JP5793841B2 - Method for improving the specific activity of flavin adenine dinucleotide-dependent glucose dehydrogenase - Google Patents

Description

  The present invention relates to a method for improving the specific activity of a flavin adenine-dependent glucose dehydrogenase (hereinafter also referred to as FADGDH), the gene encoding the flavin adenine-dependent glucose dehydrogenase variant, the flavin adenine dependency The present invention relates to a method for producing a glucose dehydrogenase variant and various applications of the flavin adenine-dependent glucose dehydrogenase variant to a glucose measurement reagent.

  In recent years, the incidence of diabetes has been increasing year by year, and it is estimated that the total number of people who are said to be diabetic reserves will be more than 10 million in Japan alone. In addition, interest in lifestyle-related diseases has increased greatly, and opportunities for self-management of blood glucose levels are increasing. Against this backdrop, development of technology for blood glucose self-monitoring is an important technology for diabetics to manage their daily blood glucose levels. With regard to blood glucose measurement technology, practical use is progressing in many ways, and electrochemical sensing is advantageous in terms of reducing the amount of specimen, shortening measurement time, and downsizing the apparatus.

  As a sensing technique in blood glucose measurement technology, an enzyme using glucose in blood as a substrate is used. An example of such an enzyme is glucose oxidase (EC 1.1.3.4). Glucose oxidase has the advantage of high specificity to glucose and high stability to heat. The blood glucose sensor using glucose oxidase is measured by the electron generated in the process of oxidizing glucose and converting it to D-glucono-δ-lactone through the mediator, and glucose oxidase is a proton generated by the reaction. There is a problem in that the measured value is affected because it is easy to pass to the dissolved oxygen.

  In order to avoid such problems, pyrroloquinoline quinone-dependent glucose dehydrogenase (hereinafter also referred to as PQQGDH) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is used as an enzyme for blood glucose sensors. It is used. PQQGDH is advantageous in that it is not affected by dissolved oxygen, but it has a disadvantage that it has poor substrate specificity, and acts on saccharides other than glucose such as maltose and lactose, thereby impairing the accuracy of measurement values.

Therefore, a flavin adenine-dependent glucose dehydrogenase (hereinafter referred to as FADGDH is referred to as FADGDH) as a glucose dehydrogenase (hereinafter also referred to as GDH) that is not affected by dissolved oxygen and has excellent substrate specificity. Is attracting attention. FADGDH is described in Non-Patent Documents 1 to 6, and has been known for a long time.
Patent document 1 describes the gene sequence and amino acid sequence of FADGDH derived from Aspergillus terreus.
Patent Document 2 describes FADGDH derived from Aspergillus oryzae, and Patent Document 3 describes modified FADGDH in which FADGDH is modified by modifying Aspergillus oryzae-derived FADGDH.

WO2004 / 058958 JP2007-289148 JP2008-237210A WO2006 / 101239

Biochim Biophys Acta. 1967 Jul 11; 139 (2): 265-76. Biochim Biophys Acta. 1967 Jul 11; 139 (2): 277-93. Biochim Biophys Acta. 146 (2): 317-27 Biochim Biophys Acta. 146 (2): 328-35 J Biol Chem (1967) 242: 3665-3672. Appl Biochem Biotechnol (1996) 56: 301-310

  The present invention is to modify the known FADGDH as described above to provide an enzyme more suitable for glucose measurement.

Known FADGDH described in Patent Document 2 or Patent Document 3 has a low specific activity as an enzyme for clinical testing, and has a drawback that a high concentration of enzyme must be added in clinical testing. Therefore, the present inventors further studied a method for modifying these at the gene level to improve the specific activity before the modification.
As a result, the present inventors have found that the specific activity of modified FADGDH obtained by substituting an amino acid at a specific position of FADGDH with another amino acid has improved, and completed the present invention.

