medium temperature can be changed as appropriate within the range in which the bacteria grow and produce modified FADGDH, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culture temperature varies somewhat depending on conditions, the culture may be terminated at an appropriate time in consideration of the time when the modified FADGDH reaches the maximum yield, and is usually about 6 to 48 hours. The medium pH can be appropriately changed within the range in which the bacteria grow and produce the modified protein, but is preferably about pH 6.0 to 9.0.

  Although the culture solution containing the cells producing the modified FADGDH in the culture can be collected and used as it is, generally, when the modified FADGDH is present in the culture solution according to a conventional method, filtration, centrifugation, etc. Thus, it is used after separating the modified protein-containing solution and the microbial cells. When the modified protein is present in the microbial cell, the microbial cell is collected from the obtained culture by a technique such as filtration or centrifugation, and then the microbial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, If necessary, a modified FADGDH is solubilized by adding a chelating agent such as EDTA and / or a surfactant, and separated and collected as an aqueous solution.

  The modified FADGDH-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or fractionation with a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. What is necessary is just to precipitate by the precipitation method. Further, heating processing and east power point processing are also effective generation means. Purified modified FADGDH can be obtained by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

Glucose assay kit The invention also features a glucose assay kit comprising a modified FADGDH according to the invention. The glucose assay kit of the present invention comprises a modified FADGDH according to the present invention in an amount sufficient for at least one assay. Typically, the kit contains, in addition to the modified FADGDH 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 FADGDH according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

Glucose Sensor The present invention also features a glucose sensor that uses a modified FADGDH 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. Typically, the modified FADGDH 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. Put buffer in constant temperature cell and maintain at constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the modified FADGDH of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an 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.

In the present invention, the activity of FAD-dependent GDH is measured under the following conditions.
[Test example]
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
163 mM PMS solution 6.8 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 15.6 ml of the above PIPES buffer solution, 0.2 ml of DCPIP solution and 4 ml of D-glucose solution are used as reaction reagents. .
<Measurement conditions>
Prewarm 2.9 ml of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 ml of GDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control, and the absorbance change per minute (ΔOD _TEST) ). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in GDH activity is defined as the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of 200 mM D-glucose.

Activity (U / ml) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.0 is the amount of the reaction reagent + enzyme solution (ml), 16.3 is the molar molecular extinction coefficient (cm ² / micromol) under the conditions for this activity measurement, and 0.1 is the enzyme solution solution. Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

Example 1: Examination of modified FADGDH thermal stability using glucose measurement system The examination was carried out in accordance with the method for measuring FADGDH activity in the test example described above.
First, Aspergillus oryzae or Aspergillus terreus-derived modified FADGDH was dissolved in an enzyme diluent (50 mM potassium phosphate buffer (pH 5.5), 0.1% Triton X-100) so as to be about 2 U / ml. 50 ml of the product was prepared. Two pieces of this enzyme solution in 1.0 ml were prepared. For the control, two modified FADGDHs (in which 0.1 ml of distilled water was added instead of various compounds) were prepared.

  Of the two, one was stored at 4 ° C and the other was treated at 50 ° C for 15 minutes. After treatment, the FADGDH activity of each sample was measured. The activity values after treatment at 50 ° C. for 15 minutes were compared and calculated as the residual activity rate (%), with the enzyme activity of those stored at 4 ° C. as 100, respectively.

Example 2: Mutation introduction into the FADGDH gene derived from Aspergillus oryzae
After transforming a commercially available E. coli competent cell (E. coli DH5a; manufactured by TOYOBO) with a recombinant plasmid pAOGDH-S2 containing a gene encoding wild-type FADGDH (SEQ ID NO: 1), the transformant was ampicillin (50 mg / mg). obtained by inoculating a liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) containing 1 ml of Nacalai Tesque and shaking overnight at 30 ° C. Plasmids were prepared from the microbial cells by a conventional method. Mutation treatment using DiversifyTM PCR Random Mutagenesis Kit (Clontech) was performed according to the protocol using the plasmid as a template to produce a modified FADGDH mutant plasmid having the ability to produce glucose dehydrogenase. Was prepared.

Example 3: Preparation of crude enzyme solution containing modified FADGDH derived from Aspergillus oryzae A commercially available E. coli competent cell (E. coli DH5a; manufactured by TOYOBO) was transformed with the plasmid prepared in Example 2. Thereafter, the transformant was applied to an agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar; pH 7.3) containing ampicillin, and then overnight at 30 ° C. The colony obtained by shaking culture was further taken into an LB liquid medium containing ampicillin (100 μg / ml) and cultured overnight at 30 ° C. with shaking. The bacterial cells obtained by centrifugation were collected from a part of the culture solution, and the bacterial cells were disrupted with glass beads in 50 mM phosphate buffer (pH 7.0) to prepare a crude enzyme solution.

