CN1701118A - Fructosylamine oxidase - Google Patents
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Abstract
The present invention provides a fructosylamine oxidase which is obtainable by culturing Fusarium proliferatum, and purifying two types of fructosylamine oxidase (FAO) with different substrate specificities from the culture, and which is useful in the measurement of amadori compounds.
Description
Technical Field
The present invention relates to a novel fructosyl amine (fructiamine) oxidase, and more particularly, to fructosyl amine oxidase derived from Fusarium proliferatum (Fusarium proliferatum), a method for producing the same, and use thereof in the determination of Amadori compounds.
Background
The present invention relates to a method for detecting glycemia in a patient, and more particularly to a method for detecting glycemia in a patient, which comprises forming amadori compounds when reactive substances having an amino group such as proteins, peptides or amino acids coexist with reducing sugars such as glucose in blood or food, so that they bind non-enzymatically and irreversibly via amino and aldehyde groups, and then form amadori compounds via amadori rearrangement, since the rate of formation of the amadori compounds is a function of the concentration of the reactive substances, contact time, temperature, etc., various information on samples containing such reactive substances can be obtained from the amount of the amadori compounds.
In enzymatic assays, the amadori compound is determined by contacting the amadori compound with an oxidoreductase and measuring the amount of hydrogen peroxide produced or the amount of oxygen consumed. Fructosyl amino acid oxidase, one of the oxidoreductases, is usually purified from microorganisms. See, for example, JP-H06-65300B, JP-H03-155780A, JP-H07-289253A ([00319, [0037]), JP-H08-154672A (claims 2 and [0027]), JP-H11-243950A ([0037]), and JP-H05-192193A.
Enzymes derived from Corynebacterium (Corynebacterium) include those specific for α -aminosaccharide amino acids, but inactive to fructosyl lysine (hereinafter, may be referred to as "FL"), which is poor in thermostability (90% or more decrease in activity after treatment at 45 ℃ for 10 minutes), and thus lacks sufficient practical applicability (JP-H06-65300B). enzymes derived from Aspergillus (Aspergillus) include those having lower activity to FL than fructosyl valine (hereinafter, may be referred to as "FV"), but whether or not these enzymes are active to glycated protein or its hydrolysate is unknown (JP-H03-155780A). enzymes derived from Gibberella (Gibberella) include those having protected fructosyl N α -Z-lysine (hereinafter, may be referred to as "FV") which show high specificity and are active to fructosyl polylysine but inactive to fructosyl valine [ JP-H07-0031]0031 and those derived from Fusarium [ 00226]0031 and having the same activity to fructosyl valine [0027]as other enzymes derived from Fusarium [ 0027-H00326]and having the same activity to fructosyl valine [0027]as the fructosyl valine [ 7]289253, and further having the same activity to Fusarium [ 0023-4937]as the fructosyl valine [ 7]or the other enzymes derived from Fusarium [ 0023-H3-289253.
For example, although these prior enzymes are active for glycated amino acids or polylysine produced by cleavage by protease treatment or the like, they have little activity for glycated peptides glycated at the α -position, and therefore, in the case of glycated hemoglobin, in which the α -amino group of the N-terminal amino acid is glycated, it is undoubtedly necessary to release N-terminal fructosyl valine in advance.
In order to accurately measure glycated proteins using the existing fructosyl amino acid oxidase, it is generally inevitable to determine the release of glycated amino acids as a substrate for the enzyme. However, no method is provided which enables the glycated amino acid of interest to be actually released, or which provides a protease with a sufficiently high specificity to ensure this. One of the strategies to solve this problem is to use fructosyl amine oxidase reacting with the peptide itselfglycated at the N-terminal. Of particular importance is the use of fructosyl amine oxidase, which is active also on glycated peptides as cleavage products, to enable accurate determination of hemoglobin A1c (HbA1c), which is important for the control of diabetes.
Disclosure of Invention
The present invention provides a novel fructosyl amine oxidase (hereinafter, may be referred to as "FAO") for accurately and efficiently assaying amadori compounds, specifically, glycated proteins.
The present inventors have intensively studied and found that a Fusarium strain produces FAO having excellent substrate specificity, and established the present invention.
Accordingly, the present invention provides fructosyl amine oxidase derived from Fusarium multiformis.
Brief Description of Drawings
FIG. 1 shows the elution profile of cultured Fusarium multiformis protein (OD. RTM. 280nm) and active Resource Q column chromatography.
FIG. 2 is a graph showing the relationship between the activity of FAO-Q1 in a solvent and pH, wherein FAO-Q1 is one of the enzymes in the present invention.
FIG. 3 is a graph showing the relationship between the activity of FAO-Q2 in a solvent and pH, wherein FAO-Q2 is one of the enzymes of the present invention.
FIG. 4 is a graph showing the relationship between the activity of FAO-Q1 in a solvent and temperature.
Fig. 5 is a graph showing the relationship between the activity of FAO-Q2 in a solvent and temperature.
FIG. 6 is a graph showing the molecular weights of FAO-Q1 and FAO-Q2 as determined by gel filtration.
Best Mode for Carrying Out The Invention
The fructosyl amine oxidase of the invention has catalytic activity in the reaction shown in reaction scheme (I).
Wherein R is1Is- [ CH (OH)]n-CH2OH (wherein n is 5 or 6), R2Is an amino acid residue or a peptide residue consisting of 2-10 amino acids.
