JPS6012032B2 - bilirubin oxidase NS-1 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel bilirubin oxidase NS-1 (Bilimbin0ki seNS-1) produced by a microorganism.

The inventors have already applied for a patent on the method for producing this enzyme (
However, subsequent research led to the conclusion that this enzyme is a new substance. Previously, enzymes that react with bilirubin were known to exist in the brains of animals such as rats and guinea pigs.
Its enzymatic properties have not been clarified.

In addition, recently, there has been a report on a substance that has the effect of oxidizing bilirubin and producing hydrogen peroxide in bacteria of the genus Agaricus, which is a type of mushroom, in addition to animals.
No. 1151193). However, the enzymatic properties of this substance have not been clarified at all. Therefore, although the existence of bilirubin-reacting enzyme is known, the properties of the enzyme are unknown, and it has not yet been registered in the International Classification of Enzymes. However, bilirubin oxidase is highly expected to be used for quantifying pirirubin in body fluids such as blood and urine, for diagnosing liver diseases, and for decolorizing cadaver urine treated water.

The present inventors searched widely for enzyme-producing bacteria that react with bilirubin from nature, and as a result of intensive studies, they discovered that a strain belonging to the genus Myrocesium has the ability to produce an enzyme that oxidizes bilirubin. We succeeded in investigating this and were able to clarify bilirubin oxidase NS-1 for the first time.

Further searching revealed that preserved strains of the genus Myrocesium also produced the same enzyme. The present invention was completed based on these findings, and the bilirupine oxidase NS-
1-producing bacterium is Myrocesium bercaria MT-1 (Myr
othec skin mvermcaria MT-1) FE
RM-PNo. 5918 (changed to PERMBP-653) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

Further examples of the bilirubin oxidase NS-1 producing bacteria in the present invention include the following strains.

Myr.

theC skin m VermCariaIF. 6113My
r.

theCiyamaVermCariaIF. 6133M
yr.

theCi Yamam VermCaria1F. 6351M
yr.

the Ciyama VermCaria IF. 9056
Myr.

theCi skin m CinCt mountain m IF. 9
950 Myrothecimm roridum
IFO9531 then Myrothecimmver
The mycological properties of mcariaMT-1 are shown.

■ Morphological properties Hyphae are colorless to white, smooth, with septa, width 1.5-3.0
It is Buddha.

Conidiogenic structures are clustered on dark olive to black-green conidia. The conidiophore is formed from the ascoccal layer and branches repeatedly, producing 2 to 4 branches at each branch, with phialides epiphytic on the last branch, colorless to white, with septa. Phialids grow in whorls of 3 to 6, closely attached to each other in parallel layers. Cylindrical shape, 11.5 x 18.0 x 1.5
~2.0 Buddha. Conidia are phialoid conidia, spindle-shaped, 7.0-8.0 x 2.0-3.0 mm, one end is pointed, the other end is truncated, and a fan-shaped appendage is observed on the pointed tip. . It gathers as a dark olive to black-green sticky mass on the constellation. [B-Growth status■ Growth on malt extract agar medium is somewhat suppressed.

The hyphae are colorless to white and woolly.

Conidiogenic structures are clustered in dark olive to black-green conidia. A colorless secretion is formed. The back side of the colony is light yellow to light brown. ■ Potato/glucose agar growth is rapid.

The hyphae are colorless to white and woolly.

Conidiogenic structures are clustered in dark olive to black-green conidia. A colorless secretion is formed. The back side of the village is pale yellow-brown to dark yellow. (C} Physiological properties■ Optimum growth condition temperature is preferably around 30 qo, and axis is preferably 5 to 7.

■ Growth range Grows at 8°C.

It grows at 370 but not at 4500.

Fences grow between 3.5 and 9.5. Based on the above properties, this strain is classified as Deuteromycetes, as no sexual reproductive organs are observed. In addition, conidia (pycnidia m.)
, it was found that it belongs to the Hyphomycetes among the Deuteromycetes because no conidiophores (acew us) are formed. Barron, G.
L. , The Genera.

fHyphomycetes from soil,
Wmiams & Wilkins, mother ltimore
(19 spots)], ■ Phialo-type conidia are formed. ■ Forms conidia. ■Conidia are spindle-shaped and have no septa. ■The conidia become dark olive-colored to black-green sticky lumps, and are of the genus Myrotheci.
It is classified as genus m). For seeds, see Turotsuku [Tyama IMh, M. ,Thegen Melon MyrotheCju
m Tode eX Fr,MyCol,Pap,CM
River o. 130, 1-42 (1972)], ① does not form setae.

