DK158681B - EQUIPMENT FOR PYRODRUIC ACID ANALYSIS. - Google Patents

Description

in

DK 158681 B

The invention relates to a pyruvic acid analysis equipment.

The equipment is peculiar to the characterizing part of claim 1.

5 Pyruvate oxidase is a known enzyme that catalyzes a reaction of pyruvic acid, phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide and derived from a strain of Lactobacillus delbruckii. This known pyruvate oxidase enzyme has an optimum pH of about 5.5. It has been found that the enzyme pyruvate oxidase is formed in a bacterial strain B-0667 belonging to the genus Pediococcus and E-0668 belonging to the genus Streptococcus isolated from a soil sample taken in a radish field in Ohito-cho, Tagata-gun,

Shizuoka-kcn, Japan. The pyruvate oxidase prepared therefrom has a higher optimum pH of 6.5-8 relative to the aforementioned corresponding Lactobacillus enzyme and it can be used for pyruvic acid analysis in a sample containing pyruvic acid or various systems which release pyruvic acid. It has also been found that this enzyme can be used for quantitative analysis of pyruvic acid, measurement of the enzyme activity of enzyme reaction systems which form pyruvate, quantitative determination of the enzyme and the substrate thereof. Pyruvic acid can be assayed by reacting a sample containing pyruvic acid with a reaction system comprising at least 25 pyruvate oxidase, flavinadenine dinucleotide (hereinafter referred to as pAD), thiamine pyrophosphate, oxygen and phosphate, and an excellent pyruvate anode analysis and equipment is provided. .

Furthermore, it has been found that the addition of a salt which releases calcium ion, cobaltion, magnesium ion or manion ion to the reaction system results in an improvement of the assay. Addition of chromogenic or fluorescent indicator to the reaction system provides a convenient and excellent analytical equipment and a corresponding method.

According to the invention, the equipment may be peculiar to the characterizing part of claim 2.

DK 158681 B

2

The prior art according to "Methods in Enzymology, Volume 1, page 482 (1955)", "Enzymes, 2nd Edition, page 684 (1964)" and Chem. Abstracts, volumes 43, 720i and volumes 57 / 17176f mention pyruvate oxidase from Lactobacillus delbriickii, Pedio-5 coccus and Streptococcus faecalis. The pyruvate oxidase of the invention is characterized by having a pH optimum of 6.5-8. Therefore, in comparison with don kendto pyruvatoxi-daso, it is better suited for use in only 1 ysosysphores against other enzymes having optimum arrows in this area, as enzymatic reaction reactions are known to be preferable in the optimum for a zymet. pH range. For example, if with a device according to the invention will analyze glutamate-oxalacetate-transferase (GOT) or glutamate-pyruvate-transaminase (GPT3 activity in sorum samples), the optimal pif value for these two enzymes is 7.4. using the known pyruvate oxidase with optimum pH 5.5, the reaction will not proceed as smoothly as using the higher pH optimum pyruvate oxidase.

The invention will be explained in more detail in connection with the drawing, in which fig. Fig. 1 shows the optimum pH of pyruvate oxidase; Fig. 1A shows the optimum pH of the pyruvate oxidase according to the invention compared to the pyruvate oxidase of Lactobacillus delbriickii; 2 shows the heat stability of pyruvate oxidase; FIG. Figure 3 shows the pH stability of pyruvate oxidase; 4 shows the result of the analysis of pyruvic acid with oxygen electrode using pyruvate oxidase; FIG. Figure 5 shows the result of analysis of serum with oxygen electrolyte using pyruvate oxidase; 6 shows the result of analysis of pyruvic acid by coulometric method using pyruvate oxidase, 22 fig. 7 the result of quantitative analysis of serum pyruvic acid using pyruvate oxidase; 3

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FIG. 8 the result. nf analysis of ADP using pyruvate oxidase, fig. 9 the result of analysis of glycerol, triglyceride and serum triglyceride using 5 pyruvate oxidase, fig.10 the result of analysis of GPT and GOT activity using pyruvate oxidase, fig.11 and correlation diagram for analysis of GPT activity using pyruvate oxidase, 10 and Fig.12 a correlation diagram for analysis of GOT activity using pyruvate oxidase. The enzyme pyruvate oxidase catalyzes an oxidative reaction of pyruvic acid, inorganic phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide and is produced by the cultivation of pyruvate oxidase-producing microbial species selected from Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO 12219 or IFO 12317.

The isolated strain B-0667 and B-0668 above have the following taxonomic properties.