In the above reaction scheme (I), R2Is an amino acid residue or a peptide residue consisting of 2 to 10 amino acids, preferably an amino acid residue or a peptide residue consisting of 2 to 6 amino acids, more preferably an amino acid residue or a peptide residue consisting of 2 to 3 amino acids.
Form R2The amino acid(s) of (a) varies according to the amadori compound determined; but examples include valine, lysine, histidine, leucine, serine, and the like. When R is2When it is a peptide residue, it may be composed of 2 to 10 amino acids having valine or leucine at the N-terminus. More preferably the peptide is substituted by N-The terminal has 2 or 3 amino acid composition of valine, and examples include valine-histidine and valine-histidine-leucine.
When the FAO of the present invention is used for measuring HbA1c, it is preferable that the FAO has activity on α -amino-glycated valine, i.e., Fructosyl Valine (FV) as described above or a peptide having FV at the N-terminal.
The FAO of the present invention is not limited to a specific source as long as it has an enzymatic activity. For example, FAO produced by a microorganism that exhibits an enzymatic activity on a glycated amino acid and a glycated peptide as substrates, grown in a medium containing a given glycated amino acid or a glycated peptide as a sole carbon source and nitrogen source, is used in the present invention. Examples of the glycated peptide used for screening such a microorganism include a cleavage product of the glycated protein of interest. The target FAO can be obtained by culturing a microorganism in a medium containing such a glycated peptide as a sole carbon source and nitrogen source, purifying the obtained enzyme and confirming the activity. As described below, the inventors screened microorganisms from soil using fructosyl-valine-histidine-leucine (FVHL) and found that the microorganism fusarium had FVHL assimilating ability.
Since the FVHL is identical to the N-terminal sequence of the hemoglobin β -chain, it is suitable for screening FAO useful in the measurement of HbA1 c.
Thus, the FAO of the present invention can be prepared microbiologically using fusarium strains. Preferred microorganisms include Fusarium multiformis and variants thereof.
Fusarium multiformis the strain that was first isolated from soil by the present inventors according to the method described in example 1. It has been deposited at "International patent organism depositary, national institute of bioscience and human technology" Central 6, 1-1-1Higashi, Tsukuba-shi, Ibarakiken, Japan (Fusarium sp. GL2-1 strain; reception date: 9/2002; registration number: FERM P-19005), and has been transferred to international depository (conversion date: 8/11/2003; registration number: FERM BP-8451). Hereinafter, Fusarium multiformis of the present invention may be referred to as "GL 2-1" or "GL 2-1 strain".
It is possible to obtain, from the original strain GL2-1, strains with an increased activity on FVHL or other substrates by mutagenesis or genetic recombination techniques. These variants arealso useful as sources of FAO in the present invention. Derived strains include those artificially obtained by mutagenesis or those obtained by screening.
The FAO of the present invention can be produced by culturing a microorganism capable of producing FAO in a glucose-valine browning medium (hereinafter, referred to as "GV browning medium"). The GV browning medium can be obtained by autoclaving glucose and valine at 120 deg.C for 30 minutes. Examples of preferred GV browning media include media containing 1.5% glucose, 0.5% L-valine, 0.1% dipotassium hydrogen phosphate, 0.1% sodium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.01% calcium chloride dihydrate and 0.2% yeast extract.
Typically, the incubation is carried out at 25-37 deg.C, preferably 28 deg.C. The pH of the medium is between 4.0 and 8.0, preferably between 5.5 and 6.0. However, the conditions are not critical and should be appropriately adjusted according to the conditions of various microorganisms, and are not limited to the above conditions.
FAO accumulates in fungal cells when GL-2 strain is cultured under the above conditions for 12-36 hours, preferably 24 hours. The cell-free extract can be obtained by filtration followed by centrifugation, by conventional methods of collecting fungal cells. The cells may be disrupted by conventional means, for example by mechanical disruption, autodigestion with solvents, freezing, sonication, pressure and the like.
Methods for isolating and purifying enzymes are also known in the art. Can be carried out by combining an appropriate known method including ammonium sulfate salting out, organic solvent such as ethanol precipitation, ion exchange chromatography, gel filtration, affinity chromatography and the like.
For example, mycelia can be harvested from the resulting culture by centrifugation or suctionfiltration, washed, suspended in 0.1M Tris-HCl buffer (pH8.0) containing 1mM DTT, and applied to a MiniBeadBeaterTM(0.5mm glass beads) were ground (broken) and centrifuged. The supernatant was used as a cell-free extract, which was then fractionated with ammonium sulfate, dialyzed and purified by column chromatography using Resource Q column (Amersham Biosciences).
Alternatively, when the FAO is secreted or accumulated in the medium, the enzyme may be isolated and purified by a method known per se, for example, by an appropriate combination of methods including ion exchange resin treatment, activated carbon adsorption treatment, organic solvent precipitation, vacuum concentration, freeze-drying, crystallization and the like.
One of the FAOs is active on Fructosyl Valine (FV) and N- α fructosyl lysine (FZL) (hereinafter referred to as "FAO-Q1") and the other is active on FV but not active on FZL (hereinafter referred to as "FAO-Q2"). although the preparation and determination are described herein with respect to FAO-Q1 and FAO-Q2 derived from GL2-1 strain, the present invention is not limited to enzymes of a specific source, but includes any FAOs for the purposes of the present invention, which have the following physicochemical properties.
