JP2000135079A - 1,5-anhydroglucitol dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a 1,5-anhydroglucitol dehydrogenase highly specific to 1,5-anhydroglucitol (1,5-AG). SOLUTION: This (1,5-AG) dehydrogenase has the following physicochemical properties: (1) having the optimum pH around pH 9; (2) having stability in the range of pH 6-10; (3) having the optimum temperature at 40-45 deg.C; (4) having >=50% remaining activity after incubation at 70 deg.C for 10 min; (5) capable of utilizing an oxidized nicotinamide adenine dinucleotide; (6) not recognizing glucose as a substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a 1,5-AG dehydrogenase using 1,5-anhydroglucitol (hereinafter, referred to as "1,5-AG") as a substrate, and the enzyme. And a method for quantifying 1,5-AG using the enzyme, and a DNA encoding the enzyme.

[0002]

2. Description of the Related Art 1,5-AG is present in a living body at a certain concentration or higher in healthy individuals, but is significantly reduced in diabetic patients. It is used as one of important test items in clinical tests, and its utility value is extremely high.

[0003] Conventionally known 1,
As a method of quantifying 5-AG, a method of colorimetric quantification of hydrogen peroxide generated by allowing pyranose oxidase to act on a sample containing 1,5-AG using a peroxidase coloring system (Japanese Patent Publication No. 5-41238) Gazette) is known.

[0004] As another method, an oxidase having 1,5-AG oxidizing ability is acted on to measure the amount of consumed oxygen and the reduced form of an electron acceptor or the oxidized 1,5-AG reaction product. Methods and the like are known. Among them, JP
No. 62-79780, Pycnopora, Coriolus,
Japanese Patent Application Laid-Open No. H10-155479 discloses 1,5-AG dehydrogenase produced by Pseudomonas bacteria, and 1,5-AG dehydrogenase produced by Agrobacterium bacteria. Is also known to be able to be used for the measurement of 1,5-AG.

[0005]

The conventionally known enzyme using 1,5-AG as a substrate cannot be said to have high specificity for 1,5-AG.

Japanese Patent Application Laid-Open No. Sho 62-79780 discloses 1,5-AG
In addition to 1,5-AG, it has been described that dehydrogenase has activity on glucose, galactose, sorbose, xylose, sorbitol, mannitol, xylitol, arabitol, and erythritol carbohydrates.

JP-A-10-155479 discloses 1,5-A
It is described that G dehydrogenase has activity on sorbose and glucose other than 1,5-AG.

When used for the quantification of 1,5-AG,
Even if the enzyme acts on 1,5-AG, if the enzyme has low substrate specificity as described above, the merit of the enzyme may not be fully utilized. Such a low specificity of the conventionally known enzyme is a further problem depending on various circumstances derived from the sample to be measured.

The blood glucose concentration of a diabetic patient is higher than that of a healthy person, and is about 60 to 110 mg / dl for a healthy person, whereas the blood glucose concentration of a diabetic patient is 110 to 1000 m / dl.
It shows a high value of g / dl.

On the other hand, the concentration of 1,5-AG in blood is about 1.5 to 2.8 mg / dl in a healthy person, whereas
In a diabetic patient, a low value of about 0.15 to 0.2 mg / dl is shown. Since the difference between the glucose concentration and 1,5-AG is about 1000-fold in diabetic patients, 1,5-AG
When a conventional dehydrogenase is used in the measurement of the enzyme by an enzymatic method, a pretreatment operation for removing or modifying glucose is required.

As a pretreatment method, there is a method of removing carbohydrates other than 1,5-AG contained in a sample using an ion exchange column or the like. However, this method requires a complicated separation operation, and particularly a large number of methods. It takes a very long time to process a sample. In addition, in the method of modifying sugars such as glucose by phosphorylation, the optimal conditions for the phosphorylation reaction of glucose and the 1,5-AG measurement conditions are different. is there.

[0012] In a method for culturing a microorganism and purifying and isolating a target enzyme, a method usually used is a method in which a substrate which specifically reacts with the target enzyme is added to a culture medium for culturing. 1,5 in the case of 1,5-AG dehydrogenase
It is a general method for producing an enzyme to culture 1,5-AG dehydrogenase-producing bacteria in a medium containing -AG.

However, when 1,5-AG is expensive and a large amount of enzyme is obtained, a large amount of 1,5-AG must be consumed, and the unit price of the separated and purified enzyme increases. Has disadvantages. If the culture has a property that a substance other than the target 1,5-AG cannot be used as a nutrient source, the nutrient source cannot be substituted with another substance.

[0014]

In order to overcome the disadvantages of the conventional enzymes, the present inventors have found that the substrate specificity for 1,5-AG is higher than that of the conventional dehydrogenase, and that it has been known. Screening was performed for bacteria that produce no 1,5-AG dehydrogenase. Then, whether or not the enzyme produced by the 1,5-AG degrading bacterium was an enzyme that can be used for the measurement of 1,5-AG was examined, and physicochemical characteristics of the enzyme were examined.

As a result, it was found that an enzyme specifically reacting with 1,5-AG as a substrate was present in a culture supernatant of a microorganism collected from soil.

When the enzyme acts on 1,5-AG in the presence of oxidized nicotinamide adenine dinucleotide (NAD) as an electron acceptor, it suggests the formation of reduced nicotinamide adenine dinucleotide (NADH). , Due to the increase in absorbance at 340 nm,
It was confirmed to be a dehydrogenase.

Further, the physicochemical properties of the separated and purified 1,5-AG dehydrogenase were examined, and it was confirmed that the enzyme was a novel enzyme which had not been known so far.

That is, the present invention provides 1,5-AG dehydrogenase having the following physicochemical properties (hereinafter, also referred to as "enzyme of the present invention"). (1) Action: catalyzes the 1,5-AG dehydrogenation reaction. (2) Substrate specificity: 1,5-AG is recognized as a substrate, but glucose is not recognized as a substrate. (3) Optimum pH: around 9.0. (4) pH stability: stable at pH 6.0 to 10.0. (5) Optimum temperature: 40-50 ° C. (6) Temperature stability: 50% or more activity remains after incubation at 70 ° C. for 10 minutes. (7) Electron acceptor: Oxidized nicotinamide adenine dinucleotide (NAD) can be used. (8) Molecular weight: The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 36,000, and the molecular weight by gel filtration is about 141,000.

The enzyme of the present invention is preferably produced by culturing a bacterium of the genus Trichoderma.
The fungus of the genus Trichoderma is preferably Trichoderma longibrachiatum KDK3003 (Tricoderma longibra
chiatum KDK3003).

The enzyme of the present invention preferably has the amino acid sequence shown in SEQ ID NO: 2.

The present invention also relates to a method for producing the enzyme of the present invention (hereinafter, also referred to as "the method for producing the present invention") and a method for quantifying 1,5-AG using the enzyme of the present invention (hereinafter, the "quantitative method for the present invention"). Also called). The production method of the present invention comprises culturing a Trichoderma bacterium producing the enzyme of the present invention in a nutrient medium,
It is characterized in that the produced enzyme of the present invention is separated and purified from the culture. The quantification method of the present invention is carried out in the presence of NAD,
NADH produced by reacting the enzyme of the present invention with a sample containing 5-AG is measured by a change in absorbance.

