JPS61115488A - Production of heat-resistant polyphenol oxidase - Google Patents

Abstract

PURPOSE:To obtain the titled compound which is an enzyme capable of oxidizing o-diphenyl to o-quinone, in high efficiency and purity, by culturing a specific microbial strain belonging to Mucor genus. CONSTITUTION:A microbial strain belonging to Mucor genus and capable of producing heat-resistant polyphenol oxidase [e.g. Mucor miehei AJ117120 (FERM P-7923), etc.] is cultured in a solid medium preferably at 40-60 deg.C for 1-10 days, and the objective substance is separated from the cultured product.

Description

[Detailed Description of the Invention] [Field of Application] Polyphenol oxidase is called by various names such as phenolase-tyrosinase, laccase, krenolase, catechol oxidase, etc.
-Cleaned from enzymes that oxidize to quinone.

When a substance is applied, changes in physical properties such as increased viscosity, solidification, and film formation occur. A change in taste may also occur. Examples of possible specific applications include the use of sumac as a solidification accelerator and in the production of tea and cocoa. Polyphenol oxidase has been known to exist in plants, animals, and microorganisms, but all of them are unstable. Furthermore, it has the disadvantage of low activity, which means that it has not been applied to food processing, etc. at present.

[Problem that the invention seeks to solve]

Conventionally known polyphenol oxidases are unstable as mentioned above.
The optimum temperature for activity of ) is 40°C, and in order to prevent contamination with bacteria during enzyme treatment, it is necessary to find an enzyme that is optimal at even higher temperatures. In order to carry out enzyme treatment in a further short time, enzyme preparations with higher activity than conventional ones are desired.

[Means for solving problems]

In view of the above-mentioned circumstances, the present inventors conducted a wide search in nature to obtain polyphenol oxidase, which has a wide range of uses and has an optimum temperature at high temperatures and is highly active. We discovered that polyphenol oxidase can be produced that exhibits high purity, and developed a method to efficiently produce polyphenol oxidase that exhibits high purity and activity at high temperatures by producing polyphenol oxidase by culturing it in liquid or solid state, and purifying it. completed.

Mucor Mayhei used in the present invention is DonaldG.
, Cooney Ra1ph, Emerson”
ThermophyllcFungi' an a
ccount of their biologr.

Activities and C15ssifica
tlon+ (W-HFFreeman) Next, details of culturing this strain and collecting heat-stable polyphenol oxidase in the present invention will be described.

The culture method may be a commonly used liquid culture method or a solid culture method, and may be cultured at a culture temperature of 40 to 60° C. for 1 to 10 days with shaking or stationary culture.

In the liquid culture medium, carbon sources include glucose, sucrose, starch, organic acids, etc., and nitrogen sources include peptone, yeast extract, (NH4)2S04. Inorganic salts such as NH4N05, KH2PO4, K2HPO4, MgSO4, C
aCl2, etc., and Zn", Fe" as necessary.
+ On” *Trace metals such as Mn+ and polyphenols are added as appropriate.

An anchor is used for solid culture, and inorganic salts and polyphenols are added as necessary.

To collect phenoloxygose from the culture obtained by this method, obtain a sterilizing solution in the case of liquid culture or an aqueous extract in the case of solid culture. The crude enzyme can be obtained by solvent precipitation with alcohol or the like.

In order to obtain a more highly purified specimen, purification can be carried out by ultrafiltration, gel filtration chromatography, adsorption chromatography, ion exchange chromatography, isoelectric point fractionation, or a combination of these techniques.

be.

1. As a result of measuring the enzyme activity at the optimal temperature, 25 to 70°C as shown in Fig. 1, the highest activity was observed at 50°C.

2. Thermal stability, shown in Figure 2.

40-6 in 0.05' phosphate buffer (pH 5,0)
Treatment was carried out at 5°C for 5 minutes, and the residual activity at each temperature showed extremely excellent stability as shown in Figure 1.

3.Optimum-1 Shown in Figure 3.

The optimum value in Maccbain buffer is around 5.0.

4. pi4 stability, shown in Figure 4.

Although it was left for 24 hours at 30°C in mazukubain buffer, p
i (stable between 4.0 and 10.0).

5. Substrate specific phenol O guaiacol O) guaia hydroxyquinone ゛
1.1 Orthophenylenediamine 1
．． 7 Pyrogallol 0 Pyrocatechol 3.4NN Dimethylparaphenylenediamine 100.0 Activity measurement was performed using phenol; modified Klnd-King method; other substrates were measured using their respective absorption values (guaiacol 436 mμ, parahydroquinone 250 mμ, orthophenylenediamine,
440 mμ, pyrogallol, 437 mμ, pyrocatechol, 395 mμ, and dimethylnoclaphenylenediamine, 550 mμ), and expressed as a relative value when the activity against dimethyl paraphenylenediamine is set as 100.