Hereinafter, the enzyme of the present invention derived from GL2-1 strain will be described in more detail.
FAO-Q1
1) The activity on fructosyl valine is almost the same or higher compared to fructosyl lysine;
2) the optimum pH for the enzymatic reaction was 7.5;
3) the optimal temperature for enzyme stability is about 30-40 ℃; and
4) the molecular weight was approximately 39kDa when assessed by SDS-PAGE and approximately 39.4kDa when assessed by gel filtration.
FAO-Q2
1) No detectable activity on fructosyl lysine but activity on fructosyl valine;
2) the optimum pH for the enzymatic reaction is 7;
3) the optimal temperature for enzyme stability is about 30-40 ℃; and
4) the molecular weight was approximately 49kDa when assessed by SDS-PAGE and approximately 58kDa when assessed by gel filtration.
Common characteristics of these two classes of enzymes are described below.
1. General Induction Properties
They are inducible enzymes that can be induced by FVHL and in a medium containing FVHL as sole carbon and nitrogen source.
2. Reaction specificity and substrate specificity
The enzyme partially purified from the culture of strain GL2-1 provided an active fraction Q1 and a fraction Q2, which were retained for different times in Resource Q column chromatography, as described in example 2 (1). Each fraction containing the enzyme is referred to herein as "FAO-Q1" and "FAO-Q2", respectively. As described above, FAO-Q1 had almost the same activity against FV and FZL and FVL. On the other hand, FAO-Q2 was active against N-terminally valine-glycated FV and FVH, FVHL, but was inactive against FVL.
pH and temperature conditions
Determination of optimum pH
According to the above method for measuring activity, the enzyme reaction was carried out under various pH conditions at a pH of 3.5 to 10.0.
The buffer solutions used were 100mM acetate buffer solution having a pH range of 3.5 to 6.0, 100mM potassium phosphate buffer solution having a pH range of 6.0 to 8.0, 100mM Tris-HCl buffer solution (pH range of 7.0 to 9.0), and 100mM glycine-sodium hydroxide buffer solution having a pH range of 9.0 to 10.0. As shown in FIGS. 2 and 3, it was revealed that the optimum pH of the FAO-Q1 of the present invention was about 7.5 at 30 ℃ and that the optimum pH of the FAO-Q2 of the present invention was about 7.0 at 30 ℃.
Enzyme-stable optimum temperature determination
The temperature condition of the enzyme was determined by incubating FAO-Q1 or FAO-Q210 minutes in 0.1M Tris-HCl buffer (pH8.0) at a temperature of between 30 and 65 ℃ and measuring the enzyme activity under usual conditions. The measurement results are shown in FIGS. 4 and 5. These figures show that the optimum temperature for enzyme stabilization is between 30 ℃ and 40 ℃.
4. Potency assessment
Titration of the enzyme may be carried out by methods known in the art (e.g.kinetic methods), for example as described in example 1 (3). In this method, hydrogen peroxide is produced by the reaction of FAO with a glycated amino acid or peptide, and the measurement of hydrogen peroxide is based on the absorbance (505nm) of the quinone dye produced in the presence of hydrogen peroxide. Calculation of the amount of hydrogen peroxide produced per minute (. mu.mol) as the molar absorbance of the quinone pigment (5.6X 10)3M-1cm-1) On the basis of the enzyme activity units (U) and the values thus obtained.
The method for measuring the activity is not limited to the above method, and the enzyme activity of the FAO of the present invention may be measured by other methods including an end-point method, a method based on measuring oxygen absorption, and the like.
Determination of the Michaelis constant
In the above-mentioned process of measuring titer,the Michaelis constant of each substrate can be measured by measuring the initial reaction rate while keeping the conditions concerning the enzyme concentration, pH, temperature, etc. constant and changing the substrate concentration alone.
Among FAOs of the present invention, FAO-Q1 showed almost the same activity against FV and FZL, and thus can be widely used for analysis of Amadol compounds. On the other hand, FAO-Q2 showed activity against FV but not against FZL, and thus could be used for selective analysis of glycohemoglobin. Furthermore, FAO-Q2 is active against the N-terminal sequences of glycohemoglobin (FVH and FVHL). Therefore, only the N-terminal glycosylation of the hemoglobin molecule can be measured without measuring the internal glycosylation (. epsilon. -position), thereby enabling a more accurate measurement of HbA1 c.
When the amadori compound such as a glycated protein is measured using the FAO of the present invention, a sample containing the amadori compound is contacted with the FAO of the present invention, and the amount of consumed oxygen or the amount of generated hydrogen peroxide can be measured according to a known method. Any sample may be used, and examples include those derived from living bodies such as blood (e.g., whole blood, plasma or serum), urine, and foods such as soy sauce and the like. Blood is a particularly preferred sample.
When the FAO of the present invention is used, appropriate reaction conditions such as pH and temperature are selected for various enzymes. That is, when FAO-Q1 is used, the reaction is carried out at a pH of about 6.5 to 12, preferably about 7 to 8, more preferably about 7.5, and a temperature in the range of 30 ℃ to 40 ℃.
When FAO-Q2 is used, the reaction may be carried out at a pH in the range of about 6 to 10, preferably about 6.5 to 8, more preferably about 7; the temperature is in the range of 30-40 ℃. However, the conditions may be changed depending on the substrate or other reaction conditions, etc., and are not limited thereto.
