Patent Application
20110053194
The present invention relates to a transformant in which a flavin adenine dinucleotide-binding glucose dehydrogenase (also referred to as “FAD-GDH” in the present specification) gene has been introduced, and a method for producing FAD-GDH using the transformant.
In recent years, a simplified self blood glucose meter using an electrochemical biosensor has been widely used. The biosensor has an electrode and an enzyme reaction layer formed on an insulating substrate. Examples of an enzyme used herein include glucose dehydrogenase (GDH) and glucose oxidase (GO). A method of using GO is easily affected by dissolved oxygen in a measurement sample, and such a problem that the dissolved oxygen gives an adverse effect on a measurement result has been pointed out.
On the other hand, as an enzyme that does not receive an effect of dissolved oxygen and acts on glucose in absence of NAD (P), GDH (PQQ-GDH) having pyrroloquinoline quinone (PQQ) as a coenzyme has been known (for example, see Patent Documents 1 to 3). However, PQQ-GDH has problems such that (1) PQQ is easily dissociated from an enzyme, (2) selectivity to glucose is low, and (3) since it is generally present in a membrane fraction, extraction and isolation operations thereof are performed with difficulty.
By the way, in recent years, a case of developing hypoglycemia, etc. in a patient who used a simplified self blood glucose meter in a hospital has been reported. As a result of an examination and analysis thereafter, it was revealed that such a developing case was caused by PQQ-GDH which was reacted to maltose contained as an infusion component and a higher value than an actual value was shown (Pharmaceuticals and Medical Devices Safety Information No. 206, October 2004, the Pharmaceutical and Medical Safety Bureau in the Ministry of Health, Labour and Welfare). In view of such a circumstance, development of a blood glucose meter that specifically acts on glucose and has low reactivity particularly to maltose has been eagerly desired.
In addition, as an enzyme that does not receive an effect of dissolved oxygen and acts on glucose in absence of NAD (P), other than PQQ-GDH, a glucose dehydrogenase having a flavin adenine dinucleotide as a coenzyme (that is, FAD-GDH) has been known. FAD-GDH has been obtained respectively from Aspergillus oryzae (Non-patent Documents 1 to 4) and Aspergillus terreus (Patent Document 4) so far. However, production amounts of the FAD-GDH are hardly recognized as sufficient production amounts supplied to industrial applications.
The study group of the present inventors focused on FAD-GDH having a flavin adenine dinucleotide as a coenzyme for the purpose of obtaining novel FAD-GDH capable of more accurately measuring a glucose level and a producing microorganism thereof. As a result of broadly screening microorganisms as objects, Aspergillus oryzae excellent in productivity of FAD-GDH was obtained. Further, they succeeded in purifying FAD-GDH that the fungus strain produced and also succeeded in determination of its various characteristics. The determined various characteristics revealed that the FAD-GDH is a novel enzyme. It was also revealed that the FAD-GDH is excellent in selectivity for glucose and at the same time is hardly affected from dissolved oxygen present in a reaction system, and thus is capable of more accurately measuring a glucose level in a sample. As a result of further progress of the studies, the present inventors succeeded in determining an amino acid sequence of FAD-GDH that the above described fungus strain has and a genetic sequence encoding the amino acid sequence. These results of the studies have been already filed in an international application (International Application No. PCT/JP2007/060694).
In view of the above described backgrounds, an object of the present invention is to highly efficiently produce a large amount of novel FAD-GDH capable of more accurately measuring a glucose level.
The present inventors made intensive studies in order to solve the above described problems. That is, they analyzed a gene encoding Aspergillus oryzae BB-56-derived FAD-GDH and found out that a specific portion of a base sequence gives effects on an optimal pH of a FAD-GDH enzyme, pH stability, temperature stability, substrate specificity, and FAD-GDH production ability of a microorganism, in other words, a functional aspect and productivity of the enzyme. As a result of producing a transformant of a microorganism on the basis of the above finding, the inventors succeeded in obtaining a transformant with extremely high productivity of FAD-GDH and thus achieved completion of the present invention. The present invention is stated as follows.
[1] A transformant, in which a DNA encoding a flavin adenine dinucleotide-binding glucose dehydrogenase selected from the group consisting of the following (a) to (f) has been introduced:
(a) a DNA encoding the amino acid sequence of SEQ ID NO: 20;
(b) a DNA consisting of the base sequence of SEQ ID NO: 19;
(c) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity;
(d) a DNA encoding the amino acid sequence of SEQ ID NO: 34;
(e) a DNA consisting of the base sequence of SEQ ID NO: 33; and
(f) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity.
[2] The transformant according to [1], which is obtained by transforming a host microorganism belonging to the genus Aspergillus or the genus Saccharomyces.
[3] The transformant according to [1], which is obtained by transforming Aspergillus oryzae.
[4] The transformant according to [1], which is obtained by transforming Saccharomyces cerevisiae.
[5] A method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase, including the following steps (1) and (2):
(1) a step of culturing the transformant according to any of [1] to [4] under the condition of producing a protein encoded by the DNA; and
(2) a step of recovering the produced protein.
[6] A glucose measurement method, wherein glucose in a sample is measured using a flavin adenine dinucleotide-binding glucose dehydrogenase produced in the production method according to [5].
[7] A glucose measuring reagent, containing a flavin adenine dinucleotide-binding glucose dehydrogenase produced in the production method according to [5].
[8] A glucose measuring kit, containing the glucose measuring reagent according to [7].
A “DNA encoding a protein” in the present specification refers to a DNA capable of obtaining the protein when the DNA is expressed, that is, a DNA having a base sequence corresponding to an amino acid sequence of the protein. Therefore, degeneracy of codons is also taken into consideration.
The term “isolated” in the present specification is used exchangeably to “purified”. “Isolated” when used in relation of the enzyme of the present invention (FAD-GDH), the term refers to a state in which, in the case where the enzyme of the present invention is derived from natural materials, components other than the enzyme are not substantially contained in the natural materials (in particular, contaminating protein is not substantially contained). Specifically, for example, in the isolated enzyme of the present invention, a content of contaminating protein is about less than 20% of the total, preferably less than 10%, more preferably less than 5%, and further more preferably less than 1% in weight conversion. On the other hand, the term “isolated” when the enzyme of the present invention is prepared in a genetic engineering technique refers to a state in which other components derived from a used host cell, a culture liquid, and the like are not substantially contained. Specifically, for example, in the isolated enzyme of the present invention, a content of contaminants is about less than 20% of the total, preferably less than 10%, more preferably less than 5%, and further more preferably less than 1% in weight conversion. In addition, as long as a different meaning from the above definition is not clearly specified, when “a flavin adenine dinucleotide-binding glucose dehydrogenase” simply described in the present specification, the wording means “a flavin adenine dinucleotide-binding glucose dehydrogenase in a state of being isolated”. The terms of “FAD-GDH” and “enzyme” used in substitution for the flavin adenine dinucleotide-binding glucose dehydrogenase are also defined in the same manner.
“Isolated” as used for a DNA refers to a state in which, in the case of a DNA that is originally naturally present, typically, the DNA is separated from a nucleic acid that is coexistent in the natural condition. Provided that such a DNA may contain a part of other nucleic acid components such as an adjacent nucleic acid sequence in the natural condition (e.g., a sequence in a promoter region and a terminator sequence). For example, in the state of being “isolated” in the case of a genome DNA, other DNA components that are coexistent in the natural condition are not substantially contained. On the other hand, in the state of being “isolated” in the case of a DNA prepared in a genetic engineering technique, such as a cDNA molecule, the DNA preferably does not substantially contain cell components, a culture liquid, and the like. In the same manner, in the state of being “isolated” in the case of a DNA prepared through chemical synthesis, the DNA preferably does not substantially contain a precursor (raw material) such as dNTP and chemical substances used in synthetic processes. As long as a different meaning from the above definition is not clearly specified, when a “DNA” is simply described in the present specification, the term means a DNA in the state of being isolated.