Furthermore, the present invention provides a DNA encoding a part of the enzyme of the present invention. This DNA has the base sequence shown in SEQ ID NO: 1.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme of the present invention cannot recognize glucose, which is the most abundant saccharide other than 1,5-AG, in a living body as a substrate, but cannot recognize 1,5-AG as a substrate. It is a dehydrogenase that shows specific activity. In the present specification, sugars and sugar alcohols having optical isomers mean D-type unless otherwise specified.

First, the physicochemical properties of the enzyme of the present invention will be described. (1) Action The catalyst catalyzes the dehydrogenation of 1,5-AG. The dehydrogenation reaction takes place in the presence of an electron acceptor. (2) Substrate specificity: 1,5-AG is recognized as a substrate, but glucose is not recognized as a substrate. More specifically, under the conditions described in Examples below, no activity was detected for glucose, sorbitol, xylitol and mannitol, and for glyceraldehyde, 1,
Assuming that the activity against 5-AG is 100%, the activity is about 50%. (3) Optimum pH: around 9.0. (4) pH stability: stable at pH 6.0 to 10.0. (5) Optimum temperature: 40-50 ° C. (6) Temperature stability: 50% or more activity remains after incubation at 70 ° C. for 10 minutes. (7) Electron acceptor: Oxidized nicotinamide adenine dinucleotide (NAD) can be used. Whether or not it can be used as an electron acceptor can be confirmed by the method described in Examples below. (8) Molecular weight: The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 36,000, and the molecular weight by gel filtration is about 141,000.

The optimum pH, pH stability, optimum temperature,
The temperature stability and the molecular weight can be measured by the methods described in Examples below.

The enzyme of the present invention is preferably produced by culturing a bacterium of the genus Trichoderma.
The fungus of the genus Trichoderma is preferably Trichoderma longibrachiatum KDK3003 (Tricoderma longibra
chiatum KDK3003). The bacteria of the genus Trichoderma producing the enzyme of the present invention and the conditions for culturing the same will be described with reference to the production method of the present invention.

The enzyme of the present invention preferably has the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 is obtained from Trichoderma longibrachiatum KD
This is deduced from the nucleotide sequence of the partial cDNA of the enzyme of the present invention obtained from K3003. For enzymes,
In general, it is well known that there can be mutations in the amino acid sequence that do not substantially affect the activity of the enzyme between species or individuals, and that such mutations can be artificially generated, Preferred enzymes of the present invention also include those having an amino acid sequence having such a mutation in the amino acid sequence shown in SEQ ID NO: 2. That is,
In the amino acid sequence shown in SEQ ID NO: 2, it has an amino acid sequence having a deletion, substitution or insertion of one or several amino acids, and has the above-mentioned properties (1) and (2) of the enzyme of the present invention (preferably, (1) to (8) are not impaired.

The enzyme of the present invention can be obtained by the production method of the present invention described below.

The production method of the present invention comprises the steps of: culturing Trichoderma spp. That produces the enzyme of the present invention in a nutrient medium;
It is characterized in that the produced enzyme of the present invention is separated and purified.

The Trichoderma bacterium producing the enzyme of the present invention is screened for Trichoderma bacterium using 1,5-AG assimilation ability as an index.
It can be obtained by screening using the dependent 1,5-AG dehydrogenation activity as an index.

The ability to assimilate 1,5-AG can be evaluated by converting 1,5-AG to glucose. Although the evaluation method is not particularly limited, for example, the production of glucose in a culture filtrate obtained by culturing the bacterium to be screened in a medium using 1,5-AG as a sole carbon source is determined as glucose oxidase. , A method in which resting cells are allowed to act on 1,5-AG in a buffer to detect the production of glucose in the buffer by thin-layer chromatography, and a combination thereof.
Specific conditions for these methods include the conditions described in Example 1 below.

NAD-dependent 1,5-AG of cell-free extract
The dehydrogenation activity can be evaluated by crushing cells and centrifuging the supernatant to obtain a cell-free extract, adding NAD and 1,5-AG thereto, and measuring the change in absorbance at 340 nm. The conditions for cell disruption and absorbance measurement were NA
There is no particular limitation as long as the D-dependent 1,5-AG dehydrogenation activity can be measured, and the conditions described in Example 1 below can be used.

Examples of the bacteria obtained by the screening method described above include Trichoderma longibrachiatum KD
K3003 (Trichoderma longibrachitaum KDK3003). This bacterium was deposited internationally on August 10, 1998 under the Budapest Treaty with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Address: 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture 305-0046, Japan). (Deposit number: F
ERM BP-6458).

The production method of the present invention includes Trichoderma longibrachiatum as long as the ability to produce the enzyme of the present invention is maintained.
A mutant strain of KDK3003 (Trichoderma longibrachitaum KDK3003) can also be used. Known methods can be used as the mutagenesis method. In addition, a spontaneous mutation may cause a mutant strain. Among the mutant strains, those retaining the ability to produce the enzyme of the present invention can be selected by the above-described screening method.

The nutrient medium used in the production method of the present invention is not particularly limited as long as the microorganism of the genus Trichoderma grows and produces the enzyme of the present invention. Examples of the medium include a carbon source, a nitrogen source, and other micronutrients that are commonly used for culturing Trichoderma bacteria. As a carbon source,
Examples thereof include 1,5-AG, glucose, and glycerol, but glucose is preferably used because it is inexpensive and provides good enzyme production. Examples of the nitrogen source include ammonium sulfate, ammonium chloride, sodium nitrate, and the like. However, it is preferable to use sodium nitrate because it provides good enzyme production.

When the above-mentioned Trichoderma genus producing 1,5-AG dehydrogenase was cultured in a medium not containing 1,5-AG, it was confirmed that 1,5-AG dehydrogenase was produced. Was. Therefore, it is possible to use inexpensive glucose as a carbon source without adding expensive 1,5-AG to the medium.
It was confirmed that G dehydrogenase could be produced. Therefore, in the production method of the present invention, it is preferable to use a nutrient medium containing no 1,5-AG. The fact that 1,5-AG is not contained means that 1,5-AG is not added to the medium, as apparent from the above, and the 1,5-AG originally contained in the nutrient medium is not included. May be present.

The method of separation and purification is not particularly limited, and a combination of ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, gel filtration and the like, which are used for separation and purification of proteins, may be used. Separation and purification can be performed using characteristics as an index. Preferred methods include ammonium sulfate fractionation (35-55% saturation), cation exchange chromatography (eg, DEAE Toyopeal 650M (manufactured by Tosoh Corporation)), ammonium sulfate fractionation (0-55% saturation), and hydrophobic chromatography (eg, phenyl). Sepharose (manufactured by Amersham Pharmacia Bitech)) and gel filtration (Superdex 200 pg (manufactured by Amersham Pharmacia Bitech)) are sequentially performed.

Further, the present invention relates to a reduced nicotinamide formed by allowing the enzyme of the present invention to act on a sample containing 1,5-AG in the presence of oxidized nicotinamide adenine dinucleotide (NAD) as an electron acceptor. A method for quantifying 1,5-AG by measuring adenine dinucleotide (NADH) based on a change in absorbance is also provided.

Samples containing 1,5-AG include serum,
Examples include biological fluids such as plasma and urine. When the sample has a property that hinders the measurement of absorbance, pretreatment of the sample such as centrifugation is performed as necessary.