This enzyme is considered to have little effect on monophenols.

6. Molecular weight The molecular weight was determined to be approximately 40,000 by filtration using Sephadex G-100.

7. 3c from the origin by single disk electrophoresis (5 mA/glue)
A single band was given at the W1 position.

Activity measurement method Ravin's method (HlA Ravln: Lanc
et al., 726 h1956) using dimethyl paraphenylenenoamine as the substrate.

Enzyme solution 11dKNN dimethylno! Add 2.5 d of a 1 mM solution of rough enylene diamine and react at 37°C for 60 minutes.
The absorbance of 07F1μ was measured. Enzyme unit is 550mμ
The activity that increases the absorbance of 1.0 under the above conditions was defined as 1 unit.

Ichiase is a highly stable enzyme, and as it shows its maximum activity at a higher temperature than the conventionally known polyphenol ocurasidase, it is considered to be a more suitable enzyme for practical use.

Next, the present invention will be explained in detail with reference to Examples.

Example 1 Soluble starch 1.5 t, NH4N0 30.5%, yeast extract 0.4 t, KH2PO40.1%, MgSO4
- 1001 t of culture medium containing 7H200.05 h, dimethylaniline 0.005 h, pi (6.0) was placed in a 500 d shoulder flask and sterilized in an autoclave at 120°C for 15 minutes.

This was cultured with commercially available potato dextrose agar for 2 days with shaking.

The polyphenol oxidase activity of the supernatant after culture was 4.
It was 5 units/d.

Example 2 ZnSO4”7H206,2 scraps 9/J to 100g
, FeSO4・5H206-3W/l, CuSO4・
5H200, 8"9/ 13-"SOa '7H20
Add 60g of inorganic salt water containing 6.3W/l, 1! The mixture was placed in a triangular 72-meter container and sterilized in an autoclave at 120°C for 30 minutes.

Mucor minihei AJ precultured in the same manner as in Example 1 was seeded onto the rice, stirred thoroughly, and then left to stand at 45°C for 5 days with occasional stirring. After culturing, add 1001 d of water and leave to extract at 4°C for 30 minutes.

The polyphenol oxidase activity of the extraction supernatant was 8.0 units/M.

NiH4-kg (20% saturation) was added and dissolved with stirring, and the resulting precipitate was removed by centrifugation at 11,000 rpm for 10 minutes. The obtained supernatant ammonium sulfate was further added to fl? '7
After stirring and dissolving (to achieve 50% saturation), the resulting precipitate was centrifuged at 10,000 rpm for 10 minutes.

The resulting precipitate was diluted with 0.05'lJ acid buffer p) 15.5
and dialyzed overnight against the same buffer.

This dialysate was equilibrated with 0.05 phosphate buffer.
EAE-Seven Fadex A-50 (3,2x3.20)
After adsorption, linear concentration gradient elution from O to 0.8'N5Ct of the same buffer was performed (recovery rate 95%).

Collect the active fraction/0.05'! J acid buffer-6
, 0. After dialysis, hydroxya matrix equilibrated with 0.05'lJ acid buffer p) 15.5,
It was adsorbed onto a column (1.5 x 30 cm), and linear concentration gradient elution from 0.05 to 1.0 of the same buffer was performed.

The active fractions were collected and dialyzed overnight, followed by DEAE-8ephsdex.
It was adsorbed onto A-50 (2, OX30cIn), and concentration gradient elution was performed with 0 to 0.5 MN5CL containing the same buffer. The active fractions were collected and separated using 5ephadexG-200 (
1,8 x 64 cm), and the active fraction was filtered through C
0 to 10 after adsorption to on A-8epha rose
Elution was at % methyl-α-D-mannopyranoside.

After concentrating the active fraction, further 5ephacryl S-400 (
1.8 x 64 cm) to obtain a purified enzyme sample. The total activity was 800 units, and the recovery rate was 10%.

This purified enzyme gave a single band in disk electrophoresis.

[Brief explanation of drawings]

FIG. 1 shows the optimum temperature of the thermostable prephenol oxidase of the present invention. FIG. 2 shows the heat resistance of the present invention, the thermostability of 791J phenol oxidase. FIG. 3 shows the optimum thermostable polyphenol oxidase of the present invention. FIG. 4 shows the stability of the thermostable polyphenol oxidase of the present invention.

Claims (1)

[Claims] A method for producing heat-stable polyphenol oxidase, which comprises culturing a heat-stable polyphenol oxidase-producing bacterium belonging to the genus Mucor, and collecting heat-stable polyphenol oxidase from the culture.