CA1143640A - Process for manufacture of pyruvate oxidase and its use for the analysis and kit - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Pyruvate oxidase is produced by a process which comprises culturing A pyruvate oxidase-producing microorganism belonging to the genus Pediococcus, Streptococcus or Aerococcus in a nutrient culture medium, and isolating the pyruvate oxidase thus produced from the culture. also provided by the invention disclosed herein is an analytical method for pyruvic acid in a sample containing pyruvic acid or a system which liberates this acid, which comprises treating said sample with a reaction system containing pyruvate oxidase, and measuring the consumed component or generated component.

Description

3~4(~

This in~ention relates to a process for manufacture of pyruvate oxidase and its use in the analysis~ and to a kit for analysis.
Pyruvate oxidase is a hitherto known enzyme which catalyzes the reaction from pyruvic acid, phosphate and oxygen to ~orm acetylphosphate, carbon dioxide and hydrogen peroxide and is derived from a strain of Lactobacillus derbr~ckii.
We have found that the enzyme pyruvate oxidase was produced by bacterial strains B-0667 belonging to the genus Pediococcus and B-0668 belonging to the genus Streptococcus iso-, . , lated from a soil sample collected in a radish field in Ohito-cho, Tagata-gun, Shizuoka~ken, Japan, and that the pyruvate oxidase produced therefrom could be used for pyruvic acid analysis in a sample containing pyruvic acid or various systems which liberate pyruvic acid. We have also 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, and quantitative determination of the enzyme and the substrates thereof. Pyruvic acid can be analysed by reacting a sample con-taining pyruvic acid with a reaction system comprising at leastpyruvate oxidase flavine adenine dinucleotide (hereinafter called as FAD), thiamine pyrophosphate, oxygen and phosphate, and we have established an excellent kit for pyruvate analysis and an analytical method for pyruvic acid.
Further we have found that adding a salt which liberates calcium ion, cobalt ion, magnesium ion or manganese ion to the said reaction system results in improvement of the analysis.
Addition of a chromogen or a fluorescent indicator to the reac-tion system provides a convenient and excellent analytical kit and method. Also we have completed a process for manufacture of an enzyme pyruvate oxidase.

~3~

An object of the present invention is to proYide a kit for analysis, especially for diagnostic analysis~ comprising a reaction system containing pyruvate oxidase.
Another object of the present invention is to provide an analytical method for pyruvic acid in a sample containing py-ruvic acid or a pyruvic acid liberating system, which method com-prises treatinq a sample with a reaction system containing pyru-vate oxidase and measuring the consumed component or generated component.
A further object of the present invention is to pro-vide a process for the manufacture of pyruvate oxidase which comprises culturing a pyruvate oxidase-producing microorganism belonging to the genus Pediococcus, Streptococcus or Aerococcus in a nutrient culture medium and isolating the pyruvate oxidase thus produced from the cultured cells thereof.
Other objects, features and advantages of the present invention will become apparent from a consideration of the following description, taken in connection with the accompanying drawings, which are graphs illustrating the present invention, and in which more particularly:
Figure 1 is a graph illustrating the optimum pH of pyruvate oxidase.
Figure 2 is a graph illustrating the heat stability of pyruvate oxidase.
Figure 3 is a graph illustrating the pH-stability of pyruva*e oxidase.
Figure 4 is a graph showing the result of analysis of pyruvic acid by an oxygen electrode using pyruvate oxidase.
Figure 5 is a graph showing the result of analysis of serum by an oxygen electrode using pyruvate oxidase.
Figure 6 is a graph showing the result of analysis of pyruvic acid by a colorimetric method using pyruvate oxidase.

2 -~36~0 Figure 7 is a graph showin~ the xesult of ~uantitativeanalysis of serum pyruvic acid using pyruvate oxidase.
Figure 8 is a graph showing the result of analysis of ADP using pyruVate oxidase.
Figure 9 is a graph illustxating the result of analysis of glycerol~ triglyceri:de and serum triglyceride using pyruvate oxidase.
Figure 10 is a graph illustrating the result of analysis of GPT- and GOT-activity using pyruvate oxidase.
Figure 11 (located in the sixth sheet of drawings, with Figure 7) is a correlation diagram of the analysis o~ GPT-activity using pyruvate oxidase.
Figure 12 (located in the seventh sheet of drawings, with Figure 8~ is a correlation diagram of the analysis of GOT-activity using pyruvate oxidase.
The enzyme pyruvate oxidase in this invention catalyzes an oxidative reaction from pyruvic acid, inorganic phosphate and oxygen to form acetyl phosphate, carbon dioxide and hydrogen peroxide, and is preferably manufactured by culturing a pyruvate 20 oxidase-producing microbial strain belonging to the genus Pediococcus, Streptococcus or Aerococcus, for example Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO
12219 or IFO 12317.
The isolated strain B-0667 and B-0668 hereinabove have the following taxonomical properties.