That is, the present invention has the following configuration.
Item 1.
A protein represented by any one of the following (a) to (c).
(A) A protein in which the amino acid at position 412 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 412 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 2.
(C) A protein in which the amino acid at position 408 in the amino acid sequence set forth in SEQ ID NO: 23 is substituted with another amino acid.
(D) an amino acid sequence having an amino acid sequence having at least 63.7% homology with at least one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 23, and encoding a protein having glucose dehydrogenase activity; A protein in which an amino acid at a site equivalent to any one of the group consisting of position 412 of SEQ ID NO: 1, position 412 of SEQ ID NO: 2 and position 408 of SEQ ID NO: 23 is substituted with another amino acid.
Item 2.
The protein according to Item 1, further comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted, and has glucose dehydrogenase activity Protein term 3.
The protein according to Item 1 or 2, wherein the amino acid at the same or equivalent position as any one of the group consisting of position 412 of SEQ ID NO: 1, position 412 of SEQ ID NO: 2 and position 408 of SEQ ID NO: 23 is cysteine, A protein substituted with any one selected from the group consisting of glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine and tyrosine.
Item 4.
Item 4. A gene encoding the protein according to any one of Items 1 to 3.
Item 5.
Item 5. A vector comprising the gene according to item 4.
Item 6.
Item 6. A transformant transformed with the vector according to Item 5.
Item 7.
A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to item 6 and collecting a protein having glucose dehydrogenase activity.
Item 8.
Item 4. A glucose assay kit comprising the protein according to any one of Items 1 to 3.
Item 9.
Item 4. A glucose sensor comprising the protein according to any one of Items 1 to 3.
Item 10.
The glucose measuring method containing the protein in any one of claim | item 1-3.

  According to the present invention, a novel FADGDH having high specific activity that is useful as an enzyme for clinical tests can be created, and the FADGDH can be produced industrially in large quantities.

It is the figure which compared the amino acid sequence of wild-type FADGDH derived from Aspergillus oryzae and the amino acid sequence of wild-type FADGDH derived from Aspergillus tereus.

The present invention is described in detail below.
In the present specification, the amino acid sequence is represented by one or three letters of the alphabet. The position of amino acid mutation is expressed as follows. For example, “S412L” means that S (Ser) at position 412 is replaced with L (Leu). In SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 23, the amino acid notation is numbered with methionine as 1.

One embodiment of the present invention is a protein represented by any of the following (a) to (c).
(A) A protein in which the amino acid at position 412 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 412 is substituted with another amino acid in the amino acid sequence set forth in SEQ ID NO: 2.
(C) A protein in which the amino acid at position 408 in the amino acid sequence set forth in SEQ ID NO: 23 is substituted with another amino acid.

SEQ ID NO: 1 is the amino acid sequence of wild-type FADGDH derived from Aspergillus oryzae. SEQ ID NO: 2 is an amino acid sequence of FADGDH improved in thermal stability by substituting glycine at position 163 with arginine and valine at position 551 with cysteine in the amino acid sequence of SEQ ID NO: 1. These amino acid sequences are known from Patent Document 2 or Patent Document 3.
The FADGDH of SEQ ID NO: 1 and SEQ ID NO: 2 confirms that the enzyme properties other than thermostability are not changed. For example, regarding the specific activity, all FADGDH values were about 600 U / A280, which was almost the same.

  SEQ ID NO: 23 is the amino acid sequence of wild-type FADGDH derived from Aspergillus terreus. This amino acid sequence is known from US Pat.

  When the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 1) and the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (SEQ ID NO: 23) were compared and analyzed with GENETYX software, 63.7% There is homology and the amino acid at position 412 of SEQ ID NO: 1 or 2 corresponds to the amino acid at position 408 of SEQ ID NO: 3. (See Figure 1)

Another embodiment of the present invention is a protein represented by the following (d).
(D) an amino acid sequence having an amino acid sequence having at least 63.7% homology with at least one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 23, and encoding a protein having glucose dehydrogenase activity; A protein in which an amino acid at a site equivalent to any one of the group consisting of position 412 of SEQ ID NO: 1, position 412 of SEQ ID NO: 2 and position 408 of SEQ ID NO: 23 is substituted with another amino acid.

In the present specification, the homology of amino acid sequences means a value compared with GENETYX software.
Further, in this specification, a position equivalent to position 412 of SEQ ID NO: 1 in a certain amino acid sequence is determined to be equivalent to a position corresponding to position 412 of SEQ ID NO: 1 when the sequences are compared with GENETYX software.
The GENETYX software used was version 6.1 sold by GENETYX CORPORATION.

  In the protein represented by any one of (a) to (d) above, one or several amino acids are further deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted. A protein having an amino acid sequence and having glucose dehydrogenase activity is also an embodiment of the present invention.

  Furthermore, one or several amino acids are deleted in the protein represented by any one of (a) to (d) above or the protein represented by any one of (a) to (d) above In a protein consisting of a substituted or added (inserted) amino acid sequence and having glucose dehydrogenase activity, among the group consisting of position 412 of SEQ ID NO: 1, position 412 of SEQ ID NO: 2, and position 408 of SEQ ID NO: 23 A protein in which an amino acid at the same or equivalent site is substituted with any one selected from the group consisting of cysteine, glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine, methionine, glutamine, arginine, threonine, valine and tyrosine Is also an embodiment of the present invention.

