JPH0671424B2 - 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] It has been proposed that a lignocellulosic material such as wood be inoculated with white rot fungus and cultured to decompose lignin to produce cellulose pulp (see Japanese Patent Laid-Open No. 5069469/50). 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, etc. 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,
It has also been proposed to selectively decompose only lignin (Science Vol. 221, 661-662, 198).
December 3rd 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. Both enzymes are presumed to be iron-containing enzymes, hydrogen peroxide is required for the enzyme action, and lignin model compound 4
It has been reported that the compound has properties such as the fact that it is confirmed that the phenolic hydroxyl group at the position has an action 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. Moreover, hydrogen peroxide is necessary for the enzymatic action of the conventionally known lignin-degrading enzyme, and it is intended to solve the problem of high cost 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.

Among the fungi known as wood-destroying fungi, the present inventors have used syringyl glycerol-β, a typical model compound of the main skeleton structure of kraft pulp effluent and lignin.
As a result of searching for the enzyme using the enzymatic activity of degrading -silylgyl ether as an index, the extracellular enzyme obtained from the culture of the genus Kawara was highly purified to obtain a standard product.

As a lignin-degrading enzyme-producing bacterium, Arachakaratake, which belongs to the genus Kawaratake, that is, Coriolus hirsutus (Cori
olus hirsutus) IFO4917, C. hirsutus IFO4920, C. hirsutus IFO7038, etc. can be used.

The main features 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 it requires an enzyme for its enzymatic action, and that it has a molecular weight of approximately 63,000 ± 5,000 (by SDS electrophoresis). ], 4 of the lignin model compound
The phenolic hydroxyl group at the position does not act on the compound converted to a methoxyl group, and the above-mentioned Phanerochaete chrysosporiu
It is an enzyme with completely different properties from the extracellular enzyme produced by m).

The lignin-degrading enzyme of the present invention has the following properties.

(1) Operation (i) Acts on syringyl glycerol-β-syringyl ether to produce 2,6-dimethoxyphenol.

(Ii) Reduce the molecular weight of lignin contained in the effluent from the multi-stage 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 4.5 to 5.0, and the stable pH is 6.0 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) This enzyme is a copper-containing enzyme and its aqueous solution exhibits a deep blue color.

(7) The action of this enzyme requires an enzyme.

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

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

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 the 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. From the mass spectrum, peak I was 2,
It was found to be 6-dimethoxyphenol (second
Figure).

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 having a phenolic hydroxyl group at which the lignin basic skeleton is further bonded at the 4-position is produced. Thus, a new phenolic hydroxyl group is generated by the cleavage of the β-allyl ether bond, and the lignin is subsequently decomposed by the enzyme of the present invention and the reaction proceeds.

[Means for Solving Problems II] In natural lignin, β-allyl ether bond is 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.

When the purified enzyme solution obtained in Example 1 (described later) containing the enzyme of the present invention as a main component was reacted with ground beech ground wood at 35 ° C. for 3 days, about 65% of free phenol units in lignin were separated. In addition, about 15% of all aromatic nuclei undergo a change (decomposition) that causes loss of aromaticity, and the alkyl ether-allyl ether bond also has 10
It was cleaved about%.

The enzyme of the present invention requires an enzyme during the reaction, but the reaction can be further performed in a pure enzyme atmosphere rather than in the air, and the enzymatic reaction rate can be further increased by shaking or stirring.

In addition, the present invention is characterized in that a lignin-degrading enzyme having the above-mentioned properties (1) to (7) is collected from a culture by culturing a lignin-degrading enzyme oxidase-producing bacterium, which belongs to the genus Kawaratake, in the medium. It exists in the method for producing degrading enzymes.

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. Although the culture time varies depending on the conditions, it is 5 to 10 days for liquid culture and about 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,
The enzyme-containing solution may be concentrated or may be salted out without concentration with a soluble salt such as ammonium sulfate, or a hydrophilic solvent such as methanol, ethanol, acetone,
The enzyme may be precipitated by adding isopropanol or the like.

