JPH0146117B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for quantifying L-phenylalanine using a novel enzyme, L-phenylalanine oxidase. L-phenylalanine is one of the essential amino acids and is used in foods, medicines such as amino acid infusions, and is also used in the diagnosis of L-phenylalanine metabolic disorders such as phenylketonosis. have important meaning. but,
There is no known method for easily and highly sensitively quantifying L-phenylalanine using an enzyme. Conventionally, oxidases that act on L-phenylalanine include L-amino acid oxidase and L-amino acid oxidase.
-Phenylalanine hydroxylase is known. For example, L-amino acid oxidase has the following formula for L-phenylalanine. It has the action expressed by various microorganisms,
Its presence has been reported in snake venom, rat kidney, etc. [Journal of Bacteriology (J.
Bacteriol.) Vol.121, No.2, P.656 (1975), Journal of Biological Chemistry (J.Biol.Chem.) Vol.235, P.2013 (1960)]. However, L-phenylalanine has a high substrate specificity.
Amino acid oxidase is unknown and could not be used for quantifying L-phenylalanine. In addition, L-phenylalanine hydroxylase has the following formula: It has the following effects, and its presence has been reported in rat liver, microorganisms, etc. [Journal of Biological Chemistry, Vol. 226,
P. 551 (1957)], although this enzyme has high substrate specificity for L-phenylalanine, it is coupled to other reactions via a coenzyme (tetrahydrobiopterin) and requires a complicated reaction system. In view of the current situation, the present inventors have conducted extensive research on enzymes that specifically act on L-phenylalanine, and have found that bacteria belonging to the genus Pseudomonas have extremely high specificity for L-phenylalanine. As a result of repeated research to apply this enzyme to the quantitative determination of L-phenylalanine, we discovered that the enzyme produces an enzyme with a high level of L-phenylalanine oxidase activity that has never been seen before. In the presence of oxygen, the amount of oxygen consumed or hydrogen peroxide produced in the reaction of oxidative decarboxylation and oxidative deamination by acting on L-phenylalanine in the sample,
The present inventors have discovered that L-phenylalanine can be specifically quantified simply and with high sensitivity by quantifying carbon dioxide gas, ammonia, phenylacetamide, or phenylpyruvic acid, leading to the completion of the present invention. The present invention will be explained in detail below. First, the physicochemical properties of the novel L-phenylalanine oxidase used in the present invention will be described below. Action and substrate specificity Reaction formula for oxidative decarboxylation of L-phenylalanine in the presence of oxygen to produce phenylacetamide, carbon dioxide gas and water [] The reaction represented by and the reaction formula for oxidatively deaminating L-phenylalanine in the presence of oxygen to produce phenylpyruvic acid, ammonia and hydrogen peroxide [] It has the effect of catalyzing the reaction represented by It exhibits extremely high substrate specificity for L-phenylalanine and does not act on L-lysine, L-proline, L-glutamic acid, and L-threonine. Optimal PH and Stable PH The enzymatic activity (oxygen consumption) of the novel L-phenylalanine oxidase against L-phenylalanine was measured using acetate buffer, phosphate buffer, and borate buffer as buffer solutions. As a result, as shown in FIG. 1, the optimum pH ranges from 5 to 10, and as shown in FIG. 2, the stable pH ranges from 6.5 to 10. Measurement of titer Method-1 Oxygen consumption method Place 0.27 ml of 1M potassium phosphate buffer (PH6.8) and 0.1 ml of enzyme solution in a closed reaction container and dilute the total volume with water.
The volume should be 2.6ml. Insert an oxygen electrode (manufactured by Beckman) and keep the temperature inside the reaction vessel constant at 37°C while stirring.
0.1 ml of 27mML-phenylalanine kept at 37°C in advance is added to start the reaction, and the amount of oxygen consumed is measured over time using an oxygen analyzer (manufactured by Beckman). Enzyme activity is 1 μ per minute
One unit is the activity that consumes a mole of oxygen. Method-2 Hydrogen peroxide method 1M potassium phosphate buffer (PH6.8) 0.3ml,
10mML - Phenylalanine 0.3ml, 4-aminoantipyrine (0.005%), phenol or N,
Add N-dimethylaniline (0.02%), peroxidase (4 units) and enzyme solution to bring the total volume to 3 ml.
After reacting at 37°C for 10 minutes, measure the visible absorption (550 nm) of the dye produced, and calculate the amount of hydrogen peroxide produced from the standard curve. Method-3 Determination of phenylacetamide and phenylpyruvate Add 0.8ml of 0.1M potassium phosphate buffer (PH6.8)
After adding 0.1 ml of 10 mM L-phenylalanine, 0.1 ml of enzyme solution was added, and the mixture was reacted at 37°C for 10 minutes.
Take an appropriate amount of this reaction solution and perform pre-adjusted high-performance liquid chromatography (column: LS410, column size 4 mm ID x 250 mm L, mobile phase: methanol: 0.1M potassium phosphate buffer = 10:90,
After separation at a temperature of 60°C and a detector: UV 220 nm), the height of each peak was converted from the peak height of the standard product, and the amount of phenylacetamide and phenylpyruvic acid were quantified. Suitable temperature range for action The amount of phenylacetamide and phenylpyruvic acid produced was measured by high performance liquid chromatography and was found to be 20 to 60°C as shown in Figure 3. Inactivation conditions due to pH, temperature, etc. As shown in Figure 4, it is stable when heat treated at 65℃ for 10 minutes, and when the enzyme activity (oxygen consumption method) is measured after being left overnight at 4℃ at pH 4 to 9, the pH is 6. It is stable between 5 and 9. Deactivated at pH 4 or lower Inhibition, activation and stabilization When each heavy metal ion (1.8mM) or inhibitor (1mM) was added and enzyme activity (oxygen consumption method) was measured, barium ion, cadmium ion, as shown in Table 1. , was strongly inhibited by copper ions. The relative activities in Table 1 are expressed as the ratio of the activity when metal ions or inhibitors are added to the activity when no metal ions or inhibitors are added.

