JPH0358269B2 - - Google Patents

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. Since the enzyme of the present invention has the property of acting on lignin to reduce the molecular weight or decompose it, it can be used in various steps in the manufacturing process of paper pulp using lignocellulose materials such as wood as raw materials. That is, it can be used to lower the molecular weight or decompose lignin in pulping processes, pulp bleaching processes, wastewater treatment processes, etc. Furthermore, in the saccharification of wood, the present invention can be applied to the field of so-called cellulosic biomass utilization, in which the action of cellulase is enhanced by decomposing lignin as a pre-stage treatment of saccharification. [Prior art] Lignocellulosic materials such as wood are inoculated with white rot fungi and cultured to decompose lignin.
Attempts have been made to produce cellulose pulp (see Japanese Unexamined Patent Publication No. 46903/1983). However, the white rot fungi used in this method also degrade coexisting carbohydrates, and when cellulase-deficient mutant strains are used, their original ability to degrade lignin is weakened. It has not yet become a reality. On the other hand, in order to solve these problems, attempts have been made to make the lignin-degrading enzymes of white-rot fungi act on lignocellulose materials to selectively decompose only lignin (Science Vol.
221, pp. 661-662, December 1983). This report mainly uses a lignin model compound as a substrate, and is the first in the world to isolate and purify a lignin-degrading enzyme. This enzyme is derived from Phanerochaete chrysosporium (Phanerochaete).
chrysosporium) is an extracellular enzyme produced by
The main characteristics are that it is an iron-containing enzyme, and that its molecular weight is
42,000, 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. Furthermore, two enzymes have been reported as extracellular enzymes produced by Phanerochaete chrysosporium (Federation of European Biochemical Societies Letters No. 169).
Vol. 2, 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, all 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 that The present inventors previously invented a lignin-degrading enzyme produced by a basidiomycete of the genus Corsicolor. (Special request 1986
-212888) 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 ± 5,000 [high performance liquid chromatography GRAPHIE (column product name TSK −3000SW manufactured by Toyo Soda)
4 of the lignin model compound
This enzyme has completely different properties from the extracellular enzyme produced by Phanerochaete chrysosporium, as it does not act on compounds in which the phenolic hydroxyl group at the position has become a methoxyl group. [Problems to be Solved by the Invention] The present invention is intended to solve the problem that when white rot fungi are allowed to act on lignocellulose materials, in addition to the decomposition of lignin, coexisting carbohydrates are also decomposed. It is a thing, and also,
The present invention is intended to solve the problem that hydrogen peroxide is required for the enzymatic action of conventionally known lignin-degrading enzymes, 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 lignin 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 objects will become clear from the description below. [Means for Solving the Problems] 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, which is a polymeric compound, is complex, and the chemical structure has not yet been determined, which is an obstacle, and in reality, there is very little knowledge regarding lignin-degrading enzymes. As a result of extensive research using milled lignin (MWL) and lignin model compounds as substrates, the present inventors have grown microorganisms and obtained extracellular crude enzymes from the culture through ammonium sulfate fractionation and gel filtration. , using an ion exchanger, etc., to obtain a highly refined product.
We have arrived at the present invention. That is, the present invention is a novel phenol oxidase produced from a microorganism and having the following properties. (1) Action () Acts on syringylglycerol-β-syringyl ether to produce 2,6-dimethoxyphenol. () 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 coloring substance is optimal around PH4.0 to 5.0, and its stable PH
is 7.0-8.0. (4) Optimal temperature and thermal stability The optimal effect is to change guaiacol into a colored substance at around 50°C, and it is stable under 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) This enzyme requires an enzyme for its action. (8) Molecular weight The molecular weight measured by high performance liquid chromatography is 55000±5000. Next, in order to confirm the action mechanism of this enzyme, we used syringylglycerol-β-syringyl ether (hereinafter referred to as SOS) as a lignin model compound.
The test was conducted using SOS was dissolved in 20mM phosphate buffer (PH4.0) containing a small amount of dioxane to make it 170mM.
Add 4 ml of the enzyme solution of the present invention obtained in Example 1 to 20 ml of solution.
was added aseptically and reacted at 25°C for 30 seconds for 8 hours. The obtained enzyme reaction solution was analyzed by TLC gas chromatography and mass spectrography. TLC analysis SOS spots disappeared 30 seconds after enzyme addition, and SOS was quickly decomposed. Gas chromatography and mass spectrometry analysis When the enzyme reaction solution obtained by reacting SOS and the enzyme of the present invention for 8 hours was analyzed by gas chromatography, peaks as shown in Figure 1 were observed, and the mass spectrometry showed that there were 2 peaks. , 6-dimethoxyphenol (Figure 2). From the above results, the enzyme of the present invention produces syringyl glycerol-β-syringyl ether.
It was confirmed that the allyl ether bond was cleaved. The enzyme of the present invention has no effect on substrates in which the phenolic hydroxyl group of SOS is changed to a 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 phenolic hydroxyl group-containing skeleton of lignin to cleave the β-allyl ether bond. As a result, a compound having a structure corresponding to 2,6-dimethoxyphenol and a phenolic hydroxyl group further bonded to a lignin basic skeleton at the 4-position is produced. In this way β−
Since a new phenolic hydroxyl group is generated by the cleavage of the allyl ether bond, the lignin is subsequently decomposed by the enzyme of the present invention and the reaction proceeds. In addition, since β-allyl ether bonds exist in approximately 50% of natural lignin, the enzyme of the present invention has β-allyl ether bonds.
- Cleavage of allyl ether bonds is of great significance in the decomposition of natural lignin. Example 1 containing the enzyme of the present invention as the main component (described later)
When the purified enzyme solution obtained was reacted with ground beech wood flour at 35℃ for 3 days, approximately 65% of the free phenol units in lignin were decomposed, and approximately 15% of the total aromatic nuclei were decomposed.
had undergone a change (decomposition) that caused it to lose its aromatic properties, and the alkyl-allyl ether bond had also been cleaved by about 10%. The enzyme of the present invention requires an enzyme 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 or stirring. The enzyme of the present invention has an isoelectric point and a molecular weight [high performance liquid chromatography (column product name
TSK -3000SW (manufactured by Toyo Soda)].

〔Example〕

EXAMPLES 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%, Peptone 1%, KH ₂ PO ₄ 0.15
%, MgSO ₄ 7H ₂ O 0.05%, Thiamine hydrochloride 0.0002
%, medium containing _{CuSO4.5H2O0.0016} % ₍ PH
5.0) Put 5 into a 10 volume jar fermenter,
After heat sterilizing at 120℃ for 20 minutes, the Coriolus buerncolor
Versicolor) FES1030 (K.Aoshima; Ps−4a),
IFO30340} and 28℃ for 8 days at 150 rpm (stirring speed). Culture was carried out under conditions of 5/min (aeration rate). After the culture is completed, the culture solution obtained by centrifugation is diluted with 0.9
After salting out with saturated ammonium sulfate, the mixture was dialyzed to obtain a crude enzyme solution. Next, after concentrating this crude enzyme solution by ultrafiltration,
Column chromatography was performed using Sephacryl S-200 (column: 2.4 x 100cm, eluent: 50mM Tris-HCl-100mM saline buffer PH
7.0, flow rate: 20ml/h, 1 fraction: 4ml),
Collect the active fraction and add it to 10mM phosphate buffer (PH7.0)
After adsorbing the enzyme through a DEAE Sephadex A-25 column (column: 1.5 x 45 cm, flow rate: 10 ml/h, 1 fraction: 2 ml) buffered with
Purified enzyme was obtained by elution with a phosphate buffer of pH 6.0 using an ionic strength gradient 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) Measurement method of titer: 200μ of the enzyme of the present invention is reacted at 37°C with a reaction solution consisting of 3.5ml of 1mM guaiacol solution prepared using 50mM acetate buffer (PH4.0), and the increase in absorbance at 420nm is measured. do. Enzyme activity is defined as an increase in absorbance of 0.01 per minute. (2) This enzyme acts on syringylglycerol-β-syringyl ether to produce 2,6-dimethoxyphenol. generate. (3) Acting on syringic acid, an elimination reaction of the carboxyl group occurs, producing 2,6-dimethoxy-p-benzoquinone and radicals accompanying the dehydrogenation reaction of the phenolic hydroxyl group of syringic acid,
Subsequently, 6-methoxy-4- was formed by radical polymerization.
(2',6'-dimethoxy-4'-carboxyphenoxy)benzoquinone (1,2) is produced. (4) Acts on guaiacol to produce a colored substance with maximum absorption around 420 nm. (5) 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. (6) Optimal PH The results of measuring the enzyme activity of the enzyme of the present invention using 50mM acetate buffer (PH3-5.5), 50mM phosphate buffer (PH5-9), and 50mM glycine buffer (PH8-11). As shown in Figure 3, the optimum pH is recognized to be around 4 to 5.0. (7) PH stability The enzyme of the present invention was left at 50°C for 30 minutes in 50mM acetate buffer (PH3-5.5), 50mM phosphate buffer (PH5-9), and 50mM glycine buffer (PH8-11) to determine enzyme activity. was measured. The results are shown in Figure 4, and the PH
Stability is around PH7-8. (8) Optimal temperature The activity of the enzyme of the present invention was measured by carrying out the enzyme reaction under different temperature conditions, as shown in FIG. 5, and the optimal temperature was found to be around 50°C. (9) Thermal stability 30-70℃ in 50mM phosphate buffer (PH7.0)
The enzyme of the present invention was left at each temperature for 10 minutes, and the enzyme activity was measured. The results are shown in FIG. 6, and in terms of its thermostability, the enzyme of the present invention is stable up to 50°C. (10) Influence of various substances The results of measuring the enzyme activity of the enzyme of the present invention by adding various substances are as follows. Note that the concentration added was 1 mM.

[Table] (11) Molecular weight () Approx. 55000±5000 [High performance liquid chromatography (column TSK 3000SW manufactured by Toyo Soda)
() approx. 63000±5000 [SDS-polyacrylamide electrophoresis method (molecular weight marker; manufactured by LKB,
Measured in the molecular weight range 12,300 to 78,000) () approx. 62,000 ± 10,000 [Sephacryl S-200
(Measured by gel filtration method using Fumarmacia)] (12) The isoelectric point is around 3 (measured by electrophoresis using Cervalite) (13) This enzyme is a copper-containing enzyme, and the aqueous solution is It has a blue color. (14) Oxygen is required for the action of this enzyme. (15) Amino acid composition A certain amount of enzyme (approximately 5 mg) was dissolved in 2 ml of 6N hydrochloric acid, and the enzyme was hydrolyzed by heating at 110°C for 24 hours in a vacuum sealed tube. , 835 type).
The results are as follows.

【table】 [Brief explanation of drawings]

Figure 1 is the gas chromatogram of the enzyme reaction solution, Figure 2 is the peak and mass spectrum of the existing standard, Figure 3 is the optimal pH, Figure 4 is the PH stability, Figure 5 is the optimal temperature, FIG. 6 is a graph showing thermal stability.

Claims (1)

[Claims] 1. Phenol oxidase produced from microorganisms and having the following properties: (1) Action: () Acts on syringyl glycerol-β-syringyl ether to produce 2,6-dimethoxyphenol. () 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 coloring substance is optimal around PH4.0 to 5.0, and its stable PH
is 7.0-8.0. (4) Optimal temperature and thermal stability The optimal effect is to change guaiacol into a colored substance at around 50°C, and it is stable under 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) This enzyme requires an enzyme for its action. (8) Molecular weight The molecular weight measured by high performance liquid chromatography is 55000±5000. 2. A method for producing phenol oxidase, which comprises culturing a phenol oxidase-producing bacterium belonging to the genus Coriolis in a medium, and collecting phenol oxidase having the following properties from the culture. (1) Action () Acts on syringylglycerol-β-syringyl ether to produce 2,6-dimethoxyphenol. () 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 coloring substance is optimal around PH4.0 to 5.0, and its stable PH
is 7.0-8.0. (4) Optimal temperature and thermal stability The optimal effect is to change guaiacol into a colored substance at around 50°C, and it is stable under 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) This enzyme requires an enzyme for its action. (8) Molecular weight The molecular weight measured by high performance liquid chromatography is 55000±5000.