Conditions for the action of the enzyme of the present invention, such as NAD abundance, temperature, pH, enzyme amount, etc., are 1,5-A
There is no particular limitation as long as the amount of NADH changes according to the amount of G. Usually, the concentration of NAD is 0.01 to 10 m.
M, a temperature of 20 to 70 ° C., a pH of 5 to 11, and an enzyme amount of 0.001 to 20 U / ml. 1 U of the enzyme is as defined in the examples below.

The wavelength at which the absorbance is measured is not particularly limited as long as the change in the absorbance at that wavelength is specific to the change in the amount of NADH, but is usually 340 nm. Since an error may occur due to a delay in the reaction at the start of the reaction, the first time (eg, 50 seconds) from the start of the reaction
It is preferable to measure the amount of change in absorbance from when elapses to when a second time (for example, 100 seconds) elapses.

Since the enzyme of the present invention is specific to 1,5-AG, according to the quantification method of the present invention, even if physiologically high concentration of glucose (for example, 110 mg / dl or more) exists, its influence is not affected. It is possible to quantify 1,5-AG at a low concentration (for example, 0.2 mg / dl or less) under physiological conditions without undergoing the reaction. Therefore, in particular, since it is not necessary to remove or modify glucose in a sample derived from a diabetic patient,
It can be applied advantageously.

A kit for quantifying 1,5-AG containing the enzyme of the present invention for use in the quantification method of the present invention can also be provided. This kit is particularly suitable for application to diabetic samples for the reasons mentioned above.

The kit usually contains reagents such as NAD and a buffer in addition to the enzyme of the present invention. The enzyme of the present invention may be immobilized on a solid support. Examples of the solid support include a polymer gel, beads, and a plate. The immobilization can be performed by a known method such as a carrier binding method, a cross-linking method, an entrapment method, and a composite method. In addition, the reference material (1,
5-AG) may be included in the kit. The enzyme of the present invention, NA
D and the standard substance may not be in a solution state, in which case it is preferable to include a solvent for dissolution in the kit.

The present invention relates to a partial cDNA of the enzyme of the present invention derived from the strain KDK3003 having the nucleotide sequence shown in SEQ ID NO: 1.
Also provide. This cDNA can be used for obtaining a full-length cDNA (a DNA encoding the enzyme of the present invention). For example, a cDNA library prepared from the KDK3003 strain can be screened by hybridization using a probe prepared based on the nucleotide sequence of this cDNA. The 5 ′ RACE method can also be performed based on the nucleotide sequence of this cDNA.

DNA encoding the obtained enzyme of the present invention
Can be used for producing the enzyme of the present invention. That is, an appropriate host is transformed with the DNA encoding the enzyme of the present invention, the resulting transformant is cultured, and the enzyme of the present invention can be obtained from the culture. As a method of transformation,
There is a method in which a DNA encoding the enzyme of the present invention is incorporated into an expression vector compatible with a host, and the obtained expression vector is introduced into the host. The host may be any of prokaryotic cells and eukaryotic cells, and specific examples include Escherichia coli and yeast. The enzyme of the present invention can be obtained from the culture by a separation and purification method as described above for the production method of the present invention.

[0047]

The present invention will be described below in more detail with reference to examples.

[0048]

[Example 1] Acquisition of a bacterium capable of producing 1,5-AG dehydrogenase (1) Screening A soil sample was added to 5 ml of a culture medium having the following composition, and the temperature was 30 ° C
For 2 days (300 reciprocations per minute). 0.01
ml of the culture was transferred to a fresh medium and cultured under the same conditions. Then, a pure culture was obtained by inoculating the culture on an agar plate of the same medium.

The composition of the culture medium was 3 g (NH_Four)_TwoS
O_Four, 1gK_TwoHPO_Four, 1g NaH_TwoPO _Four, 0.5 gM
gSO_Four・ 7H_TwoO, 0.1 g CaCl_Two・ 2H_TwoO, 5g
1,5-AG, 1 ml vitamin mixture and 10 ml metal
A solution containing 1,000 ml of distilled water (pH 5.5)
Met. The composition of the vitamin mixture is 1 mg thiamine.
HCl, 2 mg riboflavin, 2 mg Ca pantothene
Acid, 2mg pyridoxine / HCl, 0.1mg bioti
1 mg p-aminobenzoic acid, 2 mg nicotinic acid and
0.1 mg of folic acid in 100 ml of distilled water
Was. The composition of the metal solution was 11.7 g MnSO_Four・ 3H
_TwoO, 2.2 g ZnSO_Four・ 7H_TwoO, 0.4g CuSO_Four
・ 5H_TwoO, 0.28 g CoCl_Two・ 2H_TwoO, 0.26
gNaMoO_Four・ 2H_TwoO, 0.4gH_ThreeBO_ThreeAnd 0.0
6 g KI was contained in 1000 ml of distilled water.

196 cultures were isolated from 32 different soil samples. The isolated microorganisms grew using 1,5-AG as the sole carbon source and included 73 fungi, 46 yeasts and 77 bacteria.

The isolated microorganism was treated with 5 ml of 1,5-AG
The cells were shake-cultured at 30 ° C. for several days in a medium (a culture medium in which 5% yeast extract was used instead of the vitamin mixture and the metal solution). Bacteria and yeast cells,
The cells were collected by centrifugation at 8,000 × g for 5 minutes. Fungal hyphae were collected by filtration through filter paper. Remove cells and mycelium
It was washed with 85% KCl, suspended in a 0.1 M Tris-HCl buffer (pH 8.0), and crushed with a mini-beads beater model 3110BX (manufactured by Nippon Lambda). The crushed liquid was centrifuged at 8,000 × g for 10 minutes to remove cell debris, thereby obtaining a cell-free extract.

The oxidase activity of the cell-free extract was measured by a colorimetric method conjugated to POD. That is, 100 μmol Tris-HCl (pH 8.0),
4.5 μmol 4-aminoantipyrine, 6.0 μmo
1 phenol, 6.0 units peroxidase and 2
Using a reaction mixture containing μmol 1,5-AG in a total volume of 3 ml, the oxidase activity was determined by measuring the production of a quinone dye at 30 ° C. by absorbance at 505 nm (Hitachi U-3300 spectrophotometer). However, 1,5-A
No G-specific enzyme activity was observed. 1,5-A
Since the structure of G is similar to that of glucose,
It was predicted that screening for 1,5-AG-specific enzyme activity would be difficult. Thus, for the isolates,
The activity of converting AG into glucose was detected by quantifying glucose in the culture filtrate.

Glucose in the culture filtrate was measured by the glucose oxidase (GOD) method. That is, 100 μ
molMES-Na (pH 5.7), 4.5 μmol4
-Aminoantipyrine, 6.0 μmol phenol,
6.0 units POD, 100 units GOD, 5 μm
A reaction mixture containing ol1,5-AG and a 50 μl sample in a total volume of 3 ml was incubated at 30 ° C. for 30 minutes, and the absorbance at 505 nm of the reaction mixture was measured. as a result,
Glucose was detected in the culture filtrate of seven isolates containing two fungi.

