JPH0746989B2 - Lignin degrading enzyme and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel lignin degrading enzyme and a method for producing the same. The enzyme of the present invention acts on lignin and has the property of degrading or degrading it.
It can be used in various processes in the paper pulp manufacturing process using a lignocellulosic material such as wood as a raw material. For example, it can be used for lowering or degrading the molecular weight of lignin in the pulping process, pulp bleaching process, wastewater treatment process and the like. Further, in the saccharification of wood, it can be applied to the field of so-called cellulosic biomass utilization in which cellulase action is enhanced by decomposing lignin as a treatment before saccharification.

[Prior Art] A method has been proposed in which a lignocellulosic material such as wood is inoculated with a white rot fungus and cultured to decompose lignin to produce cellulose pulp (Japanese Patent Laid-Open No. 50-46903).
(See the official gazette). However, the white-rot fungus of this method also decomposes coexisting carbohydrates, and when a cellulase-deficient mutant strain is used, there is a problem that the original lignin degrading power is weakened and the like. It has not been done.

On the other hand, in order to solve such a problem, lignin-degrading enzyme of white-rot fungus acts on the lignocellulosic material,
Attempts have been made to selectively decompose only lignin (Science Vol. 221, 661-662, 1983).
December year). This enzyme is an extracellular enzyme produced by Phanerochaete chrysosporium, and its main characteristics are that it is an iron-containing enzyme, that it has a molecular weight of 42,000, and that it requires hydrogen peroxide for its action. It has been confirmed that the 4-position phenolic hydroxyl group of the lignin model compound acts on the compound having a methoxy group.

In addition, Phaneroc chrysosporium
haete chrysosporium) as an extracellular enzyme produced by
Two enzymes have been reported (Federation of 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, and it is presumed that each enzyme is an iron-containing enzyme, that hydrogen peroxide is required for the enzyme action, and phenol at the 4-position of the lignin model compound It has been confirmed that the functional hydroxyl group acts on the compound having an ethoxy group.

[Problems to be Solved by the Invention] The present invention is intended to solve the problem of causing the decomposition of coexisting carbohydrates in addition to the decomposition of lignin that occurs when white-rot fungi act on a lignocellulosic material. . Further, the present invention intends to solve the problem that hydrogen peroxide is necessary for the enzymatic action of a conventionally known lignin-degrading enzyme, which causes a problem of cost increase in industrial application.

Therefore, an object of the present invention is to provide a novel lignin degrading enzyme and a method for producing the same, and another object is to provide a novel enzyme mainly for reducing or degrading lignin in a lignocellulosic material and a method for producing the same. Especially. Another object is to propose a novel enzyme that does not depend on hydrogen peroxide and a method for producing the same.

[Means I for Solving Problems] The present invention relates to a novel lignin degrading enzyme and a method for producing the same.

Lignin is known to be well decomposed by basidiomycetes called wood-destroying fungi. However, since the chemical structure of lignin, which is a high molecular compound, is complicated and its chemical structure has not been determined even now, the fact is that there is very little knowledge about lignin-degrading enzymes.

As a result of intensive studies using bleached kraft pulp effluent and lignin model compounds as substrates, the present inventors have found that they are different from the types of bacteria that have been studied in the past, and are one species of wood-destroying fungi. A certain moss mushroom (Lentites betulina) is proliferated, and the extracellular enzyme obtained from the culture is highly purified using ammonium sulfate fractionation, ion exchanger, etc. The present invention has been reached.

Kaigaratake (Lenzites betul)
ina)) is a bacterium belonging to the genus Lentites, and is the only strain belonging to the genus Lentites, which is called the Japanese name Kaigaratake, scientific name Lentites betulina, and is a genus of one genus.

The main characteristics of the lignin-degrading enzyme of the present invention are that it is a copper-containing enzyme, that its isoelectric point is around 3.5, that oxygen is required for its enzymatic action, and that its molecular weight is 65,000 ± 5000 (by SDS electrophoresis). That is, the phenolic hydroxyl group at the 4-position of the lignin model compound does not act on the compound having a methoxyl group.
It is an enzyme with completely different properties from the extracellular enzyme produced by Phanerochaete chrysosporium.

The lignin-degrading enzyme of the present invention is a novel lignin-degrading enzyme having the following properties, which is produced from Pleurotus ostreatus.

(1) Action (i) Syringyl Glycerol-acts on β-syringyl ether to produce 2,6-dimethoxyphenol.

(ii) Reduce the molecular weight of lignin contained in the effluent from the multistage bleaching process of chemical pulp production.

(2) Substrate specificity Acts on syringyl glycerol-β-syringyl ether, but acts on compounds in which the 4-position phenolic hydroxyl group of syringyl glycerol-β-syringyl ether becomes a methoxyl group. do not do.