Example 4: Screening for mutants with improved thermostability Glucose dehydrogenase activity was measured using the crude enzyme solution of Example 3 by the activity measurement method described above. In addition, the crude enzyme solution was heat-treated at 50 ° C. for 15 minutes, and then glucose dehydrogenase activity was measured to obtain three variants with improved thermostability. Plasmids encoding these three variants were named pAOGDH-M1, pAOGDH-M2, pAOGDH-M3, and pAOGDH-M4.

  In order to identify the mutation site of pAOGDH-M1, pAOGDH-M2, pAOGDH-M3, and pAOGDH-M4, the base sequence of the gene encoding the glucose dehydrogenase was determined using a DNA sequencer (ABI PRISMTM 3700 DNA Analyzer; manufactured by Perkin-Elmer). In pAOGDH-M1, the 162nd serine described in SEQ ID NO: 2 is proline, in pAOGDH-M2, the 167th serine is proline, the 471st lysine is arginine, and in pAOGDH-M3, the 180th alanine is glycine and the 551th valine. In alanine and pAOGDH-M4, it was confirmed that the 120th lysine was substituted with glutamic acid, the 167th serine with proline, and the 369th lysine with arginine. The results are shown in Table 1.

  Using the pAOGDH-S2 plasmid as a template, the synthetic oligonucleotide of SEQ ID NO: 3, designed to replace the 160th glycine with a plurality of amino acids, a synthetic oligonucleotide complementary thereto, and the 161st tryptophan into a plurality of amino acids Synthetic oligonucleotide of SEQ ID NO: 4 designed to be substituted and a synthetic oligonucleotide complementary thereto, synthetic oligonucleotide of SEQ ID NO: 5 designed to substitute the 162nd serine with a plurality of amino acids, and a synthetic oligo complementary thereto A synthetic oligonucleotide of SEQ ID NO: 6 designed to replace the nucleotide, 163rd glycine with multiple types of amino acids, and a synthetic oligonucleotide complementary thereto, and a SEQ ID NO: designed to replace the 164th serine with multiple types of amino acids 7 synthetic oligonucleotides Synthetic oligonucleotide of SEQ ID NO: 8 designed to replace leotide and its complementary synthetic oligonucleotide, 165th leucine with plural kinds of amino acids and synthetic oligonucleotide complementary thereto, and 166th alanine as plural kinds of amino acids Synthetic oligonucleotide of SEQ ID NO: 9 designed to substitute for and synthetic oligonucleotide complementary thereto, synthetic oligonucleotide of SEQ ID NO: 10 designed to substitute the 167th serine with a plurality of amino acids, and synthesis complementary thereto Oligonucleotide, synthetic oligonucleotide of SEQ ID NO: 11 designed to replace 168th glycine with plural kinds of amino acids and synthetic oligonucleotide complementary thereto, sequence designed to substitute 169th asparagine with plural kinds of amino acids Number 12 Synthetic oligonucleotide and complementary synthetic oligonucleotide, synthetic oligonucleotide of SEQ ID NO: 13 designed to replace 170th leucine with multiple amino acids, synthetic oligonucleotide complementary thereto, and 171st serine The synthetic oligonucleotide of SEQ ID NO: 14 designed to be substituted with the amino acid of SEQ ID NO: 14 and a complementary synthetic oligonucleotide thereof, and the synthetic oligonucleotide of SEQ ID NO: 15 designed to substitute the 172nd valine with a plurality of amino acids and complementary thereto Synthetic oligonucleotide, designed to replace the 329th valine with a plurality of amino acids, the synthetic oligonucleotide of SEQ ID NO: 16 and a synthetic oligonucleotide complementary thereto, the 330th leucine with a plurality of amino acids Sequence number 17 synthetic oligonucleotides and synthetic oligonucleotides complementary thereto, 331st alanine designed to substitute a plurality of amino acids, SEQ ID NO: 18 synthetic oligonucleotide, 551th valine to be substituted with a plurality of amino acids Based on the designed synthetic oligonucleotide of SEQ ID NO: 19 and a synthetic oligonucleotide complementary thereto, QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) is used to perform a mutation operation according to the protocol, and has the ability to produce glucose dehydrogenase A modified FADGDH mutant plasmid was prepared, and the plasmid was prepared in the same manner as described above.

  After transforming a commercially available E. coli competent cell (E. coli DH5a; manufactured by TOYOBO) with the plasmid prepared in Example 4, a crude enzyme solution was prepared in the same manner as in Example 3.