(Transformant in which a DNA Encoding FAD-GDH has been Introduced)
The first aspect of the present invention provides a transformant in which a DNA encoding novel FAD-GDH has been introduced. In the transformant of the present invention, (a) a DNA encoding an amino acid sequence of SEQ ID NO: 20; (b) a DNA consisting of a base sequence of SEQ ID NO: 19; (c) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity; (d) a DNA encoding an amino acid sequence of SEQ ID NO: 34; (e) a DNA consisting of a base sequence of SEQ ID NO: 33; or (f) a DNA having a base sequence homologous to the base sequence of SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity is present as an exogenous molecule.
The amino acid sequence of SEQ ID NO: 20 is an amino acid sequence of FAD-GDH that Aspergillus oryzae BB-56 has, which was succeeded in identification by the present inventors. A specific example of a DNA encoding the amino acid sequence of SEQ ID NO: 20 is a DNA consisting of a base sequence of DNA SEQ ID NO: 19. Herein, when modification is performed on a part of a DNA encoding a certain protein, generally, a protein encoded by the DNA after modification may have functions equal to those of the protein encoded by the DNA before modification. That is, modification of a DNA sequence does not substantially give an effect on functions of a protein to be encoded and the functions of the protein to be encoded may be kept before and after modification. Thus, in one embodiment of the present invention, a DNA (c) having a base sequence homologous to the base sequence of SEQ ID NO: 19 and encoding a protein having a FAD-GDH activity is introduced. The “homologous base sequence” herein refers to a base sequence that is partly different from a nucleic acid of SEQ ID NO: 19 but is not substantially affected by the difference on the function (FAD-GDH activity is indicated herein) of a protein encoded by the nucleic acid.
A specific example of the homologous DNA includes a DNA that hybridizes under a stringent condition for a base sequence complementary to the base sequence of SEQ ID NO: 19. The “stringent condition” herein refers to a condition that a so-called specific hybrid is formed and a nonspecific hybrid was not formed. Such a stringent condition is known to a skilled person and can be set in reference to, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987). As the stringent condition, an example includes a condition such that a hybridization liquid (50% formamide, 10×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of denatured salmon sperm DNA, and a 50 mM phosphate buffer (pH 7.5)) was used and incubated at about 42° C. to about 50° C., and then washed at about 65° C. to about 70° C., using 0.1×SSC, and 0.1% SDS. An example of more preferable stringent condition is a condition of using 50% formamide, 5×SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 1×Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg/ml of denatured salmon sperm DNA, and a 50 mM phosphate buffer (pH 7.5) as a hybridization liquid.
Other specific examples of the homologous DNA include a DNA consisting of a base sequence having the base sequence of SEQ ID NO: 19 as a base and containing substitution, deletion, insertion, addition or inversion of one base or plural bases, and encoding a protein having a FAD-GDH activity. Substitution, deletion, etc. of bases may be generated in plural sites. “Plural” herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although it depends on a position or a kind of an amino acid residue in a cubic structure of a protein encoded by the DNA. Such a homologous DNA as described above can be obtained by modification of a DNA having the base sequence of SEQ ID NO: 19 so as to contain substitution, deletion, insertion, addition and/or inversion of bases, utilizing, for example, a restricted enzyme treatment, a treatment with exonuclease, DNA ligase, or the like, an introduction method of site-directed mutations (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), an introduction method of random mutations (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York), or the like. In addition, a homologous DNA can also be obtained by other methods such as ultraviolet irradiation.
Other examples of the homologous DNA include a DNA in which difference of bases as described above is recognized due to polymorphism typically represented by SNP (single nucleotide polymorphism).
A DNA to be introduced for the transformant of the present invention can be prepared into a state of being isolated by use of a standard genetic engineering technique, molecular biological technique, biochemical technique, or the like, in reference to sequence information disclosed in the specification or attached sequence listing. Specifically, the DNA can be prepared by suitably using an oligonucleotide probe primer capable of hybridizing specifically to the DNA of the present invention from a genome DNA library or a cDNA library of suitable mould fungi or yeast fungi, or an extraction liquid of fungus bodies of mould fungi or yeast fungi. An oligonucleotide probe primer can be easily synthesized using a commercially available automation DNA synthesizer, or the like. For a production method of a library used to prepare the gene of the present invention, for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York can be referred to.
When the DNA is a gene having the base sequence of SEQ ID NO: 19, for example, it can be isolated by use of a hybridization method in which whole or a part of the base sequence or its complementary sequence is used as a probe. Furthermore, the DNA can be amplified and isolated by use of a nucleic acid amplification reaction (for example, PCR) using a synthesized oligonucleotide primer designed so as to hybridize specifically to a part of the base sequence.
By the way, FAD-GDH that was succeeded in identification by the present inventors (an enzyme having an amino acid sequence of SEQ ID NO: 20, herein referred to as “the present enzyme”) has the following properties (for details, see International Application No. PCT/JP2007/060694 described above). First, the present enzyme catalyzes a following reaction, that is, a reaction of generating glucono-β-lactone by oxidizing a hydroxyl group of glucose in the presence of an electron receptor. The molecular weight of the present enzyme by SDS-polyacrylamide gel electrophoresis is about 100 kDa, and the molecular weight of the present enzyme by gel filtration is about 400 kDa (tetramer).
On the other hand, the present enzyme is excellent in substrate specificity and selectively acts on D-glucose. Specifically, FAD-GDH of the present invention has an extremely low reactivity to maltose and also has a very low reactivity to D-fructose, D-mannose, D-galactose, and the like. Specifically, when a reactivity to D-glucose is assumed to be 100%, reactivities to these substrates are all 5% or less. In a preferable embodiment of the present invention, a reactivity to maltose is 1% or less or is not substantially recognized. In a more preferable embodiment, both of a reactivity to D-fructose and a reactivity to D-galactose are 1% or less or are not substantially recognized. The present enzyme having excellent substrate specificity as described above is preferable as an enzyme for accurately measuring a glucose level in a sample. That is, according to the present enzyme, even when impure substances such as maltose and galactose are present in the sample, a desired glucose level can be more accurately measured. Therefore, the present enzyme can be recognized as being suitable for an application (typically, measurement of a glucose level in blood) in which presence of such contaminants in a sample is expected or concerned, and also considered to be applicable to various applications including the application, that is, have high versatility.
As described above, the present enzyme was obtained from an Aspergillus oryzae BB-56 strain. Note that the fungus strain is deposited in an appointed deposit institution as described below and is easily available.
Deposit institution: Incorporated Administrative Agency, Patent Microorganisms Depositary, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan)
Date of deposit: May 17, 2006
Accession number: NITE BP-236
It was revealed that the present enzyme obtained from Aspergillus oryzae BB-56 further has the following properties (for details, see International Application No. PCT/JP2007/060694 described above).
(4) Optimal pH: around 7
(5) Optimal temperature: around 60° C.
(6) pH stability: stable within the range from pH 3.0 to 7.0
(7) Temperature stability: stable at 40° C. or less
(8) Km value: Km value for D-glucose is about 8 mM
(9) Isoelectric point (pI): around 6.4
As described above, it is revealed that the present enzyme has selectivity and high affinity to glucose, is also excellent in stability, and suitable for applications and practical uses to a glucose sensor, and the like.
The transformant of the present inventor is typically prepared through transformation using a DNA to be introduced, or a DNA construct containing the DNA (e.g. a form of a vector). In the case of transformation with a vector, an expression vector is preferably used. The “expression vector” refers to a vector that can introduce a nucleic acid inserted therein into a desired cell (host cell) and is capable of expressing in the cell. The expression vector generally includes a promoter sequence necessary for expression of an inserted nucleic acid. Further, the expression vector preferably contains an enhancer sequence that promotes expression. An expression vector containing a selective marker can also be used. When such an expression vector is used, presence of absence (or its degree) of introduction of the expression vector can be confirmed utilizing a selective marker.
Insertion of the gene of the present invention into a vector, insertion of a selective marker gene (when required), insertion of a promoter (when required), and the like can be performed using a standard recombinant DNA technique (for example, a known method of using a restricted enzyme and DNA ligase, which can be referred to Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York).
