JP3587959B2 - Modified pyranose oxidase, modified pyranose oxidase gene, novel recombinant DNA and method for producing modified pyranose oxidase - Google Patents

Description

（６） Ｋｍ値
ラインウエーバー・バークのプロットによるＫｍ値の測定を行なった。グルコースに対するＫｍ値は、０．７ ｍＭ、１，５−アンヒドロ−Ｄ−グルシトールに対するＫｍ値は、１５ ｍＭであった。
【００２２】
（実施例４）
本発明の酵素の反応における優位性を示すため、本酵素と野生型酵素（特開平８−２０５８６１号公報記載）の一定量を、一定量の基質と反応させ、その反応経過を経時的に追うことで両者の比較を行なった。この測定においては、生成される過酸化水素を発色試薬により発色させ、５００ ｎｍの吸光度により定量した。ここで用いる基質（グルコース及び１，５−アンヒドロ−Ｄ−グルシトール）の濃度は希薄であるため、１０〜４０分の反応で完全に分解され、ＯＤ _５００ ｎｍは最終的に一定値に達することとなる。従って、ＯＤ _５００ ｎｍが一定値に達するまでの時間が短ければ短いほど、その基質特異性及び酵素反応性は優れているといえる。
【００２３】
（１）グルコースに対する反応性
実施例３の（１）力価の測定法において、グルコースを０．０２７ ｍｇ、ピラノース・オキシダーゼを１ Ｕ加え、３７℃下でＯＤ _５００ ｎｍを測定した。その結果を図５に示した。図５において、本発明の酵素が反応開始後１０分で一定値に達しているのに対し、野生型酵素は、反応開始後１５分においても一定値に達していないことがわかる。この結果は、本酵素が野生型酵素より基質特異性、基質との反応性の点で優れていることを示す。
（２） １，５−アンヒドロ−Ｄ−グルシトールに対する反応性
実施例３の（１）力価の測定法において、１，５−アンヒドロ−Ｄ−グルシトールを０．０２７ ｍｇ、ピラノース・オキシダーゼを３０ Ｕ加え、３７℃下でＯＤ _５００ ｎｍを測定した。その結果を図６に示す。図６において、本発明の酵素が反応開始後２０分で一定値に達しているのに対し、野生型酵素は、反応開始後３０分においても一定値に達していないことがわかる。この結果は、本酵素が野生型酵素より基質特異性、基質との反応性の点で優れていることを示す。
【００２４】
【発明の効果】
本発明によれば、改変ピラノース・オキシダーゼ、特に、Ｋｍ値、耐熱性及びｐＨ安定性に優れたピラノース・オキシダーゼが効率よく製造できる。特にＫｍ値が小さいため、測定時間の短縮化、使用酵素量の低減化を図ることができ、本発明は、産業上有用である。
【００２５】
【配列表】

【図面の簡単な説明】
【図１】本発明の酵素の安定ｐＨを示す図である。
【図２】本発明の酵素の至適ｐＨを示す図である。
【図３】本発明の酵素の至適温度を示す図である。
【図４】本発明の酵素の温度安定性を示す図である。
【図５】本発明の酵素と野生型酵素の反応性の違いを示すため、一定量の酵素で一定量のグルコースを分解する過程を経時的に追った図である。
【図６】本発明の酵素と野生型酵素の反応性の違いを示すため、一定量の酵素で一定量の１，５−アンヒドロ−Ｄ−グルシトールを分解する過程を経時的に追った図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a modified pyranose oxidase, a modified pyranose oxidase gene, a novel recombinant DNA, and a method for producing a modified pyranose oxidase, which are useful for an enzymatic assay of glucose and 1,5-anhydro-D-glucitol.
[0002]
[Prior art]
Pyranose oxidase is an enzyme that uses glucose as an optimal substrate and catalyzes the reaction of oxidizing the glucose to produce glucosone, and can be used for enzymatic measurement of glucose in foods, body fluids and the like. In addition, by acting on 1,5-anhydro-D-glucitol, it can be used for an enzymatic measurement method of 1,5-anhydro-D-glucitol, which is regarded as a diagnostic marker for diabetes. .