In the GENETYX software that performs amino acid sequence homology comparison, etc.
(1) the amino acid sequence set forth in SEQ ID NO: 1,
(2) the amino acid sequence set forth in SEQ ID NO: 2,
(3) the amino acid sequence set forth in SEQ ID NO: 23, or
(4) 63.7% or more homology with at least one or more of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 23, preferably 70% or more, more preferably 90% or more, even more preferably A protein having an amino acid sequence having a homology of 95% or more and having glucose dehydrogenase activity,
The modified FADGDH is obtained by modifying any one of the above, and its specific activity is improved as compared with FADGDH before modification.

  In the present application, the specific activity means an activity per certain amount of protein. An increase in specific activity means an increase in activity per protein per certain amount.

  The FADGDH variant of the present invention is preferably a FADGDH variant whose specific activity is improved by 10% or more compared to that before the modification. More preferably, the FADGDH variant is improved by 20% or more compared with that before the alteration. More preferably, the FADGDH variant is improved by 30% or more compared with that before the alteration.

  The specific activity measurement method is determined using the FADGDH activity measurement method described below.

The FADGDH variant of the present invention can be prepared by various known means.
Below, the manufacturing method of the FADGDH modification which modified | denatured FADGDH derived from the wild type Aspergillus oryzae shown by sequence number 1 is illustrated. Although a manufacturing method is not specifically limited, It can manufacture in the procedure as shown below.
As a method for modifying the amino acid sequence constituting flavin adenine-dependent glucose dehydrogenase, 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 flavin adenine-dependent glucose dehydrogenase variant is transferred to a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the flavin adenine-dependent glucose dehydrogenase variant. 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.

  In addition, the gene encoding the FADGDH variant, the vector containing the gene, and the transformant transformed with the vector obtained in these processes are also included as embodiments of the present invention.

The gene that is the basis for modification of the modified FADGDH of the present invention is not particularly limited, but it is desirable to use FADGDH derived from the genus Aspergillus. More preferably, it is FADGDH derived from Aspergillus oryzae or Aspergillus terreus.
Examples of FADGDH derived from Aspergillus oryzae include the protein represented by SEQ ID NO: 1 or SEQ ID NO: 2.
Examples of FADGDH derived from Aspergillus terreus include the protein represented by SEQ ID NO: 23.

  One embodiment of the present invention is a gene encoding the protein described in any of the above.

  As a gene encoding the protein before modification, for example, as a gene encoding the protein represented by SEQ ID NO: 1, the base sequence represented by SEQ ID NO: 24 can be exemplified. Moreover, as a gene which codes the protein represented by said sequence number 2, the base sequence represented by sequence number 25 can be illustrated. Moreover, as a gene encoding the protein represented by SEQ ID NO: 23 (FADGDH derived from Aspergillus terreus), the base sequence represented by SEQ ID NO: 26 can be exemplified.

The gene of the present invention is a gene sequence encoding the protein before modification, for example,
(1) position 412 of SEQ ID NO: 1,
(2) position 412 of SEQ ID NO: 2,
(3) position 408 of SEQ ID NO: 23, or
(4) A portion encoding an amino acid at a site equivalent to any one of the group consisting of position 412 of SEQ ID NO: 1, position 412 of SEQ ID NO: 2, and position 408 of SEQ ID NO: 23 may encode another amino acid. Has been replaced.

  The gene of the present invention may include a modified codon usage for the purpose of improving GDH expression, for example.

  In one embodiment of the present invention, a vector having the above gene, a transformant transformed with the vector, culturing the transformant, and collecting a protein having glucose dehydrogenase activity are obtained. It is a production method.

  For example, the above GDH gene is inserted into an expression vector (many things such as plasmids are known in the art), and an appropriate host (many things such as E. coli are known in the art). Transform. After culturing the obtained transformant and collecting the bacterial cells from the culture solution by centrifugation or the like, the bacterial cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, such as EDTA A water-soluble fraction containing GDH can be obtained by adding a chelating agent or a surfactant and solubilizing. Alternatively, the expressed GDH can be secreted directly into the culture medium by using an appropriate host vector system.