A. Observations on various media grown at 30 ° C for 2 days:

Strain B-0667 Strain B-0668

Tryptosoja fluid weak growth, homogeneous weak weight, homogeneous hazy, later woolly hazy, later woolen precipitation tig precipitation 30

Tryptosoja agar - weak growth, pale yellow - weak growth, pale yellow oblique gray, no luster, no pale gray, no luster, production of soluble no production of pigment soluble pigment 35 Tryptosoja agar colony, small and flat colony, small and flat plate 4

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gelatin plate growth along cut line, growth along cut no fluidization of line, no flowable-gelatin making gelatin BCP milk no change no change (14 days) 5 B. Microscopic observation:

Strain B-0667 Strain B-0668

Shape spherical, ovoid, pair spherical, ovoid, pair tetraform or short tetraform or short chain chain size 10: 0.5 - 1.0 x 0.5 - 1.0 µL> m 0.8 - 1.0 x 1.0 - 1 , 2 pm motility - - trace: gram staining + 4 acid resistant - - 15 color C. Physiological properties:

Strain B-0667 Strain B-0668 growth temperature

45 ° C

20 37 C + + 30 ° C + + 26 ° C + + 10 ° C + + 5 ° C + or (+) + or (+) 25 - halogen tolerance

NaCl 10% + 6.5% + ~ 5.0% + + 1.0% + + 30 0% + OF sample for intrac ivative behavior in facultative anae- facultative anae oxygen: robustly robust; rntrodukt; in on 25 indole formation 5

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hydrogen sulphate formation: gelatin hydrolysis starch hydrolysis esculin hydrolysis: + + 5 accto formation MK sample: catalase: oxidase urease (SSR): 10 urcase (Christensen): utilization of citric acid (Christensen):

Sugar formation from sugar: adonitol 25 l (- l ·) -arabinose cell obi ose + + dulcitol - - meso-erythritol fructose + + 20 fueose galactose + + glucose + + glycerol inositol 25 in now 1 in η lactose + + maltose + + mannitol + mannose + + 20 melizitose melibiosis + raffinose + L (+) - rhamnose salicin (+) 25 L-sorbose

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Table continued 6

Sugar formation from sugar: sorbitol - - starch - - sucrose + + trehalose + + 5 xylose - tolerance at 60 ° C for + 30 minutes

In consulting Bergey's Manual of Determinative Bacteriology, 8th edition 1974, and S.T. Cowan and K.J. Steel, Manual for the Identification of Medical Bacteria, Cambridge Press, 1974, refers to strain B-0667 and B-0668 with the above taxonomic properties, especially gram positive cocci, catalase and oxidase negative, glucose fermentative acid formation and no sugar gas formation ( glucose), as belonging to the genus Pediococcus and the genus Streptococcus.

Comparison of these strains with the above identification references is as follows: In the table: + = positive more than 85% - = negative more than 85% 20 d = varies among strains or species.

strain strain strain genus B-0667 B-0668 Pediococcus Streptococcus growth at 45 ° C + d 25 tolerance at 60 ° C for 30 min. + d glycerol (acid formation) ·· - d arabinose (acid formation) - - + d 30 halogen tolerance (NaCl 10%) + - + Accordingly, strain B-0667 should be designated as the genus Pediococcus or Streptococcus. When consulting the aforementioned Manual for the Identification of Medical Bacteria and J.Gen.

Microbiol., 26, 185-197 (1961). The taxonomic properties 35 of strain B-0667 are almost identical to those of Pediococcus urina-equi, but the properties described in Bergey's 7

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Manual of Determinative Bacteriology, 8th edition, 1974 is a bit different from there. Therefore, strain B-0667 is referred to as Pediococcus genus and is termed Pediococcus sp. B-0667th

The strain B-0668 is similar to the genus Streptococcus rather than 5 to the genus Pediococcus. Further consultation of the Manual for the Identification of Medical Bacteria results in strain B-0668 similar to Streptococcus fnocium var. durans, but no taxonomic properties are described in Bergey's Manual, so comparison in detail is impossible. The strain B-0668 10 is therefore referred to as Streptococcus sp. B-0668th

Strains B-0667 and B-0668 are deposited in the Institute of Microbial Industry and Technology, Agency of Industrial Science and Technology, M.I.T.I., Japan, under the respective numbers FERM-P No. 4438 and Ferm-P No. 4439.

In one embodiment of the present invention, the above Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO-12219 or Aerococcus virians IFO-12317 in a conventional medium for enzyme production.