Conventionally, pyranose oxidase derived from Coriolus versicolor (described in JP-A-8-205861) has a Km value for glucose of 1.37 mM, heat resistance of about 50 ° C., and stable pH of pH 4 0.0 to 8.0. Accordingly, there has been a need for pyranose oxidase that exhibits a lower Km value, is more stable to higher temperatures, and has broader pH stability. Further, it was expected that the Km value for not only glucose but also 1,5-anhydro-D-glucitol was small.
[0003]
[Problems to be solved by the invention]
The present invention has been made to solve such problems of the prior art, and it is an object of the present invention to provide a modified pyranose oxidase which is superior to the prior art and a means for producing the same.
[0004]
[Means for Solving the Problems]
Therefore, the present inventors further studied in view of the above-mentioned object, and as a result of genetically modifying the pyranose oxidase gene derived from Coriolus versicolor (described in JP-A-8-205861), the Km value, the heat resistance and the pH stability were improved. The present inventors have found that a gene encoding a modified pyranose oxidase having improved properties, a modified pyranose oxidase having improved Km value, heat resistance and pH stability, and the like can be obtained, and have completed the present invention.
[0005]
That is, the first invention of the present application is the following modified pyranose oxidase (a),
(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having pyranose oxidase activity
The second invention of the present application is the following modified pyranose oxidase (a),
(A) a protein in which the amino acid at position 542 is substituted in the amino acid sequence described in SEQ ID NO: 1 and which has pyranose oxidase activity
The third invention of the present application is the following modified pyranose oxidase (a),
(A) a protein in which the amino acid at position 542 in the amino acid sequence of SEQ ID NO: 1 has been substituted with lysine, and which has pyranose oxidase activity;
A fourth invention of the present application is a modified pyranose oxidase having the following physicochemical properties,
(1) Action: oxidizes glucose to glucosone.
(2) Substrate specificity: specifically acts on glucose, but also acts on galactose, L-sorbose, D-xylose, and 1,5-anhydro-D-glucitol.
(3) Stable pH: pH 3.5 to 11.0 by treatment at 50 ° C. for 30 minutes.
(4) Optimum pH: around pH 6.5
(5) Optimum temperature: around 55 ° C
(6) Temperature stability: stable up to about 55 ° C
A fifth invention of the present application is a modified pyranose oxidase gene encoding the following protein (a):
(A) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and having pyranose oxidase activity
A sixth invention of the present application is a modified pyranose oxidase gene encoding the following protein (a):
(A) a protein in which the amino acid at position 542 is substituted in the amino acid sequence described in SEQ ID NO: 1 and which has pyranose oxidase activity
A seventh invention of the present application is a modified pyranose oxidase gene encoding the following protein (a):
(A) a protein in which the amino acid at position 542 in the amino acid sequence of SEQ ID NO: 1 has been substituted with lysine, and which has pyranose oxidase activity;
An eighth invention of the present application is a novel recombinant DNA characterized in that the pyranose oxidase gene is inserted into a vector DNA,
A ninth invention of the present application is a transformant or a transductant containing the recombinant DNA,
A tenth invention of the present application is a method for producing a modified pyranose oxidase, which comprises culturing the above-mentioned transformant or transductant in a medium and collecting the modified pyranose oxidase from the culture.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The modified pyranose oxidase of the present invention can be obtained, for example, as follows.
First, a recombinant plasmid pPRME10 (described in JP-A-8-205861) containing the isolated pyranose oxidase gene derived from Coriolus versicolor ps4a strain was prepared from E. coli XL1-Blue (pPRME10) (FERM BP-4831), for example. Extraction and purification are performed by using QIAGEN (manufactured by Funakoshi).
The vector DNA that can be used in the present invention is not limited to the above-mentioned plasmid vector DNA, but may be other ones such as bacteriophage vector DNA and plasmid vector DNA. Specifically, pUC118 (manufactured by Takara Shuzo) or the like is preferable.
[0007]
Next, a pyranose oxidase gene consisting of an amino acid sequence in which one or more, preferably several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having pyranose oxidase activity Any method may be used to obtain the recombinant plasmid DNA, for example, a point mutation such as a method for randomly converting the recombinant plasmid DNA using a chemical mutagen such as hydroxylamine or nitrous acid or a PCR method, or a commercially available kit Site-directed mutagenesis, which is a well-known technique for generating site-specific substitution or deletion mutations utilizing the following: selective cleavage of the recombinant plasmid DNA, followed by removal or addition of the selected oligonucleotides , Ligation method; oligonucleotide mutagenesis method, etc. It is.