~ ~ 3 -~3~
_ ~ ~ ' o ~ o . R ~ v 0 3 ,~ _~ O

. ~ D ~ .~
S v ~ v E~ ~ ~

0 t,~ rC

3 C4 3 ~ 8 ~ c c b ~ ~ , o ~
o R 8 ~ ~ ~
v e v ~ v v ~ o o~ ~ 3 ~ 3 t) u) ~ , ~; ~ ~ ~ a ~ 0 ~ ~ ~ 0 ~ C
O ~ ~ X ~ O o ~ U

O 3 3 C ~~ ~ ~
C .. ..
,0 ~C ~ _ ~3 R n~
O ~ -æ &

~3 ~3~0 B. Microscopic observation:

_ Strain B-0667 Strain B-0668 Shape: spherical, ovoid, pairs, spherical, ovoid, pairs, tetra-shaped or short tetra-shaped or short chain. chain.
Size: 0.5 - 1.0 x 0.5 - 1.0 ~ 0.8 - 1.0 x 1.0 - 1.2 Motility: _ _ Spore: _ _ Gram's stain: + +
Acid-fa8t stain: .. .

C. Physiological properties:
_ ........................ , ~ Strain B-0667 Strain B-0668 .. .
Growth temperature: 45C _ 37C + +
30~C ~ +
; 26~C + +
. 10C + +
5C i or (~) + or ~+) Halotolerance: NaCl 10~ + ~

5.0~ + ~
1.0% ~ +
0% + _ OF-test: fermentative fermentative:
Behavior in oxygen: facultative anaerobic facultative anaerobic Nitrate reduction: _ _ Indole formation: _ _ Hydrogen sulfate formation: _ _ i Gelatin hydrolysis:

~'1~''~
,~

~3~

Table continued:

St:rch hydrolysis:
Esculin hydrolysis: +
Acetoin formation: _ MR-test: _ Catalase: _ O~idase: _ Urease (SSR): _ Urease (Christensen): _ Utilization of citric acid (Christensen): _ Acid ormation from sugar:
adonitol: _ L(+)-arabinose: _ cellobiose: +
dulcitol: _ meso-erythritol: _ fructose: +
fucose: _ galactose:
glucose: +
glycerol: _ inositol: _ inulin: _ lactose: +
raltose: +
mannitol: +
mannose: +
melezitose: _ melibiose: _ "7'`
-~ ~ 6 -~3~;~0 _ . . ..
raffinose: _ L(+~-rhamnose: _ salicin: ~) _ L-sorbose: _ _ sorbitol: _ _ starch: _ sucrose: ~
trehalose: + +
xylose: _ _ ¦ tolerance at 60C for 30 min. _ i ~

.
Consulting "Bergey's Manual of Determinative Bacteriol-ogy, 8th Ed., 1974" and "Cowan, S. T. and Steel, K. J., Manual for the Identification of Medical Bacteria, Cambridge Press, 1974", the strains B-0667 and B-0668 having the taxonomical properties hereinabove, especially Gram positive cocci, catalaseand oxidase negative, fermentative a~id formation from glucose, and no gas formation from sugar (glucose), are referred to as belonging to the genus Pediococcus and the genus Streptococcus.

Comparison of these strains with the identification manual of the above references is as follows.
In the table:~ _ positive more than 85%.
- ~ negative more than 85%.

d ~ varies among ~trains or species.
~_ _ _ . .
. strain B-0667 strain B-0668 genus genu~
. ._ _ ~ _ .__. _ . Pediococcus StreDtococcus growth at 45DC _ _ + d tolerance at 60C
for 30 min. _ ~ _ d glycerol 30tacid formation' _ _ _ d arabinose .
(acid formation) _ _ + d halotolerance _ ~NaCl 10~ _ +
. ~............................... ............... ........ .............. ... .... -. .,~

~3~

Hence the strain B-0667 will be referred to as the genus Pedlococcus or Streptococcus. Consulting the above 'IManual for the Identification of Medical Bacteria" and "J. Gen.Microbiol 26, 185-197 (1961)", the taxonomic properties of the strain B-0667 were almost identical with those of Pediococcus urina-equi, how-ever the characteristics described in "Bergey's Manual of Deter-minative Bacteriology, 8th Ed., 1974" were slightly different therefrom. Therefore the strain B-0667 is referred to as the genus Pedioco;ccus and designated as Pediococcus sp. B-0667 The strain B-0668 resembles the genus Streptococcus rather than the genus Pediococcus. Further consulting the "Manual for the Identification of Medical Bacteria", the strain B-0668 resembles Streptococcus faecium var. durans, however, no taxo-_ _ nomic properties were described in the l'Bergey's Manual" and thereby it is impossible to make a detailed comparison. The strain B-0668, therefore, is referred to as Streptococcus sp.
B-0668.
The strains B-0667 and B-0668 were deposited for perma-nent collection in the Institute for Microbial Industry and Technology, Agency of Industrial Science and Technology, M. I. T.
I., Japan, as deposition numbers FERM-P No. 4438 and FERM-P No.
4439, respectively.
In an embodiment of the present invention, the above Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317 are cultured in a conventional medium for enzyme production. Cultivation can be made by conventional liquid culture and submerged aeration culture is preferable for industrial production.
A conventional medium for microorganisms is preferably used. As for the carbon sources, assimilable carbon sources such as glucose, sucrose, lactose, maltose, fructose, molasses, py-ruvic acid or the like are preferably used. Assimilable nitrogen 3~

