JP2008228740A - Glucose dehydrogenase derived from aspergillus oryzae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently produce glucose dehydrogenase derived from Aspergillus oryzae and to provide the more practical glucose dehydrogenase. <P>SOLUTION: The glucose dehydrogenase is efficiently produced by using a glucose dehydrogenase gene isolated from the Aspergillus oryzae and the more practical glucose dehydrogenase is obtained. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to glucose dehydrogenase derived from Aspergillus oryzae and a method for producing it by genetic recombination.

  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 a problem, for example, NAD (P) -dependent glucose dehydrogenase (EC 1.1.1.47) or pyrroloquinoline quinone-dependent glucose dehydrogenase (EC 1.1.5.2 (formerly EC1. 1.9.17)) is used as an enzyme for blood glucose sensors. These are superior in that they are not affected by dissolved oxygen, but the former NAD (P) -dependent glucose dehydrogenase has poor stability and the complexity of requiring the addition of a coenzyme. On the other hand, the latter pyrroloquinoline quinone-dependent glucose dehydrogenase has poor substrate specificity and acts on saccharides other than glucose, such as maltose and lactose, and thus has the disadvantage of impairing the accuracy of measured values.

Non-Patent Documents 1 to 4 report glucose dehydrogenase derived from Aspergillus oryzae, but do not report the glucose dehydrogenase gene. Non-patent documents 1 to 4 do not describe production of glucose dehydrogenase derived from Aspergillus oryzae by genetic recombination.
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

Patent Document 1 discloses an Aspergillus-derived flavin-binding glucose dehydrogenase. This enzyme is superior in that it has excellent substrate specificity and is not affected by dissolved oxygen. Regarding thermal stability, the residual activity rate is about 89% after treatment at 50 ° C. for 15 minutes, and it is said that the stability (hereinafter also referred to as heat resistance) is excellent. Patent Document 2 reports the gene sequence and amino acid sequence thereof.
WO 2004/058958 WO 2006/101239

  Recently, the complete genome sequence of Aspergillus oryzae has been determined. However, there is no information as to which part of the sequence encodes glucose dehydrogenase.

  An object of the present invention is to provide a blood glucose sensor enzyme that is more practically advantageous, and a method for efficiently producing the enzyme. More specifically, it is to establish a method for stably producing a large amount of glucose dehydrogenase by specifying, obtaining and using a glucose dehydrogenase gene derived from Aspergillus oryzae.

In order to achieve the above-mentioned object, the present inventors estimated and obtained a glucose dehydrogenase gene derived from Aspergillus oryzae using the database of National Center for Biotechnology Information (NCBI), and used the gene to aspergillus from Escherichia coli.・ The inventors have found that glucose dehydrogenase derived from oryzae can be obtained, and have arrived at the present application.
According to the present invention, it is possible to efficiently produce glucose dehydrogenase and obtain a more practical glucose dehydrogenase by using a glucose dehydrogenase gene isolated from Aspergillus oryzae.

That is, the present invention has the following configuration.
[Claim 1]
A protein comprising the amino acid sequence set forth in SEQ ID NO: 4 [Claim 2]
The gene of either of the following (a) or (b).
(A) a gene encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 (b) a DNA comprising the base sequence set forth in SEQ ID NO: 5
[Section 3]
A recombinant vector comprising the gene according to Item 2.
[Claim 4]
Item 4. A transformant transformed with the recombinant vector according to Item 3.
[Section 5]
Item 5. The transformant according to Item 4, wherein the host is Escherichia coli.
[Claim 6]
Item 6. A method for producing a protein having glucose dehydrogenase activity, comprising culturing the transformant according to Item 4 or 5 in a nutrient medium and collecting a protein having glucose dehydrogenase activity.

  According to the present invention, glucose dehydrogenase can be produced efficiently. It has also become easier to make molecular biological improvements in order to obtain more practical glucose dehydrogenase.

  In order to achieve the above object, the present inventors have found a gene DNA presumed to be glucose dehydrogenase (also referred to as GDH) using the NCBI database.

  The DNA (gene) having the base sequence described in SEQ ID NO: 1 contains a DNA (gene) encoding glucose dehydrogenase derived from the Aspergillus oryzae RIB40 strain predicted from the NCBI database, and having no intron removed. Gene sequence.

DNA (gene) having the base sequence described in SEQ ID NO: 2 is obtained by removing introns from SEQ ID NO: 1.

The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 represents the entire sequence of the glucose dehydrogenase gene predicted from the NCIB database.