  Using the above crude enzyme solution, glucose dehydrogenase activity was measured by the activity measurement method described above. The crude enzyme solution was heat-treated at 50 ° C. for 15 minutes, and then glucose dehydrogenase activity was measured to obtain 16 types of mutants with improved thermostability. Plasmids encoding these 16 variants were pAOGDH-M4, pAOGDH-M5, pAOGDH-M6, pAOGDH-M7, pAOGDH-M8, pAOGDH-M9, pAOGDH-M10, pAOGDH-M11, pAODH-M11, pAODH They were named as M13, pAOGDH-M14, pAOGDH-M15, pAOGDH-M16, pAOGDH-M17, pAOGDH-M18, and pAOGDH-M19.

  pAOGDH-M4, pAOGDH-M5, pAOGDH-M6, pAOGDH-M7, pAOGDH-M8, pAOGDH-M9, pAOGDH-M10, pAOGDH-M11, pAOGDH-M12, pAOGDH14-M12, pAOGDH-M12 As a result of determining the nucleotide sequence of the gene encoding glucose dehydrogenase with DNA sequencer (ABI PRISMTM 3700 DNA Analyzer; manufactured by Perkin-Elmer) to identify the mutation site of M16, pAOGDH-M17, pAOGDH-M18, and pAOGDH-M19, DAO -In M5, the 160th glycine described in SEQ ID NO: 2 is proline, and in pAOGDH-M6, the 163rd glycine is lysine, pA In GDH-M7, 163rd glycine is leucine, in pAOGDH-M8, 163rd glycine is arginine, in pAOGDH-M9, 167th serine is alanine, in pAOGDH-M10, 167th serine is proline, and 167th in pAOGDH-M11. Serine is arginine, pAOGDH-M12 has 167th serine as valine, pAOGDH-M13 has 171st serine as proline, pAOGDH-M14 has 551st valine as alanine, pAOGDH-M15 has 551st valine as cysteine, pAOGDH In M16, the 551st valine is threonine, in pAOGDH-M17, the 551st valine is glutamine, in pAOGDH-M18, the 551st valine is serine, pAOGDH-M1. In 551 valine it was confirmed to be substituted with tyrosine. The results are shown in Table 2.

Example 5: Production of multiple mutants and thermostability Using a plasmid of pAOGDH-M10 as a template, a synthetic oligonucleotide of SEQ ID NO: 20 designed to replace the 160th glycine with a plurality of amino acids and a complementary synthesis thereto Oligonucleotide, synthetic oligonucleotide of SEQ ID NO: 21 designed to replace the 161st tryptophan with multiple types of amino acids and a synthetic oligonucleotide complementary thereto, sequence designed to replace the 162nd serine with multiple types of amino acids Synthetic oligonucleotide No. 22 and complementary synthetic oligonucleotide, synthetic oligonucleotide SEQ ID No. 23 designed to replace the 163rd glycine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, 164th serine Multiple amino acids A synthetic oligonucleotide of SEQ ID NO: 24 designed to be substituted and a synthetic oligonucleotide complementary thereto, and a synthetic oligonucleotide of SEQ ID NO: 25 designed to substitute the 165th leucine with a plurality of amino acids and a synthetic oligo complementary thereto A synthetic oligonucleotide of SEQ ID NO: 26 designed to substitute nucleotides, 166th alanine with plural kinds of amino acids, and a synthetic oligonucleotide complementary thereto, and SEQ ID NO: designed to substitute 168th glycine with plural kinds of amino acids 27 synthetic oligonucleotides and synthetic oligonucleotides complementary thereto, the synthetic oligonucleotide of SEQ ID NO: 28 designed to replace the 169th asparagine with a plurality of amino acids, the synthetic oligonucleotide complementary thereto, and the 170th leucine A synthetic oligonucleotide of SEQ ID NO: 29 designed to be substituted with a plurality of types of amino acids and a synthetic oligonucleotide complementary thereto, a synthetic oligonucleotide of SEQ ID NO: 30 designed to be substituted with a plurality of types of amino acids, and a synthetic oligonucleotide of SEQ ID NO: 30 Complementary synthetic oligonucleotide, the synthetic oligonucleotide of SEQ ID NO: 31 designed to replace the 172nd valine with a plurality of types of amino acids and the synthetic oligonucleotide complementary thereto, the 329th valine with a plurality of types of amino acids The synthetic oligonucleotide of SEQ ID NO: 32 designed in this way and a synthetic oligonucleotide complementary thereto, the synthetic oligonucleotide of SEQ ID NO: 33 designed to substitute the 330th leucine with a plurality of amino acids, and a synthetic oligonucleotide complementary thereto, 331st a Synthetic oligonucleotide of SEQ ID NO: 34 designed to replace lanine with multiple types of amino acids, synthetic oligonucleotide of SEQ ID NO: 35 designed to replace 551th valine with multiple types of amino acids and synthetic oligonucleotides complementary thereto Based on the above, a change FADGDH mutant plasmid having the ability to produce glucose dehydrogenase is prepared according to the protocol using QuickChangeTMSite-Directed Mutagenesis Kit (manufactured by STRATAGENE), and the plasmid is similarly prepared by the above method. Prepared.