sources such as peptone, meat extract, yeast extract, casein hydxolysate or the like can be used. Various inorganic salts such as phosphates~ carbonates, sulfates, or salts of magnesium, calcium, potassium, iron, manganese or zinc can be used.
The culturing temperature can be selected within the range for growth of microbial cells and production of pyruvate oxidase, and is preferably from 2S-37C. The culturing time can be altered depending on conditions and is terminated when the pyruvate oxidase production is substantially complete, and usually ranges from 18-48 hours.
Pyruvate oxidase exists in the cells of microorganisms.
To separate pyruvate oxidase from cultured cells, the cultured mass is filtered or centrifuged to collect the cells, and disrupted by treatment with mechanical means or an enzymatic pro-cess such as lysozyme. Further if necessary pyruvate oxidase is solubilized by adding ethylene-diaminetetraacetic acid (EDTA) and a surfactant such as "Triton X-100" (trade mark) or "Adecatol SO-120" (trade mark) to separate the enzyme. The thus-obtained solution of pyruvate oxidase is treated with or without concen-tration, and thereafter the enzyme is precipitated by salting outwith addition of a soluble salt such as ammonium sulfate or sodium chloride. Low molecular weight 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 enzyme solution thus ob-tained is treated by vacuum concentration and lyophilization to produce powdered pyruvate oxidase. Further purification can be achieved by conventional purification methods for proteins and enzymes such as adsorption chromatography, ion-exchange chroma-tography or gel~filtration.

Pyruvate oxidase produced by the present invention hasthe following physico-chemical properties, in which abbreviations are used as follows.

_ g _ Pediococcus sp. B-0667; abbreviated as B-0667 ____ .
Streptococcus sp. B~0668; abbreviated as B-0668 .
Aerococcus viridans IFO-12219: abbreviated as IFO-12219 _ . .. ..
Aerococcus viridans IFO-12317: abbreviated as IFO-12317 (1) Enzyme action:
The enzyme catalyzes oxida-tive reaction from pyruvic acid, inorganic phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide.
CH3COCOOH ~ HOPO3 + 02-~CH3COOP03 + C02 + H202 (2) Optimum pH:
The effect of pH on pyruvate oxidase activity is measured. Phosphate buffer solutions of pH 6~8 are used for the assay. The results are shown in Fig. 1 in which 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 IFO-12317: pH 6.8~7.5 Slight variations are observed in phosphate concentra-tion and in the kind of metallic ion.
(3) Heat stability:
Heat stability of the enzyme is determined by incuba-ting in 10 mM phosphate buffer (pH 6.5) containing 10 ~M FAD at 0, 40, 50, 60 and 70C for 10 minutes according to the method of enzyme assay. As shown in Fig. 2 the enzymes obtained from B~0667, IFO-12219 and IFO-12317 are slightly activated at 40 C
and denatured over 60C. The enzyme obtained from B-0668 is not activated at 40C and almost denatured over 60C.

(4) pH stability:

To each enzyme solution (0.1) ml is added 0.2 M phos-phate buffer for pH 6-8 (0.9 ml) or 0~2 M of Tris-HCl buffer for pH 7-9 (0.9 ml) each containing 10 ~M FAD, and the solutions were ~ ,.i~l `

~3~0 allowed to stand for 10 minutes at 40C. 20 ~1 of enzyme solu-tion are taken and the enzyme activity is determined. As shown in Fig. 3, the enzyme obtained from B~0667, IFO-12219 and IFO-12317 is most stable at about pH 7 and that of B-0668 is stable at an acidic pH.

(5) Effect of several substances:
1) The effect of several substances on the enzyme activity is examined by adding 5 mM of the substances indicated below instead of MgC12 in the assay system.

Substance added _ Relative activity (%) . . . . _ _ _ _ . _ No addition 25.4 72.0 42O0 50.3 MgC12 100 100 100 100 CaC12 69.4 75.0 83.4 78.7 MnC12 129.1 102.7 116.2 111.0 CoC12 81.3 81.1 85.0 84.0 BaC12 20.6 58.8 23.9 28.8 ZnC12 16.0 38.2 14.9 22.8 As shown hereinabove, the enzyme is inhibited by EDTA
++ ++ ++ ++
and activated by Mg , Ca , Mn and Co 2) Effect of eliminationof the following substances from the assay system on the enzyme activity is shown below. 0.1 M
dimethylglutarate-NaOH buffer is used in case of phosphate eli-mination.