  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. Further, purified GDH can be obtained by performing gel filtration using an adsorbent or a gel filter, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

These can proceed according to the following literature, for example.
(A) Protein Experiment Protocol Volume 1 Functional Analysis, Volume 2 Structural Analysis (Syujunsha) Supervised by Yoshifumi Nishimura and Shigeo Ohno (b) Revised Protein Experiment Notes Extraction and separation and purification (Yodosha) Masato Okada, Kaori Miyazaki
Editing (c) How to proceed with protein experiments (Yodosha) Masato Okada, Kaori Miyazaki It can also be carried out by editing or the methods exemplified below.

  The prepared DNA having the genetic information of the protein is transferred into the host microorganism in a state of being linked to the vector.

Suitable vectors are those constructed for gene recombination from phages or plasmids that can grow independently in a host microorganism. 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, pKK223-3, and pBluescript when Escherichia coli is used as a host microorganism. Of these, pBluescript or the like that retains a promoter that can be recognized in Escherichia coli upstream of the cloning site is preferable.
In addition, a suitable host microorganism is not particularly limited as long as the recombinant vector is stable, can self-propagate and can express a foreign gene. In Escherichia coli, 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 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 DH5α; manufactured by Toyobo) may be used. When yeast is used as the host, the lithium method or electroporation method is used. When filamentous fungi are used, the protoplast method or the like is used.

  In the present invention, methods for obtaining a gene encoding GDH include the following methods. Using Aspergillus oryzae genome sequence information, a predicted GDH gene can be found. Next, mRNA is prepared from Aspergillus oryzae cells and cDNA is synthesized. The cDNA thus obtained is used as a template to amplify the GDH gene by the PCR method, and this gene is bound and closed with DNA ligase or the like at the blunt end or sticky end of both DNAs to construct a recombinant vector. After transferring the recombinant vector to a replicable host microorganism, a recombinant microorganism containing a gene encoding GDH is obtained using the marker of the vector.

  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 the recombinant may be carried out by searching for a microorganism that simultaneously expresses the marker of the vector and the GDH activity. 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 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 carrying a GDH gene once selected to a recombinant vector that can be replicated in another microorganism can be carried out by using a restriction enzyme or PCR method from the recombinant vector carrying the GDH gene. It can be easily carried out by collecting the DNA that is the GDH gene and binding it to other vector fragments. Further, for transformation of other microorganisms with these vectors, a competent cell method using calcium treatment, an electroporation method, a protoplast method, or the like can be used.

  The GDH gene of the present invention may have a DNA sequence in which a part of each amino acid residue of the translated amino acid sequence of the gene is deleted or substituted as long as it has glucose dehydrogenase activity. It may have a DNA sequence in which other amino acid residues are added or substituted.

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

  The culture form of the host microorganism as a transformant may be selected in consideration of the nutritional physiological properties of the host. In many cases, it is advantageous to use liquid culture and industrially perform aeration and agitation culture. However, in consideration of productivity, it may be advantageous to use a filamentous fungus as a host and perform the solid culture.

  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, 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 bacteria grow and produce GDH, but is preferably about 20 to 37 ° C. Although the culture time varies slightly depending on the conditions, the culture may be completed at an appropriate time in consideration of the time when GDH reaches the maximum yield, and is usually about 6 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacteria grow and produce GDH, but is preferably in the range of about pH 6.0 to 9.0.

  Although the culture solution containing the cells producing GDH in the culture can be collected and used as it is, generally, when GDH is present in the culture solution according to a conventional method, filtration, centrifugation, etc. It is used after separating the GDH-containing solution and the microbial cells. When GDH 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 destroyed by a mechanical method or an enzymatic method such as lysozyme. Further, if necessary, a chelating agent such as EDTA and a surfactant are added to solubilize GDH and 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, gel filtration using Sephadex gel (manufactured by GE Healthcare Bioscience), DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience), octyl Sepharose CL-6B (manufactured by GE Healthcare Bioscience) And purified by column chromatography such as) to obtain a purified enzyme preparation. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

In the present invention, FADGDH activity is measured under the following conditions.
[Test example]
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
24 mM PMS solution 2.0 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 21.9 ml of the above PIPES buffer, 1.0 ml of DCPIP solution, 2.0 ml of PMS solution, 4.5 ml of D-glucose solution To make a reaction reagent.

<Measurement conditions>
Pre-warm 3 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 / micromole) under the conditions for this activity measurement, and 0.1 is the solution of the enzyme solution. Amount (ml), 1.0 indicates the optical path length (cm) of the cell.

Glucose Assay Kit The present invention also features a glucose assay kit comprising a modified FADGDH according to the present 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.