The microorganisms that were positive in the GOD test were subjected to a resting cell reaction to further select. That is, 100 μmol Tris-HCl (pH 8.0),
The reaction mixture containing 2 μmol 1,5-AG and 0.1 g resting cells in a total volume of 1 ml was reacted by shaking at 30 ° C. for 1 to 3 hours. The reaction mixture was loaded on silica gel (25 TLC plate 20 × 20, manufactured by Merck) and phenol /
TLC was performed using water (4: 1, v / v) as a developing solvent. Spots were detected by spraying with 20% sulfuric acid or 0.5 ml anisaldehyde / 0.5 ml sulfuric acid / 9 ml 95% methanol / several drops of acetic acid. Sulfuric acid was used for detecting reducing sugars, and anisaldehyde / sulfuric acid / methanol solution was used for detecting carbohydrates. As a result, it was found that the 11-3 strain had an activity of converting 1,5-AG to glucose in a resting cell reaction.

The cell-free extract prepared from the mycelium of the strain 11-3 was used for the enzymatic reaction with 1,5-AG, but the reaction from 1,5-AG to glucose observed in the resting cell reaction was performed. Did not occur with cell-free extract and 1,5-AG alone. Reducing coenzymes and cofactors (NADH, NADPH, FADH and GSH) and α
-Ketoglutaric acid and FAD, FMN, GSS
G and other coenzymes and cofactors involved in the redox reaction such as PMS were used in the enzymatic reaction, but in all cases both glucose and other reaction products were TLC
Was not detected in the reaction mixture. Cu ²⁺ , Fe
Addition of metal ions such as ²⁺ , Mg ²⁺ and Zn ²⁺ also had no effect.

Since there was a possibility that the enzyme was bound to the cell membrane, the cell debris after preparing the cell-free extract was
, But no glucose was detected in the reaction mixture.

Further, using the cell-free extract of the strain 11-3, the enzyme activities were measured for NAD, NADH, NADP and NA.
DPH was measured spectrophotometrically at 340 nm with coenzyme. As a result, an increase in absorbance was observed in the reaction mixture containing NAD, 1,5-AG and the cell-free extract. Therefore, the NAD-dependent dehydrogenase activity of 1,5-AG was confirmed in the cell-free extract of strain 11-3.

(2) Examination of 1,5-AG dehydrogenase producing ability (2-1) Examination of carbon source Regarding the producing ability of 1,5-AG dehydrogenase,
It was examined whether the enzyme could be produced by other carbon source media and the enzyme activity could be maintained.

As another carbon source of 1,5-AG, 1,4-AG
AG, glucose, glycerol and combinations thereof were selected and added to the above culture medium so that each concentration was 0.5%. One platinum loop of the 11-3 strain was inoculated into each medium (5 ml), and shaking culture was performed at 30 ° C. for 2 days.

The enzyme activity in the culture supernatant was measured by incubating a reaction solution having the following composition at 30 ° C. That is, the generated NADH is measured for the change in absorbance at 340 nm between 50 seconds and 100 seconds after the start of the incubation, and the measured change is used as a relative value, or the extinction coefficient of NADH of 6220 M ^-1 cm ^-1 is used. It was converted to the amount of enzyme activity. One unit (U)
It was defined as the amount of enzyme that produced 1 μmol of NADH per minute.

Tris-HCl (pH 8) 33 mM β-NAD (manufactured by Oriental Yeast) 1.67 mM 1,5-AG (manufactured by Wako Pure Chemical Industries) 0.083% Enzyme solution (culture supernatant) 50 μl

The growth was measured by measuring the wet weight of the hyphae, and
The value per hit was used as an index.

Table 1 shows the measurement results. As a carbon source other than 1,5-AG, the same or higher enzyme activity was obtained using glucose. Therefore, expensive 1,5
It was found that it was possible to substitute inexpensive glucose without using -AG as an enzyme induction medium. For this reason, subsequent experiments decided to use glucose as a carbon source.

[0064]

[Table 1] Table 1 Effect of carbon source 源 Carbon source Proliferation (g / L) Activity ( %) ────────────────────────────── 1,5-AG 24 100 1,4-AG 8 32 glucose 30 112 glycerol 28 68 glycerol + 1,5-AG 36 74 glycerol + 1,4-AG 16 98 @ AG: Anhydro-D-glucitol

(2-2) Examination of nitrogen source (NH_Four)_TwoSO_FourInstead of polypeptone, beef
Kistract, NH_FourCl, NH_FourNO_ThreeAnd NaNO_ThreeTo
Was used to inhibit the production of 1,5-AG dehydrogenase by strain 11-3.
The effects of nitrogen sources were also investigated. As shown in Table 2, 11-3
The strain is NaNO _ThreeWhen cultured in medium containing
Activity and proliferation on 1,5-AG were observed.

[0066]

Table 2 Effect of nitrogen source 窒 素 Nitrogen source Proliferation (g / L) Activity ( %) ────────────────────────────── (NH ₄ ) ₂ SO ₄ 34 100 NH ₄ Cl 30 87.6 NH ₄ NO ₃ 26 59.9 NaNO ₃ 30 150 Beef extract 46 32.2 Polypeptone 52 25.6──────────────────────────────

(2-3) Time course 11-3 strains were transferred to a 10 L jar fermenter (speed: 50
0-rpm, air: 5.0 l / min) using 1,5-A
The cells were cultured in 7 l of a culture medium containing glucose and NaNO ₃ instead of G and (NH ₄ ) ₂ SO ₄ , respectively, and the time course of hyphal growth and the specific activity of 1,5-AG dehydrogenase was examined (FIG. 1). In FIG. 1, open circles indicate the hyphae wet weight, solid circles indicate the enzyme activity, and solid squares indicate the specific activity. The amount of protein for calculating specific activity was quantified by the Bradford method using a BIO-RAD protein assay kit.

Growth and specific activity increased up to 40 hours and reached a maximum at the beginning of steady state. 1,5-AG dehydration crude enzyme activity was detected transiently.

(3) Identification of Strain 11-3 Strain 11-3 is a filamentous fungus, and the colony turns green. Under a microscope, many phyllo-type conidia were observed at the apex of the branched phialide. The conidia were agglutinated rather than chained. From these observations, it is confirmed that the 11-3 strain belongs to the genus Trichoderma.

The identification of strain 11-3 was carried out by using Centa, the Netherlands.
alureau voor Schimmelcul
tures (CBS), Trichoderma Longibrachiatum (Trichodermalon)
gibrachiatum Rifai).

The 11-3 strain was obtained from Trichoderma longibrachitaum KDK3003.
3003) and the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Address: 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture 305-0046, Japan) based on the provisions of the Budapest Treaty from August 10, 1998. Deposited (Deposit No .: F
ERM BP-6458).

[0072]

Example 2 Production and Characteristic Evaluation of 1,5-AG Dehydrogenase KDK3003 strain was cultured in an 8 L medium at 28 ° C. for 2 days using a 10 L jar fermenter. The composition of the medium was ₃ g NaNO ₃ , 1 g KH ₂ PO ₄ , 0.5 g Mg
_{SO 4 · 7H 2 O, 0.1gCaCl} 2 · 2H 2 O, a 5g yeast extract and 4g glucose was one containing distilled water 1000 ml (pH 5.5).