The amount of FAO used in the analysis can be appropriately selected depending on the method employed in the measurement; however, it is usually 0.1 unit/ml or more, preferably 1 to 100 units/ml. As the buffer, Tris-HCl and the like can be used.
When a glycated protein is assayed using the FAO of the present invention, it is preferable that the protein is cleaved in advance so that it releases amino acid or peptide residues. The methods include chemical and enzymatic methods known in the art. However, since the FAO of the present invention, particularly FAO-Q2, is active not only on glycated amino acids but also on glycated peptides, which are degradation products of glycated proteins, the measurement can be performed with high accuracy even if the cleavage treatment is not perfect.
Accordingly, the present invention also provides a method for determining amadori compounds in a sample using the above FAO (FAO-Q1 or FAO-Q2).
The FAO used in the assay method of the present invention can be prepared by culturing Fusarium multiforme (FERM BP-8451), producing the FAO in a nutrient medium, and isolating and purifying the produced FAO of the present invention from the medium. The FAO thus obtained, i.e., naturally occurring FAO, may have naturally occurring modifications and mutations as long as it meets the object of the present invention. In addition, contaminants originating from the separation and purification steps may accompany the enzyme in addition to the enzyme, as long as the accuracy and reliability of the assay are not affected.
The FAO of the present invention can also be prepared according to recombinant DNA techniques. That is, a recombinant protein corresponding to FAO-Q1 orFAO-Q2 can be produced by a conventional method using a nucleic acid sequence encoding SEQ ID NO: 4 or 6.
Accordingly, the present invention provides a polypeptide comprising SEQ ID NO: 4 or 6, or a pharmaceutically acceptable salt thereof.
As used herein, the term "FAO (including FAO-Q1 and FAO-Q2)" refers to an enzyme isolated from a naturally occurring microorganism and an enzyme isolated from a recombinantly obtained microorganism, unless otherwise specifically indicated.
The present invention also provides a DNA encoding the FAO of the present invention.
The DNA of the invention preferably encodes a polypeptide comprising SEQ ID NO: 4 or 6, more preferably, a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 5.
Methods for producing recombinant proteins by recombinant DNA techniques are known in the art. For example, recombinant proteins with the desired activity are prepared as follows: the FAO of the present invention is isolated and purified from the culture by introducing the DNA of the present invention into an appropriate host, culturing the transformant thus obtained. It is easily understood by those of ordinary skill in the art that the recombinant FAOs of the present invention obtained by this method are not limited to those having the amino acid sequences shown in SEQ ID nos. 4 and 6, but rather comprise proteins having amino acid sequences derived from the sequences according to conventional methods and fragments of the amino acid sequences shown in SEQ ID nos. 4 and 6, as long as they conform to the above definition.
The preparation of recombinant FAO can be carried out according to known methods. For example, expression vectors that allow expression of FAO in various hosts can be constructed by inserting DNA encoding FAO downstream of a promoter of an appropriate expression vector. The expression vector can then be used to transform a suitable host cell. Examples of host cells include microorganisms [ prokaryotes (bacteria such as Escherichia coli and Bacillus subtilis) and eukaryotes (such as yeast), animal cells or cultured plant cells. Suitable host-vector systems are known for each host and can be expressed using the host cells by methods described in the literature (e.g., molecular cloning: A laboratory Manual, Cold spring harbor laboratory Press) or by known methods.
Transformation of a host cell with an expression vector can also be carried out by methods described in the literature (e.g., molecular cloning, supra) or by methods known in the art.
The culture of the obtained transformant may be carried out in an appropriate medium selected from known media or freshly prepared media. The medium usually contains a carbon source (e.g., glucose, methanol, galactose, fructose, etc.), an inorganic or organic nitrogen source (e.g., ammonium sulfate, ammonium chloride, sodium nitrate, peptone, casein hydrolysate, etc.). Other nutrients such as inorganic salts (e.g., sodium chloride, potassium chloride), vitamins (e.g., vitamin B1) and antibiotics (e.g., ampicillin, tetracycline, kanamycin) may optionally be added to the medium. For mammalian cells, Eagle's medium is preferred.
The transformant is usually cultured at pH6.0 to 8.0, preferably pH7.0, at a temperature of 25 to 40 ℃ and preferably 30 to 37 ℃ for 8 to 48 hours. When the produced FAO appears in the culture solution or its filtrate (supernatant), the cultured medium is separated by filtration or centrifugation. The FAO can be purified from the filtrate/supernatant by conventional methods commonly used for the isolation and purification of naturally occurring or synthetic proteins, including dialysis, gel filtration, affinity column chromatography using anti-FAO monoclonal antibodies, column chromatography with a suitable adsorbent, high performance liquid chromatography, and the like. When the obtained FAO is present in the periplasm or cytoplasm of the cultured transformant, the cells are harvested by filtration or centrifugation, and the cell walls and/or cell membranes are disrupted by sonication and/or lysozyme treatment to obtain cell debris. The fragments are then dissolved in a suitable aqueous solution such as Tris-HCl buffer. FAO can be purified from solution according to the methods described previously. If an enzymatically active fragment is desired, the FAO may be obtained by treating it with an enzyme such as a restriction enzyme or an exonuclease. Thus, FAOs can be efficiently produced by recombinant techniques using appropriate host cells.
The following examples further illustrate the invention.