  Hereinafter, the present invention will be specifically described by way of examples.

Example 1 Preparation of Modified FADGDH Gene A recombinant plasmid pAOGDH-M76 containing a gene encoding FADGDH (encoding the protein represented by SEQ ID NO: 2) is commercially available in E. coli competent cells (E. coli). E. coli DH5α (manufactured by TOYOBO) was applied to an agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar; pH 7.3) containing ampicillin. And cultured at 30 ° C. overnight. The obtained transformant was fed with a liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) containing ampicillin (50 mg / ml; manufactured by Nacalai Tesque), and 30 Cultured with shaking at 0 ° C. overnight. A plasmid was prepared from the obtained cells by a conventional method.
Using the plasmid as a template, threonine at position 16 (in Examples 1 to 5, the position from the N-terminus in SEQ ID NO: 2 is also synonymous with “site” and “position” is also synonymous) is substituted with a plurality of amino acids. The designed synthetic oligonucleotide of SEQ ID NO: 3 and a synthetic oligonucleotide complementary thereto, the synthetic oligonucleotide of SEQ ID NO: 4 designed to replace the 17th serine with a plurality of amino acids, and a synthetic oligonucleotide complementary thereto, 48 A synthetic oligonucleotide of SEQ ID NO: 5 designed to substitute the second valine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, and a synthesis of SEQ ID NO: 6 designed to substitute the 49th threonine with a plurality of amino acids Oligonucleotide and its complementary synthetic oligonucleotide and its complementary synthetic oligonucleotide A synthetic oligonucleotide of SEQ ID NO: 7 designed to replace the 58th phenylalanine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, and a sequence designed to replace the 90th threonine with a plurality of amino acids Synthetic oligonucleotide number 8 and its complementary synthetic oligonucleotide, SEQ ID NO: 9 designed to substitute the 96th methionine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, 97th alanine The synthetic oligonucleotide of SEQ ID NO: 10 designed to substitute a plurality of amino acids and the synthetic oligonucleotide complementary thereto, and the synthetic oligonucleotide of SEQ ID NO: 22 designed to substitute the 412th serine with a plurality of amino acids And the phase Synthetic oligonucleotide, the synthetic oligonucleotide of SEQ ID NO: 11 designed to replace the 420th proline with multiple types of amino acids and the complementary synthetic oligonucleotide, the 421st phenylalanine, to be replaced with multiple types of amino acids The designed synthetic oligonucleotide of SEQ ID NO: 12 and the synthetic oligonucleotide complementary thereto, and the synthetic oligonucleotide of SEQ ID NO: 13 designed to replace the 423rd arginine with a plurality of amino acids and the synthetic oligonucleotide complementary thereto, 499 A synthetic oligonucleotide of SEQ ID NO: 14 designed to substitute the second alanine with a plurality of amino acids and a synthetic oligonucleotide complementary thereto, and a synthesis of SEQ ID NO: 15 designed to substitute the 500th asparagine with a plurality of amino acids Oh A synthetic oligonucleotide complementary to it, a synthetic oligonucleotide complementary to it, a synthetic oligonucleotide of SEQ ID NO: 16 designed to replace the 503rd serine with a plurality of amino acids, a synthetic oligonucleotide complementary thereto, and a 506th asparagine A synthetic oligonucleotide of SEQ ID NO: 17 designed to substitute for an amino acid and a synthetic oligonucleotide complementary thereto, and a synthetic oligonucleotide of SEQ ID NO: 18 designed to substitute a plurality of amino acids for the 507th proline and complementary thereto Synthetic oligonucleotide, designed to replace the 508th valine with multiple types of amino acids and the synthetic oligonucleotide of SEQ ID NO: 19 and its complementary synthetic oligonucleotide, the 537th valine with multiple types of amino acids Sequence number Based on the synthetic oligonucleotide of SEQ ID NO: 21 designed to substitute a plurality of amino acids for the 546th valine with a synthetic oligonucleotide of 0 and a complementary synthetic oligonucleotide thereof, QuickChangeTMSite- Using Directed Mutagenesis Kit (manufactured by STRATAGENE), mutation was performed according to the protocol to produce modified FADGDH.