The present inventors predicted that DNA (gene) encoding a protein having glucose dehydrogenase activity derived from Aspergillus oryzae can be easily identified from Non-Patent Documents 1 to 4 and NCBI database.
Furthermore, it was thought that a recombinant vector containing the gene can be produced, a transformed transformant can be produced, and a protein encoded by the gene expressed by the transformant can be easily purified.

  Specifically, although the amino acid sequence part and base sequence part which code for the flavin-binding glucose dehydrogenase derived from Aspergillus oryzae are not specified in the NCBI database, the present inventors described in Non-Patent Documents 1 to 4. Culturing Aspergillus oryzae with reference to the prepared methods and known techniques, purifying GDH from the culture supernatant using various chromatography, analyzing its terminal amino acid sequence, etc. to produce a probe, and glucose dehydrogenase Attempts were made to isolate the gene encoding.

  Similarly, the present inventors independently obtained a microorganism belonging to Aspergillus terreus and attempted to isolate a gene encoding glucose dehydrogenase.

The inventors of the present invention have made various studies. In the usual purification method using salting out, chromatography, etc., the GDH standard that can be clearly confirmed on SDS-PAGE with high purity from the culture supernatant of Aspergillus oryzae strain TI. It turned out that it was difficult to obtain goods. It was inferred that the sugar chain that would be bound to the enzyme protein was one of the causes that made purification and confirmation difficult. Therefore, it was determined that cloning using a partial amino acid sequence, which is one of the common methods for gene acquisition, must be abandoned.
For this reason, gene acquisition was extremely difficult with many trials and errors, but as a result of intensive studies, a gene encoding a flavin-binding glucose dehydrogenase derived from Aspergillus oryzae was isolated and the present invention was completed. It came to.
Details thereof will be described later in Examples 1 to 3.

  One embodiment of the present invention includes a gene consisting of the DNA of any one of the following (a), (b), (c) and (d), or a gene encoding the following protein (e) or (f): It is a method for producing a protein having glucose dehydrogenase activity, which comprises culturing a transformant in a nutrient medium using a recombinant vector and collecting a protein having glucose dehydrogenase activity.

(A) The DNA (gene) having the base sequence set forth in SEQ ID NO: 5 is the entire DNA (gene) encoding a protein having glucose dehydrogenase activity derived from the Aspergillus oryzae TI strain described later, as identified by the present inventors. Indicates the sequence. Also,
(C) DNA (gene) that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 5 and that encodes a protein having glucose dehydrogenase activity, Applicable to the present invention.

(B) The DNA (gene) having the base sequence set forth in SEQ ID NO: 8 contains DNA (gene) encoding a protein having glucose dehydrogenase activity derived from the Aspergillus oryzae TI strain described later, and does not remove introns. Genomic gene sequence. Also,
(D) DNA comprising a region that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 8 and which encodes a glucose dehydrogenase activity is also included in the present invention. Applicable.

(E) The gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 4 represents the entire sequence of DNA (gene) encoding a protein having glucose dehydrogenase activity derived from the Aspergillus oryzae TI strain described later. Also,
(F) A DNA (gene) encoding a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added (inserted) in the amino acid sequence shown in SEQ ID NO: 4 and having glucose dehydrogenase activity It can be applied to the present invention.

  The DNA (gene) of the present invention may include a modified codon usage to improve the expression of the present GDH.

  For example, the GDH gene derived from Aspergillus oryzae described above 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). Is transformed). 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, Edited by Kaoru Miyazaki (c) How to proceed with protein experiments (Yodosha) Masato Okada, Kaoru Miyazaki, or the following method can be used.

  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. Using the cDNA thus obtained as a template, the GLD gene is amplified by the PCR method, and this gene is bound to the vector at the blunt end or sticky end of both DNAs with DNA ligase or the like 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 bases in DNA, for example, a commercially available kit (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Selection Kit; manufactured by Stratagene, QuickChange SiteDirected MutagenStained MutaseNeste, etc.) Use of the chain reaction method (PCR) is mentioned.

  The 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, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

  The culture temperature can be appropriately changed within the range in which the 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, glucose dehydrogenase activity is measured under the following conditions.
<Reagent>
50 mM PIPES buffer pH 6.5 (including 0.1% Triton X-100)
14 mM 2,6-dichlorophenolindophenol (DCPIP) solution 1M D-glucose solution 15.8 ml of the above PIPES buffer, 0.2 ml of DCPIP solution, and 4 ml of D-glucose solution are mixed to obtain a reaction reagent.