[0008]
One or more, preferably several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1, and the protein having pyranose oxidase activity includes, for example, glutamic acid residue in pyranose oxidase. And those substituted with other amino acid residues. And other amino acids include, for example, lysine and the like. Specific examples thereof include those in which the glutamic acid residue at position 542 in the amino acid sequence of the protein has been substituted with a lysine residue.
The pyranose oxidase gene consisting of an amino acid sequence in which one or more, preferably several amino acids are substituted in the amino acid sequence shown in SEQ ID NO: 1 and encoding a protein having a pyranose oxidase activity includes, for example, pyranose oxidase. Examples include those in which a base encoding a glutamic acid residue in oxidase is replaced with a base encoding another amino acid residue. Examples of the base encoding another amino acid include, for example, a base encoding lysine. Specific examples thereof include those in which a base encoding a glutamic acid residue at position 542 in the amino acid sequence of the protein is replaced with a base encoding a lysine residue.
[0009]
The recombinant DNA after the above treatment is purified using a desalting column, QIAGEN (manufactured by Funakoshi) or the like to obtain various recombinant DNAs.
The various recombinant DNAs thus obtained are used to transform or transduce, for example, E. coli K12, preferably E. coli JM109 (manufactured by Toyobo), XL1-Blue (manufactured by Funakoshi) and the like. A transformant or a transductant containing the recombinant DNA having the pyranose oxidase gene fragment can be obtained.
For example, in the case of a transformant, a modified product having improved heat resistance, Km value, etc., is obtained from the obtained transformant (containing therein recombinant plasmid DNA containing various pyranose oxidase genes). To obtain a strain that produces pyranose oxidase, the following method can be used.
[0010]
First, the obtained transformant is cultured on an agar medium to form colonies, and each colony is replicated on a filter. The filter is placed on an agar medium (including an induction substrate) to culture each colony, and various modified pyranose oxidases contained in the recombinant plasmid DNA are induced and produced. After heat-treating the filter under appropriate conditions, the filter is incubated on an assay plate prepared so that pyranose oxidase activity can be detected, and a mutant showing residual activity is selected. The selected mutants are liquid-cultured together with a wild-type strain (XL1-Blue (pPRME10)), various pyranose oxidases are induced and produced, and the resulting culture is subjected to ultrasonic disruption. Using the crude enzyme extract, the Km value is measured, and a transformant showing an excellent Km value is selected.
The base sequence of the modified pyranose oxidase gene inserted into the recombinant DNA of the superior transformant obtained as described above is determined by, for example, the dideoxy method (Methods in Enzymology, 101, 20-78, 1983). Can do it.
[0011]
A transformant or transductant having a modified pyranose oxidase-producing ability obtained as described above, preferably, to produce a modified pyranose oxidase using a strain belonging to the genus Escherichia, as follows: Can do it.
In order to culture the above microorganism, the microorganism may be cultured by a usual solid culture method, but it is preferable to culture by using a liquid culture method as much as possible. Examples of a medium for culturing the microorganism include, for example, potassium dihydrogen phosphate, dihydrogen phosphate, and the like in one or more nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat koji leaching solution. One or more inorganic salts such as potassium, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added. The initial pH of the medium is appropriately adjusted to 7 to 9. The cultivation is preferably carried out at 25 to 42 ° C. for 6 to 24 hours by aeration and agitation deep culturing, shaking culturing, static culturing and the like.
[0012]
After completion of the cultivation, the modified pyranose oxidase can be collected from the culture using conventional enzyme collecting means. The cells are separated from the culture by, for example, filtration, centrifugation, etc., and washed. It is preferable to collect the modified pyranose oxidase from the cells. In this case, the cells can be used as they are, but a method of destroying the cells using various disrupting means such as an ultrasonic crusher, a French press, a dynamill, etc., or a cell cell wall using a cell wall lysing enzyme such as lysozyme. It is preferable to collect the modified pyranose oxidase from the cells by, for example, a method of dissolving the enzyme, or a method of extracting the enzyme from the cells using a surfactant such as Triton X-100. In order to isolate the modified pyranose oxidase from the thus obtained crude enzyme solution, a method used for ordinary enzyme purification can be used. For example, it is preferable to carry out an appropriate combination of ammonium sulfate precipitation, organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, electrophoresis, isoelectric precipitation, and the like.