Example 2 Preparation of crude enzyme solution containing modified FADGDH and comparison of culture titer The obtained modified FADGDH was transformed into a commercially available E. coli competent cell (E. coli DH5α; manufactured by TOYOBO) and contained ampicillin. The cells were cultured on LB agar medium at 37 ° C. for 16 hours. Thereafter, a single colony of modified FADGDH was ingested into LB liquid medium containing ampicillin and cultured overnight at 30 ° C. with shaking. The bacterial cells obtained by centrifugation are collected from a part of the culture solution, and a crude enzyme solution is prepared by crushing the bacterial cells with glass beads in 50 mM phosphate buffer (pH 7.0). did.

  Using the prepared crude enzyme solution, GDH activity was measured by the activity measuring method described above. Table 1 shows the results of screening. The modified site was compared with the culture titer, and substitution of 412 site confirmed that the effect of improving the culture titer over FADGDH before modification was confirmed.

Example 3 Optimization of amino acid at 412 sites A plurality of single colonies modified from the 412 site prepared in Example 2 were ingested into an LB liquid medium, and a plasmid was extracted by a conventional method. 412 sites of the extracted plasmid were identified using a DNA sequencer (ABI PRISMTM 3700 DNA Analyzer; manufactured by Perkin-Elmer), and modified FADGDH substituted with 19 kinds of amino acids was obtained. PAOGDH-S412A, a plasmid having a modified FADGDH in which the 412 site is replaced with A, a plasmid having a modified FADGDH in which the plasmid having the modified FADGDH in which the 412 site is replaced with C is replaced with pAOGDH-S412C, and the 412 site in the E. pAOGDH-S412E, a modified FADGDH in which the plasmid having modified FADGDH in which the 412 site is replaced with F is replaced with pAOGDH-S412F, a plasmid having the modified FADGDH in which the 412 site is replaced with H, and a modified FADGDH in which the 4A site is replaced with pAOGDH-S412H PAOGDH-S412I, a plasmid having a modified FADGDH in which the 412 site is replaced with K has a pFAOGDH-S412K, a modified FADGDH in which the 412 site is replaced with L Modification of plasmid having pAOGDH-S412L, pAOGDH-S412L, 412 site replaced by M with pFAOGDH-S412M, pADOGDH-S412M, 412 site replaced with N, pAOGDH-S412N PAOGDH-S412Q in which the plasmid having type FADGDH is replaced with R at the 412 site is replaced with pAOGDH-S412R in which the plasmid having 412 site is replaced with T, and the plasmid having the modified FADGDH in which 412 site is replaced with T PAOGDH-S412V, the 412 site of the plasmid having the modified FADGDH replaced with Y, and the pAOGDH-S412Y, 412 site of the plasmid having the modified FADGDH replaced with Y. PAOGDH-S412D, a plasmid having the modified FADGDH in which the 412 site is substituted with P in the plasmid having the modified FADGDH replaced with pAOGDH-S412P, a plasmid having the modified FADGDH in which the 412 site is replaced with W as pAOGDH-S412W Named.

Example 4 Comparison of culture titer of FADGDH variant substituted with 412 sites 19 FADGDH variants substituted with 412 sites obtained in Example 3 were mixed with 50 ml LB medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl, 0.02% adecanol LG-126, ampicillin 100 μg / ml; pH 7.3) was cultured with shaking at 30 ° C. for 46 hours. Thereafter, a crude enzyme solution was prepared in the same manner as in Example 2, and the culture titer was measured. Table 2 shows the results of the culture titer of the FADGDH variant. Modified FADGDH with 412 sites replaced with A Modified FADGDH with 412 sites replaced with D Modified FADGDH with 412 sites replaced with P All modified variants except modified FADGDH with 412 sites replaced with W The culture titer was improved by 50% or more compared to FADGDH before modification.
As shown in Table 2, as a result of comparing the culture titer of the FADGDH variant when cultured for 50 hours at 30 ° C. in a 50 ml LB medium / 500 ml Sakaguchi flask, all the variants except S412A, S412D, S412P and S412W The culture titer was improved compared to the double mutant of SEQ ID NO: 2 (described as “FADGDH variant 1” in Table 2).