Cultivation can be done by conventional liquid cultivation, and sub-mother aeration culture is preferred for industrial production. Preferably, a conventional medium for microorganisms can be used. As carbon sources can be used assimilable carbon sources such as glucose, sucrose, lactose, maltose, fructose, molasses and pyruvic acid. Assimilable nitrogen sources such as peptone, meat extract, gas extract and casein hydrolyzate can be used. Various inorganic salts such as phosphate, carbonate and sulfate of magnesium, calcium, potassium, iron, manganese or zinc can be used.

The culture temperature may be selected in the range of culture of microbial cells and production of pyruvate oxidase, for steps of 25-37 ° C. The culture time may change depending on the conditions and terminate when pyruvate oxidase production is virtually complete, usually 18-48 hours.

Pyruvate oxidase exists in the cells of microorganisms.

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In order to separate pyruvate oxidase from the cultured cells, the cultured mass is filtered or centrifuged to collect cells, and it is digested by treatment with mechanical agents or an enzymatic process such as a lysozyme. If necessary, pyruvate oxidase is solubilized by the addition of ethylenediaminetetraacetic acid (EDTA) and a surfactant such as Triton X-100 (registered trademark) or Adecatol SO-120 (registered trademark) to separate the enzyme. The thus obtained solution 10 of pyruvate oxidase is treated with or without evaporation and then the enzyme is precipitated by salting out by the addition of a soluble salt such as ammonium sulphate or sodium chloride. Low molecular impurities are removed by dialysis. Furthermore, impurities in the solution of pyruvate oxidase are preferably removed by adsorption chromatography, ion exchange chromatography or gel filtration. The benzyl solution thus obtained is treated by vacuum evaporation and lyophilization to produce powdery pyruvate oxidase. Further purification can be achieved by conventional purification methods for proteins and enzymes such as adsorption chromatography, ion exchange chromatography or gel filtration.

Pyruvate oxidase produced by the process of the invention has the following physicochemical properties, using the following abbreviation: 25 Pediococcus sp B-0667 is abbreviated B-0667

Streptococcus sp. B-0668 is abbreviated B-0668

Aerococcus viridans IFO- -12219 is abbreviated IFO-12219

Aerococcus viridans IFO-12317 is abbreviated IFO-12317 (1) Enzyme action:

The enzyme catalyzes oxidative reaction of pyruvic acid, inorganic phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide: ch3cocooh + hopo "~ + o2 _ ^ ch3coopo" + co2 + h2o2 9

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(2) Optimum pH:

The effect of the pH on the activity of pyruvate oxy-dase is measured. Phosphate buffer solutions of pH 6-8 are used for the study. The results are shown in Fig. 5, where the optimum pH is as follows: B-0667: pH 6.3-7.5 B-0668: pH 7.5-8.5 IFO-12219: pH 7.0-8.0 1FO-12317: pH 6.8-7.5 10 Small variations are observed by phosphate concentration and species of metal ion.

(3) Heat stability:

The heat stability of the enzyme is determined by incubation in 10 mM phosphate buffer (pH 6.5) containing 10 µM 15 FAD at 0, 40, 50, 60 and 70 ° C for 10 minutes following the method of enzyme determination. As seen in FIG. 2, the enzymes obtained from B-0667, IFO-12219 and IFO-12317 are slightly activated at 40 ° C and denatured above 60 ° C. The enzyme obtained from B-0668 is not activated at 40 ° C and is denatured almost above 20 60 ° C.

(4) pfl stability:

To each enzyme solution (0.1 ml), add 0.2 M phosphate buffer for pH 6-8 (0.9 ml) or 0.2 M of Tris-HCl buffer for pH 7-9 (0.9) and each containing 10 µM FAD followed by standing for 10 minutes at 40 ° C. 20 filters of enzyme solution are taken and the enzyme activity determined. As can be seen in Table 3, the enzyme obtained from B-0667, IFO-12219 and IFO-12317 is most stable at ca. pH 7, and the enzyme obtained from B-0668 is stable at an acidic pH.

30 (5) Effect of various substances: 1) The effect of various substances on the enzyme activity is investigated by adding 5 mM of the substances listed below instead of MgCl2 in the assay system.