■The length of conidia is less than 10 Buddhas. ■The conidial surface is
Not striped. ■Fialid is cylindrical. ■Does not form peripheral hyphae with spherical tips. ■Conidia are spindle-shaped with a fan-shaped appendage attached to one end. Due to the above properties, this strain is Myrotheci
mmvermcaria).

In the present invention, a suitable medium for culturing the bilirubin oxidase NS-1 producing strain includes a carbon source, a nitrogen source,
Both synthetic and natural media can be used as long as the medium contains appropriate amounts of minerals and other nutrients.

In addition, since the bilirubin oxidase NS-1 of the present invention is a copper enzyme, it is more preferable to add copper sulfate, for example, 5
Addition of legs increases enzyme production by about 70%. Although the medium may be liquid or solid, it is usually industrially advantageous to use a liquid medium. For culture using liquid soil, a static culture method, a concentrated culture method, an aeration culture method, etc. can be selected as appropriate, but deep aeration culture is advantageous for culturing in large quantities. Cultivation temperature is 20-35 p.m.
Culture for 48 to 11 feeding hours.

In this way, a large amount of pirirupine oxidase NS-1 is produced in the culture solution.

And the accumulated production of bilirupine oxidase NS-1
is purified by the following method. First, isolate the cells from the culture solution, add ammonium sulfate to 0.8 saturation, and leave it at 5°C overnight.
The precipitated portion is dissolved and thoroughly dialyzed using ion-exchanged water at 5°C.

After the diafiltration is completed, activated carbon is added in an amount of 0.7% to the diafiltration solution, and the mixture is filtered through a filter. The pH of the transfer liquid was adjusted to 7.4, and 0.2% of activated carbon was added and filtered.

Add this citrus liquid to 0. After adjusting the pH to 9.3 with carp ammonia water,
QAE-Sephad pre-equilibrated with 0.019 Na2C03-NaHC03 buffer (pH 9.3)
Ex A-50 column chromatography was applied to adsorb bilirubin oxidase NS-1, and after washing,
Gradient elution was performed with the same buffer (0.015-0.3M), and the active fraction was desalted and concentrated using an Amicon membrane with a molecular weight of 1000, and then equilibrated with 0.03M Na2C03-NaHC03 buffer. Bilirupine oxidase NS charged onto a 100 column and eluted
-1 active fractions were collected and freeze-dried to obtain purified bilirubin oxidase NS-1. This purified product was uniform in polyacrylamide gel disk electrophoresis. Next, the measurement of bilirubin oxidase activity in the present invention will be described.

0.2M Tris-HCl buffer (p
H8.4) Solution 2 of bilirubin 5 and 9 dissolved in 250 mg
．． 0 and the enzyme preparation 0.2 are placed in a cell, and after reacting at 370, the initial velocity is measured.

That is, the absorbance at 44 m is followed over time using a photometer, and the amount of enzyme that oxidizes 1 micromole of bilirubin per minute is defined as 1 unit. The proof that the enzyme of the present invention is an oxidase will be described below. The reaction of the enzyme of the present invention does not proceed under anoxic conditions.

Furthermore, the Warburg manometer has a behavior of absorbing oxygen. Therefore, oxidase (0 phantom d
Ase) or oxygenase (0 ygeMse).

Therefore, bilirubin, pyrogallol (Pyrogallo)
The reaction product was confirmed when the enzyme of the present invention was applied to 1) as a substrate. '1} Effect on pirirubin Bilirubin (manufactured by Sigma, crystalline product) 6-9 is 0.2
After reacting a solution of 3.0% dissolved in 5% M Tris-HCl buffer (pH 8.2) and 0.1% enzyme solution (12% blood/scream) for 10 minutes, unreacted pirirubin was removed. The aqueous layer was applied to a Sephadex G-15 column.

The purple fraction passed through the column, but the blue substance remained adsorbed on the Cephadex. Therefore, the Sephadex was taken out from the column and immersed in methanol, and the adsorbed material was eluted at 5° C. for about 5 hours without exposing it to light. Thereafter, the Cephadex was removed by sieving, and the cephadex was subjected to visible spectrum and thin layer chromatography.