  Using the plasmid of pAOGDH-M15 as a template, the QuickChangeTM Site-Directed Mutagenesis Kit (STRATAGENE) based on the synthetic oligonucleotide of SEQ ID NO: 36 designed to replace the 163rd glycine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto. And a modified FADGDH mutant plasmid having the ability to produce glucose dehydrogenase was prepared according to the protocol, and the plasmid was similarly prepared by the above method.

  After transforming a commercially available E. coli competent cell (E. coli DH5a; manufactured by TOYOBO) with the plasmid prepared in Example 4, a crude enzyme solution was prepared in the same manner as in Example 3.

  Using the above crude enzyme solution, glucose dehydrogenase activity was measured by the activity measurement method described above. The crude enzyme solution was heat-treated at 50 ° C. for 15 minutes, and then glucose dehydrogenase activity was measured to obtain 57 variants with improved thermostability. Plasmids encoding these 57 variants were designated as pAOGDH-M20, pAOGDH-M21, pAOGDH-M22, pAOGDH-M23, pAOGDH-M24, pAOGDH-M25, pAOGDH-M26, pAOGDH-M27, pAODH-M27, pAODH M29, pAOGDH-M30, pAOGDH-M31, pAOGDH-M32, pAOGDH-M33, pAOGDH-M34, pAOGDH-M35, pAOGDH-M36, pAOGDH-M37, pAOGDH-M38, pAOGDH-M38, pAOGDH-M38, pAOGDH-M38, pAOGDH-M38 -M42, pAOGDH-M43, pAOGDH-M44, pAOGDH-M45, pAOGDH-M46, pAOGDH-M47, pA GDH-M48, pAOGDH-M49, pAOGDH-M50, pAOGDH-M51, pAOGDH-M52, pAOGDH-M53, pAOGDH-M54, pAOGDH-M55, pAOGDH-M56, pAOGDH-M57, pAOGDH-M57, pAOGDH-M57 M60, pAOGDH-M61, pAOGDH-M62, pAOGDH-M63, pAOGDH-M64, pAOGDH-M65, pAOGDH-M66, pAOGDH-M67, pAOGDH-M68, pAOGDH-M69, pAOGDH-M69, pAOGDH-M69, pAOGDH-M69, pAOGDH-M69, pAOGDH-M69 They were named as pAOGDH-M73, pAOGDH-M74, pAOGDH-M75, and pAOGDH-M76.