Transformation can be performed by calcium phosphate co-precipitation, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci. U.S.A. 81, 7161-7165 (1984)), lipofection (Felgner, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)), Microinjection (Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. U.S.A. 73, 366-370 (1976)), the Hanahan method (Hanahan, D., J. Mol. Biol. 166, 557-580 (1983)), the lithium acetate method (Schiestl, R. H. et al., Curr. Genet. 16, 339-346 (1989)), the protoplast-polyethylene glycol method (Yelton, M. M. et al., Proc. Natl. Acad. Sci. 81, 1470-1474 (1984)), or the like.
Examples of a host cell include bacterial cells such as E. coli, mould fungi cells (e.g., Aspergillus oryzae, and Aspergillus niger), and yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris, and Schizosaccharomyces pombe).
Preferably, microorganisms belonging to the genus Aspergillus or microorganisms belonging to the genus Saccharomyces are used as a host. Examples of microorganisms belonging to the genus Aspergillus include Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, and Aspergillus awamori, and Aspergillus oryzae of a gene supply species that is considered to have a structure necessary for correct expression of an enzyme protein is the most preferable. On the other hand, examples of microorganisms belonging to the genus Saccharomyces include Saccharomyces cerevisiae, Pichia pastoris and Schizosaccharomyces pombe, and Saccharomyces cerevisiae is the most preferable.
The second aspect of the present invention provides a method of producing FAD-GDH using the above described transformant. In the production method of the present invention, first, the transformant is cultured under the condition of producing a protein encoded with a gene introduced therein (step (1)). A culture method and culture conditions are not particularly limited as long as a desired enzyme is produced. That is, a method and culture conditions suitable for culture of a transformant in use can be appropriately set on condition of producing the present enzyme. In the following, a medium, a culture temperature, and a culture time are exemplified as the culture conditions.
As a medium, any medium may be used as long as it is a medium capable of growing a transformant in use. For example, carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, bees wax, and organic acids, further, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or nitrogen sources such as peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, and meat extracts, and furthermore, substances added with inorganic salts such as potassium salt, magnesium salt, sodium salt, phosphate salt, manganese salt, iron salt, and zinc salt can be used. Vitamins, amino acid, and the like may be added to a medium in order to promote growth of the transformant in use. The pH of the medium is adjusted to, for example, about 3 to 8, and preferably about 5 to 7, and the transformant is cultured in an aerobic condition at a culture temperature of generally about 10 to 50° C., and preferably about 25 to 35° C., for 1 to 15 days, and preferably 3 to 7 days. As a culturing method, examples such as shake culture, aerobic submerged culture with a Jar fermentor can be used.
After the culture step, a produced protein (that is, FAD-GDH) is recovered (step (2)). When FAD-GDH can be recovered from the culture liquid, for example, the culture supernatant is treated by filtration and centrifugation to remove insoluble substances, and then, separation and purification are performed suitably combining with condensation with an ultrafilter membrane, salting out such as ammonium sulfate precipitation, dialysis, various chromatographies, and the like to thus obtain desired FAD-GDH. On the other hand, in the case of recovering from a fungus body, for example, a fungus body is crushed with a pressure treatment, an ultrasonic treatment, or the like, and then, separation and purification are performed in the same manner as described above to thus obtain desired FAD-GDH. In addition, the above described series of steps (fracture of fungus body, separation, and purification) may be performed after recovering a fungus body from a culture liquid previously by filtration, centrifugation, and the like.
Fractionation is performed using a FAD-GDH activity as an index as a general rule in each purification step, proceeding to the next step. However, the rule is not applied in the case that suitable conditions can be already set by a preliminary test, and the like.
Purity of FAD-GDH is not particularly limited and, for example, FAD-GDH can be purified so as to be in a state of having a relative activity of 50 to 160 (U/mg), and preferably 100 to 160 (U/mg). Further, FAD-GDH may be a liquid form or a solid form (including a powder state) in the final form.
A further aspect of the present invention relates to an application use of FAD-GDH produced in the above described production method. In this aspect, first, a glucose measurement method using FAD-GDH is provided. In the glucose measurement method of the present invention, a glucose level in a sample is measured utilizing an oxidation-reduction reaction with FAD-GDH. The present invention is used in, for example, a measurement of a blood sugar level, a measurement of a glucose concentration in foods (such as seasonings and beverages), and the like. The present invention may be further used to examine a fermentation degree in production steps of fermented foods (e.g., vinegar) or fermented beverages (e.g., beer and alcohols). FAD-GDH is not required to be added with a coenzyme in measurement since FAD that is a coenzyme is constantly combined with GDH, and thus, a simple and easy measurement system can be constructed.
The present invention further provides a reagent for glucose measurement including FAD-GDH produced in the above described production method. The reagent is used in the above described glucose measurement method of the present invention.
The present invention is furthermore provides a kit for performing the glucose measurement method of the present invention (glucose measuring kit). The kit of the present invention includes a reaction reagent, a buffer solution, a glucose standard solution, and the like as optional elements, in addition to the reagent for glucose measurement containing FAD-GDH produced in the above described production method. In addition, an instruction manual is generally accompanied with the glucose measuring kit of the present invention.
The present invention will be specifically described below in reference to examples, and the invention is not limited to the examples. In the examples, products made by TAKARA BIO INC. and Toyobo Co., Ltd. were used as restricted enzymes and other genetic manipulation enzymes, as otherwise particularly stated. Note that reaction conditions, and the like of the enzymes were in accordance with attached instruction manuals. Determination of a base sequence was performed with an ABI PRISM™ 310 Genetic Analyzer and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Co.), and a PCR reaction was performed with a GeneAmp PCR System 9600 (Perkin Elmer Japan Co., Ltd.). These apparatuses, and the apparatuses and products for genetic manipulation used in the examples were operated in accordance with recommended conditions in instruction manuals attached to the respective products as otherwise particularly stated.
According to the following procedure, Aspergillus oryzae BB-56 was cultured to produce FAD-GDH. 50 ml of a medium for preliminary culture, which was constituted with the components below, was dividedly charged in a 300 mL-volume conical flask and sterilized at 121° C. and 0.12 MPa for 20 minutes. The medium was cooled to room temperature, and Aspergillus oryzae BB-56 was then inoculated and cultured at 30° C. and 200 rpm for 3 days.
Yeast extract (Becton, Dickinson Company) 0.2% (w/v)
Soybean peptone (DMV Co.) 1.0% (w/v)
Glucose (Wako Pure Chemical Industries, Ltd.) 2.0% (w/v)
KH2PO4 (Wako Pure Chemical Industries, Ltd.) 0.1% (w/v)
MgSO4.7H2O (Sigma-Aldrich Japan K.K.) 0.05% (w/v)
Medium pH 5.7
FAD-GDH was produced with a medium for main culture constituted with the components below. 50 ml of the medium for main culture was dividedly charged in a 300 mL-volume conical flask and sterilized at 121° C. and 0.12 MPa for 20 minutes. The medium was cooled to room temperature, 1 mL of the culture liquid of the above preliminary culture was then inoculated, and the main culture was performed at 30° C. and 200 rpm for 4 days to produce FAD-GDH.
Glucose 15.0% (w/v)
Meast P1G (ASAHI FOOD & HEALTHCARE CO., LTD.) 3.0% (w/v)
Soybean peptone 6.0% (w/v)
KH2PO4 0.3% (w/v)
K2HPO4 (Wako Pure Chemical Industries, Ltd.) 0.2% (w/v)
4 mM Hydroquinone (Wako Pure Chemical Industries, Ltd.)