The results showed that there was an absorption maximum near 66m and 37m, and that the thin layer chromatography spot was single, and that the standard biliverdin (Sig) used as a control
Since the Rf value is the same as that of Pyrrubin (Molecular formula: C33 & 6N406, manufactured by MA), the reaction solution contains
) is an oxide of pyliverdine (molecular formula C33 ratio 4N4
06) was found to exist. '21 Action on pyrogallol The enzyme of the present invention also reacts with pyrogallol, although the degree of reaction is 1/1 that of bilirubin.

Pyrogallol aqueous solution (5 of 9/') 5.0 ml and enzyme solution (26.6 ml/') of 0.3 ml were reacted in the same manner as in {1} above, and the light-colored color precipitated in the reaction solution. After drying the crystals, IR and NMR spectra were taken. The results were published in The Ndrjch Library of Infrared Svektra.
oflnfraredSpectra) and The Aldrich Library of NMR Spectra (T
heNdrichLibra and ofNM crotch Spectr
When compared with reference a), bulbrogarin (Purpu
It was found that rogallin) was produced.
Purbrogalin (molecular formula C6 is 03) is pyrogallol (
Molecular formula C,. day 8Q) peroxidase (Perox
It is also a product when reacted with inuse)・ratio02.
From the above results, the enzyme of the present invention is an oxidase that catalyzes an oxidation reaction using molecular oxygen as an electron acceptor, and more specifically, it is a bilirubin oxidase that oxidizes bilirubin to biliverdin.

The present bilirubin oxidase NS-1 has the following properties. '1} Action: Oxidizes bilirubin in the presence of molecular oxygen, producing a green substance, a pale green substance, and then a pale purple substance, but does not produce hydrogen peroxide.

Biliverdin is included in the product. 2) Substrate specificity: 6.84mM of various substrates (dissolved in 0.2 phosphate buffer, pH 8.0) 10' and the present bilirubin oxidase NS-1 enzyme solution (2. blood/secretion)1. 0 skin 370
I reacted with

Table 1 shows the relative activity when pirirupine is set as 100. Table 1 Substrate specificity (husband) Slight reaction, {3} Temperature stability: This enzyme was dissolved in 0.1M phosphate buffer (
After treatment at each temperature for 15 minutes in pH 8.0), residual activity was measured.

As shown in FIG. 1, at 5000, more than 90% remained, but at 7000, the activity was lost (FIG. 1). ■ Optimum temperature: At pH 8, activity was measured at each temperature.

As a result, the activity at 4000 was the highest (Fig. 2). '5' pH stability: This enzyme solution was 370, 6 at each pH.
After standing for 0.6 hours, the pH was returned to 8 and the residual activity was measured.

As a result, it was stable from spot 6 to bait 10 (3rd
figure). t61 Shihaku: Since crystalline bilirubin does not fall in the acidic or neutral range, albumin-bound bilirubin (
The reaction was carried out at 37°C in the range of 6 to 10 using Bilirubin Control (trade name, manufactured by Dade Corporation) as a substrate.

As a result, fences 6 and 7 were found to be optimal for reaction (Fig. 4). '
71 Molecular weight: The molecular weight was measured using a Cephadex G-100 (manufactured by Pharmacia) column (2.5 skins in diameter x 92 skins in length) with 3 MMN equilibrated with CQ/NaHC03 buffer (pH 9.3).

As a result, the molecular weight of this enzyme was approximately 52,000. The standard protein used is cytochrome C (molecular weight 12,400
), oval albumin (molecular weight 43,000), bovine serum albumin (molecular weight 66,000), and y-globulin (molecular weight 160,000) were used.

[8- Isoelectric point: LKB focusing electrophoresis device (capacity 1
10 [), Ampholine (manufactured by LKB, pH 3.5
~5.0) under cooling (0°C) at 600V, 48
After time phoresis, the fraction was fractionated into 2.5 fractions and the isocenters were determined.

The isodew point of this enzyme was PI=4.1. [9} Disk electrophoresis: 7% polyacrylamide gel (pH 9
．． 5) using Davis' method (B.J. Davis,
Ann. N. Y. Acad. Sci. 121 404 (
1964)). Two tubes were run, one was stained, the other was cut into 2 willow units, activity was measured, and the position of the electrophoresis layer was determined.

As a result, the enzyme protein formed a single band, which coincided with the active position. 'IQ Amino acid analysis: This enzyme was hydrolyzed with carp constant boiling point hydrochloric acid at 110 qo under reduced pressure for 24, 48, and 72 hours, and then the hydrolyzate was dried under reduced pressure to sufficiently remove the hydrochloric acid. and dissolved in citrate buffer (pH 2.2).