[Table] Molecular weight The molecular weight of this enzyme was approximately 67,000 as measured by the gel filtration method using high performance liquid chromatography (G3000sw x 1, Toyo Soda Kogyo Co., Ltd.).
In the gel filtration method using Sephadex G-200,
It is 140000. From these results, it is considered that this enzyme is composed of two identical subunits with the same molecular weight. Homogeneity Acrylamide gel with 7.5% pore size (PH
As a result of performing acrylamide disc electrophoresis in a conventional manner using 9.4), a single band was observed as shown in FIG. Isoelectric point: 4.83 as measured by disk gel focusing electrophoresis. Amino acid analysis values: aspartic acid 55, threonine 28, serine 34,
Glutamic acid 41, glycine 49, alanine 72, 1/2
- cystine 3-4, valine 47, methionine 6, isoleucine 24, leucine 50, tyrosine 30, phenylalanine 21, lysine 17, histidine 13, arginine 30, proline 34, tryptophan 14. For example, when enylalanine oxidase is used to act on L-phenylalanine under the reaction conditions of 0.1M potassium phosphate buffer (PH6.8), reaction temperature 37°C, and 10 minutes, L-phenyl For every mole of alanine, 1 mole of oxygen is required, 0.85 mole of phenylacetamide, 0.85 mole of carbon dioxide according to the reaction formula [], and 0.15 mole of phenylpyruvic acid, 0.15 mole according to the reaction formula [] of ammonia, producing 0.15 moles of hydrogen peroxide. However, for substrates other than L-phenylalanine, the proportions of acidic decarboxylation and oxidative deamination in the presence of oxygen are different, and the proportion of oxidative deamination to the total reaction is shown in Table 2. As shown in column A. Under the above conditions, the substrate specificity of the enzyme of the present invention was determined by the relative activity (%) of each substrate for L-phenylalanine.
As shown in Table 2, this enzyme has substrate specificity completely different from that of conventional L-amino acid oxidases, and exhibits extremely high L-phenylalanine oxidase activity. It is different from L-amino acid oxidase, and is recognized as a new enzyme.