Example 1: screening and identification of FAO-producing microorganisms
(1) Screening of FAO-producing microorganisms
Glycation of VHL can produce fructosyl valine-histidine-leucine (vhvl) with the same sequence as the N-terminus of the glycohemoglobin β chain.
The FVHL-assimilating microorganism can be isolated from soil using a medium containing FVHL as a sole carbon source and nitrogen source (FVHL medium). The collected soil was put into a test tube (diameter: 16.5 mm) containing 5ml of FVHL medium, and cultured at 30 ℃ for 48 hours (300rpm) with shaking.
| FVHL medium | |
| FVHL Dipotassium hydrogen phosphate Sodium dihydrogen phosphate Magnesium sulfate heptahydrate Calcium chloride dihydrate Vitamin mixture* Metal solution** Distilled water | 5g 1g 1g 0.5g 0.1g 0.1％(v/v) 1.0％(v/v) q.s |
| Total volume | 1000ml |
*Vitamin mixture
| Thiamine hydrochloride Riboflavin | 1mg 2 |
| Calcium pantothenate
Vitamin B6HCl
Biotin
Para-aminobenzoic acid
Nicotinic acid
Folic | 2 2 0.1 1 2 0.1 q.s. |
| Total volume | 100ml |
**Metal solution
| Manganese sulfate trihydrate Zinc sulfate heptahydrate Copper sulfate pentahydrate Cobalt chloride dihydrate Sodium molybdate dihydrate Boric acid Potassium iodide Distilled water | 1.7g 2.2 0.4 0.28 0.26 0.4 0.06 q.s. |
| Total volume | 1000ml |
As a result, thirteen strains capable of assimilating FVHL were obtained, and then cultured to evaluate the activity in the following manner to select microbial strains having FAO activity.
(2) Culturing and preparing cell-free extract
Each of the 13 strains obtained in the above (1) was cultured in a glucose-valine (GV) browning medium, and a crude extract solution was prepared therefrom.
| GV browning medium | |
| Glucose L-valine Dipotassium hydrogen phosphate Sodium dihydrogen phosphate Magnesium sulfate heptahydrate | 1.5％(w/v) 0.5 0.1 0.1 0.05 |
| Calcium chloride dihydrate Yeast extract | 0.01 0.2 |
The cells were incubated in a test tube (16.5 mm diameter) containing 5ml of GV browning medium at 30 ℃ for 24 hours. Filtering the culture solution with a filter to obtain cell-free extract, and subjecting the mycelium to Mini-BeadBeaterTM(0.5mm glass beads) were broken and the mixture was centrifuged (4 ℃, 10,000Xg, 10 min), which was then used as a crude enzyme solution.
(3) FAO Activity assay
The FAO activity of the crude enzyme solution can be determined by the rate method described previously. The time course of hydrogen peroxide generation in the reaction mixture below can be determined by colorimetry and FAO activity evaluated.
| Tris-HCl buffer (pH8.0) 4-aminoantipyrine Phenol and its preparation FV Peroxidase enzymes Crude extract (cell-free extract) | 100μmol 4.5μmol 6μmol 5μmol 6 units 1ml |
| Total volume | 3ml |
The mixture except the enzyme solution was equilibrated at 30 deg.C (3 ml in total). After the addition of the enzyme solution, the time course of absorbance at 505nm was measured. The molar absorbance of the generated benzoquinone dye (5.6X 10)3M-1cm-1) The amount of hydrogen peroxide produced per minute (. mu.mol) was calculated on the basis thereof, and the obtained value was taken as the unit of enzyme activity (U). As a result, a strain having FAO was obtained.
(4) Identification of strains
Fungal Properties
The microorganisms were inoculated on Potato Dextrose Agar (PDA), Oat Agar (OA), or 2% malt agar (MEA) plates, and incubated at 25 ℃ for 8 weeks while observing the characteristics of the fungus. The color of the colonies is described in accordance with the teaching of Komerup&Wanscher (1978).
Visual characterization of colonies
The colony edges were smooth, slightly convex upwards.
Aerial hyphae are villous and the color of the colony surface is white to reddish from white (11A 1-2). After eight weeks, no significant change in the degree of color development or surface color was observed due to conidia insertion.
The colony back color almost coincides with the front color; the colonies were observed to be pale grayish redon PDA or MEA after a long incubation period (11A 3). A small clear exudate generation was observed on the PDA or OA plates.
Characteristic observation of colony microscopic
Mediasporium and microconidia were observed.
The microconidia is of the type conidiogenous, with a conidiophore structure resembling Acremonium (Acremonium). Conidia were almost straight and occasionally split into two branches, and aerial hyphae were observed throughout. The tip is composed of one or two cells, and the adhesive forms a block structure. The shape varies from elliptical to spindle-shaped, and the surface varies from smooth to slightly rough.
The macrocarris spore is morphologically similar to Fusarium and consists of 3 to 6 cells, a moon shape. The surface is smooth with sufficient cells. Many short aerial hyphae were observed from thick to thin in the middle. The cell wall is weak and the surface of most large conidia is incomplete.
In view of the above results, the microorganisms were classified in the genus Fusarium on the basis of the classification schemes described by Arx (1974), Domish (1993) and Malloch (1981). There are also similar morphological genera such as Cylindrocarpon, Candelaberella, Monacrosporium, Trichophoron, etc. The microorganisms which may be present according to the invention differ from these genera, for example in that the macroconidium is moon-shaped, that the aerial hyphae do not form a ring and that a microcirconium is present, in accordance with the definition of Fusarium as described in "genes of the class Hyphomycetes" (Carmichael et al, 1980).