  pAOGDH-M20, pAOGDH-M21, pAOGDH-M22, pAOGDH-M23, pAOGDH-M24, pAOGDH-M25, pAOGDH-M26, pAOGDH-M27, pAOGDH-M28, pAHGDH-M28, pAOGDH30 M32, pAOGDH-M33, pAOGDH-M34, pAOGDH-M35, pAOGDH-M36, pAOGDH-M37, pAOGDH-M38, pAOGDH-M39, pAOGDH-M40, pAOGDH-M41, MDA44H pAOGDH-M45, pAOGDH-M46, pAOGDH-M47, pAOGDH-M48, pAOGDH-M49, pAO DH-M50, pAOGDH-M51, pAOGDH-M52, pAOGDH-M53, pAOGDH-M54, pAOGDH-M55, pAOGDH-M56, pAOGDH-M57, pAOGDH-M59, pAOGDH-M59, pAOGDH-M59, pAOGDH-M59, pAOGDH-M59 M62, pAOGDH-M63, pAOGDH-M64, pAOGDH-M65, pAOGDH-M66, pAOGDH-M67, pAOGDH-M68, pAOGDH-M69, pAOGDH-M70, pAOGDH-M71, pAOGDH-M71, pAOGDH-M71, pAOGDH-M71 DNA sequencer (ABI PRISMTM 3700D) was used to identify the mutation sites of pAOGDH-M75 and pAOGDH-M76. As a result of determining the base sequence of the gene encoding glucose dehydrogenase using A Analyzer (manufactured by Perkin-Elmer), the 160th glycine described in SEQ ID NO: 2 is glutamic acid in pAOGDH-M20, the 167th serine is proline, and the pAOGDH-M21 The 160th glycine is isoleucine, the 167th serine is proline, the pAOGDH-M22 is the 160th glycine is the serine glutamine, the 167th serine is the proline, and the pAOGDH-M23 is the 160th glycine is the glutamine and the 167th serine is the proline In pAOGDH-M24, 162nd serine is alanine and 167th serine is proline, and in pAOGDH-M25, 162nd serine is cysteine and 167th serine is proline. In pAOGDH-M26, the 162nd serine is aspartic acid and the 167th serine is proline, in pAOGDH-M27 the 162nd serine is glutamic acid and the 167th serine is proline, and in pAOGDH-M28 the 162nd serine is phenylalanine. The 167th serine is proline, in pAOGDH-M29, the 162nd serine is histidine and the 167th serine is proline, in pAOGDH-M30, the 162nd serine is leucine and the 167th serine is proline, and in pAOGDH-M31 is the 163rd serine. Glycine is aspartic acid and the 167th serine is proline. In pAOGDH-M32, the 164th serine is phenylalanine and the 167th serine is proline. In pAOGDH-M33, the 164th serine is Phosphorus is threonine, 167th serine is proline, pAOGDH-M34 is 164th serine is tyrosine and 167th serine is proline, 165th leucine is alanine and 167th serine is proline, pAOGDH-M36 The 165th leucine is isoleucine, the 167th serine is proline, the pAOGDH-M37 is the 165th leucine asparagine, the 167th serine is proline, and the pAOGDH-M38 is the 165th leucine is proline and the 167th serine is proline. In pAOGDH-M39, the 165th leucine is valine and the 167th serine is proline, and in pAOGDH-M40, the 166th alanine is cysteine and the 167th serine is proline, pAOGD -In M41, 166th alanine is isoleucine and 167th serine is proline, in pAOGDH-M42, 166th alanine is lysine and 167th serine is proline, and in pAOGDH-M43, 166th alanine is leucine and 167th serine Is proline, in pAOGDH-M44, the 166th alanine is methionine, the 167th serine is proline, in the pAOGDH-M45, the 166th alanine is proline, the 167th serine is proline, and in the pAOGDH-M46, the 166th alanine is serine 167th serine is proline, pAOGDH-M47 is 167th serine as proline and 169th asparagine is lysine, and pAOGDH-M48 is 167th serine as proline. Lagine is proline, in pAOGDH-M49, the 167th serine is proline in the proline, the 169th asparagine is tyrosine, in the pAOGDH-M50, the 167th serine is proline, the 169th asparagine is tryptophan, and in the pAOGDH-M51, the 167th serine is proline. 170th leucine is cysteine, 167th serine is proline in pAOGDH-M52, 170th leucine is phenylalanine, 167th serine is proline in pAOGDH-M53, 171th leucine is isoleucine, 167 in pAOGDH-M54 The serine is proline and the 171st leucine is lysine. In pAOGDH-M55, the 167th serine is proline and the 171st leucine is methionine, and pAOGDH-M. In 56, the 167th serine is proline and the 171st leucine is glutamine. In the pAOGDH-M57, the 167th serine is proline and the 171st leucine is valine. In the pAOGDH-M58, the 167th serine is proline and the 172nd valine is In alanine and pAOGDH-M59, the 167th serine is proline and the 172nd valine is cysteine, in pAOGDH-M60, the 167th serine is proline and the 172nd valine is glutamic acid, and in pAOGDH-M61, the 167th serine is proline. The 172nd valine is isoleucine. In pAOGDH-M62, the 167th serine is proline and the 172nd valine is methionine. In the pAOGDH-M63, the 167th serine is proline and the 172nd valine is Thein, pAOGDH-M64, 167th serine is proline, 172nd valine is glutamic acid, pAOGDH-M65, 167th serine is proline, 172nd valine is tryptophan, and pAOGDH-M66, 167th serine is proline The valine is tyrosine, pAOGDH-M67 is 167th serine to proline, 329th valine is glutamine, pAOGDH-M68 is 167th serine to proline, 331st alanine is cysteine, and pAOGDH-M69 is 167th Serine is proline and 331st alanine is aspartic acid. In pAOGDH-M70, 167th serine is proline and 331st alanine is isoleucine, and 167th in pAOGDH-M71. Phosphorus is proline, 331st alanine is lysine pAOGDH-M72, 167th serine is proline, 331st alanine is leucine, AOGDH-M73 is 167th serine is proline, 331st alanine is methionine, pAOGDH-M74 It was confirmed that the 167th serine was replaced with proline and the 331st alanine was replaced with valine. The results are shown in Table 3.

Example 6: Acquisition of modified FADGDH derived from Aspergillus oryzae As modified FADGDH-producing bacteria, commercially available E. coli competent cells (p. DH5a (manufactured by TOYOBO) was transformed. The obtained transformant was cultured in a TB medium at a culture temperature of 25 ° C. for 24 hours using a 10 L jar fermenter. The cultured cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 6.5), treated with nucleic acid, and centrifuged to obtain a supernatant. A saturated amount of ammonium sulfate was dissolved therein to precipitate the target protein, and the precipitate collected by centrifugation was redissolved in 50 mM phosphate buffer (pH 6.5). Then, gel filtration using a G-25 Sepharose column, hydrophobic chromatography using an Octyl-Sepharose column and a Phenyl-Sepharose column (both elution conditions were extracted with a 25% saturated to 0% ammonium sulfate concentration gradient), and Ammonium sulfate was removed by gel filtration using a G-25 Sepharose column to obtain a modified FADGDH sample. As shown in Table 4, it was confirmed that the thermal stability of the purified sample was also improved.