[0013]
The physicochemical properties of the pyranose oxidase thus obtained are as follows.
(1) Action
Glucose is oxidized to glucosone.
(2) Substrate specificity
It acts specifically on glucose, but also on galactose, L-sorbose, D-xylose and 1,5-anhydro-D-glucitol.
(3) Stable pH
Stable at pH 3.5 to 11.0 by treatment at 50 ° C. for 30 minutes.
(4) Optimum pH
The optimum pH is around pH 6.5.
(5) Optimal temperature
The optimum temperature is around 55 ° C.
(6) Temperature stability
Stable up to about 55 ° C.
(7) Km value
The Km value for glucose is 0.7 mM and the Km value for 1,5-anhydro-D-glucitol is 15 mM.
As a result of comparing and examining the above physicochemical properties with known pyranose oxidase, the optimum pH was around 6.5, the optimal temperature was around 55 ° C, and the treatment was performed at 50 ° C for 30 minutes like the present enzyme. This enzyme was identified as a novel enzyme, since no enzyme that is stable in the range of pH 3.5 to 11.0 even after the test is known.
[0014]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
(Example 1)
(1) Mutation operation
First, a recombinant plasmid pPRME10 (described in JP-A-8-205861) containing a pyranose oxidase gene derived from Coriolus versicolor ps4a strain was obtained from Escherichia coli XL1-Blue (pPRME10) (FERM BP-4831) by QIAGEN (Funakoshi). ) Was extracted and purified. Using the recombinant plasmid pPRME10, E. coli XL1-RED (manufactured by STRATAGENE; which is prone to cause errors in replication of the plasmid and mutate during propagation) was used. M. Transformation was performed according to the method of Morrison (Methods in Enzymology, 68, 326-331, 1979) to obtain a transformant. 200 colonies of the obtained transformant were inoculated into 10 ml of TY medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl, pH 7.0), and incubated at 37 ° C for 18 minutes. The cells were cultured with shaking for an hour. After the culture, the culture was centrifuged at 6000 rpm for 10 minutes to collect the cells to obtain cells. The recombinant plasmid containing the mutation was extracted from the cells using QIAGEN tip-100 (manufactured by Funakoshi) and purified. Using this plasmid, M. Escherichia coli XL1-Blue (manufactured by Funakoshi) was transformed according to the method of Morrison (Methods in Enzymology, 68, 326-331, 1979) to obtain a transformant having a mutated plasmid. The 6000 transformants were screened for strains having improved heat resistance and Km value by the method described in item (2).
[0015]
(2) Screening for strains with improved thermostability and Km value
The above transformant obtained by the mutation procedure (1) was cultured on a TY agar medium at 37 ° C. overnight to form colonies, and each colony was replicated on a filter (Hybond N; manufactured by Amersham). The filter was placed on a TY medium agar medium containing 1 mM isopropyl-β-D-thiogalactopyranoside and cultured at 30 ° C. for 4 hours to induce and produce various modified pyranose oxidases. The filter was sealed in a plastic bag, heat-treated in a water bath at 65 ° C. for 30 minutes, and assay plates (33 mM glucose, 0.5 U / ml peroxidase, 33 mM glucose, prepared to detect pyranose oxidase activity) (1% agar containing 0.01% dianisidine) and incubated at 37 ° C. As a result, four strains had residual activity and exhibited brown coloration.
The resulting four transformants and the wild-type strain (XL1-Blue (pPRME10)) were subjected to liquid culture in 2 ml of TY medium (containing 1 mM isopropyl-β-D-thiogalactopyranoside) for each colony. Then, the gene contained in the plasmid was expressed, and various pyranose oxidases were induced and produced. After the culture, the obtained culture was subjected to ultrasonic crushing, and the Km value for glucose and 1,5-anhydro-D-glucitol was measured using the crude enzyme extract. As a result, the mutant XL1-Blue (pPROD-542K) produced a modified pyranose oxidase having the smallest Km value for both glucose and 1,5-anhydro-D-glucitol.
Escherichia coli XL1-Blue (pPROD-542K) has been deposited as FERMBP-5988 with the National Institute of Biotechnology and Industrial Technology.