Modified FADGDH with 412 sites replaced with F Modified FADGDH with 412 sites replaced with I Modified FADGDH with 412 sites replaced with L Modified FADGDH with 412 sites replaced with V Replaced 412 sites with K The modified FADGDH thus obtained was inoculated into a 50 ml-1 / 2 LB medium containing ampicillin and cultured at 30 ° C. overnight. Thereafter, each culture solution was inoculated into a 6 L LB medium prepared in a 10 L jar fermenter, cultured at 30 ° C. for 84 hours, and a part of the culture solution was sampled. Thereafter, a crude enzyme solution was prepared in the same manner as in Example 2, and the culture titer was measured. Table 3 shows the results of the culture titer when the FADGDH variant was cultured in a 10 L jar fermenter. As a result, the modified FADGDH in which the 412 site was replaced with F had a culture titer improved by 20% over the FADGDH before the modification, and the modified FADGDH in which the 412 site was replaced with I had a 50% greater culture titer than the FADGDH before the modification. The modified FADGDH in which the 412 site is replaced with L is 30% higher in culture titer than FADGDH before the modification, and the modified FADGDH in which the 412 site is replaced with V is 50% more cultured than the FADGDH before the modification. As a result, the modified FADGDH in which the 412 site was replaced with K improved the culture titer by 20% over the FADGDH before the modification.
As shown in Table 3, the results of comparison of the culture titers of the FADGDH variants (S412L, S412I, S412L, S412V, S412K) when cultured for 6 hours in 6L LB medium / 10 m ³ -jar fermenter, 30 ° C. The culture titers of all the variants were improved by 20% or more compared to the double variant of SEQ ID NO: 2 (described as “FADGDH variant 1” in Table 2).

Example 5 Preparation of modified FADGDH preparation and comparison of specific activity Modified FADGDH was cultured in LB medium at a culture temperature of 30 ° C. for 84 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 a Phenyl-Sepharose column (the elution conditions are both 25% saturated to 0% ammonium sulfate concentration gradients are used to extract peak fractions), and further a G-25 Sepharose column Ammonium sulfate was removed by gel filtration with a modified FADGDH preparation.

Using the above method, preparations of modified FADGDH in which 412 sites were replaced with I and modified FADGDH in which 412 sites were replaced with V were prepared, and the specific activity was measured. As shown in Table 4, these modified FAGGDHs improved in 30% specific activity compared to FADGDH before modification.
As shown in Table 4, the enzyme was purified from the FADGDH variant (S412I, S412V) and the specific activity was compared. As a result, the specific activity of the FADGDH variant was double mutant of SEQ ID NO: 2 (in Table 2, “FADGDH modification”). It was improved by about 30% as compared with “Body 1”.

Example 6 Production of modified FADGDH gene derived from Aspergillus terreus Commercially available E. coli DH5α, a recombinant plasmid pATGDH containing the gene (SEQ ID NO: 26) encoding FADGDH derived from Aspergillus terreus; manufactured by TOYOBO ) 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 overnight at 30 ° C. Cultured. The obtained transformant was ingested in a liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl; pH 7.3) containing ampicillin (50 mg / ml; manufactured by Nacalai Tesque), and 30 Cultured with shaking at 0 ° C. overnight. A plasmid was prepared from the obtained cells by a conventional method.
When the amino acid sequence of wild-type FADGDH derived from Aspergillus oryzae and the amino acid sequence of wild-type FADGDH derived from Aspergillus tereus were compared and analyzed with GENETYX software, there was 63.7% homology and SEQ ID NO: 1 Alternatively, it was revealed that the amino acid at the 412 site in 2 corresponds to the amino acid at the 408 site in SEQ ID NO: 23. (See Figure 1)
Therefore, position 408 of FADGDH derived from Aspergillus tereus is phenylalanine, which is different from serine at position 412 of FADGDH derived from Aspergillus oryzae, but comparatively examined specific activity by substituting this 408 position phenylalanine with a site-specific amino acid. Decided to do.
Sequence designed to replace phenylalanine at position 408 (in Examples 6-8, the position from the N-terminus in SEQ ID NO: 23. “Site” and “position” are also synonymous) using the plasmid as a template. The synthetic oligonucleotide of No. 27 and its complementary synthetic oligonucleotide, or the synthetic oligonucleotide of SEQ ID NO: 28 designed to substitute for isoleucine and its complementary synthetic oligonucleotide, or a sequence designed to substitute for valine Based on the synthetic oligonucleotide No. 29 and its complementary oligonucleotide, mutation was performed using Quick Change Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) according to the protocol, and wild type phenylalanine Produced three types of modified FADGDH expression plasmids substituted with serine, isoleucine or valine.