Medium pH 6.0
FAD-GDH was purified under the following conditions. Fungus bodies and other solid contents in the culture liquid were removed by diatomite filtration of the culture liquid obtained as described above. The purified liquid obtained through the operation was desalinated and concentrated with an ultrafilter membrane. The concentrated liquid was subjected to salting out with 90% saturated ammonium sulfate and the centrifuged supernatant was desalinated and concentrated with an ultrafilter membrane. The desalinated and concentrated liquid was applied to a CM Sepharose Fast Flow (Amersham Biosciences Co.) equilibrated with a 10 mM Mcllvaine buffer solution at pH 5.5 to adsorb FAD-GDH to a column. The column was washed with a 10 mM Mcllvaine buffer solution at pH 5.5 and FAD-GDH was eluted with a 10 mM Mcllvaine buffer solution containing 0.1 M of NaCl at pH 5.5 to recover a FAD-GDH active fraction. The recovered active fraction was concentrated with an ultrafilter membrane and applied to Octyl-Sepharose equilibrated with a 10 mM K2HPO4—NaOH buffer solution containing 2.5 M of ammonium sulfate at pH 6.5 to adsorb FAD-GDH to a column. The column was washed with a 10 mM K2HPO4—NaOH buffer solution containing 2.5 M of ammonium sulfate and FAD-GDH was then eluted by changing a concentration of ammonium sulfate in the buffer solution to 2.0, 1.5, 1.0 and 0.5 M in stages. The active fraction of FAD-GDH was recovered, and desalinated and concentrated with an ultrafilter membrane. The desalinated and concentrated liquid was applied to DEAE Sepharose™ CL-6B equilibrated with a 20 mM K2HPO4—NaOH buffer solution at pH 6.5 and FAD-GDH was eluted with a 20 mM K2HPO4—NaOH buffer solution at pH 6.5. This fraction was desalinated and concentrated with an ultrafilter membrane to thus obtain a purified enzyme preparation of FAD-GDH.
A partial amino acid sequence of Aspergillus oryzae BB-56-derived FAD-GDH was identified as follows.
3 mL of the purified FAD-GDH solution obtained in Example 2 was charged in a dialysis tube and immersed in purified water to perform dialysis at 4° C. for one night. This dialyzed sample was supplied to glycerol density gradient gel electrophoresis. Energization was performed at 5° C. at a low voltage of 400 V for 48 hours with a column volume of 110 mL using a carrier ampholyte (Pharmalyte 3-10 for IEF (Amersham Biosciences Co.)). After energization, the sample was recovered and a pH and a FAD-GDH activity of each fraction were measured to collect an active fraction.
The purified FAD-GDH obtained in the above described (1) was supplied to SDS-PAGE using a gel, PAG Mini “DAIICHI” 7.5 (Daiichi Pure Chemicals Co., Ltd.). Using a transcription buffer solution 1 (0.3 M tris (pH 10.4), 20% methanol), a transcription buffer solution 2 (30 mM tris (pH 10.4), 20% methanol), and a transcription buffer solution 3 (40 mM 6-aminohexanoic acid (pH 7.6), 20% methanol) as transcription buffer solutions, the gel after migration was transcribed to a PVDF membrane. The transcribed membrane after transcription was stained with Coomassie Brilliant Blue R-250 to detect and cut out a band corresponding to FAD-GDH. The cut out membrane was used as an analysis sample of an N-terminated amino acid sequence. In the same manner, the gel after SDS-PAGE was stained with Coomassie Brilliant Blue R250 to detect and cut out a band corresponding to FAD-GDH. The cut out gel was used as an analysis sample of an internal amino acid sequence. As a result of the analyses, the N-terminated amino acid sequence and the internal amino acid sequence were found out.
Homology in N-terminated amino acid sequences were searched as described below. All CDS sequences among the genetic information of Aspergillus oryzae RIB40 disclosed in JP 2005-176602 A were obtained from applicants of JP 2005-176602 A, homology search with N-terminated amino acid sequences found out in Example 3 was performed using database software, Kiroku Ver. 3 (World Fusion Co., Ltd.). As a result, high homology was recognized with an amino acid sequence (SEQ ID NO: 20494 in the sequence listing in JP 2005-176602 A) which has homology to a glucose oxidase precursor. In this sequence (SEQ ID NO: 20494 in the sequence listing in JP 2005-176602 A), a region corresponding to an analysis of an internal amino acid sequence of FAD-GDH identified in Example 3 was also present. From these facts, it was revealed that SEQ ID NO: 20494 (SEQ ID NO: 1) in the sequence listing in JP 2005-176602 A corresponds to an amino acid sequence of FAD-GDH.
Aspergillus oryzae BB-56 was cultured at 30° C. for one night using a shaking flask containing 100 ml of a YPD medium (1% yeast extract, 2% peptone, 2% glucose, pH 6.5), and then, the culture liquid was filtrated to obtain a fungus body. The fungus body was frozen and dried, and about 0.3 g of the dried fungus body was crushed with one spoonful of a scoopula of sea sand using a mortar, and suspended in 12 ml of an extraction buffer (1% hexadecyltrimethylammonium bromide, 0.7 M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% β-mercaptoethanol). After rotation stirring at room temperature for 30 minutes, an equivalent amount of a solution of phenol:chloroform:isoamyl alcohol (25:24:1, v/v) was added thereto and stirred, and thereafter centrifugation (1,500 g, 5 minutes, room temperature) was performed to obtain a supernatant. An equivalent amount of a solution of chloroform:isoamyl alcohol (24:1, v/v) was added to this supernatant and stirred, and thereafter centrifugation (1,500 g, 5 minutes, room temperature) was performed to obtain a supernatant. An equivalent amount of isopropanol was gently added to this supernatant, and centrifugation (20,000 g, 10 minutes, 4° C.) was performed. The obtained precipitate (deposited chromosome DNA) was washed with 70% ethanol and vacuum dried to obtain a chromosome DNA. The chromosome DNA was dissolved in 4 ml of TE (composition), thereto was added 200 μl of 10 mg/ml RNase A (Sigma-Aldrich Japan K.K.), and the mixture was incubated at 37° C. for 30 minutes. Then, 40 μl of a solution of 20 mg/ml Proteinase K, recombinant, PCR Grade (Roche Diagnostics K.K.) was added and incubated at 37° C. for 30 minutes, and an equivalent amount of a solution of phenol:chloroform:isoamyl alcohol (25:24:1, v/v) was then added. After stirring, the resultant solution was centrifuged (1,500 g, 5 minutes, room temperature) to obtain a supernatant. This washing operation was performed twice, then, an equivalent amount of a solution of chloroform:isoamyl alcohol (24:1, v/v) was added to the supernatant and stirred, and centrifugation (1,500 g, 5 minutes, room temperature) was performed. To the obtained supernatant, 1/10 volume of 3M NaOAc (pH 4.8) and twice the volume of ethanol were added, and a chromosome DNA was deposited at −80° C. The deposited chromosome DNA was centrifuged (20,000 g, 20 minutes, 4° C.) to thus recover as a precipitate. The recovered chromosome DNA was washed with 70% ethanol and then vacuum dried. The dried chromosome DNA was dissolved in 400 μl of a TE solution to thus obtain about 1 mg/ml of a chromosome DNA solution.
Synthetic primers of FG-F and FG-R (SEQ ID NO: 3, 4) corresponding to a sequence of 5′ and 3′ ends of the Aspergillus oryzae RIB40-derived gene sequence (SEQ ID NO: 2) having the whole chain length of 3226 bp, which encodes the amino acid sequence (SEQ ID NO: 1) of SEQ ID NO: 20494 in the sequence listing of JP 2005-176602 A were synthesized.
The Aspergillus oryzae BB-56-derived chromosome DNA prepared in Example 5 was used as a template, and a PCR reaction was performed. TAKARA LA Taq was used in the reaction, and the composition of the reaction solution was in accordance with a standard technique described in an attached instruction for use. Primers (FG-F, FG-R) were added in an each amount of 0.4 μM so that a template genome has a concentration of 10 ng/μl, and a PCR reaction was performed. The reaction cycle was as follows. After reacting at 94° C. for 2 minutes, reactions of (i) to (iii) constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 60° C. for 30 seconds, and (iii) a reaction at 72° C. for 3.5 minutes were repeated 35 times, and lastly, a reaction at 72° C. for 10 minute was performed. After completion of the reactions, the reaction solution was preserved at 4° C.
The PCR reaction solution of Example 7 was supplied to agarose electrophoresis, and an amplified fragment (3241 bp) and primers were separated to be extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.). The extracted amplified fragment was sub-cloned to a SmaI site of a vector pUC19 (TAKARA BIO INC.) to obtain pFG2 (
A high producing strain of Aspergillus oryzae BB-56-derived FAD-GDH was obtained as follows.