_ . _ _ .. _ ...... .. . . ... ..... .. . _ .
Substance eliminated Relative activity (%) No elimination 100 100 100 100 thiaminepyrophosphate 0 0 0 0 FAD 33.9 100 32.7 41.7 thiaminepyrophosphate and FAD 0 0 0 0 ph_sphate 0 0 0 ~3~0 As a result, the enzyme requires thiaminepyrophosphate and FAD as cofactor and phosphate as substrate.
Further oxygen consumption in the enzvme reaction is measured by an oxygen-electrode, and the oxygen is consumed in proportion to the enzyme activity (formation of hydrogen peroxide).
The results are shown as follows:

Oxygen consumption Reaction product (~ mole/min.) (~ mole/min.) H2O2 acetylphosphate .
10 B-0667 0.042 0.042 0.038 B-0668 0.022 0.0213 0.020 IFO-122190.040 0.038 0.037 IFO-123170.035 0.036 0.034 . . .
Assays are per~ormed as follows:

Oxygen consumption: dissolved oxygen meter (Trade mark;
YSI-dissolved oxygen meter Model-53) Acetyl phosphate: F. Lipmann et al, J. Biol. Chem., 134, 463-464 (1940).
Hydrogen peroxide: a method using N,N-dimethylaniline, 4-aminoantipyrine and horseradish peroxidase As hereinabove explained, the enzyme obtained from the above four strains is referred to as pyruvate oxidase and flavine protein.
The assay method of pyruvate oxidase of the present 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 MgC12 50 ~l 30 10 mM thiaminepyrophosphate 20 ~1 peroxidase (45 U/ml) 0.1 ml 1 mM FAD 10 ~1 distilled water 0.22 ml ~,,~,. ..

~3~

The above reaction mixture (1.0 ml) is pxe-incubated at 37 C for 3 minutes. To this solution is added the enzyme solution (20 ~1) and incubated at 37C for 10 minutes. 0.1 M citrate buf-fer (pH 6.0, 2 ml) containing 0.1 M EDTA is added to terminate the reaction. The violet color formed is measured by a colorimetric method at 565 nm.
A unit (1 unit, 1 U) of enzyme activity is defined as the activity which generates 1 ~mole of hydrogen peroxide per minute.
In order to activate the pyruvate oxidase reaction system, FAD, thiaminepyrophosphate and phosphate are added.
Further for activation of the enzyme, an ion-liberating salt which liberates calcium ion, cobalt ion, magnesium ion or man-ganese ion, in the form of chloride is preferably added thereto.
An indicator such as a coloring indicator or a fluorescent indi-cator for hydrogen peroxide is preferably selected.
The amount and ratio of components in the enzyme reac-tion system can be selected for substantial enzyme reaction and will be varied according to the amount of pyruvate, temperature ar~d time of the enzyme reaction. For example, 1-20 U of pyruvate oxidase, 0.1-20 n moles of FAD, 0.05-0.5 ~ mole of thiaminepyro-phosphate, 1-10 ~ moles of inorganic phosphate and 0.05-10 ~moles of ion liberating salt p~r test is preferably used. Pyruvate oxidase can be in a microcapsulated form or in an immobilized form of covalent linkage with an organic or inorganic carrier or adsorbed on a carrier. The molar ratio of indicator for hydrogen peroxide is at least an equimolar or excess amount of generated hydrogen peroxide. In the case of the peroxidase, o.5-20 U per test is preferably used. These components of the enzymatic reac-tion mixture are preferably used by dissol~ing in the buffer ata suitably ad~usted pH.

~3~

The thus prepared enzymatic xeaction system is applied for analysis of pyruvic acid. Any samples which contain pyru;
vate can be analysed. For example, pyruvic acid itself, pyruvic acid in serum or urine and pyruvic acid forming enzyme reaction systems such as lactic acid and lactate dehydrogenase (LDH)~ ADP
and pyruvate kinase, and glycerol, glycerol kinase and pyruvate kinase can be mentioned. Further detailed examples of the enzy-matic reactions which form pyruvic acid and which can be assayed as are follows:
(1) Assay of lactic acid or LDH activity:
lactate LDH -~ pyruvate + NADH2 (2) Assay of ADP or pyruvate kinase (PK) activity:
ADP ~ phosphoenolpyruvate > ATP + pyruvate (3) Assay of glutamate-pyruvate-transaminase (GPT) activity or ~-ketoglutarate:

alanine + ~-ketoglutarate GPT >pyruvate + glutamate (4) Assay of glutamate-oxaloacetate-transaminase (GOT) activity:
aspartate + ~-ketoglutarate OT ~ oxaloacetate +

glutamate oxaloacetate decarboxylase +
oxaloacetate ~ pyruvate (5) Assay of glycerol or glycerophospho kinase (GK) activity:
glycerol + ATP GK -~ glycerol 3-phosphate + ADP
ADP + phosphoenolpyruvate K _~ pyruvate + ADP