35 10

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Relative Activity (%) __

Additive _ B-0667 B-0668 IFO-12219 IFO-12317 no addition 25.4 72.0 42.0 50.3 5 EDTA 0 0 0 0

MgCl2 100 100 100 100

CaCl₂ "69.4 75.0 83.4 78.7

MnCl2 129.1 102.7 116.2 111.0

CoCl2 81.3 81.1 85.0 84.0 10 BnCl2 20.6 58.8 23.9 28.8

ZnCl 2 16.0 38.2 14.9 22.8

As seen above, the enzyme is inhibited by EDTA and active. ++ _ ++. ++ ++ veres of Mg, Ca, Mn and c o 2) The effect of elimination of the following substances from

The analysis system on enzyme activity is shown below. 0.1 M

dimethylglutarate-NaOH buffer is used in the case of phosphate elimination.

Eliminated substance _ Relative activity (%) _ B-0667 B-0668 IFO-12219 IFO-12317 20 No elimination 100 100 100 100

Thi nminpyrophosphate 0 0 0 0 FAD 33.9 100 32.7 41.7

Thiamine Pyropliosphnt and KAI) 0 0 0 0 Phosphate 0000 25 It appears that the enzyme requires thiamine pyrophosphate and FAD as cofactor and phosphate as substrate.

The oxygen consumption of the enzyme reaction is measured with an oxygen electrode and the oxygen is consumed in relation to an enzyme activity (hydrogen peroxide formation). The results 30 are as follows:

Oxygen Reaction Product (pmol / min.

(pmol / min.) H2 ° 2 acetyl phosphate B-0667 0.042 0.042 0.038 B-0668 0.022 0.0213 0.020 IFO-12219 0.040 0.038 0.037 IFO-12317 0.035 0.036 0.034 35 11

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The analyzes are performed as follows:

Oxygen consumption: Dissolved oxygen meter (Trade name: YST dissolved oxygen meter model 53).

Acethyl phosphate: F. Lipmann et al., J. Biol. Chem., 5, 134, 463-464 (1940).

Hydrogen Peroxide: Method using N, N-dimethylaniline, 4-aminoantipyrine and horseradish oxidase.

As explained above, the enzyme obtained from the four above strains is referred to as pyruvate oxidase and flavin protein. The method of analysis for pyruvate oxidase according to the invention is as follows: 0.5 M potassium pyruvate 0.1 ml 0.5 M phosphate buffer (pH 7.0) 0.2 ml 0.2% 4-aminoantipyrine 0.1 ml 0.2% N N-dimethylaniline 0.2 ml 0.2 M MgCl 2 50 pl 10 mM thiamine pyrophosphate 20 yl peroxidase (45 units / ml) 0.1 ml 1 mM FAD 10 jiliter 20 distilled water 0.22 ml

The above reaction mixture (1.0 ml) is preincubated at 37 ° C for 3 minutes. To this solution is added the enzyme solution (20 µl) and incubated at 37 ° C for 10 minutes. 0.1 M citrate buffer (pH 6.0, 2 ml) containing 0.1 M EDTA is added to quench the reaction. The violet color formed is measured by colorimetric method at 665 nm.

One unit of enzyme activity is defined as the activity which produces 1 pmol of hydrogen peroxide per liter. minute.

To activate the pyruvate oxidase reaction system, FAD, thiamine pyrophosphate and phosphate are added. To activate the enzyme, additional ion-releasing salt is preferably added which releases calcium ion, cobalt ion, magnesium ion or manganese ion in the form of chloride. Preferably, an indicator such as a color indicator or fluorescence indicator for hydrogen peroxide is selected.

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The amount and proportion of components of the enzyme reaction system can be selected for mainly enzyme reaction and will vary according to the amount of pyruvate, temperature and time of the enzyme reaction.

5 For example. For example, preferably 1-20 units of pyruvate oxidase, 0.1-20 nmol FAD and 0.05-0.5 ρηοΐ thiamine pyrophosphate, 1-10 µmol inorganic phosphate and 0.05-10 µmol ion-releasing salt can be used. attempt. Pyruvate oxidase may be in microencapsulated form or immobilized form of covalent bond with organic or inorganic carrier or adsorption with carrier. The molar ratio of indicator of hydrogen peroxide is at least an equimolar ratio or excess of hydrogen peroxide formed. In the case of peroxidase, preferably 0.5-20 units per ml are used. attempt. These 15 components of enzymatic reaction mixture are used stepwise by dissolving in the suitably adjusted arrow value of the buffer.