(1) Amplification of Orotidylate Decarboxylase (pyrG) Gene in PCR Method
Sequence information of a pyrG gene of Aspergillus oryzae RIB40 was obtained from JP 2005-176602 A, and synthesis primers of PYR-F and PYR-R (SEQ ID NO: 21, 22) corresponding to 5′ and 3′ ends of the sequence were formed. A PCR reaction was performed using the chromosome DNA derived from Aspergillus oryzae BB-56 prepared in Example 5 as a template. TAKARA LA Taq was used in the reaction, and the composition of the reaction was in accordance with a standard technique described in an attached instruction for use. The reaction solution was prepared so that the primers of PYR-F and PYR-R were contained in an each amount of 0.4 μM and a chromosome DNA of the template has a concentration of 10 ng/μl, and the PCR reaction was performed. In the PCR reaction condition, after reacting at 94° C. for 2 minutes, reactions of (i) to (iii) constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 58° C. for 30 seconds, and (iii) a reaction at 72° C. for 2 minutes were repeated 35 times, and lastly, a reaction at 72° C. for 10 minute was performed. After completion of the reactions, the reaction solution was preserved at 4° C.
The pyrG gene fragments (1884 bp) amplified by supplying the above described PCR reaction solution to agarose electrophoresis and the primers were separated and extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.). Both ends of the extracted pyrG gene fragment were digested with restricted enzymes, SalI and HindIII. The DNA fragments treated with the restricted enzymes and pFG2 treated with SalI and HindIII were ligated using a DNA Ligation Kit Ver. 2.1 to produce pFGP2 (
The above described transformant of E. coli was cultured and a plasmid pFGP2 was extracted from the fungus body. The extracted pFGP2 was digested with restricted enzymes, HpaI and HindIII, then supplied to agarose electrophoresis to separate about 5 kbp of a DNA fragment, and extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.). As expected, it was confirmed that 5 kbp of the DNA fragment was a DNA fragment formed by linking an Aspergillus oryzae BB-56-derived FAD-GDH gene and pyrG.
(4) Acquisition of Aspergillus oryzae BB-56-Derived Uridine Requiring Strain
Aspergillus oryzae BB-56 was cultured in a YPD medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar, pH 6.5) and spores were attached thereon. The attached spores were suspended in an aqueous 0.9% NaCl solution containing 0.01% Tween 80 to obtain a spore suspension. Ultraviolet ray was irradiated to the spore suspension to thereby induce a mutation, and the spore suspension was then spread on a minimum medium containing 5-fluoroorotic acid (5-FOA) (0.052% KCl, 0.152% KH2PO4, 0.6% NaNO3, 0.1% trace element solution (0.004% Na2B4O7.10H2O, 0.04% CuSO4.5H2O, 0.08% FePO4.nH2O, 0.103% MnSO4.5H2O, 0.08% Na2MoO4.2H2O, 0.8% ZnSO4.7H2O), 0.2% uridine, 1% glucose, 0.052% MgSO4.7H2O, 0.1% 5-FOA, 2% agar, pH 6.5) to obtain a 5-FOA resistant strain. Presence or absence of orotate phosphoribosyltransferase activity and orotidylate decarboxylase activity of the 5-FOA resistant strain were confirmed by a method of Belser, et al. (Methods in Enzymology Vol. 51 P.135) and an orotidylate decarboxylase activity-defected mutant strain showing an uridine requiring property was obtained.
The uridine requiring strain derived from Aspergillus oryzae BB-56 obtained above was cultured in an YPD medium containing uridine (1% yeast extract, 2% peptone, 2% glucose, 0.2% uridine, pH6.5) at 30° C. for one night and the culture liquid was then passed through a glass filter to obtain a fungus body, and the fungus body was suspended in a protoplast preparation liquid (0.8 M NaCl, 10 mM sodium phosphate buffer, pH 6.0, 3 mg/ml Yatalase™, 0.3 mg/ml Novozymes 234 (Novo Nordisk Pharma Ltd.)) and treated at 30° C. and 78 rpm for 1 hour and 30 minutes to perform a protoplastization treatment. The protoplast suspension was filtrated with a filter and the filtrate was centrifuged (780 g, 5 minute, room temperature) to obtain a precipitate. The precipitate was suspended in 10 mL of 0.8 M NaCl and the precipitate obtained by centrifugation (780 g, 5 minutes, room temperature) was suspended in 10 ml of 0.8 M NaCl. The number of protoplasts in the suspension was measured by microscopic observation and then centrifuged (780 g, 5 minutes, room temperature), and the precipitate was suspended in Sol I (0.8 M NaCl, 10 mM CaCl2, 10 mM Tris-HCl, pH 8.0) to prepare about 2×108/ml of a protoplast solution. The protoplast solution was added with ⅕ volume of a polyethylene glycol (PEG) solution (25% PEG 6000, 50 mM CaCl2, 10 mM Tris-HCl, pH 8.0) to be mixed. 200 μl of the mixed solution was dividedly charged, thereto was added 13 μl of a solution (19 μg/μl) of 5 kbp of the gene fragment prepared above, which is obtained by linking a FAD-GDH gene and a pyrG gene, and the solution was suspended and then stood still in ice for 30 minutes. 1 mL of a PEG solution (25% PEG6000, 50 mM CaCl2, 10 mM Tris-HCl, pH 8.0) was added to the suspension, and the mixture was suspended and stood still at room temperature for 15 minutes. Thereto was further added 8.5 mL of SolI, and the mixture was suspended and centrifuged (780 g, 5 minutes, room temperature) to obtain a precipitate, and the precipitate was suspended in 1.2 mL of SolI to obtain a protoplast suspension. 0.2 mL of the suspension was transferred to a petri dish, and thereafter a regenerating medium (0.052% KCl, 0.152% KH2PO4, 0.6% NaNO3, 0.1% trace element solution, 21.86% sorbitol, 1% glucose, 0.052% MgSO4.7H2O, 2% agar, pH 6.5) was poured to be solidified. After culture at 30° C. for 5 days to 7 days, the grown transformant was isolated into a single fungus with a minimum medium (0.052% KCl, 0.152% KH2PO4, 0.6% NaNO3, 0.1% trace element solution, 1% glucose, 0.052% MgSO4.7H2O, 2% agar, pH 6.5) to thus obtain a transformant of Aspergillus oryzae introduced with about 5 kbp of a ligated DNA fragment composed of an Aspergillus oryzae BB-56-derived FAD-GDH gene and pyrG.
The transformant obtained in Example 9 was cultured and productivity of FAD-GDH was confirmed.
A medium for preliminary culture constituted with the components below was dividedly charged in a conical flask and sterilized. Aspergillus oryzae BB-56 and the transformant obtained in Example 9 were respectively inoculated to the above described medium and preliminary culture was performed at 30° C. and 200 rpm for 3 days.
Yeast extract (Becton, Dickinson Company) 0.2% (w/v)
Soybean peptone (DMV Co.) 1.0% (w/v)
Glucose (Wako Pure Chemical Industries, Ltd.) 2.0% (w/v)
KH2PO4 (Wako Pure Chemical Industries, Ltd.) 0.1% (w/v)
MgSO4.7H2O (Sigma-Aldrich Japan K.K.) 0.05% (w/v)
pH 5.7
A medium for main culture constituted with the components below was dividedly charged in a conical flask and sterilized. After cooling, 1 mL of the culture liquid after preliminary culture described above was inoculated and main culture was performed at 30° C., 200 rpm for 4 days. After completion of the culture, the culture liquid was filtrated and a FAD-GDH activity of each filtrate was measured.
Glucose 15.0% (w/v)
Meast P1G (ASAHI FOOD & HEALTHCARE CO., LTD.) 3.0% (w/v)
Soybean peptone 6.0% (w/v)
KH2PO4 0.3% (w/v)
K2HPO4 (Wako Pure Chemical Industries, Ltd.) 0.2% (w/v)
4 mM Hydroquinone (Wako Pure Chemical Industries, Ltd.)
pH 6.0
FAD-GDH catalyzes a reaction of producing D-glucono-β-lacton by oxidizing a hydroxyl group of glucose in the presence of an electron receptor. A FAD-GDH activity was measured in the following reaction system.