Example 7: pH stability In order to know the pH stability of the purified preparation obtained in Example 6, a buffer solution in the range of pH 3.5 to 8.5 (pH 3.5 to 6.3: 0.1 M acetic acid) Buffer, pH 6.3 to 7.3, 0.1 M PIPES buffer, pH 7.3 to 8.5: 0.1 M Tris-HCl buffer, pH 6.0 to 7.7: 0.1 M phosphate buffer) Using these, each GDH was diluted to an enzyme concentration of 1 U / ml. The diluted solution was incubated at 25 ° C. for 16 hours to compare the activity before and after the incubation. A graph showing the activity remaining rate after incubation with respect to the activity before incubation is shown in FIG. As shown in FIG. 1, it was confirmed that the width of the pH stable region was widened.

Example 8: Mutation introduction into the FADGDH gene derived from Aspergillus tereus
We succeeded in cloning the GDH gene derived from Aspergillus oryzae and obtained the base sequence information. In addition, screening using thermal stability as an index is performed to identify amino acid sites that are effective in improving thermal stability. Therefore, the predicted amino acid sequence of Aspergillus oryzae-derived GDH is aligned with the predicted amino acid sequence of Aspergillus oryzae-derived GDH, and in Aspergillus oryzae-derived GDH, it corresponds to a site that was effective in improving thermal stability in Aspergillus oryzae FADGDH. Amino residues were identified.

  A synthetic oligonucleotide designed to substitute the 116th lysine with a plurality of amino acids using the recombinant plasmid pAtGDH-s2-7 prepared in Experimental Example 6 as a template, a synthetic oligonucleotide complementary thereto, and a 159th glutamine A synthetic oligonucleotide designed to substitute a plurality of amino acids with a synthetic oligonucleotide complementary thereto, a synthetic oligonucleotide designed to substitute the 161st glutamic acid with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, Synthetic oligonucleotide designed to replace the 164th asparagine with multiple amino acids and its complementary synthetic oligonucleotide, and complementary to the synthetic oligonucleotide designed to replace the 166th threonine with multiple amino acids Synthetic oligonucleotide, synthetic oligonucleotide designed to substitute the 167th threonine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, and synthetic oligonucleotide designed to substitute the 175th glycine with a plurality of amino acids Synthetic oligonucleotide designed to replace 325th serine with multiple amino acids and synthetic oligonucleotide complementary to it, designed to replace 327th serine with multiple amino acids Synthetic oligonucleotide designed to replace the 365th glutamine with a plurality of amino acids, a complementary oligonucleotide complementary thereto, and a 547th valine A synthetic oligonucleotide designed to be substituted with a mino acid and a complementary synthetic oligonucleotide thereof were subjected to a mutation operation using the QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) according to the protocol, and a commercially available E. coli competent cell ( E. coli DH5a (TOYOBO) was transformed and applied to an agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar; pH 7.3) containing ampicillin. And then cultured at 30 ° C. overnight.

Example 9: Preparation of crude enzyme solution containing modified FADGDH derived from Aspergillus terreus The obtained colonies were further taken into an LB liquid medium containing ampicillin (100 µg / ml) and cultured with shaking at 30 ° C overnight. The bacterial cells obtained by centrifugation were collected from a part of the culture solution, and the bacterial cells were disrupted with glass beads in 50 mM phosphate buffer (pH 7.0) to prepare a crude enzyme solution.

Example 10: Screening of mutants with improved thermostability Using the crude enzyme solution of Example 9, glucose dehydrogenase activity was measured by the activity measurement method described above. The crude enzyme solution was heat-treated at 50 ° C. for 15 minutes, and then glucose dehydrogenase activity was measured.

  The sequence of these FADGDH gene sequences was confirmed using a DNA sequencer (ABI PRISM ™ 3700 DNA Analyzer; manufactured by Perkin-Elmer). Table 5 shows the residual activity ratio (%) after the heat treatment relative to the activity before the heat treatment. From this result, it was possible to obtain Aspergillus terreus-derived glucose dehydrogenase with improved thermal stability using a recombinant.

Moreover, about the substrate specificity with respect to various saccharides, the enzyme activity was measured according to said activity measuring method. The dehydrogenase activity value when glucose was used as the substrate solution and the dehydrogenase activity value when the saccharide (for example, maltose) of the same molar concentration was used as the substrate solution instead were measured and glucose was used as the substrate. The relative value when the measured value in this case was 100 was determined.

  The substrate specificity of the various mutants obtained above was good.

Example 11: Mutagenesis into FADGDH gene Transformation of commercially available E. coli competent cells (E. coli DH5; manufactured by TOYOBO) with a recombinant plasmid pAOGDH-S2 containing a gene (SEQ ID NO: 1) encoding wild type FADGDH After that, the transformant was fed with a liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) containing ampicillin (50 g / ml; manufactured by Nacalai Tesque). Plasmids were prepared from cells obtained by culturing overnight at 30 ° C. by a conventional method. Using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) based on the synthetic oligonucleotide of SEQ ID NO: 49 designed to replace the 53rd glycine with a plurality of amino acids using the plasmid as a template Then, according to the protocol, a mutation operation was performed to prepare a modified FADGDH mutant plasmid having the ability to produce glucose dehydrogenase, and the plasmid was similarly prepared by the above method.