【table】

[Table] Next, specific means for producing the novel L-phenylalanine oxidase will be described below. First, the production raw material may be of any origin, but for example, it is preferable to use a strain of microbial origin, especially a strain belonging to the genus Pseudomonas, which has the ability to produce a novel L-phenylalanine oxidase. It is particularly advantageous in manufacturing. Specific examples of strains belonging to the genus Pseudomonas include Pseudomonas sp.P-501,
Variants or mutant strains of this bacterium can also be used. The above-mentioned Pseudomonas sp.P-501 is a strain newly isolated by the present inventor from soil, and its mycological properties are as shown below. The mycological properties were generally determined according to the methods described in the Manual of Microbiological Methods (published by McGraw-Hill Book Company, 1959). (a) Morphology Microscopic observation (cultivated on broth agar medium at 30℃ for 16 hours) Cell shape and size: 0.5-1.0 x 1.5-
Pleomorphism of 3.0 micron rod cells: Not observed. Motility: Has polar hairs and is motile. Presence or absence of spores: Not formed. Gram staining: negative. Anti-acidity: negative. (b) Growth status in each culture medium Juice agar plate culture At 30°C for 24 hours, colonies are yellow-green, with a slightly rough surface, dull luster, and opaque. It has a yellow-green diffusible pigment. Juicy agar slant culture Growth is good and colonies are yellow-green in color. Meat juice liquid culture Grows well and uniformly, with turbidity. Meat juice gelatin puncture culture Grows slightly and liquefies in 4 days at 30℃. Litmus milk: slightly alkaline. (c) Physiological properties Nitrate reduction: Positive. Denitrification reaction: Negative. MR test: Negative. VP test: negative. Indole production: negative. Hydrogen sulfide formation: Negative. Hydrolysis of starch: Negative Use of citric acid: Use of Christensen's medium. Utilization of inorganic nitrogen sources: Positive. Pigment production: Produces a yellow-green water-soluble pigment. Urease: Negative. Oxidase: Positive. Catalase: Positive. Growth range: Optimum pH 5-8, optimal temperature 25-8
30℃. Attitude towards oxygen: aerobic. O-F test: Oxidizing. Production of acids and gases from sugars