(3) Optimum pH and pH stability The optimum pH is around pH 4.0 to 5.0, and the stable pH is 7.5 to 9.0.

(4) Optimum temperature and thermal stability The optimum temperature is around 60 ℃, and it is stable to heat up to 50 ℃.

(5) The isoelectric point is around 3.5.

(6) Molecular weight 65,000 ± 5000 (by SDS electrophoresis) (7) This enzyme is a copper-containing enzyme, and its aqueous solution exhibits deep blue color.

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

In order to confirm the action mechanism of this enzyme, a test was conducted using syringyl glycerol-β-syringyl ether (hereinafter referred to as SOS) as a lignin model compound.

200 mM containing 0.4 M SOS dissolved in a small amount of dioxane
A reaction solution containing 0.4 ml of the enzyme solution of the present invention obtained in Example 1 in 4 ml of sodium acetate buffer (pH 4.5) was reacted at 25 ° C for 30 seconds and 8 hours.

The obtained enzyme reaction solution was subjected to TLC, gas chromatography,
It was analyzed by mass spectrometry.

TLC analysis The SOS spot disappears 30 seconds after addition of the enzyme, and SOS is rapidly decomposed.

Gas chromatography and mass spectroscopic analysis When an enzyme reaction solution obtained by reacting SOS with the enzyme of the present invention for 8 hours was analyzed by gas chromatography, a peak as shown in Fig. 1 was observed, and peak I was 2, from the mass spectrum. It was found to be 6-dimethoxyphenol (Fig. 2).

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

The enzyme of the present invention has no effect on a substrate in which the phenolic hydroxyl group of SOS is changed to a methoxyl group. From this, it is considered that the enzyme of the present invention specifically acts on the substrate having a phenolic hydroxyl group.

[Action] The mechanism by which the enzyme of the present invention acts on natural lignin is considered as follows.

The enzyme of the present invention specifically acts on the skeleton of lignin having a phenolic hydroxyl group to cleave the β-allyl ether bond. As a result, a compound having a structure corresponding to 2,6-dimethoxyphenol and further having a phenolic hydroxyl group bonded to the lignin substrate skeleton at the 4-position is produced. Thus, a new phenolic hydroxyl group is generated by the cleavage of the β-allyl ether bond, so that the lignin is subsequently decomposed by the enzyme of the present invention and the reaction proceeds.

[Means for Solving Problems II] In the natural lignin, β-allyl ether bonds have about
Since it is present at 50%, it is extremely significant that the enzyme of the present invention cleaves the β-allyl ether bond in the decomposition of natural lignin.

Although the enzyme of the present invention requires oxygen during the reaction, the reaction rate in the pure oxygen atmosphere is preferably shaken and stirred in the atmosphere rather than in the air, whereby the enzyme reaction rate can be further increased.

The present invention also cultivates a lignin-degrading enzyme-producing bacterium, which is composed of a moss mushroom (Lentites beturina) belonging to the genus Lenzites, in a medium, and collects the lignin-degrading enzyme having the properties (1) to (8) from the culture And a method for producing a lignin-degrading enzyme.

The culture form of the above-mentioned bacterial cells may be either liquid culture or solid culture. As the nutrient source for the medium, those commonly used for culturing microorganisms can be widely used.
The carbon source may be any assimilable carbon source, and for example, wood flour, glucose, choucloth, lactose, molasses and the like are used. In particular, it is advantageous to culture in a wood flour medium containing a lignocellulosic component because the lignin-degrading enzyme of the present invention can be produced in high purity. As the nitrogen source, any available nitrogen compound may be used, and for example, peptone, meat extract, soybean powder, casein hydrolyzate and the like are used. Others, phosphate, sulfuric acid, salt, magnesium,
Salts of calcium, potassium, sodium, copper, manganese, zinc and the like are used as necessary. Especially, this enzyme is a copper-containing enzyme, and addition of a copper salt is effective.

The culturing temperature can be appropriately changed within a range in which the bacterium grows and produces the lignin-degrading enzyme, but it is preferably about 23 to 27 ° C. The culture time varies depending on the conditions, but it is 5 to 10 days for liquid culture and 1 to 3 months for solid culture.

Then, the lignin-degrading enzyme is collected from the thus obtained culture. Since this enzyme is mainly secreted outside the cells, the cells should be collected in liquid culture in order to collect this enzyme. Using the culture solution removed by centrifugation, etc., or the extract solution extracted from the culture in solid culture,
If the enzyme-containing solution is concentrated, or salted out without concentration with a soluble salt such as ammonium sulfate, or if the enzyme is precipitated by adding a hydrophilic solvent such as methanol, ethanol, acetone, or isopropanol. good.

The precipitate can then be dissolved in water or buffer and dialyzed through a semipermeable membrane to remove low molecular weight impurities. Further, the lignin-degrading enzyme is purified by chromatography with an adsorbent or a gel filter. Further, the enzyme solution obtained by these means is concentrated under reduced pressure,
The lignin-degrading enzyme is obtained by purification by ultrapermeabilization and further freeze-drying.

[Examples] The present invention will be described below with reference to Examples.

Example 1 Glucose 3%, Peptone 1%, KH ₂ PO ₄ 0.15%, MgSO ₄
・ 7H ₂ O 0.05%, thiamine hydrochloride 0.0002%, CuSO ₄ 5H ₂ O0.0
5 l of a medium (pH 5.0) containing 016% was placed in a 10 l jar fermenter and sterilized by heating at 120 ° C. for 20 minutes, and then the oyster mushroom {Lenzites betulin (Lenzites betulin
a)} and 28 rpm, 8 days, 50 rpm (stirring speed). 5l /
Culture was performed under the condition of minute (aeration amount).

After completion of the culture, the mixture was passed through a cloth to remove the bacteria to obtain a crude enzyme solution.

Next, this crude enzyme solution was mixed with 10 mM potassium phosphate buffer (pH 7.
It was passed through a DEAE Toyopearl column (20 × 5 cm) buffered with 0). Enzymes are adsorbed, so after washing away unadsorbed impure proteins with the same buffer, it contains 30% ammonium sulfate
Elution was performed with 10 mM potassium phosphate buffer (pH 7.0). The active fractions were collected, the impure protein precipitated with 50% saturated ammonium sulfate was removed by centrifugation, and the portion precipitated with 70% saturated ammonium sulfate was collected by centrifugation. It was dissolved in a small amount of water and dialyzed against 2 l of distilled water. The dialysis solution is changed several times a day,
After dialysis overnight, it was concentrated by ultrafiltration (Amicon, PM-10). The procedure of adding 30 mM potassium phosphate buffer (pH 7.0) and performing an ultrafiltration was repeated twice, and then loaded on a DEAE Toyopearl column (1.5 × 20 cm) equilibrated with the same buffer. After washing with 400 ml of the same buffer, elution was performed with the same buffer of 45 mM, and 2.2 ml was fractionated. A portion of each of the active fractions was subjected to polyacrylamide gel electrophoresis to examine homogeneity, and uniform fractions were collected based on the results of electrophoresis.
The homogeneity of this fraction was confirmed by polyacrylamide gel electrophoresis containing SDS.

Next, the assay method and properties of the enzyme of the present invention will be described.

(1) Measurement of titer 200 mM containing 0.4 mM SOS dissolved in a small amount of dioxane
A reaction solution containing 0.4 ml of the enzyme solution of the present invention in 4 ml of a sodium acetate buffer solution (pH 4.5) is kept at 30 ° C., and the increase in absorbance at 290 mm is measured.

Enzyme activity is defined as a unit of 0.01 increase in absorbance per minute.

(2) This enzyme acts on syringyl glycerol-β-syringyl ether to give 2,6-dimethoxyphenol. To generate.

(3) Reaction on silingaic acid to cause elimination reaction of carboxyl group to generate radicals associated with dehydrogenation reaction of 2,6-dimethoxy-p-benzoquinone and silingic acid's phenolic hydroxyl group, followed by radical polymerization To produce 6-methoxy-4- (2 ', 6'-dimethoxy-4'-carboxyphenoxy) benzoquinone (1,2).

(4) Acting on syringyl glycerol-β-syringyl ether, syringyl glycerol-β-
It does not act on compounds in which the 4-position phenolic hydroxyl group of syringyl ether has become a methoxyl group.

(5) Optimal pH 50 mM acetate buffer (pH 3 to 5.5), 50 mM phosphate buffer (pH 5
~ 9), the enzyme activity against the enzyme of the present invention was measured using 50 mM Tris-HNO ₃ buffer (pH 8-9), and the results are shown in Fig. 3, and the optimum pH is recognized to be around 4.0-5.0. .

(6) pH stability 50 mM acetate buffer (pH 3 to 5.5), 50 mM phosphate buffer (pH 5
.About.9), the enzyme of the present invention was allowed to stand in 50 mM glycine buffer (pH 8 to 10) at 50.degree. C. for 30 minutes to measure the enzyme activity.

The results are shown in Fig. 4 and the pH stability is pH.
It is around 7.5-9.