[0016]
(3) Analysis of mutation site
The following analysis was performed on the modified pyranose oxidase gene contained in the superior mutant strain obtained as described above in order to confirm which site of the gene was replaced by the mutation operation. The recombinant DNA contained in the mutant strain Escherichia coli XL1-Blue (pPROD-542K) was purified using QIAGEN Plasmid Midi Kit (Funakoshi). The nucleotide sequence of the modified pyranose oxidase gene containing the mutation contained in the recombinant DNA was determined using a Deletion Kit for Kilo-Sequence (manufactured by Takara Shuzo) and a 370 DNA Sequencing System (manufactured by Applied Biosystems). As a result, when the amino acid sequence encoding the nucleotide sequence was compared with the amino acid sequence of wild-type pyranose oxidase (described in SEQ ID NO: 1), the glutamic acid residue (GAG) at position 542 was replaced with a lysine residue (AAG). (See Japanese Patent Application Laid-Open No. 8-2055861, the nucleotide sequence of wild-type pyranose oxidase).
[0017]
(Example 2)
100 ml of TY medium was placed in Sakaguchi Kolben and sterilized at 121 ° C. for 10 minutes. Escherichia coli XL
One loopful of loop was inoculated from a stock slant of 1-Blue (pPROD-542K), and this was shake-cultured at 37 ° C for 14 hours with a shaker to obtain a seed culture.
Next, 100 ml of the seed culture solution was inoculated into a 30-L jar fermenter containing 20 L of the TY medium containing 1 mM isopropyl-β-D-thiogalactopyranoside (same conditions as in the sterilization conditions), and rotated at 300 rpm. The cells were cultured at a volume of 10 L / min at 30 ° C. for about 16 hours. After completion of the culture, the pyranose oxidase activity was measured based on the production of hydrogen peroxide, and was found to be 0.3 U / ml.
After completion of the culture, cells were collected from 40 L of the culture solution (20 L * 2 times) using an ultrafiltration membrane (AHV-3010, manufactured by Asahi Kasei Corporation), washed with tap water, washed with tap water, and then cultured. Was concentrated to about 10 L.
Step 1 (adjustment of crude enzyme solution): 5 g of egg white lysozyme, 1 L of 0.55 M EDTA 2Na (pH 8.0), 0.1 L of potassium phosphate buffer (pH 8.0) 0) 1 L was added, mixed, and allowed to stand at 37 ° C. for 24 hours, and then lysed by freeze-thawing method. A 5% aqueous protamine solution (pH 8.0) was added dropwise to the lysate with stirring to remove nucleic acids. The supernatant was dialyzed and concentrated against a 10 mM potassium phosphate buffer (pH 7.5) containing 50 mM potassium chloride using an ultrafiltration membrane to obtain about 500 ml of a crude enzyme solution. .
Step 2 (heat treatment): The dialysate (about 500 ml) was heated to 50 ° C. in a hot water bath and kept for 30 minutes to thermally denature contaminating proteins. After the heat treatment, denatured proteins were removed using a cooling centrifuge (Hitachi CR22 model). The resulting supernatant (490 ml) was dialyzed against a 10 mM potassium phosphate buffer (pH 7.0) containing 50 mM potassium chloride using an ultrafiltration membrane to obtain about 500 ml of an enzyme solution. .
[0018]
Step 3 (DEAE-cellulose treatment): About 1 kg of wet weight of DEAE-cellulose was added to the above-mentioned treatment liquid (about 500 ml), mixed and allowed to adsorb the enzyme, and then containing 50 mM potassium chloride. The DEAE-cellulose is washed with a 10 mM potassium phosphate buffer (pH 7.0), and the enzyme is then washed with a 10 mM potassium phosphate buffer (pH 7.0) containing 0.5 M potassium chloride. Was eluted. The obtained eluate was dialyzed and concentrated against a 10 mM potassium phosphate buffer (pH 7.0) containing 50 mM potassium chloride using an ultrafiltration membrane to obtain about 500 ml of an enzyme solution.
Step 4 (QAE-Sephadex A-50 column chromatography): The enzyme solution (500 ml) is adsorbed on a QAE-Sephadex A-50 column (7.8 × 40 cm) and contains 0.26 M potassium chloride. The column of QAE-Sephadex A-50 was washed with 10 mM potassium phosphate buffer (pH 7.0), and then 10 mM potassium phosphate buffer (pH 7.0) containing 0.28 M potassium chloride. To elute the enzyme.
By the above purification operation, 150 mg (about 3000 U, 20 U / mg protein) of an enzyme preparation having a purity of 90% or more was obtained.