【table】

[Table] (d) Properties Can grow using DL-arginine as the sole carbon source. Comparing the taxonomic properties of this bacterium with the above-mentioned novel L-phenylalanine oxidase-producing ability with the classification in the 8th edition (1974) of the ``Birgies Manual of Determinative Bacteriology,'' The strain is negative in Gram staining, is an aerobic nonspore bacillus, has flagella, is motile, and is positive for catalase and oxidase, etc.
It belongs to the genus Pseudomonas and is considered to be closely related to Pseudomonas marginata and Pseudomonas cepacia, but its isolation source and physiological properties such as mesacon membrane, D-tartaric acid, rhamnose, tryptamine and other carbon sources are not available. There are differences. For the above reasons, this bacterium was used as Pseudomonas sp.P.
It was named −501. In addition, Pseudomonas sp.P-501 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 5887. Next, the production of L-phenylalanine oxidase using the above novel bacteria capable of producing L-phenylalanine oxidase will be described. In the present invention, the medium used for culturing the above-mentioned novel bacteria capable of producing L-phenylalanine oxidase includes a medium used for culturing microorganisms belonging to the genus Pseudomonas. As a nitrogen source for the medium, any available nitrogen compound or one containing it may be used.
For example, yeast extract, peptone, meat extract, soybean,
One or more organic or inorganic nitrogen sources such as amino acids, ammonium sulfate, potassium nitrate, urea, etc. are used. Examples of carbon sources include glucose, galactose,
Examples include carbohydrates such as xylose and organic acids such as levulinic acid. As inorganic salts, potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, sodium chloride, etc. were used as appropriate, and various organic substances, inorganic substances, vitamins, etc. necessary for bacterial growth or enzyme production were added as necessary. is suitably used as a medium. The bacteria may be cultured by solid culture, but it is usually preferable to use a liquid culture method, and the bacteria are cultured aerobically by shaking culture, stirring culture, aeration culture, or the like. Industrially, it is preferable to use a liquid medium and perform submerged culture with aeration and agitation. The culture temperature is 10-40°C, preferably around 25-35°C. The pH during culturing is preferably 5 to 8. The culture time varies depending on the culture format, but is 14 to 72 hours. Extraction of the enzyme from the culture thus obtained,
For purification, general enzyme extraction and purification methods can be used. For example, after separating bacterial cells from a culture by an appropriate method, the bacterial cells are ground in the presence of a trifle, a method using a hydrolytic enzyme such as lysozyme, a method using ultrasonic energy, and an osmotic shock. or by shaking or standing in the presence of a surfactant such as Triton X-100, toluene, etc.
Alternatively, after the enzyme is excreted from the bacterial cells by autolysis or the like, the solution is treated with an appropriate operation such as filtration or centrifugation to remove solids and obtain a bacterial cell extract; Extract with water, buffer, or a suitable solvent to obtain the crude enzyme solution as it is.
In addition, the crude enzyme powder can be obtained by subjecting the extract to a suitable method such as freeze-drying, salting out with ammonium sulfate, or organic solvent precipitation using alcohols or acetones. You can also get it. In order to obtain a further purified enzyme preparation from the above-mentioned crude enzyme solution or crude enzyme powder, for example, gel filtration method using Cephadex or biogel, adsorption elution method using ion exchanger, adsorption elution method using hydroxyapatite, sucrose density The method of the present invention can be achieved by appropriately selecting and combining a sedimentation method such as gradient centrifugation, an affinity chromatography method using phenylcephalose 4B, a fractionation method using a molecular sieve membrane or a hollow fiber membrane, etc. Purified enzyme preparations can be obtained that can be used in Furthermore, the present enzyme can be obtained by similarly treating the culture solution obtained when autolysis is performed by increasing the culture time. In the method of the present invention, the preparation form and degree of purification of the novel L-phenylalanine oxidase are
- It is appropriately selected depending on the type of oxygen absorption or detection of the product produced by the decomposition reaction of phenylalanine by the enzyme. In other words, L-phenylalanine does not contain any substances that significantly interfere with the decomposition reaction of L-phenylalanine and/or the detection system for the product.
It can be used even if contaminants are present in the phenylalanine oxidase enzyme preparation. Depending on the quantitative method, the enzyme may be prepared as a soluble enzyme or as an adsorbed or insolubilized enzyme on an appropriate carrier. The enzymatic reaction conditions for L-phenylalanine oxidase can be quantified in the range of PH4.5 to 11, preferably in the range of PH6 to 10, in the case of the enzyme produced by a strain belonging to the genus Pseudomonas. The reaction time is usually about 10 to 20 minutes, and the temperature conditions are 70℃ or less.