[Mathematical Formula 1]
D-Glucose+PMS→D-glucono-β-lacton+reduced PMS (1)
2Reduced PMS+NTB→2PMS+Diformazan (2)
PMS in the formula represents phenazine methanesulfate and NTB represents nitrotetrazorium blue.
In the reaction (1), reduced PMS was produced along with oxidization of glucose, and further in reaction (2), diformazan produced by reduction of NTB with reduced PMS was measured at a wavelength of 570 nm.
An enzyme activity was calculated with the calculating formula below.
Vt in the formula represents a total liquid amount, Vs represents a sample amount, 20.1 represents an absorbance coefficient per 0.5μ mole of diformazan (cm2/0.5μ mole), 1.0 represents a light path length (cm), and Df represents a diluting multiple, respectively.
2.55 mL of a 50 mM PIPES-NaOH buffer solution containing 0.22% (w/v) Triton X-100 at pH 6.5, 0.09 mL of a 1 M D-glucose solution, 0.2 mL of a 3 mM PMS solution, and 0.1 mL of a 6.6 mM NTB solution were mixed, the temperature was kept at 37° C. for 5 minutes, and 0.1 mL of the above described culture filtrate was then added thereto to initiate a reaction. Diformazan having an absorption at 570 nm is produced along with progress of an enzyme reaction. An increase of an absorbance at 570 nm per 1 minute was measured and a FAD-GDH activity was measured (
Deposit institution: Incorporated Administrative Agency, Patent Microorganisms Depositary, National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan)
Date of deposit: Oct. 22, 2007
Accession number: NITE BP-438
FAD-GDH production abilities of 9 kinds of Aspergillus oryzae including Aspergillus oryzae BB-56, that is, Aspergillus oryzae BB-56, Aspergillus oryzae RIB40, Aspergillus oryzae IAM2603, Aspergillus oryzae IAM2609, Aspergillus oryzae IAM2628, Aspergillus oryzae IAM2683, Aspergillus oryzae IAM2736, Aspergillus oryzae IAM2706, and Aspergillus oryzae NBRC30113 were compared. Culture and measurement of FAD-GDH activities were conducted in accordance with the methods in Example 1 and Example 10. As a result, although culture liquids derived from Aspergillus oryzae RIB40 and Aspergillus oryzae IAM2609 were seemed to have FAD-GDH activities very weakly, the activities were a detection limit or less (
From comparison between the base sequences of Aspergillus oryzae BB-56-derived FAD-GDH found out in Example 8 and an Aspergillus oryzae RIB40-derived gene sequence (SEQ ID NO: 2) having a whole chain length of 3226 by and encoding an amino acid sequence (SEQ ID NO: 1) of SEQ ID NO: 20494 in the sequence listing shown in JP 2005-176602 A, it was revealed that there is a nonhomologous region of 18 bases (6 amino acids) between the both sequences in regions that encode amino acids and do not contain intron (
The above result suggested that the revealed 18 bases (6 amino acid residues) give certain effects on productivity of FAD-GDH and stability of an enzyme.
(1) Amplification of Aspergillus oryzae RIB40-Derived FAD-GDH Gene
Sequence information of an Aspergillus oryzae RIB40-derived FAD-GDH gene was obtained from gene information disclosed in JP 2005-176602 A to prepare synthetic primers, FG-E22 (SEQ ID NO: 23) and FG-R-Sal (SEQ ID NO: 24), which correspond to sequences of 5′ and 3′ ends. FG-E22 has a recognition site of a restricted enzyme, EcoT22I, and FG-R-Sal has a recognition site of a restricted enzyme, SalI. A PCR reaction was performed using an Aspergillus oryzae RIB40-derived chromosome DNA prepared in Example 11 as a template. TAKARA LA Taq was used in the reaction. The PCR reaction was prepared so that each of primers of FG-E22 and FG-R-Sal has a concentration of 0.4 μM and a template genome has a concentration of 10 ng/μl. In the reaction, after reacting at 96° C. for 2 minutes, reactions of (i) to (iii) constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 53° C. for 30 seconds, and (iii) a reaction at 72° C. for 3 minutes and 30 seconds were repeated 35 times, and lastly, a reaction at 72° C. for 10 minutes was performed. After completion of the reactions, the reaction solution was preserved at 4° C. The PCR amplified fragment was digested with restricted enzymes, EcoT22I and SalI, and then supplied to agarose electrophoresis to separate the PCR amplified fragment and the primers, and the PCR amplified fragment, that is, an Aspergillus oryzae RIB40 strain-derived FAD-GDH gene fragment was extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.).
A PCR reaction was performed using pKF-PCSPb containing a high expression promoter gene fragment disclosed in JP 2003-319786 A as a template. TAKARA LA Taq was used in the reaction. Each synthesized primer of synthesized taaPf (SEQ ID NO: 25) and taaEcoT22R (SEQ ID NO: 26) was added so as to have a concentration of 1 μM and a template plasmid was added so as to have a concentration of 0.5 ng/μl to perform the PCR reaction. In the reaction cycle, after reacting at 94° C. for 2 minutes, reactions constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 60° C. for 30 seconds, and (iii) a reaction at 72° C. for 2 minutes were repeated 30 times, and lastly, a reaction at 72° C. for 10 minutes was performed. After completion of the reactions, the sample was preserved at 4° C. The PCR amplified fragment was digested with restricted enzymes, EcoRI and EcoT22I, and then supplied to agarose electrophoresis to separate the PCR amplified fragment and the primers, and the PCR amplified fragment, that is, a high expression promoter gene fragment was extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.).
(3) Preparation of Plasmid pCS-FG
The Aspergillus oryzae RIB40-derived FAD-GDH gene, the high expression promoter gene fragment, and plasmid pUC118 (TAKARA BIO INC.) treated with EcoRI and SalI, which were prepared as described above, were ligated using a DNA Ligation Kit Ver. 2.1, and pCS-FG constructing plasmid pCS-FG was introduced in an E. coli DH5 strain competent cell to obtain a transformant of E. coli. The transformant was cultured to obtain plasmid pCS-FG using a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich Japan K.K.).
(4) Preparation of Plasmid pCFPT
A PCR reaction was performed using pCS-FG prepared as described above as a template. KOD-plus was used in the reaction. Each of synthetic primers, FGH-F (SEQ ID NO: 27) and FGH-R (SEQ ID NO: 28) was added to have a concentration of 0.4 μM and a pCS-FG template plasmid was added to have a concentration of 0.5 ng/μl so that the PCR reaction solution was prepared. In a reaction cycle, after reacting at 96° C. for 2 minutes, reactions (i) to (iii) constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 54° C. for 30 seconds, and (iii) a reaction at 68° C. for 3 minutes and 30 seconds were repeated 30 times and, lastly, a reaction at 68° C. for 10 minutes was performed. After completion of the reactions, the sample was preserved at 4° C. The PCR amplified fragment was digested with a restricted enzyme, HindIII, and then supplied to agarose electrophoresis to separate the PCR amplified fragment and the primers, and the amplified fragment was extracted from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.). On the other hand, a vector pPTRI (TAKARA BIO INC.) was digested with HindIII and then 5′-dephosphorylated with an alkaline phosphatase (E. coli C75). The 5′-dephosphorylated vector and the above described PCR amplified fragment were ligated using a DNA Ligation Kit Ver. 2.1 to construct plasmid pCFPT. The pCFPT was introduced in an E. coli DH5 competent cell to obtain a transformant of E. coli. The transformant was cultured and plasmid pCFPT was extracted using a QIAfilter™ Plasmid Maxi Kit (QIAGEN K.K.).