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 [Experiment 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
A medium (pH 5.0) 5 containing 016% was placed in a 10-volume jar fermenter and sterilized by heating at 120 ° C. for 20 minutes, and then Arakawakawatake (Coriolus hirstas (Coriolus h
irsutus) IFO4917, (K.Aoshima; Ps-4a)} and inoculated 28
C, 8 days, 150 rpm (stirring speed). 5 / min (aeration amount)
The culture was performed under the conditions of.

After the completion of the culture, the product was passed through a cloth to remove the bacteria and a crude enzyme solution was obtained.

Next, this crude enzyme solution was mixed with 10 mM potassium phosphate buffer (pH 7.
It was passed through a DEME Toyopearl column (20 × 5 cm) buffered with 0). Since the enzyme is adsorbed, unpurified impure protein was washed away with the same buffer, and then eluted with 10 mM potassium phosphate buffer (pH 7.0) containing 30% ammonium sulfate. Collect the active fractions, 5
Impure proteins that precipitated with 0% saturated ammonium sulfate were removed by centrifugation and the 70% saturated ammonium sulfate precipitated portions were collected by centrifugation. It was dissolved in a small amount of water and dialyzed against distilled water 2. The dialysate was exchanged several times a day, dialyzed overnight, and then concentrated by ultrafiltration (Amicon, PM-10). After 60 mM potassium phosphate buffer (pH 7.0) was added and the operation of ultrafiltration was repeated twice, it was placed on a DEAE Toyopearl column (1.5 × 20 cm) equilibrated with the same buffer. 400 ml of the same buffer
After washing with, the elution was performed with 80 mM of the same buffer, and 5 ml fractions were 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. This fraction is
The homogeneity 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) Method for measuring 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 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
˜9), the enzyme of the present invention was allowed to stand in 50 mM Tris-HNO ₃ buffer (pH 8 to 9) at 50 ° 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 6-9.

(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 10 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. 6, 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 63,000 ± 5,000 [SDS-polyacrylamide gel electrophoresis (molecular weight marker; LKB, molecular weight range 12,300 to 7
8,000)] (11) The isoelectric point is around 3.5 (measured by isoelectric focusing 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 [Experiment 2] An enzyme solution was prepared under the same conditions as in Experiment 1 using C. hirsutus IFO4917 instead of C. hirsutus IFO4917 in Experiment 1. The resulting enzyme showed the same properties as in Experiment 1.

Craft 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.
Adjust the pH to 5 and add 10 ml of this solution to 1 ml of the enzyme solution obtained in Experiment 2.
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 a gel hypercurve when the pulp of the enzyme of the present invention is applied to bleaching wastewater.

Claims (2)

[Claims] 1. A ligning-degrading enzyme produced from Acacia chinensis belonging to the genus Coriolus and having the following properties: (1) Operation (i) Acting 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 multi-stage bleaching process of chemical pulp production. (2) Substrate specificity It 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 4.5 to 5.0, and the stable pH is 6.0 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) This enzyme is a copper-containing enzyme and its aqueous solution exhibits a deep blue color. (7) The action of this enzyme requires an enzyme. 2. A lignin-degrading enzyme-producing bacterium, which belongs to the genus Kawaratake, is cultivated in a medium, and the culture has the following properties: (1) action (i) action on syringylglycerol-β-syringylether. To produce 2,6-dimethoxyphenol. (Ii) Reduce the molecular weight of lignin contained in the effluent from the multi-stage bleaching process of chemical pulp production. (2) Substrate specificity It 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 4.5 to 5.0, and the stable pH is 6.0 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) This enzyme is a copper-containing enzyme and its aqueous solution exhibits a deep blue color. (7) The action of this enzyme requires an enzyme. A method for producing a lignin-degrading enzyme, comprising collecting a lignin-degrading enzyme having: