JP4836017B2 - Method for high expression of glucose dehydrogenase from filamentous fungus in recombinant - Google Patents

Description

  The present invention relates to a method for highly expressing a glucose dehydrogenase derived from a filamentous fungus in a recombinant.

  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.

References 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.

  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.

  However, the production of flavin-binding glucose dehydrogenase (also referred to as FAD-dependent glucose dehydrogenase) is extremely difficult even using recombinant DNA technology. In fact, in the recombinant Escherichia coli K12 strain of FAD-dependent glucose dehydrogenase disclosed in Patent Document 2, the level was as low as 0.09 U / ml. The Escherichia coli K12 strain is the most widely used host for recombinant production, is a host that is widely used industrially from the viewpoint of ease of culturing, safety, and the like because of the simple recombination operation. Therefore, a method for efficiently producing FAD-dependent glucose dehydrogenase by a recombinant using E. coli K12 as a host has been desired.

WO 2004/058958 WO 2006/101239

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

  An object of the present invention is to provide an enzyme for blood glucose sensor that is more practically advantageous. More specifically, it is to obtain and use a large amount of glucose dehydrogenase (GDH) derived from filamentous fungi having excellent production cost as well as substrate specificity.

In order to achieve the above object, the present inventors first identified and obtained a glucose dehydrogenase gene using genomic information of Aspergillus oryzae. And it discovered that glucose dehydrogenase derived from Aspergillus oryzae could be obtained from E. coli using this gene. In addition, the amino acid sequence was estimated from the genome information, and it was found that an amino acid sequence that seems to be a signal peptide exists in the N-terminal region of the amino acid sequence.
Since the signal peptide becomes a signal for transition to the periplasmic space and there is a possibility that the expression level may be limited in the space of the space, the examination of deleting the function of the signal peptide was conducted. As a result, the production cost can be reduced to 1/10.

That is, the present invention has the following configuration.
(1) In a method for recombinantly producing glucose dehydrogenase derived from filamentous fungi (hereinafter also referred to as GDH), the expression level of the target enzyme is mutated by introducing a mutation into the signal peptide sequence present in the N-terminal region. A method for producing GDH, characterized in that it is higher than before.
(2) In a method for recombinantly producing GDH derived from filamentous fungi, the expression level of the target enzyme is reduced before mutation introduction by deleting or replacing part of the amino acid sequence of the signal peptide present in the N-terminal region. The method for producing GDH as set forth in (1), wherein the method is increased as compared with the above.
(3) Among the amino acid sequences described in any of SEQ ID NOs: 2 and 4, these amino acid sequences are present by deleting a part or all of the amino acid sequence of MLFSLAFLSALSLATASPAGRA present at the N-terminus thereof The method for producing GDH as described in (1), wherein the expression level is increased as compared with the case of performing.
(4) By performing 1 to 22 amino acid substitutions or / and amino acid insertions into the amino acid sequence of MLFSLAFLSALSLATASPAGRA present at the N-terminal of the amino acid sequences described in SEQ ID NOs: 2 and 4, The method for producing GDH as described in (1), wherein the expression activity is increased as compared with the case where the amino acid sequence is present.
(5) By increasing the expression level compared to the case where these amino acid sequences are present by deleting some or all of the amino acid sequences of MLGKLSFLSALSLAVAATLSNSTSA present at the N-terminus of glucose dehydrogenase derived from filamentous fungi, Alternatively, the GDH production method according to (1), wherein the expression activity is increased by performing 1 to 25 amino acid substitutions and / or amino acid insertions as compared to the case where the original amino acid sequence is present.
(6) In a filamentous fungus-derived glucose dehydrogenase (GDH), a GDH gene in which a part or all of a DNA sequence encoding a signal peptide present at the N-terminus thereof is substituted or / and deleted is encoded (1) to ( A DNA sequence used in any production method of 5).
(7) A recombinant vector comprising the DNA sequence of (6).
(8) A transformant obtained by introducing the recombinant vector according to (7) into a host.
(9) A GDH protein produced using the transformant according to (8).
(10) A composition comprising the GDH protein according to (9).
(11) A method for measuring glucose concentration using the composition according to (10).
(12) A glucose sensor comprising the composition according to (11).

According to the present invention, it has become possible to efficiently produce glucose dehydrogenase and obtain a more practical glucose dehydrogenase.
According to the present invention, an amino acid sequence is deduced from a glucose dehydrogenase gene isolated from Aspergillus or Penicillium, a signal peptide region is predicted, and a DNA sequence encoding part or all of the signal peptide is deleted. In addition, by subjecting the amino acid sequence encoded by the signal peptide to amino acid substitution or amino acid insertion, it is possible to efficiently produce glucose dehydrogenase in a recombinant, and more practical glucose from the viewpoint of industrial use. It became possible to obtain dehydrogenase.

FIG. 1 is a diagram showing prediction of a signal peptide cleavage site of Aspergillus oryzae-derived FAD-GDH. FIG. It is a graph which shows the relationship between the culture | cultivation phase of oryzae wild type GDH (with signal peptide), microbial turbidity (OD), pH, and a GDH activity value. FIG. It is a graph which shows the relationship between the culture | cultivation phase of an oryzae mutant type GDH-S2, microbial turbidity (OD), pH, and a GDH activity value. FIG. It is a graph which shows the relationship between the culture | cultivation phase of oryzae mutant type GDH-S3, microbial turbidity (OD), pH, and a GDH activity value. FIG. It is a graph which shows the relationship between the culture | cultivation phase, cell turbidity (OD), pH, and GDH activity value in culture temperature of 28 degreeC and Kd * P0.5 of Teleus wild type GDH (expected signal peptide sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase, cell turbidity (OD), pH, and GDH activity value in culture temperature of 28 degreeC and Kd * P0.75 of Teleus wild type GDH (expected signal peptide sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase, cell turbidity (OD), pH, and GDH activity value in culture temperature of 28 degreeC and Kd * P1.0 of Teleus wild type GDH (expected signal peptide sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase and cell turbidity (OD), pH, and GDH activity value in culture | cultivation temperature of 28 degrees C of Teleus wild type GDH (expected signal peptide arrangement | sequence excision) and Kd * P1.5. FIG. It is a graph which shows the relationship between the culture | cultivation phase and cell turbidity (OD), pH, and GDH activity value in the culture temperature of 20 degreeC and Kd * P0.5 of Teleus wild type GDH (expected signal peptide sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase and cell turbidity (OD), pH, and GDH activity value in the culture temperature of 23 degreeC and Kd * P0.5 of Teleus wild type GDH (expected signal peptide sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase and cell turbidity (OD), pH, and GDH activity value in the culture temperature of 26 degreeC and Kd * P0.5 of Teleus wild type GDH (expected signal peptide arrangement | sequence excision). FIG. It is a graph which shows the relationship between the culture | cultivation phase and cell turbidity (OD), pH, and GDH activity value in the culture temperature of 29 degreeC and Kd * P0.5 of Teleus wild type GDH (expected signal peptide sequence excision).

  One embodiment of the present invention is a method for recombinantly producing filamentous fungal-derived GDH by introducing a mutation into a signal peptide sequence present in the N-terminal region thereof, thereby reducing the expression level of the target enzyme before introducing the mutation. This is a method for producing GDH characterized by an increase in comparison.

  As a result of diligent efforts to obtain a large yield of GDH protein, the present inventors first achieved the above-mentioned object by first using the genomic DNA of Aspergillus oryzae in the previous invention as the genetic DNA that is the source of the modification. Has been used to find a genetic DNA presumed to be glucose dehydrogenase (hereinafter referred to as GDH).

  The present inventors have found a gene DNA presumed to be FAD-dependent glucose dehydrogenase (hereinafter referred to as GDH) using the NCBI database.

  The present inventors predicted that DNA (gene) encoding a protein having GDH activity can be easily identified from Non-Patent Documents 1 to 4, NCBI database, and the like. 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, Aspergillus oryzae is cultured with reference to the methods and known techniques described in Non-Patent Documents 1 to 4, and GDH is purified from the culture supernatant using various chromatographies. A probe was prepared by analyzing the sequence and the like, and an attempt was made to isolate a gene encoding GDH.

  The present inventors have made various studies. In the usual purification method using salting-out, chromatography, etc., a GDH preparation that can be clearly confirmed on SDS-PAGE with high purity from Aspergillus oryzae culture supernatant. It turned out to be difficult to obtain. 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. However, as a result of intensive studies, a gene encoding GDH derived from Aspergillus oryzae was isolated. Details thereof will be described later in Examples.

  In addition, the present inventors also studied various methods for obtaining GDH genes of other origins, and isolated a gene encoding GDH derived from Penicillium lilacinochinulatum and a gene encoding GDH derived from Aspergillus terreus. Details thereof will be described later in Examples.

  The gene DNA of the present invention may include a modified codon usage so as to further improve the expression of the present GDH.

  The signal peptide exists as an extended peptide having 15 to 30 residues at the N-terminus of the mature protein. The peptide sequence has a hydrophobic amino acid region, serves as a lead for attachment of the nascent polypeptide chain to the endoplasmic reticulum membrane and passage through the membrane, and is cleaved after passage through the membrane.

  Although the GDH recombinant derived from a filamentous fungus in the present invention is not particularly limited, a GDH recombinant using Aspergillus genus, Penicillium genus or the like as a GDH gene supply source can be exemplified. Preferred is Aspergillus genus GDH, and more preferred is Aspergillus oryzae. Most preferably, GDH which has an amino acid sequence described in any of SEQ ID NOs: 4 and 10 can be exemplified.

  For these GDH recombinants, a gene encoding the GDH is obtained by PCR, this gene is inserted into an expression vector, a transformant transformed into an appropriate host is cultured, and centrifuged from the culture solution. After collecting the cells by separation, etc., the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme, and if necessary, solubilized by adding a chelating agent such as EDTA or a surfactant. Thus, it can be obtained as a water-soluble fraction containing GDH. Alternatively, the expressed GDH can be secreted directly into the culture medium by using an appropriate host vector system.

In the present invention, the function of the signal peptide can be lowered by deleting or replacing part of the amino acid sequence of the signal peptide present in the N-terminal region of GDH.
For example, in GDH having the amino acid sequence described in SEQ ID NO: 4, the function of the signal peptide can be lowered by deleting a part or all of the amino acid sequence of MLFSLAFLSALSLATASPAGRA present at the N-terminus thereof. Alternatively, it is possible to perform 1 to 22 amino acid substitutions and / or amino acid insertions.
Specific examples of the substitution position include a signal peptide cleavage site. Preferably, the function can be reduced by substituting alanine corresponding to the C-terminus of the signal peptide with another amino acid.

  Whether or not the expression level of the target enzyme has increased compared to the state in which the signal peptide sequence is present can be confirmed by comparing the total activity value per 1 ml of the culture solution before and after introducing the mutation into the sequence. Further, the modification of the signal peptide sequence can be confirmed by the N-terminal amino acid sequence using Edman degradation.

  It has been found that the expression level of GDH protein is improved by deleting the function of the signal peptide present in the N-terminal region.

  PSORT and SignalP software are often used as signal peptide prediction tools. Each can be used on the web from the address “psort.nibb.ac.jp/” or “www.cbs.dtu.dk/services/SignalP-2.0/”.

  When the signal peptide cleavage position of the amino acid sequence (SEQ ID NO: 2) deduced from the GDH gene derived from Aspergillus oryzae was predicted using SignalP software, the position between the 16th alanine and the 17th serine, or the 22nd It was predicted that there was a high possibility of cleavage between alanine and the 23rd lysine (FIG. 1).

  For example, an Aspergillus oryzae-derived 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 Escherichia coli is known in the art) Cultivate the transformant transformed in (1)), collect the cells from the culture solution by centrifugation, etc., and then destroy the cells by mechanical methods or enzymatic methods such as lysozyme. Thus, a water-soluble fraction containing GDH can be obtained by adding a chelating agent such as EDTA, a surfactant or the like to solubilize. 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 FADGDH can be obtained by performing gel filtration with 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 (Shujunsha) Nishimura Yoshifumi, Ohno Shigeo Supervision (b) Revised Protein Experiment Notes Top Extraction and Separation 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 present invention further includes a vector containing a gene encoding GDH and a transformant transformed with the vector.

  The prepared DNA having genetic information of the protein is transferred into the host microorganism in a state of being linked to the vector, and becomes a transformant that produces the modified protein.

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

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

  In the present invention, methods for obtaining a gene encoding GDH derived from filamentous fungi include the following methods. First, the predicted GDH gene of the target filamentous fungus can be obtained by referring to the sequence information of the GDH gene of Aspergillus oryzae. MRNA is prepared from the filamentous fungus and cDNA is synthesized. The cDNA thus obtained is used as a template to amplify the GDH gene by PCR, and the gene is closed by binding the vector with a DNA ligase or the like at the blunt or sticky ends of both DNAs to construct a recombinant vector. After transferring the recombinant vector to a replicable host microorganism, a microorganism carrying the recombinant vector containing the gene encoding GDH is obtained using the marker of the vector.

  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, which is a transformant, may be selected in consideration of the nutritional physiological properties of the host, and in many cases, the culture is performed in liquid culture. Industrially, aeration and agitation culture is advantageous.

  As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, lactose, molasses, pyruvic acid and the like are used. The nitrogen source may be any available nitrogen compound. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline extract, and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary.

  The culture temperature can be appropriately changed within the range in which the 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. In the cultivation of a filamentous fungus-derived GDH recombinant, it is particularly important to control the pH of the medium, and it is desirable to control the pH to be 7.1 to 7.3 or less, and the pH is in the range of 6.0 to 7.3. It is particularly preferable to control the culture with In this way, by controlling the pH and culturing, it is possible to prepare a large amount of filamentous fungal GDH protein.

However, although GDH derived from filamentous fungi retains high stability in an acidic region at pH 7.0 or lower, it becomes unstable at pH 7.1 to 7.3 or higher, and a rapid decrease in activity is observed.
Therefore, in the present invention, it is preferable to control the pH during culture to 7.3 or less in recombinant production of glucose dehydrogenase (GDH) derived from filamentous fungi.

  At the time of culture production of the filamentous fungus-derived GDH recombinant, the rate of decrease in this activity is reduced by 10% or more compared to the original activity value due to the increase in pH by 0.2. It was about 30% lower. Therefore, in the present patent, when the pH is increased by 0.2, the pH of the culture solution before the decrease in activity in a phenomenon where the pH decreases by 10% or more compared to the original activity value, specifically, 7 The effectiveness of pH control at .3 or less, preferably 7.1 or less was estimated and confirmed by actual examination.

  In recombinant culture, pH control is generally performed in consideration of the death of bacterial cells and degradation of the target protein. However, it is common to maintain a neutral pH. There is no example that clarifies the necessity of pH control below neutral, such as glucose dehydrogenase derived from filamentous fungi.

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

Test Example 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 1 M 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. 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 factor} / (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.

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

  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. The intron is removed from the gene sequence. SEQ ID NO: 2 is the corresponding amino acid sequence.

  The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 represents the entire sequence of the glucose dehydrogenase gene predicted from the NCIB database.

  The DNA (gene) having the base sequence set forth in SEQ ID NO: 3 represents the entire sequence of the DNA (gene) encoding the protein having glucose dehydrogenase activity derived from the Aspergillus oryzae TI strain described later, as identified by the present inventors. . SEQ ID NO: 4 is the corresponding amino acid sequence. A DNA (gene) 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: 3 and that encodes a protein having glucose dehydrogenase activity is also present. It is included in the scope of the invention.

  The gene encoding a protein comprising the amino acid sequence described 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, 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 is included in the scope of application of the present invention.

  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. (Experimental 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.