Example 12 Preparation of Crude Enzyme Solution Containing Modified FADGDH Plasmid pAOGDH-S2 prepared in Example 2 was used to transform a commercially available E. coli competent cell (E. coli DH5; manufactured by TOYOBO), and then transformed. The body was applied to an agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar; pH 7.3) containing ampicillin and then cultured overnight at 30 ° C. with shaking. The obtained colonies were further taken into an LB liquid medium containing ampicillin (100 μg / ml) and cultured with shaking at 30 ° C. overnight. The bacterial cells obtained by centrifugation were collected from a part of the culture solution, and the bacterial cells were disrupted with glass beads in 50 mM phosphate buffer (pH 7.0) to prepare a crude enzyme solution.

Example 13: Screening of mutants with improved substrate specificity Using the crude enzyme solution of Example 3, the activity was measured using glucose and xylose as substrates by the activity measuring method described above. Improved mutants were obtained. Plasmids encoding these seven variants were designated as pAOGDH-M1, pAOGDH-M2, pAOGDH-M3, pAOGDH-M4, pAOGDH-M5, pAOGDH-M6, and pAOGDH-M7.

  pAOGDH-M1, pAOGDH-M2, pAOGDH-M3, pAOGDH-M4, pAOGDH-M5, pAOGDH-M6, pAOGDH-M7 DNA sequencer (ABI PRISMTMlerMelE 3mDNAerP As a result of determining the base sequence of the gene encoding dehydrogenase, the 53rd glycine described in SEQ ID NO: 2 is cysteine in pAOGDH-M1, the 53rd glycine described in SEQ ID NO: 2 is histidine in pAOGDH-M2, and the sequence is histidine in pAOGDH-M3. The 53rd glycine described in No. 2 is lysine, pAOGDH-M4 is 53th glycine described in SEQ ID NO: 2 is methionine, and pAOGDH-M5 is 53rd glycine described in SEQ ID NO: 2. Asparagine, 53 th glycine of SEQ ID NO: 2 described pAOGDH-M6 threonine, 53 th glycine of SEQ ID NO: 2 described pAOGDH-M7 it was confirmed to be replaced by valine. The results are shown in Table 1.

Example 14: Preparation of a mutant with improved substrate specificity and / or thermal stability Using the plasmid pAOGDH-M2 prepared in Example 13 as a template, SEQ ID NO: designed to replace the 164th serine with proline 50 synthetic oligonucleotides, synthetic oligonucleotides complementary thereto, pAOGDH-M5 as a template, the synthetic oligonucleotide of SEQ ID NO: 50 designed to replace the 164th serine with proline, and the complementary synthetic oligonucleotides The synthetic oligonucleotide of SEQ ID NO: 51, which was designed to replace 163rd glycine with arginine, using pAOGDH-M2 as a template, and a synthetic oligonucleotide complementary thereto, and further designed to replace 551th valine with cysteine. Synthetic oligonucleotide of SEQ ID NO: 52 A modified FADGDH having excellent substrate specificity and / or thermal stability is prepared by performing a mutation operation in the same manner as in the method of Example 2 on the basis of leotide and a synthetic oligonucleotide complementary thereto. The plasmid was prepared in the same manner as above. In order to identify the mutation site, the nucleotide sequence of the gene encoding glucose dehydrogenase was determined with a DNA sequencer (ABI PRISMTM 3700 DNA Analyzer; manufactured by Perkin-Elmer) in the same manner as in Example 4. As a result, pAOGDH-M8 described SEQ ID NO: 2 The 53rd glycine in histidine, the 167th serine in proline, pAOGDH-M9 described in SEQ ID NO: 2 as 53rd glycine in asparagine, the 167th serine in proline, and pAOGDH-M10 as described in SEQ ID NO: 2 It was confirmed that the 53rd glycine was replaced with asparagine, the 163rd glycine was replaced with arginine, and the 551st valine was replaced with cysteine. When activity was measured using glucose and xylose as substrates by the same activity measurement method as in Example 4, it was confirmed that pAOGDH-M8, pAOGDH-M9, and pAOGDH-M10 had improved substrate specificity. In order to measure thermal stability, the crude enzyme solutions of pAOGDH-M8, pAOGDH-M9, and pAOGDH-M10 were prepared in the same manner as in Example 3, and glucose dehydrogenase activity was measured by the activity measurement method described above. The crude enzyme solution was heat treated at 50 ° C. for 15 minutes, and then glucose dehydrogenase activity was measured. It was confirmed that pAOGDH-M8, pAOGDH-M9, and pAOGDH-M10 have improved thermal stability. The results are shown in Table 2.