The enzymatic reaction system thus prepared can be used to analyze pyruvic acid. Any sample containing pyruvate can be analyzed. Eg. pyruvic acid itself, serum or urine pyruvic acid, and pyruvic acid-forming enzyme reaction systems such as lactic acid and lactase dehydrogenase (LDH), ADP and pyruvate kinase and glycerol, glycerol kinase and pyruvate kinase. Further examples in units of enzymatic reactions which form pyruvic acid and which are subject to analysis are the following:

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(1) Analysis of lactic acid or LDH activity: lactate LDH γ pyruvate + NAD1

NAD

(2) Analysis of ADP or pyruvate kinase (PK) activity: 5 ADP + phosphoenol pyruvate _PK_ ^ ATp + pyruvate (3) Analysis of glutamate pyruvate transaminase (GTP) activity or cctoglutarate: alanine + Λ -cotoglutarate -GPT_ ^ pyruvate + 9lutamate (4) Analysis of glutamate oxaloacetate transaminase (GOT) activity: asparaginate + A. -ketoglutarate -G ° T_x, xaioaCetate + glutamate,,. oxaloacetate decarboxylase, oxaloacetate -—- * - γ pyruvate 15 + CO 2 (5)

CK

glycerol. + ATP-glycerol-3-phosphate + ADP

PK

ADP + phosphenolpyruvate - 7pyruvate + ATP

2 (6) Analysis of triglyceride:. n lipase or lipoprotein lipase, ηη triglyceride - Ec Ec γ γ glycerol - -3-phosphate

+ ADP

PK

ADP + phosphoenolpyruvate pyruvate + ATP

(7) Analysis of creatinine or creatinine phosphikinase

(CPK) O

,. . creatinase,.

Creatinjn ---------- - -----> creatin

CPK

creatine + ATP --- ^ creatine phosphate + ADP

ADP + phosphoenolpyruvate -¾ pyruvate + ATP

(8) Analysis of myokinase:

ATP + AMP 2 ADP

PK

ADP + phosphoenol pyruvate> pyruvate + ATP (9) Analysis of fatty acid or thiokinase activity: fatty acid + CoA + ATP t ^ locin - ^ - s ^ acyl-CoA + 35 AMP + PPi AMP + ATP niYokinase ^ 2 ADp 14

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PK

ADP + phosphoenolpyruvate -> pyruvate + ATP

Diaso enzyme reaction is merely illustrative, and these pyruvate-forming reactions can be found in combination of enzyme and its substrate, e.g. a biological sample. As exemplified above, the assay can be performed not only for the analysis of pyruvate, but also for the analysis of enzyme, enzyme activity or substrate.

An assay is performed by incubation with a sample and reagent mixture. Preferably, the poultry mixture is an equipment 10 of the necessary reagents. For analysis, consumed component or component formed is measured. Measuring the amount of oxygen consumption with the aid of dissolved oxygen apparatus is a preferred method of analysis. In this case, no indicator of hydrogen peroxide is required. In the case of assay for produced or formed component, measurement of the amount of hydrogen peroxide, e.g. by means of a hydrogen peroxide electrode meter such as YSI oxidase meter or by colorimetric or fluorometric analysis with indicator of hydrogen peroxide. The assay may preferably be carried out for 10-60 minutes and at 20-40 ° C, preferably 35-37 ° C.

One hydrogen peroxide indicator is a combination of one or more chromogen or fluorescence indicators that are affected by coupling with hydrogen peroxide. Examples of such indicators are combinations of tetravalent titanium compound and xylenol orange coupled with hydrogen peroxide to form a stable red color, or combinations of phenol or N, N-dimethylaniline or homovanilic acid, 4-aminoantipyrine and peroxidase to measure color or fluorescence. 4-Aminophenazone can be replaced by 4-aminophenazone. A combination of 2,6-dichlorophenolindophenol and peroxidase and of guaiaeol and peroxidase can also be used. The indicator can be prepared in advance as a solution. Colorimetric or fluorometric analyzes are performed by measuring the absorption of appropriate wavelengths such as 563 nm.

The amount of pyruvic acid can be measured by calculating from a corresponding standard curve of oxygen consumed or hydrogen peroxide formed.

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Phosphate as a spent component or acetylphosphate as a formed component can also be analyzed by conventional methods.

As explained above, a device for assay, especially 5 diagnostic assay, comprising pyruvate oxidase with optimum pH 6.5-8 and its use for various assay methods is disclosed. Diagnostic analysis such as analysis of pyruvate in pyruvate-containing reagents or in serum or urine, analysis of enzyme activity of LDH, pyruvate kinase, GPT, GOT, glycerol kinase, lipase, lipoprotein lipase, creatinine phosphokinase, myokinase or thiokinase, and analysis of biological components such as lactate , glycerol, triglyceride, creatinine or fatty acids can be advantageously carried out by the equipment according to the invention.