Therefore, as a result of searching for microorganisms that produce GDH in addition to the above and eagerly searching for them, we found that Penicillium lilacinochinatum NBRC6231 strain produces GDH and succeeded in obtaining a purified enzyme of high purity from the culture solution of this strain. . (Experimental example 1 [2])
Next, the partial amino acid sequence was successfully determined using the enzyme. Based on the determined amino acid sequence, a part of the Penicillium lilacinochinatum NBRC6231-derived GDH gene was obtained by PCR, and the base sequence was determined (1356 bp) . (Experimental example 1 [3] [4])
Finally, based on this base sequence, the Aspergillus oryzae GDH gene was estimated and obtained from the published genome database of Aspergillus oryzae (Experimental Example 1 [5]).

<Experimental example 1>
Aspergillus oryzae-derived glucose dehydrogenase (hereinafter also referred to as AOGDH) gene estimation [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. Hereinafter, this is referred to as Aspergillus oryzae TI strain. The L dry strain of Aspergillus oryzae strain TI was inoculated on potato dextrose agar medium (manufactured by Difco) and recovered by incubating at 25 ° C. The mycelium on the restored plate was collected together with the agar and suspended in filter sterilized water. Prepare 6 L of production medium (1% malt extract, 1.5% soybean peptide, 0.1% MgSO ₄ .7 hydrate, 2% glucose, pH 6.5) in two 10 L jar fermenters, After autoclaving at 120 ° 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, then adsorbed on an Octyl-Sepharose column, and ammonium sulfate saturation 60 The fraction with GDH activity was recovered by elution with a gradient of from 0 to 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 GDH derived from Penicillium filamentous fungus Using Penicillium lilacinochinatumum NBRC6231 (purchased from National Institute of Technology and Evaluation Technology) as a GDH-producing fungus derived from Penicillium filamentous fungus, the same procedure as the above Aspergillus oryzae TI strain Then, culture and purification were performed, and a nearly uniform purified sample was obtained by SDS electrophoresis.

[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 partial sequence of GDH gene GDH derived from Penicillium lilacinoechinatum NBRC6231 purified above was dissolved in Tris-HCl buffer (pH 6.8) containing 0.1% SDS and 10% glycerol, and Glu-specific V8 was added thereto. Endoprotease was added to a final concentration of 10 μg / ml, and partial degradation was performed by 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: 15) and WGGGTKQTVRAKGALGTGT (SEQ ID NO: 16). Based on this sequence, a degenerate primer containing a mixed base was prepared, and PCR was carried out using a Penicillium lilacinoechinatum NBRC6231-derived cDNA as a template. As a result, an amplification product was obtained, which was confirmed by agarose gel electrophoresis. It was about a single band. 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 Based on the determined base sequence, 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%.

<Example 1>
Introduction of Aspergillus oryzae-derived glucose dehydrogenase (hereinafter referred to as AOGDH) gene into E. coli mRNA was prepared from Aspergillus oryzae cells and cDNA was synthesized. Two types of oligo DNAs shown in SEQ ID NOs: 5 and 6 were synthesized, and AOGDH gene (wild type) was amplified using KOD-Plus (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. This recombinant plasmid was introduced using competent high DH5α (manufactured by Toyobo). A plasmid was extracted according to a conventional method, and the base sequence of the AOGDH gene was determined (SEQ ID NO: 3). The amino acid residue deduced from the cDNA sequence was 593 amino acids (SEQ ID NO: 4).
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. . Further, mFAD-GDH was substituted with M at the N-terminal K and the same total number of amino acids as mFAD-GDH was expressed as S3. In S2, PCR is performed using the oligonucleotide of SEQ ID NO: 7 as an N-terminal primer and a combination of the primer of SEQ ID NO: 6, and a recombinant plasmid having a DNA sequence encoding S2 is constructed in the same manner. A transformant was obtained. In S3, PCR is performed using the oligonucleotide of SEQ ID NO: 8 as the N-terminal primer and a combination of the primer of SEQ ID NO: 6, and a recombinant plasmid having a DNA sequence encoding S3 is constructed. I got it. In addition, it was confirmed that the plasmid having the DNA sequence of each modified FAD-GDH has no sequence error in DNA sequencing.
SEQ ID NO: 9 shows the DNA sequence of the signal peptide deletion mutant S2 determined above. SEQ ID NO: 10 shows its corresponding amino acid sequence.
These transformants were subjected to liquid culture in TB medium for 1-2 days using a 10 L-jar fermenter. After collecting the cells in each culture phase, the cells were ultrasonically disrupted to confirm the GDH activity. The relationship between the culture phase of various transformants and the OD, pH, GDH activity is shown in Tables 1, 2, 3 and FIGS. In addition, Aspergillus oryzae wild strain was prepared from a liquid medium (1% malt extract, 1.5% soybean peptide, 0.1% MgSO ₄ .7hydrate, 2% glucose, 0.05 mM p-benzoquinone, 0.1 mM EDTA. , PH 6.5) using a 10 L-jar fermenter and culturing at 30 ° C. for 1 day, and confirming the activity inside or outside the cells, both of which are about 0.2 U / ml culture solution GDH It was active.