Preferably it is 30-65°C. Furthermore, in order to maintain the optimum pH for the enzyme reaction, it is preferable to use various buffer solutions as the enzyme reaction medium. The buffer may be any buffer as long as it can maintain the above-mentioned pH range and does not inhibit the enzymatic reaction; for example, phosphate buffer, Tris-HCl buffer, acetate buffer, borate buffer, etc. can be used. The amount of the new L-phenylalanine oxidase to be used is sufficient for the enzymatic reaction, and is usually
It is about 0.1 to 0.5 units. Quantification of L-phenylalanine by the method of the present invention detects the amount of oxygen consumed in the enzymatic reaction or the amount of reaction product hydrogen peroxide, carbon dioxide, ammonia, phenylacetamide, or phenylpyruvic acid produced. , and the specific method for detection is not particularly limited. Detection of oxygen consumption or reaction products,
Typical measurement methods will be exemplified below. The oxygen consumption can be measured according to a conventional method, such as the Warburg pressure method or the oxygen electrode method. As an application of the oxygen electrode method, there is also a method using an enzyme electrode in which L-phenylalanine oxidase is closely attached to the tip of the oxygen electrode. When using an enzyme electrode, the L-phenylalanine concentration can be determined by placing the electrode in a sample solution containing L-phenylalanine and measuring the oxygen consumption rate. Methods for bringing L-phenylalanine oxidase into close contact with the tip of the oxygen electrode include encapsulating a soluble enzyme with a semipermeable membrane, or encapsulating an enzyme immobilized chemically or physically in a suitable carrier. There are methods. Examples of immobilizing enzymes on carriers include enclosing them in acrylamyl gel, immobilizing them in organic polymer powder, etc.
Various enzyme immobilization methods can be applied. Hydrogen peroxide can be measured using a hydrogen peroxide electrode or colorimetrically using a system consisting of one or more coloring agents that react with hydrogen peroxide to change its color tone. can be mentioned. In addition, examples of this coloring agent include, for example, the reaction between xylenol orange and a tetravalent titanium compound that reacts with the generated hydrogen peroxide to form a stable red color, or phenol, N,N-dimethylaniline, 4-aminoantipyrine, and Examples include a method using a reaction using peroxidase. The coloring agent may be adjusted together with the enzyme, if necessary, and then the resulting color tone may be measured at an appropriate wavelength, and hydrogen peroxide may be quantified from the calibration curve. Ammonia can be measured using a method using an ammonium ion electrode, a colorimetric method using the indophenol method, or a method in which the decrease in β-NADH is quantified by absorption at 340 nm by coupling it with a glutamate dehydrogenase reaction. Carbon dioxide can be easily measured using a Warburg manometer, but in the case of this enzymatic reaction, oxygen absorption also occurs at the same time, so correction must be made. Alternatively, a method of measuring the radioactivity of carbon dioxide released in an enzymatic reaction using a substrate labeled with ¹⁴ C can also be used. It is also possible to use a carbon dioxide electrode. As a method for quantifying phenylacetamide or phenylpyruvic acid, it may be separated by high-performance liquid chromatography and then detected and quantified by ultraviolet absorption, or for phenylpyruvic acid, it may be determined by a general method of quantifying keto acids. For example, a method for quantifying hydrazone produced using 2,4-dinitrophenylhydrazine can be used. As described above, the method for quantifying L-phenylalanine using the new L-phenylalanine oxidase is based on the amount of oxygen consumed by the enzyme reaction or the amount of hydrogen peroxide, carbon dioxide, ammonia, phenylacetamide, or phenylacetamide produced. By measuring enylpyruvic acid, for example, quantitative measurement of L-phenylalanine in serum, quantitative measurement of L-phenylalanine in pharmaceuticals using L-phenylalanine, fermentation of L-phenylalanine. It is useful for various analyses, such as the quantitative measurement of L-phenylalanine and the activity of enzymes using L-phenylalanine as a substrate. Hereinafter, an example of implementation of the method of the present invention will be specifically explained. However, these are merely examples and do not limit the method of the present invention in any way. The novel L-phenylalanine oxidase used in each example was prepared from Pseudomonas sp. . Example 1 Add 0.27 ml of 1M potassium phosphate buffer (PH6.8) and 0.1 ml of new L-phenylalanine oxidase solution (5 units/ml) to a closed reaction vessel for a total volume of 2.6 mL of water.
ml. Insert an oxygen electrode (manufactured by Beckman) and keep the temperature inside the reaction vessel constant at 37°C while stirring. Next, standard L-phenylalanine solution (0 to 5 μmol/ml) or each sample solution kept at 37°C in advance.
Add 0.1ml, start the reaction, continue the reaction until the oxygen consumption becomes constant (within 10 minutes), and measure the oxygen consumption with an oxygen analyzer (manufactured by Beckman).
Measured at A calibration curve was created and the L-phenylalanine content in the sample was determined. The calibration curve is shown in FIG. The samples were L-phenylalanine when using a natural medium prepared from casamino acids, yeast extract, and meat extract, and a medium to which L-phenylalanine was added as shown in Table 3 based on the natural medium. The quantitative values are shown in Table 3.