Example 7 Preparation of Crude Enzyme Solution Containing Modified FADGDH Derived from Aspergillus terreus and Comparison of Culture Titers Three types of modified FADGDH expression plasmids obtained are commercially available in E. coli DH5α; TOYOBO Produced) and applied to an LB agar medium containing ampicillin, followed by culturing at 30 ° C. overnight. Thereafter, a single colony of modified FADGDH was added to a TB liquid medium containing ampicillin (1.2% polypeptone, 2.4% yeast extract, 0.4% glycerol, 1.25% dipotassium phosphate, 0.23% phosphorus). Acid potassium) and cultured with shaking at 25 ° C. for 2 days. The bacterial cells obtained by centrifugation are collected from a part of the culture solution, and a crude enzyme solution is prepared by crushing the bacterial cells with glass beads in 50 mM phosphate buffer (pH 7.0). did.

  Using the prepared crude enzyme solution, GDH activity was measured by the activity measuring method described above. The culture titer of wild type FADGDH and modified FADGDH was compared, and the effect of improving the culture titer than FADGDH before modification was confirmed by replacing 408 sites. The modified FADGDH in which F at 408 site is replaced with S has a 1.7 times higher culture titer than FADGDH before modification, and the modified FADGDH in which F at 408 site is replaced with I is more cultured than the FADGDH before modification. 3.4 times, and the modified FADGDH in which F at 408 sites was replaced with V improved the culture titer by 4.1 times compared to FADGDH before the modification.

Example 8 Production of a modified FADGDH preparation derived from Aspergillus terreus and comparison of specific activity The modified FADGDH was cultured in a TB medium at a culture temperature of 25 ° C. for 52 hours using a 2 L Sakaguchi flask. The cultured cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 6.0), 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 6.0). Then, gel filtration using a G-25 Sepharose column and ion exchange chromatography using a DEAE-Sepharose column (all elution conditions were performed by applying a 0 to 0.4 M NaCl concentration gradient to extract a peak fraction). Then, gel filtration with a G-25 Sepharose column, hydrophobic chromatography with a Phenyl-Sepharose column (all elution conditions were extracted with a 25% saturated to 0% ammonium sulfate concentration gradient), and further a G-25 Sepharose column. Ammonium sulfate was removed by gel filtration with a modified FADGDH preparation.

Standards of Aspergillus terreus-derived wild-type FADGDH, modified FADGDH in which 408 sites are replaced with S, modified FADGDH in which 408 sites are replaced with I, and modified FADGDH in which 408 sites are replaced with V Articles were prepared and the specific activity was measured. As shown in Table 6, there was no significant difference between the modified FADGDH substituted with S and the wild type FADGDH, but the modified FAGGDH substituted with I or V was different from the FADGDH before modification, respectively. 20%, 30% specific activity was improved.
Thus, in FADGDH having at least 63.7% homology with the amino acid sequence of Aspergillus oryzae-derived wild-type FADGDH (calculated by GENETYX software), the amino acid corresponding to the 412 site of SEQ ID NO: 1 or 2 It became clear that the specific activity is likely to be improved by substituting. This phenomenon is an effective technique for all homologous FADGDH, but in FADGDH derived from filamentous fungi, in particular, it is an effective site for improving specific activity, and at least in FADGDH derived from the genus Aspergillus, It is considered that a modified FADGDH that improves the activity can be produced almost certainly.

  By improving the specific activity of FADGDH, the amount of enzyme used in the glucose sensor can be reduced. If the amount of enzyme used can be reduced, the enzyme can be sufficiently dissolved even with a small amount of sample, and this can lead to improvement in measurement accuracy and reduction in measurement time.

  The improvement in the specific activity of flavin adenine-dependent glucose dehydrogenase according to the present invention makes it possible to reduce the amount of enzyme used in the production of glucose measurement reagents, glucose assay kits and glucose sensors, and to improve the measurement accuracy. It is a great place to contribute.

Claims (9)

A protein represented by any one of the following (a) to (c) .
(A) A protein in which the amino acid at position 412 is substituted with I or V in the amino acid sequence shown in SEQ ID NO: 1.
(B) A protein in which the amino acid at position 412 is substituted with I or V in the amino acid sequence shown in SEQ ID NO: 2.
(C) a protein in which the amino acid at position 408 is substituted with S, I or V in the amino acid sequence set forth in SEQ ID NO: 23 ; The protein according to claim 1, further comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added (inserted) at a position other than the position where the amino acid is substituted, and has glucose dehydrogenase activity. Protein A gene encoding the protein 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 protein having glucose dehydrogenase activity, comprising culturing the transformant according to claim 5 and collecting a protein having glucose dehydrogenase activity. A glucose assay kit comprising the protein according to claim 1 or 2 . Glucose sensor comprising a protein according to claim 1 or 2. A glucose measurement method comprising the protein according to claim 1 or 2 .

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