The strain has been preserved as "Fusarium GL2-1 strain" by International patent organism depositary, national high-grade Industrial science and technology research and hence the registration number FERM BP-8451.
Species identification (analysis of ribosomal base sequence)
The GL2-1 strain was identified by detecting the 18S ribosomal DNA (18SrDNA) sequence.
GL2-1 strain was cultured in GV medium according to the method described in (2) above, and DNA was prepared from the resulting mycelia by a conventional method. Using the obtained DNA as a template, the internal transcribed spacer sequence of rDNA was then amplified by PCR, and the base sequence was determined (Mycopathologia Vol.140P 35-491997). As a result, SEQ ID NO: 1, or a nucleotide sequence shown in the following table. The base sequence homology search indicated 100% homology with Fusarium multiforme.
Example 2: preparation and characterization of FAO Using GL2-1
(1) Partial purification of FAO
1) Culturing and preparing cell-free extract
The GL-2 strain identified in example 1 was cultured in 100ml of GV browning medium as described in example 1(2) under the same medium composition and culture conditions.
After culturing, the mycelium was collected by filtering the culture medium through a filter. The resulting mycelia (0.6g) were suspended in 0.1M Tris-HCl buffer (pH8.0) containing 1mM DTT, and applied to a Mini-BeadBeaterTM(0.5mm glass beads) and centrifuged (4 ℃, 10,000Xg, 10 minutes) to obtain the supernatant as a cell-free extract.
2) Ammonium sulfate fractionation
The cell-free extract obtained from 1) was dissolved in 50mM Tris-HCl buffer containing 1mM DTT and dialyzed against the same buffer for ammonium sulfate fractionation (30-80% saturation).
3) Resource Q column chromatography
The ammonium sulfatefraction after dialysis was subjected to chromatography under the following conditions.
Conditions of analysis
Column (volume): resource Q column (1ml) (Amersham Biosciences K.K.)
Flow rate: 1ml/min
And (3) buffer solution A: 50mM Tris-HCl buffer (pH8.0) +1mM DTT
And (3) buffer solution B: buffer A +1M sodium chloride
Elution conditions
0-5 minutes: 0% buffer B
5-35 minutes: 0-5% buffer B
35-40 minutes: 50-100% buffer B
The elution pattern and activity of Resource Q column chromatography protein (OD. RTM. 280nm) are shown in FIG. 1. When FAO activity was examined using FV as a substrate, two fractions (Q1 and Q2) were found to be active. The activity assay was carried out analogously to the method described in example 1 (3). FAO containing these fractions are referred to herein as "FAO-Q1" and "FAO-Q2".
Table 1: activity change according to purification step
| Step (ii) of | Total unit (U) | Specific activity (U/mg) | Yield (%) | |
| Cell-free extract Passing through 30-80% ammonium sulfate After fractional dialysis Resource Q | Q1 | 0.5 0.3 0.22 | 0.019 0.0199 0.3 | 100 60 44 |
| Q2 | 0.03 | 0.067 | 6 |
(2) Comparison of substrate specificity of FAO-Q1 and FAO-Q2
The substrate specificity of the enzyme (FAO-Q1, FAO-Q2) contained in the two fractions isolated by (1) and (3) above was determined. FAO activity was measured according to the method described in example 1(3) using the two fractions as enzyme solutions, respectively. FV, FVH, FVHL, FVL and FVL as substrates. The results are shown in Table 2.
Table 2: substrate specificity of FAO-Q1 and FAO-Q2
| Relative Activity (%) | ||
| FAO-Q1 | FAO-Q2 | |
| FV | 100 | 100 |
| FVH | n.d. | 2.4 |
| FVHL | n.d. | 0.6 |
| FVL | 1.1 | 0.6 |
| FVLS | n.d. | 3.3 |
| FZL | 108 | n.d. |
FV is fructosyl valine, FVH is fructosyl valine-histidine, FVHL is fructosyl valine-histidine-leucine, FVL is fructosyl valine-leucine-serine, FVL is fructosyl N- α -lysine n.d.
Table 2 clearly shows that FAO-Q1 has almost the same activity on FV and FZL, and also on FVL. Whereas FAO-Q2 is active against FV but not against FZL, and is active against FVH and FVHL in which N-terminal valine is glycated.
(3) Determination of Km value
The activity was measured to determine the Km value (Michaelis constant) of FAO-Q1 or FAO-Q2 against FV or FZL according to the method described in example 1(3) using FV or FZL as a substrate. The results are shown in Table 3.
Table 3: km values of FAO-Q1 and FAO-Q2 against FV or FZL
| FAO-Q1 | FAO-Q2 | |
| FV | 0.62 | 0.64 |
| FZL | 0.56 | n.d. |
n.d.: not detected.
The Km values of FAO-Q1 and FAO-Q2 against FV were equivalent. Furthermore, the Km value for FZL of FAO-Q1 was smaller than that for FV, indicating that the enzyme had stronger affinity for FV.
1) SDS electrophoresis
Molecular weight was determined by SDS electrophoresis using a gradient gel (gel concentration: 10-15 w/v%), when molecular weight standards (phosphatase b: 97kDa, bovine serum albumin: 68kDa, ovalbumin: 45kDa, carbonic anhydrase: 32kDa, trypsin inhibitor: 20.1kDa and α -lactalbumin: 14.4 kDa; Amersham Biosciences K.K.) were used as standard proteins of known molecular weight, the molecular weight of FAO-Q1 was approximately 39kDa, and the molecular weight of FAO-Q2 was approximately 49 kDa.
2) Gel filtration
The molecular weight was determined by a conventional method using Superdex 200 (column size: 1X 30 cm; Amersham biosciences K.K.) gelfiltration. The molecular weight of the enzyme of the present invention was calculated using a standard curve obtained from molecular weight standards (aldolase: 150kDa, bovine serum albumin: 68kDa, ovalbumin: 45kDa, chymotrypsinogen A: 25kDa, cytochrome C: 12.5 kDa; Roche Diagnostics K.K.). The results are shown in FIG. 2, indicating that the molecular weight of FAO-Q1 is about 39.4kDa, whereas the molecular weight of FAO-Q2 is about 58 kDa.
(5) Partial amino acid sequence analysis
To determine the N-terminal amino acid sequence, the purified enzyme FAO-Q2 was dialyzed against distilled water, and 40ng of the same enzyme was used as a sequencing sample for the N-terminal amino acid. The N-terminal 10 residues were analyzed using a 476A protein sequencer. The N-terminal sequence of FAO-Q2 shows a sequence similar to SEQ ID NO: 2 are identical in amino acid sequence. Furthermore, the sequence of FAO-Q1 was not determined by this method, since its N-terminus was blocked.
Example 3: cloning of FAO cDNA
Genomic DNA of GL2-1 was prepared. The cDNA of FAO can be obtained by PCR using genome DNA as a template.
(1) Genomic DNA preparation of GL2-1 Strain
Genomic DNA was prepared from GL2-1 strain according to a method comprising the following steps.
GL2-1 strain was cultured in 15ml of DP medium (1% Dextone, 1% peptone, 0.5% sodium chloride, pH7.4) for 2-3 days at 30 ℃ in liquid.
2. Fungal cells (wet weight, 0.3g) were collected by filtration through a glass filter (3 GL).
3. The obtained fungal cells were homogenized in a mortar containing liquid nitrogen, which was equipped with a pestle, further crushed by a motor or the like, and then collected into a Corning tube.
4. After addition of 2ml ice-cold TE buffer (10mM Tris-HCl (pH8.0), 1mM EDTA), the mixture was vortexed gently.
5. After adding 2ml of 50mM EDTA and 0.5% SDS solution, the mixture was stirred by rotation several times and then incubated at 37 ℃ for 30 minutes.
6. The mixture was centrifuged (3000rpm, 10 min).
7. The supernatant was treated with phenol-chloroform (3 times) while stirring with rotation.
8. 2.5 volumes of ethanol were added and the mixture was stirred several times with rotation, whereupon filamentous DNA appeared.
9. The mixture was briefly centrifuged (3000rpm, 5 minutes) to precipitate filamentous DNA. When the DNA is not filamentous, the mixture is centrifuged according to usual ethanol precipitation.
10. The pellet was dissolved in 400. mu.l of TE buffer (10mM Tris-HCl (pH8.0), 1mM EDTA; hereinafter, "TE buffer" means the same), transferred to an Eppendorf tube, added with 5. mu.l of RNase (10mg/ml), and incubated at 37 ℃ for 30 minutes.
11. After two phenol-chloroform treatments, 2.5 volumes of ethanol were added to the tube, followed by thorough stirring with rotation.
12. The filamentous DNA was transferred to a new tube with a toothpick (excess ethanol removed).
13. The DNA was dissolved in 50-100. mu.l TE buffer (gently pipetted without vortexing).
14. The DNA was quantitatively determined. DNA (1. mu.g) was run on an agarose gel to confirm bands.
(2) cDNA was prepared by PCR.
1) Preparation of partial sequences (about 200bp fragment)
The regions of high homology were searched usingthe known full amino acid sequence derived from the filamentous fungal FAOD. Based on the information obtained, the following primers were designed.
Primer and method for producing the same
A forward primer: 5 '-GGBTTYTTTCWTSGARCCNRAYGA-3'
SEQ ID NO：7
Reverse primer: 5 '-GTRCVGYRYMCCAGCAVAT-3'
SEQ ID NO：8
PCR was carried out using the above genomic DNA as a template in a standard composition reaction solution using Taq polymerase (TaKaRa Ex Taq, TAKARA BIO INC.).
PCR conditions
Primer (SEQ ID NO: 7) 0.2. mu.M
Primer (SEQ ID NO: 8) 0.2. mu.M
10 μ l of ExTaq PCR buffer (TAKARA BIO INC.)
Magnesium chloride 2.5mM
Taq polymerase (TAKARA BIO INC.) 2.5U
D.d.w (di-deionized water) was added to make the total volume 100 μ l.
One cycle at 94 ℃ for 1 minute, 35 cycles (94 ℃ for 1 minute, 50 ℃ for 1 minute, 72 ℃ for 1 minute), and one cycle at 72 ℃ for 3 minutes.