(7) Optimum temperature As a result of measuring the activity of the enzyme of the present invention by carrying out an enzymatic reaction under different temperature conditions, it is confirmed that the optimum temperature is around 60 ° C as shown in Fig. 5.

(8) Thermostability The enzyme of the present invention was allowed to stand for 60 minutes at a temperature of 30 to 70 ° C in a 50 mM phosphate buffer (pH 7.0), and the enzyme activity was measured.

The results are shown in FIG. 7, and the thermostability of the enzyme of the present invention is stable up to 50 ° C.

(9) Effect of various substances The results of measuring the enzyme activity of the enzyme of the present invention by adding various substances are as follows. The addition concentration is 1 mM.

(10) Molecular weight about 65,000 ± 5,000 [SDS-polyacrylamide gel electrophoresis (molecular weight marker; LKB, molecular weight range 12,300 to 7
8,000)] (11) Isoelectric point is around 3.5 (measured by isoelectric focusing method using ampholine) (12) This enzyme is copper-containing nitrogen, and the solution shows deep blue color.

(13) This enzyme requires an enzyme for its action.

Example 2 Kraft cooking of Douglas-fir chips by a conventional method,
Unbleached pulp having a cooking degree (kappa number) of 30 was obtained, and the pulp was subjected to multi-stage bleaching (CEHED) to obtain a bleached pulp having a whiteness of 81 points. The primary alkaline extract after chlorination was treated with 5N sulfuric acid.
The pH was adjusted to 5 and 1 ml of the enzyme solution obtained in the present invention was added to 10 ml of this preparation.
After the reaction was carried out at 37 ° C. for 5 hours, 2 ml was put on a column of Sephadex G-50 (diameter 24 × 250 mm) and eluted with distilled water. When water was added in place of the enzyme solution as a control in the same manner, lignin having a molecular weight of about 1,000 to 5,000 was reduced to a low molecular weight by the enzyme treatment, as shown in FIG.

[Brief description of drawings]

Fig. 1 shows the gas chromatogram of the enzyme reaction solution, Fig. 2 shows the mass spectrum of peak I, and Fig. 3 shows the optimum pH, 4
FIG. 5 is a graph showing pH stability, FIG. 5 is an optimum temperature, and FIG. 6 is a graph showing thermal stability. FIG. 7 is a graph of gel hypercurve when the enzyme of the present invention is applied to pulp bleaching wastewater.

Claims (2)

[Claims] 1. A lignin-degrading enzyme which is produced from Pleurotus cornucopiae (Lentites beturina) and has the following properties: (1) action (i) action on syringyl glycerol-β-syringyl ether to give 2,6-dimethoxyphenol. To generate. (ii) Reduce the molecular weight of lignin contained in the effluent from the multistage bleaching process of chemical pulp production. (2) Substrate specificity Acts on syringyl glycerol-β-syringyl ether, but acts on compounds in which the phenolic hydroxyl group at the 4-position of syringyl glycerol-β-syringyl ether becomes a methoxyl group. do not do. (3) Optimum pH and pH stability The optimum pH is around pH 4.0 to 5.0, and the stable pH is 7.5 to 9.0. (4) Optimum temperature and thermal stability The optimum temperature is around 60 ° C, which is stable to heat up to 50 ° C. (5) The isoelectric point is around 3.5. (6) Molecular weight 65,000 ± 5000 (by SDS electrophoresis) (7) This enzyme is a copper-containing enzyme, and its aqueous solution exhibits deep blue color. (8) Oxygen is required for the action of this enzyme. 2. A lignin-degrading enzyme-producing bacterium, which is composed of oyster mushroom (Lentites beturina), is cultivated in a medium, and the culture has the following properties: (1) action (i) syringyl glycerol-β-syringyl ether. To produce 2,6-dimethoxyphenol. (ii) Reduce the molecular weight of lignin contained in the effluent from the multistage bleaching process of chemical pulp production. (2) Substrate specificity Acts on syringyl glycerol-β-syringyl ether, but acts on compounds in which the phenolic hydroxyl group at the 4-position of syringyl glycerol-β-syringyl ether becomes a methoxyl group. do not do. (3) Optimum pH and pH stability The optimum pH is around pH 4.0 to 5.0, and the stable pH is 7.5 to 9.0. (4) Optimum temperature and thermal stability The optimum temperature is around 60 ° C, which is stable to heat up to 50 ° C. (5) The isoelectric point is around 3.5. (6) Molecular weight 65,000 ± 5000 (by SDS electrophoresis) (7) This enzyme is a copper-containing enzyme, and its aqueous solution exhibits deep blue color. (8) Oxygen is required for the action of this enzyme. A method for producing a lignin-degrading enzyme, comprising collecting a lignin-degrading enzyme having: