JPS61285989A - Phenol oxidase and production thereof - Google Patents

Abstract

PURPOSE:To obtain a novel enzyme which makes lignin in ligno-cellulose lowmoleculate or decompose, by cultivating n fungus belonging to the genus Coriolus, capable of producing phenol oxidase, or a white rot mold similar to it in a medium and collecting the enzyme from the culture mixture. CONSTITUTION:A fungus (Coriolus versicolor IFO30340, IFO8754, etc.) belonging to the genus Coriolus, capable of producing phenol oxidase, or a white rot mold similar to it is multiplicated and a crude extracellular enzyme obtained from the culture mixture is highly purified by using ammonium sulfate fractiona tion, gel filtration, ion exchanger, etc., to give the title product. Novel phenol oxidase having no hydrogen peroxide dependence is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel phenol oxidase and a method for producing the same. The enzyme t-X of the present invention has the property of acting on IJ genin and reducing or decomposing it into Z-lower molecules γ
Therefore, glue gnocellulose materials such as wood can be used as a raw material in various steps in the filter paper pulp manufacturing process.

In other words, it can be used to decompose lignin into low molecular weight in pulping processes, valve bleaching processes, wastewater treatment processes, etc. Furthermore, in the saccharification of wood, it can be applied to the field of so-called cellulosic biomass utilization, where the lignin decomposition ability lulase action χ is increased as a pre-stage treatment of saccharification.

(Prior Art) Attempts have been made to inoculate lignocellulosic materials such as wood with white rot fungi and culture them to decompose lignin and produce cellulose pulp (Japanese Patent Laid-Open No. 50-469
See Publication No. 03]. For reference, the white rot fungi used in this method also degrade coexisting carbohydrates, and when a cellulase-deficient mutant strain is used, the original lignin-degrading ability is weakened. It has not yet been standardized.

On the other hand, in order to solve these problems, the lignin degrading enzyme χ of white rot fungi was used to act on lignocellulose materials.
Attempts have been made to selectively decompose only lignin (Science Vol. 221, Nos. 661-662).
Page, December 1983).

This report mainly uses the lignin model compound χ as a substrate, and is the first in the world to isolate and purify a lignin degrading enzyme. This enzyme is associated with Phanerochaetechry.
sosporium) is an extracellular enzyme produced by
The main feature is that it is an iron-containing enzyme, and its molecular weight is 42,000.
0, that hydrogen peroxide is required for enzymatic action, and that it has been confirmed that it acts on compounds in which the 4-position phenolic hydroxyl group of a lignin model compound has become a methoxyl group.

In addition, Phanerochaete chrysosporium (Phan
erochaete chrysosporium)
Two enzymes have been reported as extracellular enzymes produced by (Federation Φ op European Biochemical Society Letters Vol. 169,
No. 2, pp. 247-250, 1984). One of these enzymes has a molecular weight of 41,000 or less; the other enzyme has a molecular weight of 46,000 or less;
Furthermore, both enzymes are estimated to be iron-containing enzymes, hydrogen peroxide is required for enzyme action, and they act on compounds in which the 4-position phenolic hydroxyl group of a lignin model compound has become an ethoxyl group. It has been confirmed whether it will be done or not.

The present inventors previously invented a lignin-degrading enzyme produced by a basidiomycete of the genus Corsicolor. (Patent application No. 59-21288
No. 8) The main characteristics of this lignin-degrading enzyme are that it is a copper-containing enzyme, that its isoelectric point is around 3.5, that oxygen is required for enzyme action, and that its molecular weight is approximately 53,000±5oo.
o C high performance liquid chromatography (column product name TS
K[F]-3000SW prepared by Toyo Sou], and does not act on compounds in which the 4-position phenolic hydroxyl group of the lignin model compound has become a methoxyl group, and the above-mentioned Phanerochaete chrysosporium (Phanerocbatechrysos DOri
It is an enzyme with completely different properties from the extracellular enzyme produced by M. um ).

[Problems to be Solved by the Invention J The present invention solves the problem of causing the decomposition of coexisting carbohydrates in addition to the decomposition of lignin that occurs when white rot fungi'YIJ acts on cellulose materials.
This is intended to solve the problem that conventionally known lignin-degrading enzymes require hydrogen peroxide for their enzymatic action, which leads to high costs in industrial applications. .

An object of the present invention is to provide a novel phenol oxidase and a method for producing the same, and another object of the present invention is to provide a novel enzyme that degrades or decomposes ligogenin y in glinocellulose materials and a method for producing the same. be. Another purpose is to propose a new enzyme that is not dependent on hydrogen peroxide and a method for producing the same. Other objectives of each mata will become clear from the description below.

[Means to solve problem Z]

The present invention relates to a novel phenol oxidase and a method for producing the same.

It is known that lignin is well decomposed by basidiomycetes called wood-decaying fungi. However, the chemical structure of lignin, a polymeric compound, is complex, and the chemical structure has not yet been determined.
The reality is that there is very little knowledge regarding lignin degrading enzymes.

As a result of intensive research using milled lignin (MWL) and lignin model compound χ as a substrate, the present inventors have grown microorganism Z, obtained extracellular crude enzyme t1 from the culture, ammonium sulfate fraction, gel filtration, The present invention was achieved by obtaining a standard product by highly purifying it using an ion exchanger or the like.

Specifically, the present invention is a novel phenol oxidase produced from microorganisms and having the following properties.

(1) Action (1) Acts on syringylglycerol-β-syringyl ether to produce 2,6-simethoxyphenol.

(ll Acts on guaiacol to produce a colored substance χ having maximum absorption χ near 420 fLm.

(2) Substrate specificity It acts on syringyl glycero-β-syringyl ether, but it acts on compounds in which the 4-position phenolic hydroxyl group of syringyl glycerol-β-syringyl ether has become a methoxyl group. Doesn't work.

(3) Optimum pH and pH stability The effect of converting guaiacolta into a colored substance is optimal around pH 4.0 to 5.0, and its stable pH is around 7.0 to 5.0.
It is 0 to 8.0.

(4) Optimal temperature and thermal stability The effect of converting guaiacol into a colored substance at around 50C is optimal, and it is stable to heat up to 50C.

(5) The isoelectric point is around 3.

(6) This enzyme is a steel-containing enzyme and exhibits a deep blue color in aqueous solution.

(7) Oxygen is required for the action of this enzyme.

Next, in order to confirm the mechanism of action of this enzyme, seven tests were conducted using syringylglycerol-β-syringyl ether (hereinafter referred to as SO8) as a lignin model compound.

20mM'J acid buffer containing a small amount of dioxane Z (
4 ml'l of the enzyme solution of the present invention obtained in Example 1 (added aseptically and incubated at 25C for 30 seconds,
Allowed time to react.

The obtained enzyme reaction solution YTLC gas chromatography,
Analyzed by mass spectrography.

■ TLC analysis The SoS spot disappeared 30 seconds after adding the enzyme5.
osH is rapidly decomposed.

■ Gas chromatography and mass analysis The enzyme reaction solution obtained by reacting SO8 with the enzyme of the present invention for 8 hours was analyzed by gas chromatography, and the peaks shown in Figure 1 were observed. I
i 2.6-Simethoxyphenol (Figure 2).

From the above results, it was confirmed that the enzyme of the present invention cleaves the β-allyl ether bond in syringyl glycerol-β-syringyl ether.

It should be noted that the enzyme of the present invention has no effect on the substrate in which the phenol hydroxyl group of SO8 is changed to the methoxyl group.

This suggests that the enzyme of the present invention acts specifically on substrates having phenolic hydroxyl groups.

From these points, the mechanism by which the enzyme of the present invention acts on natural lignin is considered to be as follows.

The enzyme of the present invention acts specifically on the skeleton of lignin having a phenolic hydroxyl group and cleaves the β-allyl ether bond. As a result, a compound having a structure χ corresponding to 2,6-simethoxyphenol and a phenolic hydroxyl group further bonded to a lignin basic skeleton at the 4th position is produced. In this way, phenolic hydroxyl groups are newly generated by the cleavage of the β-allyl ether bond, so that the enzyme of the present invention continues to decompose the lignin and the reaction proceeds.

Note that in natural lignin, approximately 50% of β-allyl ether bonds are present, and therefore, the ability of the enzyme of the present invention to cleave β-allyl ether bonds is extremely significant in the decomposition of natural lignin.

When the purified enzyme solution used in Example 1 (see below), which is the main component of the enzyme of the present invention, was reacted with ground beech powder at 35C for 3 days, about 65 units of free phenol units in the lignin were decomposed. Approximately 15 of the total aromatic nuclei underwent a change (decomposition) that resulted in the loss of aromatic properties, and approximately 101 of the alkyl-allyl ether bonds were also cleaved at °C.

The enzyme of the present invention requires oxygen χ for the reaction, but the reaction is preferably carried out in a pure oxygen atmosphere rather than in the air, and the enzyme reaction rate can be further increased by shaking and stirring.

The enzyme of the present invention has an isoelectric point and a molecular weight (high performance liquid chromatography (column brand name: TSK[F]
-30003W manufactured by Toyo Soda].

5 Also, during enzyme purification, the prior patented enzyme was DEAE.
After adsorbing the enzyme on a Sephadex A-25 column, 10mM at pH 4.0! The enzyme of the present invention is a novel enzyme, with the difference that it elutes with a J-citrate buffer, whereas the enzyme of the present invention does not.

The present invention also resides in a method for producing phenol oxidase, which is characterized by culturing a phenol oxidase-producing bacterium belonging to the genus Versicolor in a Z medium and collecting the phenol oxidase from the culture.

The enzyme-producing microorganism of the present invention is particularly preferably a phenol oxidase-producing microorganism belonging to the genus Corsicolor, but any similar white-rot fungi may be used.

For example, Coriolus guercicolor (Coriolus guercicolor) is a phenol oxidase-producing bacterium belonging to the Coriolus genus.
iolus versicolor) IFO3034
0, IFO8754, etc., but the present invention is not limited to the microorganisms exemplified in the specification. The culture form of the above-mentioned bacterial cells may be either liquid culture or solid culture. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon source (e.g., wood flour, glucose, sucrose, lactose, molasses, etc.).In particular, when cultured in a wood flour medium containing lignocellulose components, the phenol oxidase of the present invention 'Y
It is advantageous because it can be produced with high purity. As the nitrogen source, any available nitrogen compound may be used, such as bezuton, meat egisu, soybean flour, casein hydrolyzate, and the like. In addition, salts such as phosphate, sulfur, salt, magnesium, calcium, potassium, sodium, copper, manganese, zinc, etc. are used as necessary. In particular, this enzyme is a copper-containing enzyme, and the addition of copper salts. is valid. The culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce the phenol oxidase, but is preferably 23-27
C degree is good (and the culture time varies depending on the conditions,
Liquid culture takes 5 to 10 days, solid culture takes about 1 to 3 months. Next, phenol oxidase is collected from the culture obtained in this way, but since this enzyme is mainly secreted outside the bacterial cells, it is difficult to collect this enzyme.
ce'! In liquid culture, the culture solution removed by centrifugation or the like is used, and in solid culture, the enzyme-containing solution is concentrated using the extract extracted from the culture, or the enzyme-containing solution is concentrated (without soluble salts). , for example, by salting out using ammonium sulfate, or by salting out using a hydrophilic solvent such as methanol,
The enzyme may be precipitated by adding ethanol, acetone, isopropanol, or the like. Next, this precipitate is dissolved in water or a buffer solution, and the low molecular weight impurity Z can be removed by dialysis using a semipermeable membrane. Phenol oxidase is also purified by chromatography using an adsorbent or gel filtration agent. Furthermore, the enzyme solution obtained by these means is purified by treatments such as vacuum concentration, ultrafiltration membrane concentration, and freeze-drying to obtain phenol oxidase.

[Example J The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Glucose 3%, Buton 1, KH2PO40.15
H, MgSO4.7 H2O0.05 H, thiamine hydrochloride 0.0002 H, Cu SO4' 5. H200,
0016% Y-containing medium (pH 5,0) 51YlO
Place in a 1-volume jar fermenter and heat sterilize at 120C for 20 minutes, then add Coriolus Versicolor
FES 1030 (K, Aoshima; P
s-4a), IFo 30340) w inoculation 2
8tl:', 8 days, 150 rpm (stirring speed). 51
Culture was carried out for 7 times under the condition of /min (aeration rate).

After completion of the culture, the culture F solution obtained by centrifugation was salted out with 9 saturated ammonium sulfate, and then dialyzed to obtain a crude enzyme solution.

Next, this crude enzyme solution was concentrated by χ ultrafiltration, and then subjected to column chromatography using Sephacryl S-200'.
(Column: 2.4 x 100cWL, Eluent: 50mM Tris-HCl-100mM NaCl buffer pH 7
, 0, flow rate: 20g/h, 17 lactation: 4-), collect the active fraction and add it to 10mM phosphate buffer (pH 7,0)
DEAE Sephadex A-25 column buffered with
Column: 1.5X45cR, flow rate: 10d/h, 1 fraction: 2g) After adsorbing the enzyme, p
The purified enzyme χ was eluted with He,o phosphate buffer using an ionic strength gradient χ of 0 to 500 mM sodium chloride.

Next, the method for measuring the titer and properties of the enzyme of the present invention will be described.

(1) Method for measuring titer A reaction solution consisting of 3.5-mM guaiacol solution prepared in 50mM acetate buffer (pH 4,0)' was reacted with 200 μl of the enzyme of the present invention at χ37C, and the absorbance was 420.
Measure the increase in absorbance at 3 m.

It is assumed that the absorbance increases by 0.01 per minute due to enzyme activity at position t11.

(2) This enzyme acts on syringylglycerol-β-syringyl ether to produce 2,6-cymethoxyphenol H.

(3) Acting on syringic acid, a carboxyl group elimination reaction occurs, resulting in 2,6-simethoxy p-banzoquinone,
In addition, radical polymerization was performed by dehydrogenation of the 7-enolic aqueous group of syringic acid, followed by radical polymerization of 6-
Medoxy 4- (2', 6'-dimethoxy-4'-
Carboxyphenoxy)benzoquinone (1,2) is produced.

(4) Acts on guaiacol to produce a colored substance with maximum absorption near 420 nm.

<5) It acts on syringyl glycero-β-syringyl ether, but syringyl glycerol-
It does not act on compounds in which the 7-enolic hydroxyl group at the 4-position of β-syringyl ether has become a methoxyl group.

(6) Optimal pH 50mM acetate buffer (pH3-5.5), 50mM phosphate buffer (pH5-9), 50mM glycine buffer (pH3-5.5), 50mM glycine buffer (pH5-9),
The results of measuring the enzyme activity of the enzyme of the present invention using H8-11) are as shown in Figure 3, and the optimum pHtf
Approximately 14 to 5.0.

(71 pH stability 50mM acetate buffer g (pH 3-5.5), 50mM
The enzyme activity of the present invention vsoC was allowed to stand in phosphate buffer (pH 5-9) and 50 mM glycine buffer (pH 8-11) for 30 minutes, and the enzyme activity was measured.

The results are as shown in Figure 4, and the pH stability H
The pH is around 7-8.

(8) Optimum Temperature The activity of the enzyme of the present invention was measured by changing the temperature conditions χ and enzymatic reaction. The results are as shown in FIG. 5, and the optimum temperature was found to be around H50tT.

(9) Thermostability 30-70C in 50mM phosphate buffer (pH 7,0)
Release the enzyme of the present invention for 10 minutes at each temperature. The enzyme activity Z was measured.

The results are shown in FIG. 6, and the enzyme of the present invention is stable up to 1 soc in terms of its thermostability.

α1 Effect of various substances The results of measuring the enzyme activity of the enzyme of the present invention with the addition of various substances are as follows. Note that the added concentration is 1 mM.

αυ Molecular weight (1) Approximately 55,000 ± 5oooc Measured by high performance liquid chromatography (column TSK [F] 3000 SW manufactured by Toyo Soda) (Ill approximately 63,000 ± 5,000 (5DS-polyacrylamide electrophoresis (molecular weight marker; LKB)
manufactured by Sephacryl S
-200 (manufactured by Pharmacia)] 0 The isoelectric point is around 3 (measured by electrophoresis using Serparaite V) 3 This enzyme is a copper-containing enzyme, and the aqueous solution has a deep blue color. present.

Negative → W for the action of this enzyme! Requires accumulation.

[Brief explanation of drawings]

@Figure 1 is the gas chromatography of the enzyme reaction solution, Figure 2 is the mass of peak and existing standards, Figure 3 is the optimum pLi4 figure is the pH stability, Figure 5 is the optimum temperature, and Figure 5 is the optimum temperature. Figure 6 is a graph showing thermal stability.

Claims (1)

[Claims] 1. Phenol oxidase produced from microorganisms and having the following properties: (1) Action: (i) Acts on syringyl glycerol-β-syringyl ether to produce 2,6-dimethoxyphenol. (ii) acts on guaiacol to produce a colored substance with maximum absorption around 420 nm; (2) Substrate specificity It acts on syringylglycerol-β-syringyl ether, but it does not act on compounds in which the 4-position phenolic hydroxyl group of syringylglycerol-β-syringyl ether has become a methoxyl group. do not. (3) Optimal pH and pH stability The effect of converting guaiacol into a colored substance is optimal around pH 4.0 to 5.0, and its stable pH is 7.0.
~8.0. (4) Optimal temperature and thermal stability The effect of converting guaiacol into a colored substance at around 50°C is optimal, and it is stable to heat up to 50°C. (5) The isoelectric point is around 3. (6) This enzyme is a copper-containing enzyme, and its aqueous solution exhibits a deep blue color. (7) Oxygen is required for the action of this enzyme. 2. A method for producing phenol oxidase, which comprises culturing a phenol oxidase-producing bacterium belonging to the genus Coriolus in a medium, and collecting the phenol oxidase from the culture.