This solution was analyzed using a Hitachi KLA-5 automatic amino acid analyzer. The content of tryptophan and tyrosine is C℃odM
It was performed according to the n-Monon method. (T.W.G℃od
wjnand R. A. Morbn, Biochem.
J. 40, 628 (1964)). Cystine was quantified by oxidizing the enzyme with performic acid, and then removing the performic acid with hydrogen bromide (S.M.
oore, J. Biol. Chem. 238, 235 (
1963)), the glue was decomposed in constant boiling point hydrochloric acid at 110 qo for 1 hour, and then the hydrolyzate was dried under reduced pressure to sufficiently remove the hydrochloric acid, and then added to 0.09M citric acid buffer (pH 2.2).
dissolved in This solution was analyzed using a Hitachi KLA-5 automatic amino acid analyzer. The results are shown in Table 2.

Table 2 Amino acid composition (11) Effect of metal ions: This enzyme was dissolved in 5 plates of M Tris-HCl buffer (pH 8.3), and each metal ion was added to this at a concentration of 10-2M. , 5
Processed for minutes.

Thereafter, activity measurements were performed. In addition, for C is 10, it is 10-
Treated with 3M. As shown in Table 3, the results are significantly inhibited by Fe20. 3- Effect of metal ions (12) Effect of inhibitors, etc.: This enzyme solution was mixed with various inhibitors.
After treatment with H8 at room temperature for 1 hour, the mixture was diluted with 1M Mochiko and the activity was measured.

It is inhibited by heavy metals and potassium cyanide, sodium azide, and thiourea, which form rust bodies. (Table 4). Fourth
Table: Effects of inhibitors, etc. (13) Absorption in the visible region: Absorption in the visible region of this enzyme solution (1%) was measured.

As a result, there is no absorption maximum near 375 nm and 46 m. That is, this enzyme is FAD (F1avine
adenine nucleotide),
FMN (F1avjnemononuc1eo
It does not have the specific absorption that exists in tides. (14) Sugar content: Using 7% polyacrylamide gel (PH8.0), disk electrophoresis was performed and sugar staining (PAS staining: R.M. Necharius, T.E. all, T. . Monison, J. J. Woodlock, Ana
l. Biochem. 30148 (1969)).

As a result, the same area as the protein staining area was stained, indicating that sugar was contained in the enzyme. The bran content was determined by the phenol-sulfuric acid method and was approximately 7.8% as glucose. (15) Amount of copper: After heating and decomposing this enzyme with nitric acid, allow it to cool, add perchloric acid, and gently heat to boil.

After cooling, copper was quantified using a PerkinElmer 402 atomic absorption spectrometer. As a result, it was found that 1 mol of copper contained 1 mol of enzyme. (16) Effect of sodium chloride: As a result of measuring the activity of this enzyme solution containing various concentrations of sodium chloride, it was found that the higher the concentration, the more the activity was inhibited.

(Table 5). However, sodium chloride is removed by dialysis.
The activity recovers.

Table 5 Effects of sodium chloride Next, test examples and examples of the present invention will be shown.

Test Example 1 Myrocesium Berkaria MT-1, FERM-P Shame.
The ability to produce bilirubin oxidase NS-1 was investigated using B. 5918 and preserved strains belonging to the genus Myrocesium.

Glucose-potato soil (potato 4) was used as a medium.
Add 1 tsp of water at night, boil, cool, pass through gauze, add 1% glycose to 1 tsp of lint liquid, adjust the mottling to 5.8) and make 100 each. Using a saw, put it into a 500' Sakaguchi flask and inoculate each stock strain,
The cells were cultured at 2500 °C for various hours, and the bilirubin oxidase activity in the culture solution was examined. The results are shown in Table 6.

Table 6 As is clear from the results in Table 6, Myrothecim
mverrucaria MT-1 FERM-P
No. In addition to 5918 (changed to FERM-BP653), Mymtheci mverrucaria I
F0 6113, MMothecium verruc
arlaIF0 6133, Myrotheciu
m verrucaria IF06351, Myro
thecium verrucaria IFO 90
56, Myr.

theC skin m r. ridumIF. 9531・Myr
.