After completion of PCR, 10. mu.l of the reaction solution was subjected to agarose gel electrophoresis, and the band observed at 200bp was considered as a target fragment. The band was excised, treated with TOPOTA cloning kit (Invitrogen) according to the kit instructions, and transformed into E.coli JM 109. Optionally selecting 20 transformants, and extracting plasmids. Each plasmid was treated with restriction enzymes, and plasmids containing DNA of appropriate size were selected and sequenced. Sequencing was performed using the BigDye Terminator cycle sequencing kit and sequencer ABIPRISM3100 gene analyzer.
As a result, two base sequences (polynucleotides) were obtained, possibly corresponding to two isoenzymes (FAO-Q1 and FAO-Q2). From the putative amino acid sequence deduced from the thus determined base sequence, the amino acid sequence of the purified enzyme was confirmed. It was revealed that the above PCR-amplified DNA fragments contained a part of the genes encoding FAO-Q1 and FAO-Q2, respectively.
2) Preparation of upstream or downstream partial sequences and Total DNA
Using TaKaRa LA PCR in vitro cloning kit, the DNA sequences of the upstream and downstream regions were determined from the two 200bp fragments obtained in 1) above. The base sequences of the obtained FAO-Q1 and FAO-Q2 are respectively shown as SEQ ID NO: 3 and SEQ ID NO: 5, respectively. The deduced encoded amino acid sequences are as set forth in SEQ ID NOs: 4 and SEQ ID NO: and 6.
INDUSTRIAL APPLICABILITY
The present invention provides novel FAOs that are expected to contribute to the development of assays for amadori compounds. In particular, in the case of conventional FAOs, the activity of the enzyme to glycated peptides is equivalent to that of glycated proteins, so that glycated proteins can be measured more accurately even if the cleavage of glycated proteins is not perfect. As a result, HbA1c, which is important for controlling blood glucose level of a diabetic patient, can be accurately measured, and thus, it is useful for treating diabetes and preventing complications of the diabetic patient. Further, the DNA encoding the novel fructosyl amine oxidase of the present invention is expected to be effective in mass production of the enzyme by gene recombination techniques, thereby promoting the development of the assay for Amadol compounds.
Sequence listing
<110>Aikolai Kabushiki Kaisha
<120>fructosyl amine oxidase
<130>663979
<150>JP 2002-277214
<151>2002-09-24
<150>JP 2002-309734
<151>2002-10-24
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<170>PatentIn version 3.1
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<223>peptide designed based on peptide produced by Fusarium multiforme
<400>8
Gly Thr Arg Cys Val Gly Tyr Arg Tyr Met Cys Cys Ala Gly Cys Ala
1 5 10 15
Val Ala Thr
Claims (11)
1. A fructosyl amine oxidase derived from Fusarium multiforme.
2. Fructosyl amine oxidase from Fusarium multiformis having the following physicochemical properties:
(1) the activity on fructosyl valine is almost the same or higher compared to fructosyl lysine;
(2) the optimum pH for the enzymatic reaction was 7.5;
(3) the optimal temperature for enzyme stability is about 30-40 ℃; and
(4) the molecular weight was approximately 39kDa when assessed by SDS-PAGE and approximately 39.4kDa when assessed by gel filtration.
3. The fructosyl amine oxidase of claim 2, comprising SEQ ID NO: 4.
4. Fructosyl amine oxidase from Fusarium multiformis having the following physicochemical properties:
(1) no detectable activity on fructosyl lysine but activity on fructosyl valine;
(2) the optimum pH for the enzymatic reaction is 7;
(3) the optimal temperature for enzyme stability is about 30-40 ℃; and
(4) the molecular weight was approximately 49kDa when assessed by SDS-PAGE and approximately 58kDa when assessed by gel filtration.
5. The fructosyl amine oxidase of claim 4, comprising SEQ ID NO: 6.
6. A Fusarium proliferatum (FERM BP-8451) characterized in that it produces the fructosyl amine oxidase of any one of claims 1 to 5.
7. ADNA encoding the fructosyl amine oxidase of any one of claims 1 to 5.
8. The DNA of claim 7, comprising the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5.
9. A host cell transformed with the DNA of claim 7 or 8.
10. A method of preparing fructosyl amine oxidase, the method comprising: culturing the microorganism of claim 6 or the host cell of claim 9 in a culture medium, and recovering the fructosyl amine oxidase from the culture.
11. A method for measuring amadori compounds in a sample, characterized by the fructosyl amine oxidase of any one of claims 1 to 5.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002277214 | 2002-09-24 | ||
| JP277214/2002 | 2002-09-24 | ||
| JP309734/2002 | 2002-10-24 |
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| CN1701118A true CN1701118A (en) | 2005-11-23 |
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| CN 03825364 Pending CN1701118A (en) | 2002-09-24 | 2003-09-16 | Fructosylamine oxidase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080308A (en) * | 2010-08-06 | 2013-05-01 | 龟甲万株式会社 | Amadoriase having altered substrate specificity |
| CN111398605A (en) * | 2013-08-09 | 2020-07-10 | 龟甲万株式会社 | Composition for measuring glycated hemoglobin |
-
2003
- 2003-09-16 CN CN 03825364 patent/CN1701118A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103080308A (en) * | 2010-08-06 | 2013-05-01 | 龟甲万株式会社 | Amadoriase having altered substrate specificity |
| CN111398605A (en) * | 2013-08-09 | 2020-07-10 | 龟甲万株式会社 | Composition for measuring glycated hemoglobin |
| CN111398605B (en) * | 2013-08-09 | 2024-04-05 | 龟甲万株式会社 | Glycosylated hemoglobin measurement composition |
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