(6) Assay o~ triglyceride:
t i 1 eride lipase or lipoprotelnlipase 3 glycerol 3-phosphate + ADP
ADP + phosphoenolpyruvate ~ pyruvate+ ATP

(7) Assay of creatinine or creatinine phosphokinase (CPK):
creatinine creatininase ~ creatine creatine + ATP ~ creatine phosphate -~ ADP
ADP + phosphoenolpyruvate > pyruvate + ATP

~3~0

(8) Assay of myokinase:
ATP + AMP myOkinase > 2 ADP
ADP + phosphoenolpyruvate PK ~ pyruvate + ATP

(9) Assay of fatty acid or thiokinase activity:
fatty acid + CoA ATP thiokinase ~ acyl Coa + AMP + PPi AMP + ATP myokina9e ~ 2 ADP
ADP + phosphoenolypyruvate PK ~ pyruvate + ATP
These enzyme reactions are given only for illustration and thesepyruvate forming reactions can be found with the com-bination of enzyme and its substrate, for example in biologicalsamples. As exemplified hereinabove, the assay can be applied not only for the assay of pyruvate but also for the assay of enzymes, enzyme activity or substrates.
The assay is performed by incubation with the sample and reagent mixture. The reagent mixture is preferably a kit of necessary reagents. For assaying, the consumed component or gen-erated component is measured. Measuring the amount of oxygen consumption by a dissolved oxygen meter is preferable for the assay method. In this case no indicator for hydrogen peroxide is necessary. As for the assaying of a generated component, the measurement of the amount of hydrogen peroxide is preferable, for example by using a hydrogen peroxide electrode meter such as a YSI~oxidase meter or by colorimetric or fluorometric assay with an indicator for hydrogen peroxide. The assay is preferably carried out for a period of 10-60 minutes at 20-40C, preferably at 35-37C.
The indicator for hydrogen peroxide is a combination of one or more chromogens or fluorescers, which is effected by coupling with hydrogen peroxide. Examples of such indicators are a combination of a tetravalent titanium compound and xylenol orange which couples with hydrogen peroxide to form a stable red ~i~ color, or combinations of phenol or N,N-dimethylaniline or homo-vanillic acid~ 4-aminoantipyrine and peroxid~se for measuring color or fluorescence. 4~aminoantipyrine can be replaced by 4~aminophenazone. A combination of 2,6-dichlorophenol, indophenol and peroxidase and of guaiacol and peroxidase can also be used.
The indicator can be previously prepared as a solution. A color-imetric or fluorometric assay is performed by measuring the ab-sorption at a suitable wave length such as 565 nm.
The amount of pyruvic acid can be measured by calculating from a corresponding standard curve of consumed oxygen or gener-ated hydrogen peroxide.
Phosphate as a oonsumed oomponent or aoetylphosphate as a generated ccmponent can also be assayed by a conventional method.
As hereinabove explained, a kit for analysis, especially diagnostic analysis, cMmprising pyruvate oxidase and its use for various assay methods are provided. More particularly as illustrated hereinbefore, diagnostic analyses such as the analysis of pyruvate in a pyruv~te con~;n;ng reagent or in serum or urine, the assay of enzyme activity of LDH, pyruvate kinase, GPT, GOT, glyoerol kinase, lipase, lipoprotein lipase, creatinine, phospho-kinase, myokinase or thiokinase, and the analysis of biological components such as lactate, ADP, glycerol, triglyceride, creatinine or fatty acid can advantageously be made by the kit and method of the present invention.
The follcwing examples illustrate the embodiments of the present invention but are not to be construed as limiting the invention.
Example 1.
A medium (each 100 ml, pH 7) comprising glucose (1%), peptone (1%), yeast extract (0.5%), NaCl (0.2%), KH2PO4 (0.1%), K2HPO4 (0.1~), MgSO4 (0.05%) and CaCO3 (0.3%) in a 500 ml Erlenmeyer flask was sterilized at 120C for 20 minutes. To each medium was inoculated a strain of Pediococcus sp. B-0667 FERM-P
No. 4438, Streptococcus sp. B-0668 FERM-P No. 4439, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317, respectively and the media were shake-cultured at 30C for 24 hours, at 300 ~3~0 r.p.m. Thereafter cultured cells centrifu~ally collected were washed with 10 mM phosphate buffer (pH 6.5) and again centrifuged to collect bacterial cells. The thus obtained cells were sus-pended in the 10 mM phosphate bu~fer (10 ml~ pH 7.0) containing 0.02% lysozyme and 0.1% "Txiton X~100" and incubated at 37C for 60 minutes. The supernatant obtained centrifugally which contains pyruvate oxidase was collected. The enzyme activity of the super-natant is shown in the following table.
Strain Enzyme activity (U/ml) B-0667 0.60 B-0668 0.38 IFO-12219 0.52 IFO-12317 0.46 Example 2.
A medium (20 1.) consisting of the same components as described in Example 1 in a 30 1. Jar-fermenter was sterilized by steam. Cultured broth (200 ml) of Pediococcus sp. B-0667 FERM-P No. 4438, cultured in the same way as in Example 1 was transferred thereto, and cultured at 30C for 24 hours. The bacterial cells centrigually collected (about 100 g) were sus-pended in the lysozyme solution (0.2 mg/ml, 4 1.) there was further added "Triton X-100" (trade mark, 4 g) EDTA t3 g3 and 1 M phosphate buffer (pH 6.5, 40 ml) thereto and the mixture was stirred at 37C for 60 minutes. To the supernatant obtained centrifugally was added ammonium sulfate and the precipitate at 0.54-0.73 saturation was collected by centrifuge. The precipi-tate was dissol~ed in 10 mM phosphate buffer (pH 6.5, 1000 ml) t5160 U, recoYery; 86~), then cold acetone (0.65 ~olume) was added thereto and the impure precipiate was separated. Further acetone (0.3 volume) was added and the precipitate, which was collected by centri~u~er was dissolved in 10 mM phosphate buffer (pH 6.5~ 70 ml) (~750 Ur reco~ery 7~.2~).