The following examples illustrate embodiments of the invention.

Eksempel_T_

A medium (each 100 ml pH 7) comprising glucose (1%), peptone (1%), yeast extract (0.5%), NaCl (0.2%), KI ) (K2HPO4 (0.1%), MgSO4) (0.05%) and CaCC> 3 (0.3%) in a 500 ml conical flask are sterilized at 120 ° C for 20 minutes.

In each medium, a strain of Pediococcus sp. B-0667, FERM-P No. 4438, Streptococcus sp. B-0668, FERM-P No. 4439, Aerococcus viridans IFO-12218 or Aerococcus viridans IFO-25 12317 and grown under shaking at 30 ° C for 24 hours at 300 rpm. minute. Then, the cultured cells are collected by centrifugation and washed with 10 mM phosphate buffer (pH 6.5) and centrifuged again to assemble the bacterial cells. The cells thus obtained are suspended in 10 mM 30 phosphate buffer (10 ml, pH 7.0) containing 0.02% lysozyme and 0.1% Triton X-100 and incubated at 37 ° for 60 minutes. The obtained centrifuged supernatant containing pyruvate oxidase is collected. The enzyme activity of the supernatant is shown in the following table: 35 Strain Enzyme activity units / ml B-0667 0.60 B-0668 0.38 IFO-12219 0.52 IFO-12317 0.46 16

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Eksempel_2

A medium (20 liters) consisting of the same components as described in Example 1 in a 30 liter fermentation vessel is steam sterilized. Culture liquid (200 ml) of Pediococcus sp.

B-0667 FERM-P No. 4438 grown in the same manner as in Example 1 is transferred thereto and grown at 30 ° C for 24 hours. The bacterial cells collected by centrifugation (about 100 g) are suspended in the lysozyme solution (0.2 mg / ml, 4 liters) and further added Triton X-100 (4 g), EDTA (3 g) and 1 M phosphate buffer 10 (pH (6.5 ml, 40 ml) and stirred at 37 ° C for 60 minutes.

To the supernatant obtained by centrifugation is added ammonium sulfate and the precipitate at 0.54 - 0.73 saturation is collected by centrifugation. The precipitate is dissolved in 10 mM phosphate buffer (pH 6.5, 1000 ml) (5160 units, yield 86%), then cold acetone (0.65 volume) is added and the crude precipitate is separated. Additional acetone (0.3 volume) is added and the precipitate which is collected by centrifugation is dissolved in 10 mM phosphate buffer (pH 6.5, 70 ml) (4750 units, yield 79.2%).

20 To the solution is added ammonium sulfate and the precipitate at 0.54 - 0.70 saturation is collected by centrifugation.

After dissolving the precipitate in 10 mM phosphate buffer (arrow value 6.5), the solution is poured onto a column of Sepha-dex G-25 (registered trademark) (6.0 x 70 cm) and a fraction is collected which shows absorbance at 280 nm. The active fractions are combined and lyophilized to give a powder of pyruvate oxidase (3940 units, 758 mg, yield 65.7%). Example 3

Equipment for pyruvate analysis (for oxygen electrode): 3Q pyruvate oxidase obtained in Example 2 (same as in the following Examples) 300 units FAD 0.5 µmol thiamine pyrophosphate 10 pmol nm Cl ^ 25 µmol 0.2 M phosphate buffer (pH 7.5) 1.0 ml sucrose 0.5 g 0.2 M dimethyl glutarate NaOH buffer (pH 7.5) 5 ml 17

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The above mixture is lyophilized to prepare the equipment for analyzing pyruvic acid (for oxygen electrode measurement, 50 experiments).

Example 4 Equipment for pyruvate analysis (for colorimetric analysis):

Reagent (I) pyruvate oxidase 200 units FAD 0.5 pmol thiamine pyrophosphate 10 pmol 10 0.2 M phosphate buffer (pH 7.5) 1.0 ml sucrose 0.5 g 0.2 M dimethylglutarate NaOH buffer (7.5) 5 ml peroxidase (100 units / mg, horseradish) 2.5 mg 0.3% 4-aminoantipyrine 5 ml The above mixture is lyophilized to form reagent (I) in the pyruvate assay equipment (for colorimetric experiments).

Additional reagent (II) consisting of: 0.2% aqueous Ν, Ν-dimethylaniline containing 25 µmol 20 nm CI 2 (50 ml) and stop reagent (III) consisting of: 0.1 M citrate buffer (pH 6.0, 100 ml) containing 0.1 M EDTA is added.