<Measurement conditions>
Prewarm 2.9 ml of reaction reagent at 37 ° C. for 5 minutes. Add 0.1 ml of GDH solution and mix gently, then record the change in absorbance at 600 nm for 5 minutes using a spectrophotometer controlled at 37 ° C. with water as a control. Change in absorbance per minute from the straight line (ΔODTEST) Measure. In the blind test, a solvent for dissolving GDH is added to the reagent mixture instead of the GDH solution, and the change in absorbance per minute (ΔODBLANK) is measured in the same manner. 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) = {− (ΔODTEST−ΔODBLANK) × 3.0 × dilution factor}
/(16.3×0.1×1.0)

In the formula, 3.0 is the reaction reagent + enzyme solution volume (ml), 16.3 is the millimolar molecular extinction coefficient (cm 2 / micromole) under the present activity measurement conditions, and 0.1 is the enzyme solution volume. (Ml), 1.0 indicates the optical path length (cm) of the cell.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

  The outline of the procedure for obtaining the Aspergillus oryzae GDH gene described in Examples described later is as follows.

In order to obtain the GDH gene derived from Aspergillus oryzae, purification of GDH was attempted from the supernatant of Aspergillus oryzae and Aspergillus terreus using salting out, chromatography, etc., but it was difficult to obtain high purity GDH. Met. (Example 1 [1])
Therefore, cloning using a partial amino acid sequence, which is one of the conventional methods for gene acquisition, has to be abandoned.

Thus, as a result of earnest search for microorganisms that produce GDH other than the above, we found that Penicillium lilacinoechinatum NBRC6231 produces GDH, and succeeded in obtaining a purified enzyme of high purity from the culture solution of this strain. (Example 1 [2])
Subsequently, the partial amino acid sequence was successfully determined using the enzyme, and P.P. A part of lilacinoechinatum NBRC6231-derived GDH gene was obtained and the nucleotide sequence was determined (1356 bp). (Example 1 [3] [4])
Finally, based on this base sequence, the Aspergillus oryzae GDH gene was estimated (Example 1 [5]) and obtained from the published genome database of Aspergillus oryzae.

<Example 1>,
Estimation of Aspergillus oryzae-derived glucose dehydrogenase (hereinafter also referred to as AOGDH) gene [1] Acquisition of Aspergillus oryzae-derived GDH Aspergillus oryzae obtained from soil and stored as an L dry strain according to a standard method was used. This is hereinafter referred to as Aspergillus oryzae TI strain.
The L dry strain of Aspergillus oryzae strain TI was inoculated into potato dextrose agar medium (manufactured by Difco) and reconstituted by incubation 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, purification was attempted by various means such as ion exchange chromatography and gel filtration chromatography, but it was not possible to obtain a purified preparation that could be used for partial amino acid sequencing. It was.
In addition, we independently searched for and obtained microorganisms belonging to Aspergillus terreus, and tried purification from the culture supernatant by salting out, Octyl-sepharose, etc. in the same manner as described above, but as with Aspergillus oryzae, partial amino acid sequencing was performed. It was not possible to obtain a purified sample that could be provided. Usually, it was not possible to obtain a GDH preparation with high purity and clearly identifiable on SDS-PAGE by using a commonly used purification method. I guessed that the chain might be one of the causes. Accordingly, it has been unavoidable to abandon cloning using a partial amino acid sequence of the protein, which is one of the common methods for gene acquisition.
[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, almost by SDS electrophoresis A uniform purified sample was obtained.

[3] Preparation of cDNA The Penicillium lilacinoechinalum 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 filter paper filtration. 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.

[4] Determination of GDH gene partial sequence The above-purified NBRC6231-derived GDH was dissolved in Tris-HCl buffer (pH 6.8) containing 0.1% SDS and 10% glycerol, and the Glu-specific V8 endoprotease was dissolved therein. Was added to a final concentration of 10 μg / ml and incubated at 37 ° C. for 16 hours for partial degradation. 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: 9) and WGGGTKQTVRAKGALGGGTST (SEQ ID NO: 10). 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).

[5] Estimation of AOGDH gene A homology search is performed from the NCBI BLAST homepage (http://www.ncbi.nlm.nih.gov/BLAST/) based on the determined base sequence, and from a plurality of candidate sequences The AOGDH gene was estimated in consideration of homology with known glucose oxidase. AOGDH and P.E. The homology at the amino acid level with the GDH partial sequence derived from lilacinoechinatum NBRC6231 was 49%.