- 17 ~

~3~0 To the soluti~n was, added a~monium sulfate and the pre-cipitate at 0.54~0.70 saturation was collected centrifugally.
After dissolving the precipitate in 10 mM phosphate buffer (pH 6.5), the solution was charged on a column of "Sephadex G-25" (trade mark) (6.0 X 70 cm) and the fraction showing the absorbency at 280 nm was collected. The active frac-tions were pooled and freeze dried to obtain a powder of pyru-vate oxidase ('394a U, 758 mg, recovery 65.7%) Example 3.
A kit for pyruvate analysis (for oxygen electrode):

pyruvate oxidase obtained in Example 2 (the same as in the following examples) 300 U
FAD 0.5 ~mole thiamine pyrophosphate 10 ~moles MnC12 25 ~moles 0.2 M phosphate buffer (pH 7.5) 1.0 ml sucrose 0.5 g 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 5 ml The above mixture is lyophilized to prepare the kit for assay of pyruvic acid (for oxygen electrode measurement, 50 tests).
Example 4 A kit for pyruvate analysis (for colorimetric assay):
Reagent (I) pyruvate oxidase 200 U
FAD 0.5 ~mole thiamine pyrophosphate 10 ~moles 0.2 M phosphate buffer (pH 7.5) 1.0 ml sucrose 0.5 g 0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 5 ml peroxidase (100 U/mg, horseradish) 2.5 mg 0.3~ 4-aminoantipyrine 5 ml ~f~3 3 '~ .
~i~, ,~

~3~

The above mixture is lyophilized to prepare the reagent (I) of the kit for pyruvate analysis (for colorimetry, 50 tests).
Additional reagent (II) consisting of 0.2% aqueous N,N~dimethylaniline containing 25 ~moles MnC12 (50 ml) and a stop reagent (III) consisting of 0.1 M citrate buffer (pH 6.0, 100 ml) containing 0.1 M EDTA were attached.
Example 5.

, _ . . _ .
The kit illustrated in Example 3 was dissolved in dis-tilled water (50 ml) and an aliquot solution (1.0 ml) thereof was put in reaction vessels~ Thereto were added 5.0 mM pyruvate solution (each 0-100 ~1), or human serum 50 ~1 and 5.0 mM pyru-vate solution (each additionally 0--100 ~1), and incubated at 37 C, then the oxygen consumption was measured by a Garvanic oxy-gen electrode. The results are shown in Fig. 4.
Further, the kit shown in Example 3 was dissolved in distilled water (25 ml) and an aliquot solution tO.5 ml) thereof was put in reaction vessels, then incubated with the addition of an aqueous solution (0.5 ml) containing human serum ~0-0.5 ml) at 37 C. In Fig. 5 there is shown the result of assay of oxygen consumption measured by a Garvanic oxygen electrode.
As shown in ~ig. 4 and 5 good linear relations were observed.
Example 6.
The lyophilized reagent (I) prepared in Example 4 was dissolved by addition of reagent (II) (50 ml), and each aliquot solution (1 ml) in the small test tubes were incubated at 37 C.
Thereto was added 5 mM potassium pyruvate solution (each 0-50 ~1), or human serum (50 ~1) added with 5 mM potassium pyruvate solution (each 0-50 ~1), and incubated at 37C for 10 minutes. A stop solu-tion (2 ml~ was added thereto and the absorbency at 565 nm was measured. As sh~wn in Fig. 6, good linear relations and quanti-tative results were observed in the above assays and also in the i~,,~ .