[0019]
(Example 3)
The following items were analyzed for the enzyme of the present invention obtained in Example 2.
(1) Measurement method of titer
1.0 ml of a 0.1 M Tris-HCl buffer (pH 7.0) and a coloring solution (0.1 M Tris-HCl buffer (pH 7.0) containing 10 mM each of 4-aminoantipyrine and phenol) were added. 0.2 ml of oxidase per 100 ml of the solution (0.1 ml), 0.1 ml of 1 M glucose solution, 0.1 ml of pyranose oxidase solution and 1.6 ml of water. The enzyme-containing solution was reacted at 37 ° C., and the increase in absorbance at 500 nm was measured with a U2000 type double beam spectrophotometer (manufactured by Hitachi, Ltd.).
One unit of the enzymatic activity is defined as the amount of an enzyme that produces 1 μmol of hydrogen peroxide per minute at 37 ° C. and pH 7.0, and the titer is calculated based on the amount of the quinoimine dye produced under the above conditions. The molecular extinction coefficient is 5.3 × 10 ³ And performed according to the following equation.
Enzyme activity = (△ OD ₅₀₀ /5.3) x (3.0 / 0.1) x enzyme dilution
In addition, $ OD ₅₀₀ Is the increase in absorbance at 500 nm per unit time (1 minute).
[0020]
(2) Stable pH and optimal pH range
The stable pH is 100 mM glycine-hydrochloric acid buffer (pH 2.5-3.5), 100 mM sodium acetate-acetic acid buffer (pH 3.5-5.5), 100 mM female-sodium hydroxide buffer. Solution (pH 5.5-7.0), 100 mM Hepes-sodium hydroxide buffer (pH 7.0-8.0), 100 mM taps-sodium hydroxide buffer (pH 8.0-9.0) ), 100 mM chess-sodium hydroxide buffer (pH 9.0-10.0), 100 mM chaps-sodium hydroxide buffer (pH 10.0-11.0), 100 mM dipotassium phosphate-chloride Potassium buffer (pH 11.0-12.0), 100 mM potassium phosphate buffer (pH 6.5-8.0), 100 mM Tris-HCl buffer (pH 8.5-9.0) Used, pH 2.5- In 2.0, it was determined by measuring the residual activity of the enzyme after each for 30 minutes at 50 ° C.. The results are as shown in FIG. 1, and the stable pH of this enzyme was 3.5 to 11.0.
The optimal pH is 100 mM sodium acetate-acetic acid buffer (pH 3.5-5.5), 100 mM female-sodium hydroxide buffer (pH 5.5-7.0), 100 mM Hepes-hydroxide. Sodium buffer (pH 7.0-8.0), 100 mM taps-sodium hydroxide buffer (pH 8.0-9.0), 100 mM chess-sodium hydroxide buffer (pH 9.0-9.0) 10.0), 100 mM potassium phosphate buffer (pH 6.5 to 8.0), and 100 mM Tris-HCl buffer (pH 8.0 to 9.0). I asked for it. The results are as shown in FIG. 2, and the optimum pH of the present enzyme was around 6.5.
[0021]
(3) Optimal temperature
1.0 ml of 0.1 M Tris-HCl buffer (pH 7.0), 0.2 ml of the same coloring solution as above, 0.1 ml of 1 M glucose solution, 1.0 U / ml pyranose oxidase solution A total of 3.0 ml of the enzyme-containing solution consisting of 0.1 ml and 1.6 ml of water was reacted for 1 minute at each temperature using a thermostat, and the absorbance difference at 500 nm was measured using a U2000 type double beam spectrophotometer ( (Manufactured by Hitachi, Ltd.), and the enzyme activity was calculated. From FIG. 3, the optimum temperature of the present enzyme was about 55 ° C.
(4) Temperature stability
An enzyme preparation (400 U / ml) was mixed at a ratio of 9: 1 with 100 mM Tris-HCl buffer (pH 8.0), and these solutions were kept at the respective temperatures shown in FIG. 4 for 30 minutes. After cooling, the mixture was diluted 50-fold with a 100 mM Tris-HCl buffer (pH 7.0), allowed to stand at room temperature for 10 minutes, and the residual activity was measured. According to FIG. 4, this enzyme was stable up to about 55 ° C.