(5) Transformation to Aspergillus oryzae RIB40 Strain
Preparation of a protoplast solution of Aspergillus oryzae RIB40 was conducted in accordance with the method of Example 9. Introduction of genes into a protoplast was conducted in accordance with the method of Example 9 except that a gene to be introduced was 10 μl of a 1.8 μg/μl pCFPT solution. Regeneration of the protoplast after gene introduction was performed as follows. 0.2 mL of a protoplast suspension after gene introduction was transferred to a petri dish, and a regenerating medium (0.052% KCl, 0.152% KH2PO4, 0.6% NaNO3, 0.1% trace element solution, 21.86% sorbitol, 1% glucose, 0.052% MgSO4O4.7H2O, 0.5 μg/ml pyrithiamine, 2% agar, pH 6.5) was poured to be solidified as a plate. After culture at 30° C. for 5 days to 7 days, the grown pyrithiamine resistant strain was isolated into a single fungus with a minimum medium containing 0.5 μg/ml pyrithiamine (0.052% KCl, KH2PO4 0.152%, 0.6% NaNO3, 0.1% trace element solution, 1% glucose, 0.052% MgSO4O4.7H2O, 0.5 μg/ml pyrithiamine, 2% agar, pH 6.5) to thus obtain a transformant #75 having a plurality of Aspergillus oryzae RIB40-derived FAD-GDH genes on a chromosome.
The transformant #75 prepared in Example 13 was cultured in accordance with the method of Example 10 to obtain a culture liquid. Fungus bodies and other solid contents in the culture liquid were removed by diatomite filtration using 5.0 L of the culture liquid. 5.4 L of the purified liquid obtained through the operation was desalinated and concentrated with an ultrafilter membrane, and at the same time, buffer-exchanged to a 10 mM McIlvaine buffer solution at pH 5.0 to prepare 600 mL of a solution having a pH of 4.7 and a conductivity of 1.34 mS/cm. The concentrated liquid was applied to SP Sepharose™ Fast Flow equilibrated with a 10 mM McIlvaine buffer solution at pH 5.0 to adsorb FAD-GDH to a column. The column was washed with a 10 mM McIlvaine buffer solution at pH 5.0 and FAD-GDH was then eluted with a 10 mM McIlvaine buffer solution containing 0.1 M of NaCl at pH 5.0 to recover active fractions of FAD-GDH. The active fractions of FAD-GDH were combined to be one fraction, desalinated and concentrated with an ultrafilter membrane, and at the same time, buffer-exchanged to a 20 mM KH2PO4-NaOH buffer solution at pH 6.5 to prepare 300 mL of a concentrated liquid having a pH of 6.3 and a conductivity of 2.80 mS/cm. The concentrated liquid was applied to DEAE Sepharose™ CL-6B equilibrated with a 20 mM KH2PO4-NaOH buffer solution at pH 6.5 and washed with a 20 mM KH2PO4—NaOH buffer solution at pH 6.5. The column pass-through and the washing liquid were all recovered and a column non-adsorbed fraction was recovered. This fraction was concentrated with an ultrafilter membrane to thus obtain a purified enzyme solution of Aspergillus oryzae RIB40-derived FAD-GDH. Productivity of an enzyme due to transformation was improved and purification from a large amount of the culture liquid was performed, thereby leading to success in narrowly obtaining 0.01 u/mL of a purified FAD-GDH solution.
Optimal pHs of Aspergillus oryzae BB-56-derived FAD-GDH prepared in Example 2 and Aspergillus oryzae RIB40-derived FAD-GDH prepared in Example 14 were compared.
2.55 mL of a 100 mM McIlvaine buffer solution containing 0.22% (w/v) Triton X-100 (pH was adjusted to 4.5, 5.0, 6.0, 7.0, or 8.0), 0.1 mL of a 1 M D-glucose solution, 0.2 mL of a 3 mM PMS solution, and 0.1 mL of a 6.6 mM NTB solution were mixed and the temperature was kept at 37° C. for 5 minutes, and then, each 150 μl of the mixed solution was dividedly charged in a 96-well microplate and 5 μl of an Aspergillus oryzae BB-56-derived or Aspergillus oryzae RIB40-derived purified FAD-GDH solution was added to initiate a reaction. Diformazan produced by an enzyme reaction was measured at an absorbance of 570 nm and a yield of diformazan per 1 minute was measured to thereby measure an enzyme activity. POWERSCAN HT (Dainippon Pharmaceutical Co., Ltd.) was used in the measurement. An optimal pH of Aspergillus oryzae BB56-derived purified FAD-GDH was around 7 (
Aspergillus oryzae BB-56-derived or Aspergillus oryzae RIB40-derived purified FAD-GDH solutions was each diluted with a 0.1 M acetate buffer solution at pH 3, the same buffer solution at pH 4, a 0.1 M McIlvaine buffer solution at pH 4.5, the same buffer solution at pH 5.0, the same buffer solution at pH 6.0, the same buffer solution at pH 7.0, the same buffer solution at pH 8.0, or a 0.1 M Na2CO3—NaHCO3 buffer solution at pH 9.5, and treated at 37° C. for 30 minutes at each pH. An each treated liquid was diluted with a 0.2 M KH2PO4—NaOH buffer solution at pH 6.5, and FAD-GDH activities were measured in the same method as Example 15. As a control, an activity of purified FAD-GDH that was diluted with a 0.1 M KH2PO4—NaOH buffer solution at pH 6.5 and cooled with ice to be preserved was also measured and, when the activity was assumed to be 100%, a relative activity was calculated. As a result, Aspergillus oryzae BB-56-derived purified FAD-GDH kept 80% or more of an activity within the pH range from 3.0 to 7.0 (
Aspergillus oryzae BB-56-derived or Aspergillus oryzae RIB40-derived purified FAD-GDH was diluted with a 50 mM PIPES-NaOH buffer solution containing 0.22% of (w/v) Triton X-100 at pH 6.5, and treated at respective specified temperatures, that is, 4° C., 30° C., 40° C., 50° C., and 60° C., for 20 minutes. After cooling in ice, a FAD-GDH activity of an each treated liquid was measured in the same method as that in Example 15. As a control, a FAD-GDH activity of an enzyme without a thermal treatment was also measured and, when the activity was assumed to be 100%, a relative activity was calculated. As a result, the Aspergillus oryzae BB-56-derived purified FAD-GDH kept approximately 100% of an activity until 40° C. (
2.55 mL of a 50 mM PIPES-NaOH buffer solution (pH 6.5) containing 0.22% (w/v) Triton X-100, 0.09 mL of a solution of a 1 M substrate (D-glucose, maltose, galactose, xylose, maltotriose, maltohexaose, or maltopentaose), 0.2 mL of a 3 mM PMS solution, and 0.1 mL of a 6.6 mM NTB solution were mixed and the temperature was kept at 37° C. for 5 minutes, and then, each 150 μl of the mixed solution was dividedly charged in a 96-well microplate, and 5 (activity) of an Aspergillus oryzae BB-56-derived or Aspergillus oryzae RIB40-derived purified FAD-GDH solution was added to initiate a reaction. Diformazan produced by an enzyme reaction was measured at an absorbance of 570 nm and a yield of diformazan per 1 minute was measured to thereby measure an enzyme activity. POWERSCAN HT (Dainippon Pharmaceutical Co., Ltd.) was used in the measurement. A relative activity for each substrate was calculated when a reaction rate for D-glucose was assumed to be 100%. The result showed that Aspergillus oryzae RIB40-derived purified FAD-GDH was acted on not only glucose but also maltose, galactose, and maltohexaose, and thus, substrate specificity thereof was lower than that of the Aspergillus oryzae BB-56-derived FAD-GDH (
As described above, the results of Examples 15 to 18 revealed that 18 base sequences (6 amino acids) that do not correspond between an Aspergillus oryzae BB-56-derived FAD-GDH gene sequence and an Aspergillus oryzae RIB40-derived FAD-GDH gene sequence give effects on pH stability, thermal stability, and substrate specificity of an enzyme.
(1) Preparation of Aspergillus oryzae BB-56 or Aspergillus oryzae RIB40-Derived cDNA
50 mL of a medium constituted with the components below was dividedly charged in a 300 mL-volume conical flask and sterilized at 121° C. for 20 minutes. After cooling, each of Aspergillus oryzae BB-56 and Aspergillus oryzae RIB40 was inoculated and cultured at 30° C. and 200 rpm for 3 days.