case, this result was coincided with the calibration curve by 22 ( .5 mM H2O2, 0 50 ~1.).
Further aliquot samples of human serum (0-0.5 ml) were put into small test tubes and adjusted to 0.5 ml hy adding dis-tilled water. Each solution (1.0 ml) of reagent (I) prepared with adding reagent (II) in Example 4 was added thereto, and in-cubated at 37C for 10 minutes. The reaction was terminated by adding stop reagent (IIIl (1.5 ml) and colorimetrically assayed at 565 nm. As shown in Fig. 7, a good quantitative result was obtained.
Example 7.

.
Reaction mixture:
0.2 M dimethylglutarate~NaOH buffer (ph 7.5) 0.2 ml

10 mM MnC12 50 ~1 0.2% N, N-dimethylaniline 0.2 ml 0.3~ 4-aminoantipyrine 0.1 ml peroxidase ~45 U/ml) 0.1 ml 10 mM ~hiamine pyrophosphate 20 ~1 0.2 M phosphate buffer (pH 7.5) 25 ~1 20 mM phosphoenolpyruvate 0.1 ml pyruvate kinase (4000 U/ml) 5 ~1 5 mM ADP 0-50 ~
The above reaction mixture was adjusted to 1.0 ml by adding distilled water, pre-incubated at 37C, then a solution of pyruvate oxidase (200 U/ml, 20 ~1) was added thereto, and incu-bated at 37C for 10 minutes. After stopping the reaction with addition of 0.1 M EDTA in 0.1 M citrate buffer (pH 6.0, 2.0 ml), the absorbency at 565 nm was measured. As shown in Fig. 8, good quantitative results of ADP assay were observed by assaying hydro-gen peroxide generated from the reaction mixture of ADP, pyruvate kinase, phosphoenol-pyruvate and others.

Also as shown in Fig. 8 good linearity was observed when 2.5 mM hydrogen peroxide were used instead of 5 mM ADP.
Example 8.
.
Triglyceride assay kit:
Reagent (I):
0.2 M dimethylglutarate-NaOH buffer (pH 7.5) 10 ml 0.3% ~-aminoantipyrine 5 ml peroxidase (100 U/mg) 2.5 mg thiamine pyrophosphate 10 ~moles 0.2 M phosphate buffer (pH 7.5) 1.25 ml phosphoenolpyruvate 100 ~moles pyruvate kinase (4000 U/ml) 0.1 ml pyruvate oxidase (200 U/ml) 2 ml lipoprotein lipase (3000 U/ml) 0.5 ml glycerol phosphokinase (300 U/ml)1.0 ml ATP 100 ~moles The above reagent was lyophilized.
Rea~ent (II):
25 ~ moles of MnCl2 in 0.2% dimethylaniline 50 ml Reagent (III): (stopper solution) 0.1 M EDTA in 0.1 M citrate buffer (pH 6.0) 100 ml _ample 9.
Reagent (I) in Example 8 was dissolved by reagent (II) and each aliquot solution (1.0 mll thereof was put in the test tubes.
Each ali~lot sample (0-50 ~l) of human serum containing 1.02 ~ mole/ml of triglyceride, 5 mM glycerol solution, 4.2 mM
triolein in 0.1~ "Triton X-100" solution or 2.5 mM hydrogen per-oxide solution, respectively~ was added thereto and incubated at 37 C for 10 minutes. As shown in Fig. 9 good linearities were observed.

~i/, o Example lQ.
A kit for assay of serum transaminase; (for 100 tests);
(1) A kit for GPT assay: (for 100 tests);
Reagent (I): lyophilized rea~ent consisting of the following:
pyruvate oxidase 400 U
FAD 500 n moles thiamine pyrophosphate 15~.~ moles L-alanine 20 m moles ~-keto~lutarate 1 m mole sucrose 1 g 4-aminoantipyrine 150 ~ moles peroxidase 450 U
0.2 M phosphate buffer (ph 7.5) 2.5 ml 0.2 M dimethylglutarate-NaOH buffer (ph 7.5) 30 ml Reagent (II): ~100 ml are used for Reagent (I)~;
42 ~ moles MnC12 in 0.2% dimethylaniline solution 210 ml Reagent (III): (st.opper solution); r2oo ml for Reagent (I), 2.0 ml per one testJ;
0.1 M EDTA in 0.2 M citrate buffer (ph 5.0) 420 ml 0 (2) A kit for GOT assay: (for 100 tests);
Reagent (I): Lyophilized reagent consisting of the following;
pyru~ate oxidase 400 U -FAD 500 n moles thiamine pyrophosphate 150 ~ moles L-aspartic acid 20 m moles ~- ketoglutarate 1 m mole oxaloacetate decarboxylase 200 U