[Table] Example 2 1M potassium phosphate buffer (PH6.8) 0.3ml, 4
- 0.005% aminoantipyrine, 0.02% N,N-dimethylaniline, 5 units of peroxidase, and a novel L-phenylalanine oxidase solution (5
Unit/ml) Add 0.1ml to make the total volume 2.9ml. Add standard L-phenylalanine solution (0-10 μmol/ml) and 0.1 ml of each sample solution, and incubate at 37°C.
After reacting for 10 minutes, the visible absorption (550 nm) of the dye produced was measured. A calibration curve was created and the L-phenylalanine content in the sample was determined. The calibration curve is shown in FIG. The sample was prepared in the same manner as in Example 1, and the results were reported in the fourth example.
Shown in the table.

[Table] Example 3 0.2 ml of L-phenylalanine-containing sample prepared in the same manner as Example 1, 1M potassium phosphate buffer (PH
6.8) 0.1ml, L-phenylalanine oxidase
Dilute 0.5 units to 1 ml with distilled water and react at 37°C for 10 minutes. To 1 ml of the reaction solution, add 0.3 ml of 1M potassium phosphate buffer and 0.03M α-ketoglutaric acid (PH7.0).
0.2ml, 2.25mMβ-NADH0.2ml, 15mMADP0.1
ml, glutamate dehydrogenase (10mg/ml)
0.05 ml was added to bring the total volume to 3 ml, and after reaction at 37°C for 20 minutes, ammonia was measured from the decrease in absorption at 340 nm. From the calibration curve prepared in advance, L-
The content of phenylalanine was measured. The calibration curve is shown in FIG. 8, and the quantitative values are shown in Table 5.

[Table] Example 4 Two Warburg manometers were prepared, and one was distilled with 0.1 ml of 1M acetate buffer (PH5.8) and 0.5 units of novel L-phenylalanine oxidase in the main chamber of the Warburg vessel. The volume was made up to 2.5 ml with water, and 0.5 ml of the L-phenylalanine-containing sample prepared in the same manner as in Example 1 was placed in the side chamber. The other is prepared in the same manner as above, except that 0.2 ml of caustic potassium solution (10%) is placed in the antechamber. After temperature equilibration for 20 min at 30 °C,
The reaction is started by transferring the L-phenylalanine-containing sample placed in the side chamber to the main chamber. After 20 to 30 minutes, measure the readings of both manometers, calculate the amount of carbon dioxide generated by Warburg's direct method, and calculate the amount of carbon dioxide generated using the known L-phenylalanine calibration curve.
-The content of phenylalanine was determined. The calibration curve is shown in FIG. 9, and the quantitative values are shown in Table 6.

[Table] Example 5 0.1ml of L-phenylalanine-containing sample prepared in the same manner as Example 1, 1M potassium phosphate buffer (PH
6.8) Dilute 0.1 ml of the novel L-phenylalanine oxidase (0.5 units) to 1 ml with distilled water, and conduct the reaction at 37°C for 10 minutes. High performance liquid chromatography (column:
LS410, Toyo Soda Kogyo Co., Ltd. Column size 4mm ID×
250mmL, Mobile phase: methanol:potassium phosphate buffer = 10:90, temperature 60℃, microwave =
Separate with 0.04), detect with UV220mm, convert each peak height from the peak height of the standard product, and quantify the amount of phenylacetamide and phenylpyruvic acid. From the calibration curve obtained from known L-phenylalanine, L-
The content of phenylalanine was determined. The calibration curve for phenylacetamide is shown in FIG. 10, the calibration curve for phenylpyruvic acid is shown in FIG. 11, and the quantitative values are shown in Table 7.