Example 5 An equipment illustrated in Example 3 is dissolved in still water (50 ml) and a portion of solution (1.0 ml) thereof is placed in a reaction vessel. Add 5.0 mM pyruvate solution (each 0-100 µl) or human serum 50 pliter and 5.0 mM pyruvate solution (each additional 0-100 µl) and incubate at 37 ° C and then measure oxygen for use with galvanic oxygen electrode. The results are shown in FIG. 4th

Further, a device of Example 3 is dissolved in distilled water (25 ml) and a portion of solution (0.5 ml) thereof is placed in reaction vessels and incubated with the addition of aqueous solution (0.5 ml) containing human serum (0-0 , 5 ml) at 37 ° C. In FIG. 5 shows the result of an analysis of oxygen consumption measured with galvanic oxygen electrode.

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As shown in FIG. 4 and 5, good linear relationships are observed.

Example 6

The lyophilized reagent 5 (I) prepared in Example 4 is dissolved by the addition of reagent (II) (50 ml) and each portion of solution (1 ml) in the small test tubes is incubated at 37 ° C. 5 mM potassium pyruvate solution (each 0-50 µl) or human serum (50 µl) is added with 5 mM potassium pyruvate solution (every 0-50 µl) and incubated at 37 ° C for 10 minutes. Stop solution (2 ml) is added and absorption is measured at 565 nm. As shown in FIG. 6, good linear relationships and quantitative results are obtained in the above analyzes, and in this case, too, this result coincides with the calibration curve at ^ 2 ° 2 ^, 5 mM I ^ C ^, 0-50 µl).

An additional sample portion of human serum (0-0.5 ml) is placed in small test tubes and adjusted to 0.5 ml by the addition of distilled water. Each solution (1.0 ml) of reagent (I) prepared by the addition of reagent (II) in Example 4 is added and incubated at 37 ° C for 10 minutes.

The reaction is quenched by the addition of stop reagent (III) (1.5 mL) and colorimetric analyzed at 565 nm. As seen in FIG. 7, a good quantitative result is obtained. Example 7

Reaction mixture: 0.2 M dimethylglutarate-NaOH buffer 0.2 ml 10 mM MnCl 2 50 µl 0.2% N, N-dimethylaniline 0.2 ml 0.3% 4-aminoantipyrine 0.1 ml peroxidase (45 units / ml 10 ml of 10 mM thiamine pyrophosphate 20 ml 0.2 M phosphate buffer (pH 7.5) 25 ml 20 mM phosphoenolpyruvate 0.1 ml pyruvate kinase (4000 units / ml) 5 ul

5 mM ADP 0-50 µU

The above reaction mixture is adjusted to 1.0 ml by the addition of distilled water and preincubated at 37 ° C, then a solution of pyruvate oxidase (200 units / ml, 20 µl) is added and incubated at 37 ° C for 10 minutes. 19

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nutter. After stopping the reaction by adding 0.1 M EDTA in 0.1 M citrate buffer (pH 6.0, 2.0 ml), the absorbance is measured at 565 nm. As seen in FIG. 8, good quantitative results for ADP analysis are obtained, by analyzing the hydrogen peroxide formed by the reaction mixture of ADP, pyruvate kinase and phosphoenol pyruvate.

Also seen in FIG. 8, a good linearity is observed when using 2.5 mM hydrogen peroxide instead of 5 mM ADP.

Example 8

Triglyceride analysis equipment:

Reagent (I): 0.2 M dimethyl glutarate NaOH buffer 10 ml (pH 7.5) 0.3% 4-aminoantipyrine 5 ml peroxidase (100 units / mg) 2.5 mg thiamine pyrophosphate 10 µm 0.2 M phosphate buffer (pH 7.5) 1.25 ml phosphoenolpyruvate 100 µmol 20 pyruvate kinase (4000 units / ml) 0.1 ml pyruvate oxidase (200 units / ml) 2 ml lipoprotein lipase (3000 units / ml) 0.5 ml glycerol phosphokinase (300 units / ml) 1.0 ml ATP 100 µmol The above reagent is lyophilized.

Reagent (II): 25 µmol MnCl 2 in 0.2% dimethylaniline 50 ml

Reagent (III): (Stop Solution) 0.1 M EDTA in 0.1 M Citrate Bucket 100 ml 30 (pH 6.0)

Example 9

The reagent (I) of Example 8 is dissolved in reagent (II) and each portion of solution (1.0 ml) thereof is placed in test tubes.