sucrose 1 g 4-aminoantipyrine 150 ~ moles peroxidase 450 U
0.2 M phosphate buffer (ph 7.5~ 2.5 ml ~3~0 0.2 ~ dimethylglutarate~N~OH buffer (ph 7.5) 30 ml Reagent (II) and Reagent (III):
The same as the above (1).
Example 11.
Reagents (I) prepared in Example 10, (1) (for GPT acti-vity assay) and (2) (for GOT activity assay), were dissolved by adding reagent (II) (100 ml) respectively. Each aliquot amount (1.0 ml) of the solution was separately put into small test tubes and pre-incubated at 37C for 5 minutes Lpre-incubated solution of reagent (I)J . The standard serum solution (20 ~1) (Calbiochem Co., trade mark: "Maxitol", containing GPT 700 K U/ml and GOT
1000 K U~ml) diluted with constant ratio was added thereto and incubated at 37C for 10 minutes. The reaction was stopped by adding stopping reagent (III) (2.0 ml) and the absorbency at 565 nm was measured. As shown in Fig. 10, both enzyme activities have linearity up to an optical density of about 1.0 (enzyme activity: about 750 K U/ml).
Example 12 To each pre-incubated solution of reagent (I) obtained in Example 11 were added 20 ~1 of human serums (45 samples) and incubated at 37 C for 20 minutes. Stopping reagent (III) (2.0 ml) was added thereto and the absorbency at 565 nm was measured.
~lso the same samples of human serums were assayed by LKB method (ultra violet absorption method, GOT assay kit and GPT
assay kit, made in LKB Corp.) and the correlation was plotted with activity of GPT and GOT.
As shown in Fig. 11, the correlation coefficient; r =
0.998 and the regression equation; y - 0.00295 x ~ 0.0032 forGP~
assay were observed.
On GOT assay, the correlation pattern is shown in Fig.
12, in which the correlation coefficient, r ~ 0.966 and the regression equation; y _ 0.00287 x ~ 0.0180 resulted.
~3 ~...,. ~

Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A kit for analysis of Pyruvic Acid comprising a reaction system which contains pyruvate oxidase which catalyses the reaction to generate acetylphosphate, carbon dioxide and hydrogen peroxide from pyruvate, inorganic phosphate and oxygen; thiamine pyrophosphate, phosphate, and a salt which liberates calcium ion, cobalt ion, magnesium ion, or manganese ion.

2. A kit as claimed in Claim 1 which also includes flavine adenine dinucleotide (FAD) and an indicator for hydrogen peroxide.

3. A kit as claimed in Claim 2 wherein the indicator for hydrogen peroxide consists of peroxidase, 4-aminoantipyrine and phenol or N,N-dimethylaniline or homovanillic acid.

4. A kit as claimed in Claim 1 wherein the pyruvate oxidase is an enzyme obtained from a culture of a pyruvate oxi-dase-producing microorganism belonging to the genus selected from the group consisting of Pediococcus, Streptococcus and Aerococcus.

5. A method for analysis of pyruvic acid or of a py-ruvate liberating system, which method comprises treating said sample with a reaction system containing pyruvate oxidase which catalyses the reaction to generate actylphosphate, carbon dioxide and hydrogen peroxide from pyruvate, inorganic phosphate and oxygen; thiamine pyrophosphate, flavine adenine dinucleotide (FAD), phosphate, and a salt which liberates calcium ion, cobalt ion, magnesium ion or manganese ion; and measuring the oxygen which is consumed or the hydrogen peroxide which is generated.

6. A method as claimed in Claim 5 wherein the said pyruvate liberating system is an enzyme reaction system which generates pyruvates.

7. A method as claimed in Claim 5 wherein the reaction system containing pyruvate oxidase also includes an indicator for hydrogen peroxide.

8. A method as claimed in Claim 7 wherein the said indicator for hydrogen peroxide consists of peroxidase, 4-amino-antipyrine, and phenol or N,N-dimethylaniline or homovanillic acid.

9. A method as claimed in Claim 5 wherein the pyruvate oxidase is an enzyme obtained from a culture of a pyruvate oxidase-producing microorganism belonging to the genus selected from the group consisting of Pediococcus, Streptococcus and Aerococcus.

10. A process for manufacture of pyruvate oxidase which comprises culturing a pyruvate oxidase-producing microorganism belonging to the genus Pediococcus, Streptococcus and Aerococcus in a nutrient culture medium and isolating the pyruvate oxidase thus produced from the cultured cells thereof.

11. A process as claimed in Claim 10 wherein the pyru-vate oxidase-producing microorganism is Pediococcus sp. B-0667 FERM-P No. 4438, Streptococcus sp. B-0668 FERM-P No. 4439, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317.