Thereafter, the culture solution was subjected to suction filtration to collect mycelia, washed, and washed with 0.1 M Tris-H containing 2 mM DTT.
Cl (pH 8.0) (130 ml) and suspended in glass beads (30 ml) using a bead beater (manufactured by Nippon Lambda).
0 ml, 0.25-0.50 mmf) to break up the mycelium. The crushed material was centrifuged at 9,000 × g for 30 minutes at 4 ° C. to remove non-crushed cells and cell debris, and the supernatant was used as a cell-free extract. The following purification operations were all performed at 4 ° C.

Ammonium sulfate was added to the cell-free extract to 35% saturation with stirring, and the pH was adjusted to 7.0 with a 35% ammonium hydroxide solution. After stirring for 1 hour, the precipitate was centrifuged at 16,000 × g for 30 minutes to remove the precipitate. The supernatant was collected, and ammonium sulfate was added for 5 minutes.
The precipitate was collected in the same manner as described above in addition to 5% saturation, and buffer A (50 mM Tr containing 2 mM DTT) was collected.
is-HCl (pH 8.0)) and dialyzed against the same buffer.

The dialysate was washed with DEAE equilibrated with buffer A.
-Applied to a Toyopearl 650M (manufactured by Tosoh Corporation) column (2.2 φ × 20 cm).
The column was washed with the same buffer, and the adsorbed protein was washed with KC.
Elution was with a 1 linear gradient (0-0.5M).
The eluted enzyme activity fraction was collected, and ammonium sulfate was added thereto with stirring so as to be 55% saturated, and the pH was increased to 35%.
It was adjusted to 7.0 with sodium hydroxide solution. After stirring for 1 hour, the precipitate was centrifuged at 16,000 × g for 30 minutes, and the supernatant was removed. The precipitate was dissolved in buffer A, dialyzed against buffer A containing 25% saturated ammonium sulfate, and then phenyl-Sepharose (Amersham Pharmacia Biotech) equilibrated with buffer A containing 25% saturated ammonium sulfate. Column) (1.0φ × 10c)
m). After washing with the same buffer, the enzyme was eluted with a linear gradient of ammonium sulfate from 25% saturation to 0% saturation (flow rate 1 ml / min). The eluted enzyme-active fraction was collected and superdex (Superde) equilibrated with buffer A containing 0.1 M NaCl.
x) The mixture was applied to a 200 pg (manufactured by Amersham Pharmacia Biotech) column (1.6 φ × 60 cm), and the enzyme was eluted with the same buffer (2 ml / min).

Table 3 shows the results of the purification. The enzyme activity and the amount of protein were measured by the method described in Example 1 (2).

[0077]

[Table 3] Table 3 Summary of purification ──────────────────────────────────── Process Total activity Total protein Specific activity Purity Yield (U) (mg) (U / mg) (fold) (%) ─────────────────────────── ───────── Cell-free extract 51 280 0.18 1 100 Ammonium sulfate fraction (35-55% saturated) 47 123 0.38 2 94 DEAE-Toyopearl 19 11.3 1.69 9 37 Ammonium sulfate fraction (0-55% saturated) ) 19 7.36 2.58 14 37 Phenyl Sepharose 5.3 2.84 1.87 10 10 Superdex 200pg 2.6 0.79 3.29 18 5.1 ───────────────────────────── ───────

The enzyme obtained after chromatography with 200 pg of Superdex was used as an enzyme for examining the physicochemical properties of the following enzymes.

(1) Examination of optimum pH and pH stability 100 m showing pH 5.5, 6.5, 7.0, and 7.5
M McIlvine, pH 7.5, 8.0, 8.
A buffer solution of 100 mM Tris-HCl showing 5, 9.0 and 100 mM glycine-NaOH having pH of 9.0, 9.5, 11.0, 12.0, 12.5 was prepared.
The enzyme activity was measured under the conditions described in Example 1 (2) except that these buffers were used instead of Tris-HCl (pH 8) so as to have the same molar concentration. FIG. 2 shows the results obtained by calculating the relative enzyme activity as a percentage when the maximum enzyme activity was taken as 100. As a result, it was found that the optimum pH of the present enzyme was around 9.

Further, the present enzyme was added to each buffer at each pH for 3 hours.
The mixture was incubated at 0 ° C. for 10 minutes, and the mixed solution after the incubation was used as an enzyme solution. The enzyme activity was measured under the conditions described in (2) of Example 1, and the relative enzyme activity when the maximum enzyme activity was taken as 100. FIG. 3 shows the results obtained by determining the activity in percent. As a result, the enzyme was found to be stable in the pH range of 6 to 10.

(2) Examination of optimum temperature and temperature stability Except that the reaction temperature is set at a temperature of 10 to 60 ° C. at intervals of 10 ° C., the enzyme activity was measured under the conditions described in Example 1, (2). FIG. 4 shows the results obtained by measuring and determining the relative enzyme activity as a percentage when the maximum enzyme activity was taken as 100.
As a result, it was found that the optimum temperature of the present enzyme was between 40 and 50 ° C.

After incubating the enzyme solution for 10 minutes at a temperature of 10 ° C. in the range of 30 ° C. to 80 ° C., the enzyme activity was measured using the enzyme solution under the conditions described in Example 1, (2). As a result of the measurement, this enzyme was found to be 70
Even at 10 ° C. for 10 minutes, 50% or more of the activity remained, confirming that the temperature stability was good (FIG. 5).

(3) Examination of Electron Receptor It was confirmed at the time of examination of the culture supernatant that this enzyme utilizes NAD as an electron acceptor. 300 μl of 10
0 mM Tris-HCl (pH 9), 25 μl of 10
mM oxidized NADP, 25 μl of 5% 1,5-A
G, 10 μl of the enzyme solution and 640 μl of water were mixed well, incubated at 30 ° C., and 340 nm
When the change in absorbance at was measured, the absorbance did not increase. Therefore, it was found that this enzyme can use NAD as a coenzyme, but cannot use NADP.

(4) Examination of molecular weight The purified enzyme was eluted using a gel filtration method using Superdex 200 pg (manufactured by Amersham Pharmacia Bitech), and the molecular weight was determined from the elution volume of a molecular weight marker (No. 104558 manufactured by Boehringer Mannheim) determined in advance. As a result, the molecular weight was 141,000 (141 kD
a).

An analysis by SDS-PAGE (sodium dodecyl sulfate / polyacrylamide gel electrophoresis) was also performed. Using the First System (Amersham Pharmacia Biotech), PastGel Gradie
Using an nt 10-15 (manufactured by Amersham Pharmacia Biotech) gel, the gel was electrophoresed at 250 V for 30 minutes, and protein staining was performed using Coomassie Brilliant Blue G-250.
I went in. As a result of determining the molecular weight from the molecular weight marker (LMW marker kit) that was simultaneously electrophoresed, 36,000 was obtained.
(36 kDa). This enzyme was expected to be a tetramer.

(5) Examination of Substrate Specificity To examine the substrate specificity for glucose, fructose, sorbitol, xylitol, mannitol, glyceraldehyde, glyceraldehyde triphosphate, and acetaldehyde, a 0.25% aqueous substrate solution was prepared. did.

The enzyme activity was measured under the conditions described in (2) of Example 1 except that the aqueous solution of the substrate was added to a final concentration of 3 mM. The activity was determined in percent and the results are shown in Table 4.