Example 15: Obtaining modified FADGDH As a modified FADGDH producing bacterium, commercially available E. coli competent cells (E. coli DH5; manufactured by TOYOBO) were transformed with pAOGDH-M8, pAOGDH-M9, and pAOGDH-M10. The obtained transformant was cultured in a TB medium at a culture temperature of 25 ° C. for 24 hours using a 10 L jar fermenter. The cultured cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 6.5), treated with nucleic acid, and centrifuged to obtain a supernatant. A saturated amount of ammonium sulfate was dissolved therein to precipitate the target protein, and the precipitate collected by centrifugation was redissolved in 50 mM phosphate buffer (pH 6.5). Then, gel filtration using a G-25 Sepharose column, hydrophobic chromatography using an Octyl-Sepharose column and a Phenyl-Sepharose column (both elution conditions were extracted with a 25% saturated to 0% ammonium sulfate concentration gradient), and Ammonium sulfate was removed by gel filtration using a G-25 Sepharose column to obtain a modified FADGDH sample. As shown in Table 3, it was confirmed that the thermal stability of the purified sample was also improved.

  According to the present invention, amino acid residues that are involved in improving thermal stability in FAD-GDH can be found, and it has been shown that this can be applied to FAD-GDH of all genera and species. In addition, the improved stability of FADGDH according to the present invention reduces the heat inactivation of the enzyme during the preparation of glucose measurement reagents, glucose assay kits, and glucose sensors, thereby reducing the amount of enzyme used and improving the measurement accuracy. It is a great place to contribute to industries such as medical-related fields.

Claims (10)

A modified FADGDH having improved thermostability as compared with a wild-type Aspergillus flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), wherein an amino acid substitution is performed at any one of the following positions (1) to (3): A modified FADGDH having
(1) In the amino acid sequence of SEQ ID NO: 2, positions 120, 160, 162, 163, 164, 165, 166, 167, 169, 170, 171, 172, 180, At least one position of the group consisting of the positions 329, 331, 369, 471, and 551.
(2) The position of the amino acid in SEQ ID NO: 46 identified by aligning the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 46, wherein positions 120, 160, 162, 163 in SEQ ID NO: 2 1st, 164th, 165th, 166th, 167th, 169th, 170th, 170th, 171st, 172th, 180th, 329th, 331st, 369th, 369th, 471st and 551th At least one location corresponding to.
(3) Amino acid sequences other than SEQ ID NO: 2 and SEQ ID NO: 46, which are identified by aligning the sequence and the amino acid sequence of SEQ ID NO: 2, positions 120, 160, 162 in the amino acid sequence of SEQ ID NO: 2 163rd, 164th, 165th, 166th, 167th, 169th, 170th, 171st, 172th, 180th, 329th, 331st, 369th, 369th, 471st and 551th A position equivalent to one of them. In the amino acid sequence of SEQ ID NO: 46, any one of the group consisting of positions 116, 159, 161, 164, 166, 167, 175, 325, 327, 365, and 547, or 2. The modified FADGDH of claim 1 having an amino acid substitution at an equivalent position. The modified FADGDH according to claim 2, comprising an amino acid sequence having any of the following mutations (1) to (11) in the amino acid sequence of SEQ ID NO: 46.
(1) Lysine (K) at position 116 is substituted with aspartic acid (D), glycine (G), leucine (L), phenylalanine (F) or glutamine (Q).
(2) Glutamine (Q) at position 159 is substituted with alanine (A), lysine (K), asparagine (N), proline (P), valine (V) or leucine (L).
(3) Glutamic acid (E) at position 161 is substituted with cysteine (C).
(4) Asparagine (N) at position 164 is substituted with tyrosine (Y), valine (V) or cysteine (C).
(5) Threonine (T) at position 166 is substituted with phenylalanine (F), tyrosine (Y) or tryptophan (W).
(6) Threonine (T) at position 167 is substituted with leucine (L), valine (V) or serine (S).
(7) Glycine (G) at position 175 is substituted with lysine (K).
(8) Serine (S) at position 325 becomes alanine (A), glycine (G), lysine (K) glutamine (Q), arginine (R), threonine (T), valine (V) or tyrosine (Y). Has been replaced.
(9) The 327th serine (S) is substituted with glutamic acid (E).
(10) Glutamine (Q) at position 365 is substituted with arginine (R).
(11) Valine (V) at position 547 is substituted with serine (S), cysteine (C), alanine (A) or glutamine (Q). The gene which codes modified FADGDH in any one of Claims 1-3. A vector comprising the gene according to claim 4. A transformant transformed with the vector according to claim 5. A method for producing modified FADGDH, wherein the transformant according to claim 6 is cultured. A glucose assay kit comprising the modified FADGDH according to any one of claims 1 to 3. A glucose sensor comprising the modified FADGDH according to any one of claims 1 to 3. The glucose measuring method containing the modified FADGDH in any one of Claims 1-3.

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