In this patent, the expression level is compared by comparing the amount of GDH activity per 1 ml culture solution.
In wild-type FAD-GDH (WT), a peak (6.6 U / ml-b) was observed after 16 to 18 hours of culture, and thereafter a decrease in activity was observed. On the other hand, S2 of modified FAD-GDH shows a peak at 22-25 hours (72-73 U / ml-b), and S3 at 20-23 hours (74-75 U / ml-b), similar to WT Thereafter, a decrease in GDH activity was observed.
When the culture titers at the respective peaks were compared, it was revealed that the GDH productivity increased by 10 times or more by deleting the amino acid sequence that seems to be a signal peptide.
Moreover, when the specific activity (U / mg) of the FADGDH purified preparation was compared before and after the deletion of the signal peptide, the wild type FADGDH (WT) was 270 U / mg, and the modified FADGDH S2 after deletion was 670 U / mg. There was a 2.5-fold increase in specific activity. It has been suggested that the specific activity is also increased by deleting the signal peptide.
Considering the increase in specific activity, the amount of activity seems to increase at least 4.4 times.

Furthermore, as a result of intensive studies, we have found that the stability of FAD-GDH derived from filamentous fungi decreases in the pH range of pH 7.1 or higher. In this study, how important is pH control at pH 7.1 to 7.3 or less, preferably pH 7.1 or less in the production culture of the recombinant FAD-GDH derived from filamentous fungi. Was revealed. It has been confirmed that a peak of GDH activity can be maintained by controlling the culture so that the pH is lower than the above value.
In the method of recombinantly producing filamentous fungus-derived glucose dehydrogenase (hereinafter also referred to as GDH), if the mutation is introduced into the signal peptide sequence present in the N-terminal region, the culture control pH is increased to 7.3. Can do.