1,5-AG dehydrogenase does not react with glucose, fructose, sorbitol, mannitol, and xylitol, unlike conventional enzymes. In addition, it shows about half the activity of glyceraldehyde as compared with the activity using 1,5-AG as a substrate. Therefore, it was suggested that the enzyme is a completely different 1,5-AG dehydrogenase from the conventionally known enzymes.

[0089]

Table 4 Substrate specificity 基質 Substrate (3 mM) Relative activity 表─────────────── 1,5-AG 100 glucose not detected fructose not detected sorbitol not detected xylitol not detected mannitol not detected glyceraldehyde 52 glyceraldehyde 3 phosphorus Acid Not detected Acetaldehyde Not detected ─────────────────────────

Under the conditions described in Example 1, (2), 1,5-
When the measurement was performed while changing the concentrations of AG and NAD, the Km values of 1,5-AG and NAD were 0.6, respectively.
7 mM and 0.50 mM.

(6) Investigation of the effects of metal ions and reagents The effects of various metal ions and reagents on enzyme activity were examined.

The metal salts shown in Table 5 were added to a reaction mixture containing no substrate solution to a final concentration of 1 mM, and the mixture was incubated at 30 ° C. for 10 minutes, and then the substrate solution was added. Table 5 shows the results obtained by measuring the enzyme activity under the conditions described in (1) and determining the relative enzyme activity as a percentage when the maximum enzyme activity was taken as 100.

[0093]

[Table 5] Table 5 Effect of metal ion 化合物 Compound relative activity (%) ──────────────無 し None 100 LiCl ₂ not detected MgCl ₂ 24 CaCl ₂ not detected MnCl ₂ not detected CoCl ₂ 57 NiCl ₂ not detected CuCl ₂ 14 ZnCl ₂ 24 BaCl ₂ not detected HgCl ₂ 21 FeCl ₂ 269 FeCl ₃ not detected ────────────────────

The enzymatic activity was measured using 1 mM Mn ²⁺ , Fe ³⁺ , N
It was completely inhibited by i ²⁺ , Ca ²⁺ , Ba ²⁺ and Li ²⁺ . On the other hand, activation was observed with Fe ²⁺ .

The reagents shown in Table 6 were used at a final concentration of 1 mM (PCM
B is 0.1 mM), and the mixture is added to the reaction mixture containing no substrate solution, incubated at 30 ° C. for 10 minutes, and then added with the substrate solution, except that the enzyme is prepared under the conditions described in Example 1, (2). The activity was measured, and the relative enzyme activity was calculated as a percentage when the maximum enzyme activity was defined as 100. Table 6 shows the results.

[0096]

[Table 6] Table 6 Effect of reagents 化合物 Compound relative activity (%) ───────────────無 し None 100 2,2-bipyridyl 57 o-phenanthroline Not detected Ethylenediaminetetraacetic acid 104 Potassium cyanide 100 Sodium azide Not detected Potassium fluoride 130 Iodoacetic acid 70 PCMB (0.1 mM) Not detected DTNB detected Phenylhydrazine 178 PCMB: p-chloromercuribenzoic acid DTNB: dithiobis (nitrobenzoic acid)

[0097] Enzyme activity was measured using PCMB, DTNB, NaN.
Strongly inhibited by ₃ and o-phenanthroline.

These results suggest that the SH group and iron ions are involved in the enzyme reaction.

(7) Examination of Reaction Products As described in Example 1, glucose production was detected in the resting cell reaction using the mycelium of the KDK3003 strain, but glucose was detected by TLC analysis of the enzyme reaction mixture. No production was detected. Since glucono-δ-lactone reductase activity was remarkably observed in the cell-free extract, detection of gluconic acid, which is a hydrolyzate of glucono-δ-lactone, was attempted using gluconate dehydrogenase. As a result, when the reaction solution after the 1,5-AG dehydrogenation reaction was used as a substrate solution, a gluconate dehydrogenase reaction was remarkably observed. Therefore, 1,5-AG
Is presumed to be converted to glucono-δ-lactone by 1,5-AG dehydrogenase.

[0100]

Example 3 1,5 using 1,5-AG dehydrogenase
Quantification of -AG 1,5-AG using 1,5-AG dehydrogenase (AGDH)
The enzymatic quantification of the amount of -AG was studied. The purified AGDH used was prepared by the method described in Example 2, except that the last gel filtration was not performed.

The serum 1,5-AG concentration of diabetic patients is generally between 0.15 and 0.2 mg / dl. So 0.2
mg / dl of 1,5-AG was used as a sample instead of a substrate solution, 2.7 U / ml of purified AGDH was used as an enzyme solution, and a potassium phosphate buffer was used instead of a Tris-HCl buffer. Was measured in the same manner as in Example 1 (2). As a result, it was found that 1,5-AG at this concentration could be detected.

Furthermore, various concentrations (0.5-2 mg / dl) of 1,5-A based on the 1,5-AG level in serum
The measurement was performed in the same manner as above using G as a sample. The same measurement was performed in the presence of glucose (110 mg / dl in the sample) and fructose (0.56 mg / dl in the sample). The results are shown in FIGS. FIG. 6 shows 1,5-AG
7 shows the results in the presence of glucose, and FIG. 8 shows the results in the presence of fructose.

As is clear from FIG. 6, a linear relationship was observed between the concentration of 1,5-AG and the formation of NADH. Furthermore, it was shown that the coexistence of glucose and fructose at concentrations comparable to physiological conditions did not affect the quantification of 1,5-AG by AGDH (FIGS. 7 and 8).

These results show that the quantification of 1,5-AG using the AGDH of the present invention is suitable for the diagnosis of diabetes.

[0105]

Example 4 Acquisition of cDNA for 1,5-AG Dehydrogenase AGDH was purified from KDK3003 strain as in Example 2. The purified enzyme was partially digested with protease V8 (manufactured by Sigma-Aldrich Japan) and subjected to SDS-PAGE.
And blotted on a PVDF membrane. The amino acid sequence of the peptide in each band was analyzed using a protein sequencer (Applied Bi
psystem model 476A). The following primers were designed based on the determined partial amino acid sequence.

From the cells obtained by culturing the KDK3003 strain for 32 hours as described in Example 2, the total RNA was subjected to the acid guanidine-phenol-chloroform method (Anal. Bioc.
162, 156-159 (1987)).
RNA was purified with an oligo (dT) -cellulose span column using a Quick Prep mRNA purification kit (manufactured by Amersham Pharmacia Biotech). From 5 mg of mRNA, Re
cDNA was synthesized using ady To-Go PCR Beads (Amersham Pharmacia Biotech) based on the manufacturer's instructions.

Using the obtained cDNA as a template, auto-hot-start PCR using AmpliWax ™ PCR Gem (manufactured by PerkinElmer Japan) was performed to maximize amplification specificity (J. Virol., 5272-5281 (199
6)). Keep all reaction components except primers at 80 ° C
After heating for 1 minute, the primer is added and then TaKaRa P
Thermocycler sequence using CR Thermal Cycler MP (Takara Shuzo) (first denaturation at 94 ° C for 1 minute, 94
35 cycles of 1 minute at 55 ° C for 1 minute and 72 ° C for 2 minutes, and a final extension at 72 ° C for 3 minutes). The PCR mixture was 6.25 μM denaturing primer (5'-ATYCCNGTBCARAAGCCNGG
N (SEQ ID NO: 3) or 5′-AAGCCNGGNGTBGAYGARGTBYT (SEQ ID NO: 4) and 3′-CVACRTGNGCNGCCTTNGCRA (SEQ ID NO: 5).