Each portion of sample (0-50 pliter) of human serum containing 1.02 pnol / ml triglyceride, 5 mM glycerol solution, 4.2 mM triolein in 0.1% Triton X-100 solution or 2.5 mM hydrogen peroxide is added. and incubated at 37 ° C for 10 minutes.

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As shown in FIG. 9, good linearity is obtained.

Example 10

A serum transaminase assay (for 100 trials): 5 (1) a GPT assay (for 100 samples):

Reagent (I): Lyophilized reagent consisting of: pyruvate oxidase 400 units FAD 500 nmol thiamine pyrophosphate 150 µmol L-alanine 20 mmol + - ketoglutarate 1 mmol sucrose 1- g 4-aminoantipyrin 150 ymole peroxidase 450 units 15 M phosphate buffer (pH 7.5) 2.5 ml 0.2 M dimethyl glutarate NaOH buffer 30 ml (pH 7.5)

Reagent (II) (100 ml is used for reagent (D): 42 µmol MnC 2 in 0.2% dimethylaniline solution 210 ml solution

Reagent (III) (stop solution) (200 ml for reagent (I), 0.2 ml for each experiment): 0.1 M EDTA in 0.2 M citrate buffer (pH 5.0) 420 ml (2) for GOT analysis (for 100 trials):

Reagent (I): Lyophilized reagent consisting of: pyruvate oxidase 400 units FAD 500 nmol thiamine pyrophosphate 150 µmol 3ø L-aspartic acid 20 mmol ft-ketoglutarate 1 mmol oxaloacetate decarboxylase 200 units sucrose 1 g 4-aminoantipyrate 150 jamol 35 peroxide (pH 7.5) 2.5 ml 0.2 M dimethylglutarate NaOH buffer (pH 7.5) 30 ml 21

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Reagents (II) and Reagents (III):

Same as above (1).

Example 11

Reagents (I) prepared in Example 1, (1) (for analysis of GPT activity) and (2) (for analysis of GOT activity) are dissolved by the addition of reagent (II) (100 ml). Each portion (1.0 ml) of the solution is placed separately in small test tubes and preincubated at 37 ° C for 5 minutes (preincubated solution of reagent (I)). The standard serum solution (20 liters)

10 (Calbiochem Co., trade name: Maxitol, containing GPT 700 K

units / ml and GOT 1000 K / ml) diluted with constant ratio are added and incubated at 37 ° C for 10 minutes. The reaction is quenched by the addition of stop reagent (III) (2.0 ml) and the absorption is measured at 565 nm.

15 As shown in FIG. 10 has both enzyme activities linear; up to the optical density of approx. 1.0 (enzyme action value: about 750 K units / ml).

Eksempel_1_2

For each preincubated solution of reagent (I) prepared in Example. 1.1 plots of human serum (45 samples) are added and incubated at 37 ° C for 20 minutes. Stop reagent (III) (2.0 ml) is added and adsorption is measured at 565 nm.

The same samples of human serum are also analyzed by the LKB method (ultraviolet absorption method, GOT analysis equipment and GPT analysis equipment manufactured by LKB Corp.) and the correlation is graphically bound to the activity of GPT and GOT.

As seen in FIG. 11, the correlation coefficient r = 0.998 and the regression equation y = 0.00295 x + 0.0032 for the GPT analysis are observed.

In the GOT analysis, the correlation pattern is shown in fig.

12, where the correlation coefficient r = 0.966 and the regression equation y = 0.00287 x + 0.0180 are obtained.

35

Claims (2)

An apparatus for pyruvic acid analysis comprising a reaction system containing pyruvate oxidase which catalyzes a reaction of pyruvic acid, phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide, said reaction system consisting of at least pyruvate oxidase, thiamine pyrophosphate, calcium ion, cobaltion, magnesium ion or manganese ion, possibly also FAD and an indicator of hydrogen peroxide, e.g. consisting of peroxidase, 4-aminoantipyrin and phe-2 quinol or Ν, Ν-dimethylaniline or homovanillic acid, characterized in that the pyruvate oxidase has an optimum pH of 6.5-8. Equipment according to claim 1, characterized in that the optimum pH 6.5-8 pyruvate oxidase is an enzyme obtained from a culture of a pyruvate oxidase-producing microorganism selected from Pediococcus sp. B-0667 FERM-P No. 4438, Streptococcus sp. B-0668 FERM-P No. 4439, Aerococcus vi ridans IFO-12219 or Aerococcus viridans IFO-12317. 20 25 30 35