[Table] The L-phenylalanine content measured by amino acid analysis in the natural medium used in Examples 1 to 5 was 1.1 micromol/ml, and the value measured by the method of the present invention approximates this value. I got the value. Reference examples KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.1%, MgSO ₄ .
_7H2O0.05 %, _FeSO4・_7H2O0.001 %, yeast extract
Pour 50 ml of a medium containing 0.2% and 1% L-phenylalanine into each 500 ml shake flask,
After sterilization at 120℃ for 10 minutes, Pseudomonas sp.P-501
(Feikoken Bacterial Serial No. 5887) was inoculated and incubated at 30℃ for 16 hours.
Shaking culture was carried out at an amplitude of 5 cm and 120 rpm. After the completion of culture, strain the culture solution 10%, suspend the resulting bacterial cells in 2000 ml of 0.01M phosphate buffer (PH7.0), add 0.5% Triton X-100, lyse, and centrifuge. The solid content is removed to obtain a crude enzyme solution. Add ammonium sulfate to the crude enzyme solution, collect the 30% saturated to 50% saturated precipitate, and dissolve it in 50 ml of 0.01M phosphate buffer (PH7.0).
Dialyze overnight against the same buffer. Next, a DEAE cellulose column (5.2 x 80
cm), elute the impurities with the same buffer containing 0.1M NaCl, and then elute with the same buffer containing 0.2M NaCl.
The active fraction was collected, and after concentration, gel filtration was performed using a Biogel A-0.5m column (2.5 x 150 cm) and the active fraction was collected. The precipitated fraction of the solution with 60% ammonium sulfate saturation was collected, dissolved in 10 ml of 0.01M phosphate buffer (PH7.0) containing 10% ammonium sulfate, and mixed with phenylcephalose CL-4B (manufactured by Pharmacia, Sweden). ) through a column (2.8 x 10 cm), and after washing the column with 0.01M phosphate buffer (PH7.0) containing 10% ammonium sulfate, 0.01M containing 1% ammonium sulfate and 50% ethylene glycol.
Elute the enzyme with phosphate buffer, collect the active fraction, and apply it again to the Biogel A-0.5m column (2.5 x 150
2.1 mg of purified enzyme preparation (yield 11.6%, specific activity 94.2 units/mg protein) was obtained by collecting the active fraction.

[Brief explanation of drawings]

Figure 1 is a diagram showing the optimal pH of the novel L-phenylalanine oxidase used in the method of the present invention, and Figure 2 is a diagram showing the residual activity after treatment at each pH for 20 hours at 4°C. It is. FIG. 3 is a diagram showing the action temperature of the novel L-phenylalanine oxidase determined by high performance liquid chromatography, and FIG. 4 is a diagram showing the thermostability at pH 6.8. FIG. 5 shows an electrophoretic diagram of the novel L-phenylalanine oxidase.
FIG. 6 to FIG. 11 are calibration curves prepared in an example of the method of the present invention.

Claims (1)

[Claims] 1 L-phenylalanine in a sample is decomposed using L-phenylalanine oxidase in the presence of oxygen, and the amount of oxygen consumed in the reaction solution or hydrogen peroxide and carbon dioxide gas produced , ammonia, phenylacetamide, or phenylpyruvic acid. 2 L-phenylalanine oxidase according to claim 1, wherein the L-phenylalanine oxidase has the following physicochemical properties.
-Method for quantifying phenylalanine. (a) Action: Reaction formula for oxidative decarboxylation of L-phenylalanine in the presence of oxygen to produce phenylacetamide, carbon dioxide gas and water [] The reaction represented by and the reaction formula for oxidatively deaminating L-phenylalanine in the presence of oxygen to produce phenylpyruvic acid, ammonia and hydrogen peroxide [] It has the effect of catalyzing the reaction represented by (b) Substrate specificity: Shows extremely high substrate specificity for L-phenylalanine, and does not act on L-lysine, L-proline, L-glutamic acid, and L-threonine. (c) Optimal PH and stable PH range: Optimal PH: 5-10 Stable PH range: 6.5-10 (d) Molecular weight: The value measured by gel filtration method using high performance liquid chromatography is about 67,000.