As a result, a fragment of about 300 bp was obtained as a PCR product. The PCR product was directly ligated into the pT7Blue-2 T vector, and the recombinant plasmid was used for ELECTRO CELL MAN.
E. coli JM109 was transformed using UPULATER 600 (manufactured by BTX) (Nucleic Acids Res., 16, 6127-6145 (1988);
Genetic Engineering-Principles and Methods vol. 1
2, pp. 275-295 (1990)). Colonies containing the plasmid were detected as white colonies on 2 × YT agar medium containing 0.1% IPTG and 2.0% X-gal. Cloning of the PCR product was confirmed by colony PCR. Plasmids obtained from the colonies were subjected to a dideoxy termination reaction using the ABI PRISM ™ BigDye ™ Terminator Cycle Sequencing Ready Reaction Kit and sequenced on an ABI PRISM 310 Genetic Analyzer.

On the basis of the determined base sequence, 3′-R
ACE was performed using a 3′-RACECore set (Takara Shuzo) to clone the 3 ′ flanking region (J. Virol., 5272-5281 (1996)). The mRNA of strain KDK3003 was primed for reverse transcriptase using an oligo dT-3 site adapter primer. The reverse transcription reaction was performed at 30 ° C. for 10 minutes, and further incubated with AMV transcriptase at 50 ° C. for 30 minutes. The second PCR
5 μM 3-site adapter primer (Takara Shuzo), 5 μM gene-specific primer (5′-CTGATCTAGAGGTACCGGATCCGCT designed based on cDNA base sequence)
GCACGTACTGCATGAACGG-3 ′ (SEQ ID NO: 6)) and the RT-PCR product as a template. PCR conditions are 94 ° C
There were 35 cycles of 1 minute, 1 minute at 65 ° C and 2 minutes at 72 ° C. The procedure for cloning and sequencing of the amplification products was performed as described above.

As a result, a fragment of about 1.2 kbp was obtained, and 1231 bp encoding 308 amino acids (SEQ ID NO: 2) was obtained.
It was found to consist of nucleotides (SEQ ID NO: 1). The deduced amino acid sequence contained two amino acid sequences determined by purified AGDH (amino acids 1 to 16 and amino acids 102 to 117 in SEQ ID NO: 2).

[0111]

The 1,5-AG dehydrogenase of the present invention comprises
Although it specifically reacts with 1,5-AG but does not react with glucose contained most in a biological component, a pretreatment operation is not required when 1,5-AG is measured by the present enzyme. Therefore, it can be advantageously applied to an automatic analyzer.

[0112]

[Sequence List] <110> Kyoto Daiichi Kagaku Co., Ltd. <120> 1,5-Anhydroglucitol dehydrogenase <130> P-6700 <150> JP 10-259311 <151> 1998-08-28 < 160> 6 <210> 1 <211> 1231 <212> DNA <213> Trichoderma longibrachitaum <220> <221> CDS <222> 1..924 <400> 1 aag cct ggg gtg gat gaa gtt ctc gtc aac atc aag tac tcc ggc gtc 48 Lys Pro Gly Val Asp Glu Val Leu Val Asn Ile Lys Tyr Ser Gly Val 1 5 10 15 tgc cac acc gac ctg cac gcc atg atg ggc gac tgg ccg ctc gac acg 96 Cys His Thr Asp Leu His Ala Met Met Gly Asp Trp Pro Leu Asp Thr 20 25 30 aag ctg ccc ctc gtc ggc ggc cac gag ggc gcg ggc gtc gtc gtg gcc 144 Lys Leu Pro Leu Val Gly Gly His Glu Gly Ala Gly Val Val Val Ala 35 40 45 cgc ggg ctg gtc aag gac atc cag atc ggc gac tac gcg ggc atc 192 Arg Gly Glu Leu Val Lys Asp Ile Gln Ile Gly Asp Tyr Ala Gly Ile 50 55 60 aag tgg ctc aac ggc tcg tgc ctc agc tgc atg tac acg Lys Trp Leu Asn Gly Ser Cys Leu Ser Cys Thr Tyr Cys Met Asn Gly 65 70 75 80 gac gag ccc ctc tgc cac aag gcc ctc ctc tcg ggc tac acc gtc gac 288 Asp Glu Pro Leu Cys His Lys Ala Leu Leu Ser Gly Tyr Thr Val Asp 85 90 95 ggc acc ttt cag gag tac gcc atc gcc aag gcg gcc cac gtc gcc cgc Thr Phe Gln Glu Tyr Ala Ile Ala Lys Ala Ala His Val Ala Arg 100 105 110 atc ccc aag gag tgc gac ctc gag tcc gtc gcg ccc atc ctg tgc gcc 384 Ile Pro Lys Glu Cys Asp Leu Glu Ser Val Ala Pro Ile Leu Cys Ala 115 120 125 ggc atc acc gtc tac aag ggc ctc aag gag tcc ctc gcc cgc ccc ggc 432 Gly Ile Thr Val Tyr Lys Gly Leu Lys Glu Ser Leu Ala Arg Pro Gly 130 135 140 cag acc atc gcc gtc gtc ggc gcc ggc ggc ctc ggc agc atc gcg 480 Gln Thr Ile Ala Val Val Gly Ala Gly Gly Gly Leu Gly Ser Ile Ala 145 150 155 160 ctg cag tac gcc aaa gca atg ggc ctg cac tcc gtc gct att gac gcc 528 Leu Gla Tyr Ayr Met Gly Leu His Ser Val Ala Ile Asp Ala 165 170 175 ggc gac gag aag cgc gac ctg tgc atg cgc ctc ggc gcc tcc gcc ttt 576 Gly Asp Glu Lys Arg Asp Leu Cys Met Arg Leu Gly Ala Ser Ala Phe 180 185 gac ttc tct acc agc aag gac ctc gtc gcc gac gtc cgc gcc gcc 624 Val Asp Phe Ser Thr Ser Lys Asp Leu Val Ala Asp Val Arg Ala Ala 195 200 205 acc ctc gac ggc gag ggc ccc cac gcc gct ctc gtt gcc 672 Thr Leu Asp Gly Glu Gly Pro His Ala Ala Leu Leu Val Ala Ala Gln 210 215 220 gag aag ccc ttc cag cag gcc acg cag tac ctc cgc tca aag ggc cgt 720 Glu Lys Pro Phe Gln Gln Ala Thr Gln Tyr Leu Arg Ser Lys Gly Arg 225 230 235 240 cct cgt ctg cat cgg att gcc cgc cgg cgc aaa gct tca agc gca cct 768 Pro Arg Leu His Arg Ile Ala Arg Arg Arg Lys Ala Ser Ser Ala Pro 245 250 255 gtc ttt gat acc gtc atc cgc atg atc acc atc aag gca gct acg tcg 816 Val Phe Asp Thr Val Ile Arg Met Ile Thr Ile Lys Ala Ala Thr Ser 260 265 270 gca acc ggc gcc gat aca cag gag gcc ctc gac ttc ttc cgg cga ggc 864 Ala Thr Gly Ala Asp Thr Gln Glu Ala Leu Asp Phe Phe Arg Arg Gly 275 280 285 ctt cat cac cgt gcc ctt cca aga cga ttg gcc tca gcc agc tgc agg 912 Leu His His Arg Ala Leu Pro Arg Arg Leu Ala Ser Ala Ser Cys Arg 290 295 300 ata ttt aca ctc tgatgcacga ggccaagatt gcgggccgct atgttgtcga 964 Ile Phe Thr Leu 305 tcctacgaga taagggcgcg accatttctt tgctgtttga tgtgtccaag agtcgatacg 1024 gtttgagttg aaaggagttg aggcaaaagc gaggttaggt tgttcactgt gggcgaattt 1084 gggagtatga ggcggtggtg gttggttggc tggtctgcgg atacacatga tgatgagatg 1144 acgatgacag atgtacatta caatgcgatg agcagttttc cttggttaat gagatatgag 1204 atgaattact accaaaaaaa aaaaaaa 1231 <210> 2 <211> 308 <212> PRT <213> Trichoderma longibrachitaum <400> 2 Lys Pro Gly Val Asp Glu Val Leu Val Asn Ile Lys Tyr Ser Gly Val 1 5 10 15 Cys His Thr Asp Leu His Ala Met Met Gly Asp Trp Pro Leu Asp Thr 20 25 30 Lys Leu Pro Leu Val Gly Gly His Glu Gly Ala Gly Val Val Val Ala 35 40 45 Arg Gly Glu Leu Val Lys Asp Ile Gln Ile Gly Asp Tyr Ala Gly Ile 50 55 60 Lys Trp Leu Asn Gly Ser Cys Leu Ser Cys Thr Tyr Cys Met Asn Gly 65 70 75 80 Asp Glu Pro Leu Cys His Lys Ala Leu Leu Ser Gly Tyr Thr Val Asp 85 90 95 Gly Thr Phe Gln Glu Tyr Ala Ile Ala Lys Ala Ala His Val Ala Arg 100 105 110 Ile Pro Lys Glu Cys Asp Leu Glu Ser Val Ala Pro Ile Leu Cys Ala 115 120 125 Gly Ile Thr Val Tyr Lys Gly Leu Lys Glu Ser Leu Ala Arg Pro Gly 130 135 140 Gln Thr Ile Ala Val Val Gly Ala Gly Gly Gly Leu Gly Ser Ile Ala 145 150 155 160 Leu Gln Tyr Ala Lys Ala Met Gly Leu His Ser Val Ala Ile Asp Ala 165 170 175 Gly Asp Glu Lys Arg Asp Leu Cys Met Arg Leu Gly Ala Ser Ala Phe 180 185 190 Val Asp Phe Ser Thr Ser Lys Asp Leu Val Ala Asp Val Arg Ala Ala 195 200 205 Thr Leu Asp Gly Glu Gly Pro His Ala Ala Leu Leu Val Ala Ala Gln 210 215 220 Glu Lys Pro Phe Gln Gln Ala Thr Gln Tyr Leu Arg Ser Lys Gly Arg 225 230 235 240 Pro Arg Leu His Arg Ile Ala Arg Arg Arg Lys Ala Ser Ser Ala Pro 245 250 255 Val Phe Asp Thr Val Ile Arg Met Ile Thr Ile Lys Ala Ala Thr Ser 260 265 270 Ala Thr Gly Ala Asp Thr Gln Glu Ala Leu Asp Phe Phe Arg Arg Gly 275 280 285 Leu His His Arg Ala Leu Pro Arg Arg Leu Ala Ser Ala Ser Cys Arg 290 295 300 Ile Phe Thr Leu 305 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <220> <221> misc_feature <222> 6, 18, 21 <223> n = a, g, toc <400> 3 atyccngtbc araagccngg n 21 <210> 4 <211> 23 <212> DNA < 213> Artificial Sequence <220> <223> Synthetic DNA <220> <221> misc_feature <222> 6, 9 <223> n = a, g, t or c <400> 4 aagccnggng tbgaygargt byt 23 <210> 5 < 211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <220> <221> misc_feature <222> 5, 11, 14 <223> n = a, g, t or c <400> 5 arcgnttccg ncgngtrcav c <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 6 ctgatctaga ggtaccggat ccgctgcacg tactgcatga acgg 44