<Example 2>
Acquisition of AOGDH gene and introduction into E. coli In order to acquire the AOGDH gene, mRNA was prepared from cells of Aspergillus oryzae TI strain, and cDNA was synthesized. Two types of oligo DNAs shown in SEQ ID NOs: 6 and 7 were synthesized, and the 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: 5). As a result, it was revealed that the amino acid residue deduced from the cDNA sequence consists of 593 amino acids (SEQ ID NO: 4). The predicted GDH from the RIB40 strain registered in the database is 588 amino acids (SEQ ID NO: 3), suggesting that the number of amino acid residues is different from that of the 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. Since the database strain RIB40 and TI strains were suggested to have different GDH gene sequences, a recombinant plasmid containing the TI strain GDH gene was used as a template to include the GDH gene sequence predicted from the database RIB40 strain sequence. A recombinant plasmid was prepared using QuickChange Site Directed Mutagenesis Kit (manufactured by Stratagene), and a transformant was obtained. 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 with respect to the cell disruption solution, the GDH activity could not be confirmed with the transformant having the GDH sequence derived from the RIB40 strain, but the transformant having the GDH sequence derived from the TI strain was cultured in the cells. A high GDH activity of 8.0 U per ml of liquid was obtained. The GDH activity of the culture supernatant of Aspergillus oryzae TI strain carried out in Example 1 was 0.2 U / ml. This result suggests that the GDH gene predicted from the RIB40 strain database sequence does not function as GDH. As long as the gene sequences of the TI strain and the RIB40 strain are compared, the RDH40 strain has the same GDH gene sequence. It was thought that the cause was partial deletion.

<Example 3>
The Escherichia coli DH5α transformant having the GDH sequence derived from the TI strain cultured in Example 2 was collected by centrifugation, suspended in 20 mM potassium phosphate buffer (pH 6.5), and then broken into French presses. GDH was extracted using a powder machine. The purified enzyme preparation (rAOGDH) was obtained by the same procedure as that of AOGDH purified in Example 1, and the characteristics were compared with the AOGDH purified enzyme preparation.
[1] Substrate specificity When a blood glucose level is measured using a blood glucose sensor, it is required to use a glucose-specific enzyme in order to avoid misdiagnosis. Therefore, the reactivity of rAOGDH to various saccharides was examined. The results are shown in FIG. rAOGDH has the same substrate specificity as AOGDH, and it has been confirmed that it does not act on maltose, which is a particular problem when infusion patients use the sensor.
[2] Maltose degradability GDH itself used in a blood glucose sensor does not act on maltose, but may also lead to misdiagnosis when an enzyme preparation or the like contains a component that decomposes maltose into glucose. That is, it is extremely important that the GDH enzyme preparation does not contain a component that decomposes maltose. Therefore, the contamination of maltose-decomposing components in the GDH purification table was examined. The contamination test for maltose-degrading enzyme was performed as follows. 50 μl of 8 mM maltose was added to 50 μl of various purified enzyme solutions prepared to 10 U / ml and reacted at 37 ° C. After completion of the reaction, the concentration of glucose contained in the reaction solution was examined using Liquitech Glucose HK Test (Roche Diagnostics). The calibration curve for calculating the glucose concentration was prepared by measuring the calibration coefficient setting glucose (manufactured by Wako Pure Chemical Industries, Ltd.) with the measurement kit. The results are shown in FIG. In the AOGDH purified sample, 90% of maltose was already decomposed into glucose after treatment at 37 ° C. for 30 seconds, and nearly 10% of maltose was decomposed after 10 minutes of reaction treatment. Therefore, the measurement was performed in the same manner using a sample obtained by highly purifying AOGDH for the purpose of partial amino acid sequence analysis in Example 1, but the degradation activity was also confirmed from maltose. On the other hand, rAOGDH showed no glucose accumulation even after 10 minutes of treatment at 37 ° C. Aspergillus oryzae has long been used in the fermentation industry and is known to produce significant amounts of carbohydrate-related enzymes such as amylase and glucoamylase. In such an environment, only GDH is highly purified. It is considered extremely difficult to purify.

From these results, when using Aspergillus oryzae-derived GDH in a blood glucose sensor, it is considered essential to use GDH prepared from a gene recombinant.

  The present invention makes it possible to produce glucose dehydrogenase derived from Aspergillus oryzae in large quantities by using recombinant Escherichia coli. In addition, the present invention makes it possible to produce glucose dehydrogenase suitable for glucose sensors and the like that does not act on maltose in a broad sense.

The substrate specificity of the GDH purification grade of this invention is shown. Each substrate concentration was set to 4 mM, and the activity with respect to glucose was set to 100. The maltose decomposition property of the GDH refinement | purification grade of this invention is shown.

Claims (1)

A protein comprising the amino acid sequence set forth in SEQ ID NO: 4

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