Glucose 15.0% (w/v)
Meast P1G (ASAHI FOOD & HEALTHCARE CO., LTD.) 3.0% (w/v)
Soybean peptone 6.0% (w/v)
KH2PO4 0.3% (w/v)
K2HPO4 (Wako Pure Chemical Industries, Ltd.) 0.2% (w/v)
4 mM Hydroquinone (Wako Pure Chemical Industries, Ltd.)
pH 6.0
Fungus bodies recovered through filtration of a culture liquid after culture was rapidly frozen with liquid nitrogen and crushed. The crushed fungus bodies were added with 2 ml of a TRIzol reagent (Invitrogen Corp.) and further crushed. The crushed fungus bodies were adjusted back to room temperature, the suspension of the crushed fungus bodies was collected in a 1.5 ml-volume tube, and whole RNA was obtained in accordance with a method described in an attached instruction manual. Then, a Micro FastTrack 2.0 Kit (Invitrogen Corp.) was used and mRNA was purified in accordance with a method described in an attached instruction manual. A Takara RNA LA PCR™ kit (AMV) ver. 1.1 was used and the mRNA was reverse-transcribed to thus obtain a cDNA solution.
(2) Preparation of cDNA of FAD-GDH
Two kinds of cDNAs prepared as described above were used as templates, and cDNAs of FAD-GDH were amplified by a PCR reaction. KOD-Plus was used in the reaction, and components of the reaction solution were in accordance with an attached instruction manual. 1 μM of each of primers of FADGDH-1 (SEQ ID NO: 29) and FADGDH-2 (SEQ ID NO: 30), and 1 μl of the cDNA solution as a template cDNA were added to the reaction system and a PCR reaction was performed. In the reaction cycle, after reacting at 94° C. for 2 minutes, reactions of (i) to (iii) constituted with (i) a reaction at 94° C. for 30 seconds, (ii) a reaction at 60° C. for 30 seconds, and (iii) a reaction at 68° C. for 2 minutes were repeated 35 times. After completion of the reactions, the reaction solution was preserved at 4° C.
The two kinds of cDNA fragments of FAD-GDH obtained as described above were respectively digested with restricted enzymes, HindIII and BamHI. The reaction solutions after the reaction with the restricted enzymes were supplied to agarose electrophoresis to separate a desired fragment and extract from an agarose gel using a GENECLEAN III Kit (Q-BIOgene Co.). These DNA fragments were each ligated with a yeast expression vector, pYES2 (Invitrogen Corp.) that was treated with restricted enzymes, HindIII and BamHI, in advance to thus prepare pYESGDH having a cDNA of Aspergillus oryzae BB-56-derived FAD-GDH and pYES2-RFGC having a cDNA of Aspergillus oryzae RIB40-derived FAD-GDH. The ligation was performed using a DNA Ligation Kit Ver. 2.1. pYESGDH and pYES2-RFGC were respectively introduced in a Saccharomyces cerevisiae INVSc strain (Invitrogen Corp.) to obtain transformants. The transformation was performed in a lithium acetate method described in an attached instruction manual.
(4) Confirmation of Base Sequence of Genetically Transduced cDNA Fragment
It was confirmed that the desired cDNA fragments were correctly introduced in the above described two kinds of transformants. The cDNA fragments of two types of FAD-GDH digested with restricted enzymes, HindIII and BamHI, which were obtained in the above described (3), were ligated with a vector pBR322 (Toyobo Co., Ltd.) treated with restricted enzymes, HindIII and BamHI to thus construct (i) pGDH having a cDNA of Aspergillus oryzae BB-56-derived FAD-GDH and (ii) pRFGC having a cDNA of Aspergillus oryzae RIB40-derived FAD-GDH. The linking was performed using a DNA Ligation Kit Ver.2.1. Each of pGDH and pRFGC was introduced in an E. coli DH5 strain competent cell to thus obtain a transformant of E. coli. These two kinds of the transformants were respectively cultured to thus obtain plasmids, pGDH and pRFGC, using a GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich Japan K.K.). The base sequences of pGDH and pRFGC were determined in accordance with the method of Example 8. As a result, it could be confirmed that pGDH was a sequence in which a promoter sequence, a terminator sequence and an intron sequence of SEQ ID NO: 19 were dropped (cDNA sequence in the upper column in
Two kinds of the transformants obtained in Example 19 were cultured to produce FAD-GDH. Culture was performed in an induction culture method with Galactose, which is recommended in an attached instruction manual. In order to verify production of FAD-GDH outside and inside fungus bodies after the culture, FAD-GDH activities of the culture supernatant and the fungus body crushed liquid were measured. Measurement of the FAD-GDH activities was performed as follows.
2.55 mL of a 50 mM PIPES-NaOH buffer solution (pH 6.5) containing 0.22% (w/v) Triton X-100, 0.09 mL of a 1 M D-glucose solution, 0.2 mL of a 3 mM PMS solution, and 0.1 mL of a 6.6 mM NTB solution were mixed and the temperature was kept at 37° C. for 5 minutes, then, 150 μl of this mixed solution was dividedly charged in a 96-well microplate, and thereto was added 5 μl of the culture supernatant or the fungus body crushed liquid, that is, a culture supernatant or a fungus body crushed liquid of a yeast transformed with a cDNA of Aspergillus oryzae BB-56-derived FAD-GDH or a yeast transformed with a cDNA of Aspergillus oryzae RIB40-derived FAD-GDH to initiate a reaction. Diformazan produced by an enzyme reaction was measured at an absorbance of 570 nm and a yield of diformazan per 1 minute was measured to thus measure a FAD-GDH activity. POWERSCAN HT (Dainippon Pharmaceutical Co., Ltd.) was used in the measurement.
As a result, a FAD-GDH activity was detected in both outside and inside of fungus bodies in the yeast transformed with an Aspergillus oryzae BB-56-derived cDNA. On the other hand, a FAD-GDH activity was not detected in either outside or inside of fungus bodies in the yeast transformed with an Aspergillus oryzae RIB40-derived cDNA (
Purification of FAD-GDH was tried from two kinds of the culture supernatants obtained in Example 20. Each of the culture supernatants was supplied to dialysis using a 10 mM acetate Na buffer solution at pH 5.0 as an external dialysate. The solution after dialysis was applied to HiTrap™ SP Sepharose Fast Flow (GE Healthcare Bio-Sciences Co., Ltd.) equilibrated with a 10 mM acetate Na buffer solution at pH 5.0 to adsorb FAD-GDH to a column. The column was washed with a 10 mM acetate Na buffer solution at pH 5.0, and FAD-GDH was then eluted with a 10 mM acetate Na buffer solution containing 0.1 M of NaCl at pH 5.0 to recover a fraction showing a FAD-GDH activity. Measurement of FAD-GDH activities was conducted in accordance with the method of Example 20.
The result showed that most of Aspergillus oryzae BB-56 derived FAD-GDH was detected from a column adsorbed eluted fraction (
Among the purified enzyme fractions obtained by the purification operation in Example 21, a fraction NO. 18 having the highest relative activity of an Aspergillus oryzae BB-56-derived FAD-GDH activity (
As described above, the results of respective examples revealed that 18 nucleic acid bases (6 amino acids) that do not correspond between a gene encoding Aspergillus oryzae BB-56-derived FAD-GDH and a gene encoding Aspergillus oryzae RIB40-derived FAD-GDH give effects on an optical pH, pH stability, thermal stability and substrate specificity of FAD-GDH, and production ability of an enzyme.
According to the transformant of the present invention, FAD-GDH excellent in substrate specificity can be produced at high efficiency and in a large amount. FAD-GDH produced by the transformant of the present invention makes it possible to measure a glucose level more accurately and is suitable for a measurement of a blood sugar level and a measurement of a glucose concentration in foods (such as seasonings and beverages).
This invention is not limited to description of the above embodiments and examples of the invention. Various modified embodiments are also included in the present invention within a range where a skilled person can easily conceive without departing from the scope of claims of the patent.
Contents of thesis, unexamined patent applications, and patent applications expressed in the present specification in their entirety were incorporated herein by reference to the whole contents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2007-308072 | Nov 2007 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2008/068011 | 10/3/2008 | WO | 00 | 10/25/2010 |