[Brief description of the drawings]

FIG. 1 shows an example of a time course of culture.

FIG. 2 shows the% activity of 1,5-AG dehydrogenase at each pH.

FIG. 3 shows the stability of 1,5-AG dehydrogenase at each pH.

FIG. 4 shows the% activity of 1,5-AG dehydrogenase at various temperatures.

FIG. 5 shows the stability of 1,5-AG dehydrogenase at various temperatures.

FIG. 6: 1,5-A using 1,5-AG dehydrogenase
The quantification of G is shown.

FIG. 7 shows the quantification of 1,5-AG using 1,5-AG dehydrogenase in the presence of glucose.

FIG. 8 shows the quantification of 1,5-AG using 1,5-AG dehydrogenase in the presence of fructose.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Masayuki Yagi 57 Kyoto Higashi-Kujo Nishiakeda-cho, Minami-ku, Kyoto

Claims (7)

[Claims] 1. A 1,5-series compound having the following physicochemical properties:
Anhydroglucitol dehydrogenase. (1) Action: catalyzes the dehydrogenation of 1,5-anhydroglucitol. (2) Substrate specificity: Recognizes 1,5-anhydroglucitol as a substrate, but does not recognize glucose as a substrate. (3) Optimum pH: around 9.0. (4) pH stability: stable at pH 6.0 to 10.0. (5) Optimum temperature: 40-50 ° C. (6) Temperature stability: 50% or more activity remains after incubation at 70 ° C. for 10 minutes. (7) Electron acceptor: Oxidized nicotinamide adenine dinucleotide (NAD) can be used. (8) Molecular weight: The molecular weight by dodecyl sulfate-polyacrylamide electrophoresis is about 36,000, and the molecular weight by gel filtration is about 141,000. 2. The 1,5-anhydroglucitol dehydrogenase according to claim 1, which is produced by culturing a bacterium of the genus Trichoderma. 3. The microorganism of the genus Trichoderma is Trichoderma longibrachiatum KDK3003 (Tricoderma locus).
3. The 1,5-anhydroglucitol dehydrogenase according to claim 2, which is ngibrachiatum KDK3003). 4. The 1,5-anhydroglucitol dehydrogenase according to claim 1, which has the amino acid sequence shown in SEQ ID NO: 2. 5. A bacterium of the genus Trichoderma which produces 1,5-anhydroglucitol dehydrogenase according to any one of claims 1 to 4, which is cultured in a nutrient medium, and from the culture,
A method for producing 1,5-anhydroglucitol dehydrogenase, comprising separating and purifying the produced 1,5-anhydroglucitol dehydrogenase. 6. The method according to claim 1, wherein the sample containing 1,5-anhydroglucitol is used in the presence of oxidized nicotinamide adenine dinucleotide (NAD). A method for quantifying 1,5-anhydroglucitol, characterized in that reduced nicotinamide adenine dinucleotide (NADH) produced by reacting hydroglucitol dehydrogenase is measured by a change in absorbance. . 7. A DN having the nucleotide sequence of SEQ ID NO: 1.
A.