<Example 2>
Introduction of Aspergillus terreus-derived glucose dehydrogenase (hereinafter referred to as ATGDH) gene into Escherichia coli The ATGDH gene is a cell of Aspergillus terreus (registered number NBRC 33026, which is registered in the Product Evaluation Technology Infrastructure and Biological Resources Division). MRNA was prepared and cDNA was synthesized. Two types of oligo DNAs shown in SEQ ID NOs: 13 and 14 were synthesized, and the ATGDH gene (gene sequence from which the expected signal peptide sequence was removed) was amplified using KOD-Plus (manufactured by Toyobo) with 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. This recombinant plasmid was introduced using competent high DH5α (manufactured by Toyobo). A plasmid was extracted according to a conventional method, and the base sequence of the ATGDH gene was determined (SEQ ID NO: 11). The amino acid residue deduced from the cDNA sequence was 568 amino acids (SEQ ID NO: 12).
These transformants were cultured in 50 ml of LB medium containing 100 μg / ml ampicillin at 30 ° C. overnight, and then again cultured in 50 ml of LB medium containing 100 μg / ml ampicillin at 30 ° C. for 8 hours. An inoculum was prepared.
The prepared inoculum was liquid-cultured in a 10 L-jar fermenter for 6 days in a TB medium containing 100 μg / ml ampicillin in the range of Kd · P 0.5 to 1.5. After collecting the cells in each culture phase, the cells were ultrasonically disrupted to confirm the GDH activity. The relationship between the culture phase and OD, pH, GDH activity is shown in Tables 4, 5, 6, 7 and FIGS. In addition, Aspergillus terreus wild strain was prepared in a liquid medium (1% malt extract, 1.5% soybean peptide, 0.1% MgSO ₄ .7hydrate, 2% glucose, 0.05 mM p-benzoquinone, 0.1 mM EDTA. , PH 6.5), and culturing at 30 ° C. for 1 day using a 10 L-jar fermenter, and confirming the GDH activity in the cells, the peak was about 0.1 U / ml culture solution.
On the other hand, in the FAD-GDH recombinant, an activity of about 9 to 21 U / ml-b was observed at the peak, and an increase in culture titer of about 100 to 200 times was observed. In addition, when the culture conditions were examined, by setting Kd · P as low as 0.5, it was about 1.5 times when Kd · P 0.75, and about Kd · P 1 to 1.5. A double culture titer was observed. In addition, when Kd · P is set to 2 or more, it has been confirmed that the culture titer is about 5 U / ml-b or less.
Regarding the culture temperature, the relationship between the culture phase and the OD, pH, GDH activity is shown in Tables 8, 9, 10, and 11 and FIGS. The culture temperature is optimally 26-28 ° C, and it is necessary to culture at least 23-28 ° C. When cultured at a temperature higher than 29 ° C, the culture titer tended to be halved.
Even when cultivating ATGDH gene recombinants, it is very important to keep the pH of the culture solution at 7.3 or lower in order to keep the enzyme activity stable. In ATGDH, culturing at a lower pH than AOGDH. It seemed necessary to quit.
The ATGDH gene is a signal peptide (MLGKLSFLSALSLAVAATLSNSTSA) (SEQ ID NO: 17) sequence part of DNA excised from the DNA sequence encoding Aspergillus tereus-derived FAD-GDH. Those containing a signal peptide sequence include: Similar to oryzae-derived FAD-GDH, the expression level of FAD-GDH was poor, and from the beginning, the culture was examined using one that does not contain a signal peptide.

  Tables 1 to 3 show the relationship between the culture phase in 10 L jar fermenter culture of various mutants, the turbidity (OD), pH, and GDH activity value of the culture solution. Table 1 corresponds to FIG. 2, Table 2 corresponds to FIG. 3, and Table 3 corresponds to FIG.

  Tables 4 to 7 show the culture phase and the cell turbidity (OD), pH, GDH activity value of Kd · P 0.5, 0.75, 1, 1.5 in 10 L jar fermenter culture, respectively. The relationship is shown. Table 4 corresponds to FIG. 5, Table 5 corresponds to FIG. 6, Table 6 corresponds to FIG. 7, and Table 7 corresponds to FIG.

  Tables 8 to 11 show the relationship between the culture phase and the microbial turbidity (OD), pH, and GDH activity values at culture temperatures of 20, 23, 26, and 30 ° C. in 10 L jar fermenter culture, respectively. Table 8 corresponds to FIG. 9, Table 9 corresponds to FIG. 10, Table 10 corresponds to FIG. 11, and Table 11 corresponds to FIG.

  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.

Claims (2)

  A method for recombinant production of Aspergillus terreus-derived FAD-dependent glucose dehydrogenase in Escherichia coli, wherein the Aspergillus terreus-derived wild-type FAD-dependent glucose dehydrogenase is derived from Aspergillus terreus registered under the deposit number NBRC 33026. A gene encoding a mutant FAD-dependent glucose dehydrogenase in which the amino acid sequence of MLGKLSFLSALSLAVAA present in the N-terminal region of the amino acid sequence of FAD-dependent glucose dehydrogenase and the amino acid sequence consisting of 8 amino acid residues following the C-terminal side are deleted Is expressed in Escherichia coli.   Amino acid residues of MLGKLFSLSLSLAVAAA present in the N-terminal region of the amino acid sequence of FAD-dependent glucose dehydrogenase derived from Aspergillus terreus registered as deposit number NBRC 33026 from wild-type FAD-dependent glucose dehydrogenase derived from Aspergillus terreus A DNA encoding a mutant FAD-dependent glucose dehydrogenase from which an amino acid sequence consisting of 8 amino acid residues following the C-terminal is deleted.