(5) Substrate specificity
Substrate specificity of the enzyme of the present invention was examined using various sugar solutions (0.1 M) shown in Table 1 in place of glucose in the reaction solution in the above titration method. Pyranose oxidase was contained in the reaction solution in an amount of 2.5 U. The activity was expressed as a relative activity when glucose was set to 100%. As shown in Table 1, this enzyme had high specificity for glucose and also acted on galactose, D-xylose, and 1,5-anhydro-D-glucitol.

(6) Km value
The Km value was measured by a Lineweaver-Burk plot. The Km value for glucose was 0.7 mM, and the Km value for 1,5-anhydro-D-glucitol was 15 mM.
[0022]
(Example 4)
In order to show the superiority in the reaction of the enzyme of the present invention, a certain amount of the enzyme and a wild-type enzyme (described in JP-A-8-205861) are reacted with a fixed amount of a substrate, and the progress of the reaction is followed with time. Thus, the two were compared. In this measurement, the produced hydrogen peroxide was colored with a coloring reagent and quantified by absorbance at 500 nm. Since the concentrations of the substrates (glucose and 1,5-anhydro-D-glucitol) used here are low, they are completely decomposed in a reaction for 10 to 40 minutes, and the OD ₅₀₀ nm will eventually reach a constant value. Therefore, OD ₅₀₀ It can be said that the shorter the time required for nm to reach a certain value is, the better its substrate specificity and enzyme reactivity are.
[0023]
(1) Reactivity to glucose
In the (1) titer measurement method of Example 3, 0.027 mg of glucose and 1 U of pyranose oxidase were added, and the OD was measured at 37 ° C. ₅₀₀ nm was measured. The results are shown in FIG. FIG. 5 shows that the enzyme of the present invention reached a constant value 10 minutes after the start of the reaction, whereas the wild-type enzyme did not reach a constant value even 15 minutes after the start of the reaction. This result indicates that the present enzyme is superior to the wild-type enzyme in terms of substrate specificity and reactivity with the substrate.
(2) Reactivity to 1,5-anhydro-D-glucitol
In (1) titer measurement method of Example 3, 0.027 mg of 1,5-anhydro-D-glucitol and 30 U of pyranose oxidase were added, and the OD was measured at 37 ° C. ₅₀₀ nm was measured. FIG. 6 shows the result. FIG. 6 shows that the enzyme of the present invention reached a constant value 20 minutes after the start of the reaction, whereas the wild-type enzyme did not reach a constant value even 30 minutes after the start of the reaction. This result indicates that the present enzyme is superior to the wild-type enzyme in terms of substrate specificity and reactivity with the substrate.
[0024]
【The invention's effect】
According to the present invention, modified pyranose oxidase, particularly pyranose oxidase excellent in Km value, heat resistance and pH stability can be efficiently produced. Particularly, since the Km value is small, the measurement time can be shortened and the amount of the enzyme used can be reduced, and the present invention is industrially useful.
[0025]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing a stable pH of the enzyme of the present invention.
FIG. 2 is a graph showing the optimum pH of the enzyme of the present invention.
FIG. 3 is a diagram showing an optimum temperature of the enzyme of the present invention.
FIG. 4 is a diagram showing the temperature stability of the enzyme of the present invention.
FIG. 5 is a diagram showing a process of decomposing a fixed amount of glucose with a fixed amount of enzyme over time to show a difference in reactivity between the enzyme of the present invention and a wild-type enzyme.
FIG. 6 is a diagram showing the process of decomposing a certain amount of 1,5-anhydro-D-glucitol with a certain amount of enzyme over time to show a difference in reactivity between the enzyme of the present invention and a wild type enzyme. is there.

Claims (5)

The following modified pyranose oxidase (a).
(a) a protein in which the amino acid at position 542 in the amino acid sequence of SEQ ID NO: 1 has been substituted with lysine, and which has pyranose oxidase activity; A modified pyranose oxidase gene encoding the following protein (a):
(a) a protein in which the amino acid at position 542 in the amino acid sequence of SEQ ID NO: 1 has been substituted with lysine, and which has pyranose oxidase activity; A novel recombinant DNA comprising the modified pyranose oxidase gene according to claim 2 inserted into a vector DNA. A transformant or a transductant containing the recombinant DNA according to claim 3. A method for producing a modified pyranose oxidase, comprising culturing the transformant or the transductant according to claim 4 in a medium, and collecting the modified pyranose oxidase from the culture.

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