It is found that theCMmci skin tum IFO9950 has the ability to produce bilirubin oxidase NS-1. Example 1 Myrothecimm vermcaria MT-1
, FERM-PNo. 5918 was inoculated into a 500M Sakaguchi flask containing 100% of the glucose potato medium described in Test Example 1, cultured in a shaking moat (15 pm) at 25°C for 72 hours, and the resulting seed culture was 1 in advance
7. Glucose potato medium sterilized in a jar of 0.0 volume and antifoam agent Adekanol LG12
6 was added at 0.07% (the amount of inoculation was 2%).

Culture temperature 2500, clear water 25 pm, ventilation rate 6 so/mi
The cells were cultured for 6 hours under the conditions of n, and after the completion of the culture, bacteria were removed to obtain a culture solution 3000.

The bilirubin oxidase activity of this product is 19. It was blood/secretion. Example 2 Myrothec skin m verrucaria IF0
6133 was treated in exactly the same manner as in Example 1, and 3,500 microliters of culture solution containing bilirubin oxidase activity of 14.0 u/' was obtained from the medium.

Example 3 Myrothecimmroridum IFO9531
The cells were treated in exactly the same manner as in Example 1, and 3300' of culture solution containing 7.5 u of bilirubin oxidase activity was obtained from the 7 days old culture medium.

Example 4 Ammonium sulfate was added to 3,000 ml of the cultured oak solution obtained in Example 1 to a saturation level of 0.8, and after standing at 5°C overnight, the precipitated portion was dissolved. Dialyze.

After the diafiltration is completed, activated carbon is added in an amount of 0.7% to the diafiltration solution, and the mixture is filtered through a filter. The pH of the transfer liquid was adjusted to 7.4, and 0.2% of activated carbon was added and filtered. Add this citrus liquid to 0. After adjusting the solution to 9.3 with carp ammonia water, the solution was 0.018 in advance.
Bilirupine oxidase was adsorbed by column chromatography on ○~AE-Sephadex A-50 equilibrated with MNa2CQ-NaHC03 buffer (pH 9.3), and after washing, the same buffer (0.015~0 .8
Elute with a boiled gradient, and collect the active fraction with a molecular weight of 10.
Desalt and concentrate with 0.00 Amicon membrane and further add 0.03 MNa.
Purified bilirubin oxidase NS-1 was obtained by charging the column chromatography column of Sephadex G-100 equilibrated with 2C03-NaHC03 buffer, collecting the eluted bilirubin oxidase active fraction, and freeze-drying it.

This purified product was uniform in polyacrylamide gel disk electrophoresis. Table 7 shows the yield of the purification process. Table 7 Purification of bilirupine oxidase NS-1

[Brief explanation of the drawing]

Figure 1 is a diagram showing the temperature stability of the pirirubin oxidase of the present invention, Figure 2 is a diagram showing the optimum temperature, Figure 3 is a diagram showing pH stability, and Figure 4 is a diagram showing the optimum pH. be. Figure 1 Figure 2 Figure 3 Figure 4

Claims (1)

[Claims] Bilirubin oxidase NS-1 having the following physical and chemical properties. (1) Action: Oxidizes bilirubin in the presence of molecular enzymes, producing a green substance, a pale green substance, and then a pale purple substance, but does not produce hydrogen peroxide. (2) Substrate specificity: Reacts with bilirubin. It reacts slightly with biliverdin, hemin, hematoporphyllin, and chlorophyllin. It does not react with hemoglobin, chlorophyll, or vitamin B_1_2. (3) Temperature and pH stability: pH 8.0 (0.1M phosphate buffer)
, more than 90% remained after treatment at 50℃ for 15 minutes, and at 70℃, 1
It becomes inactive in 5 minutes. pH 5 after treatment at 37℃ for 60 minutes
-95% or more remains between pH 10.5. (4) Optimum temperature: around 40℃. (5) Optimal pH: p
H6-7. (6) Molecular weight: Approximately 52,000 (gel filtration method) (7)
Isoelectric point: PI = 4.1 (8) Effect of metal ions: F^
It is significantly inhibited by 2^+_e. (9) Effects of inhibitors, etc.: Inhibited by potassium cyanide, sodium azide, and thiourea. (10) Visible absorption: There is no absorption maximum near 375 nm and 46 nm. (11) Contains about 7.8% sugar. (12) Contains 1 mol of copper in 1 mol. 2. Bilirubin oxidase NS-1 according to claim 1, wherein the bilirubin oxidase NS-1 producing bacterium is a bacterium belonging to the genus Myrocesium.