JP2007325604A - Method for decomposing lignin using filamentous fungus and treating agent for lignin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for decomposing lignin and a treating agent for lignin using a lignin-degrading filamentous fungus. <P>SOLUTION: Disclosed are a method for decomposing lignin characterized in that β-allyl ether bond which is a main intramolecular bond of lignin is specifically cleaved using a culture of a lignin-degrading filamentous fungus except Basidiomycetes or a treated product thereof, a treating agent for lignin, and a novel lignin-degrading filamentous fungus available thereto. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for degrading lignin using a filamentous fungus and a lignin treatment agent. More specifically, a lignin decomposition method and lignin treatment using a filamentous fungus that specifically cleaves β-allyl ether bond, which is a main intramolecular bond of lignin, and converts lignin into an industrially available low molecular weight compound. It relates to the agent.

  In recent years, its effective use has been studied as an energy source that does not deplete biomass resources such as plant waste materials and as a renewable industrial raw material. However, plants generally contain very robust polymer fibers formed by binding lignin, a natural aromatic polymer, to cellulose fibers or hemicellulose fibers, thereby maintaining the body structure. This complex chemical structure by lignin is advantageous when used as a structural material, but is an obstacle to high utilization as biomass. For this reason, most of them are discarded or used as fuel at best.

In order to decompose polymer fibers in plants, decomposition with alkali or acid or mechanical pulverization is necessary, but various problems have arisen in terms of environment and processing costs. Wood resources are also important as a raw material for pulp, but the kraft pulp manufacturing process requires the decomposition and removal (bleaching) of lignin using chlorine, and there is a need for a processing method that keeps the use of harmful chlorine low. It has been.
Therefore, attention has been paid to a lignin decomposition method as an alternative to the conventional chemical or mechanical method. For example, a lignin decomposition method using an extracellular enzyme of a white rot fungus or a lignin decomposition method using a basidiomycete is known (Journal of the Wood Society, 38 , 811-819 (1992)).

  However, the conventional lignin decomposition method using microorganisms is a low specificity system in which radicals such as manganese peroxidase and laccase, which are extracellular enzymes, are involved and lignin is completely decomposed. In order to use lignin as a biomass resource, it is indispensable to partially decompose lignin having a complicated chemical structure and keep it available without being completely decomposed. Partially decomposed lignin includes, for example, food flavoring ingredients, insecticide synthetic raw materials, pharmaceutical synthesis intermediates, fruit ripening accelerators, oil antifoaming agents, zinc coating whitening agents, herbicides, vulcanization inhibitors, etc. It can be used for various purposes.

Journal of the Wood Society, 38, 811-819 (1992)

  An object of the present invention is to provide a lignin decomposition method and a lignin treatment agent that search for microorganisms that partially decompose lignin without completely degrading lignin, and convert the lignin into biomass resources that are easy to use. is there.

  In order to solve the above-mentioned problems, the present inventors have conducted screening for measuring the fluorescence intensity of a fluorescent substance released by cleavage of a β-allyl ether bond present in a lignin model compound molecule for a large number of filamentous fungi collected from soil. A strain having a high degrading activity was selected and identified by the method, and the present invention has been reached in which the β-allyl ether bond of lignin is specifically degraded and lignin can be used as a useful resource.

That is, the present invention relates to a lignin degradation method using a filamentous fungus that cleaves the β-allyl ether bond in lignin and partially decomposes lignin, a lignin treatment agent, and a novel filamentous fungus used for them.
1) A lignin decomposition method comprising treating lignin using a lignin-degrading filamentous fungus (excluding basidiomycetes).
2) The lignin decomposition method according to 1 above, wherein the β-allyl ether bond in the lignin molecule is cleaved.
3) lignin-degrading fungus is Trichoderma (Trichoderma) sp, Arusuriniumu (Arthrinium) genus or Humicola (Humicola) the one or lignin decomposition method, wherein the bacterium belonging to the genus.
4) The lignin-degrading filamentous fungus is Trichoderma sp. F5053 strain (FERM P-17251), Arthrinium sp. F5518 strain (FERM P-17253), Humicola sp. 1) or 2 above, which is f5342 strain (FERM P-17252), filamentous fungus f5520 strain (Strain f5520) (FERM P-17254) or filamentous fungus 2BW-1 strain (strain 2BW-1) (FERM P-17266) Of lignin decomposition.
5) A lignin treating agent comprising a lignin-degrading filamentous fungus (excluding basidiomycetes).
6) lignin-degrading fungus is Trichoderma (Trichoderma) sp, Arusuriniumu (Arthrinium) genus or Humicola (Humicola) lignin treatment agent of the 5 is a bacterium belonging to the genus.
7) The lignin-degrading filamentous fungi are Trichoderma sp. F5053 strain (FERM P-17251), Arthrinium sp. F5518 strain (FERM P-17253), Humicola sp. ) Lignin according to 5 above, which is f5342 strain (FERM P-17252), filamentous fungus f5520 strain (Strain f5520) (FERM P-17254) or filamentous fungus 2BW-1 strain (strain 2BW-1) (FERM P-17266) Processing agent.
8) Trichoderma sp. (Trichoderma sp.) F5053 strain (FERM P-17251), Arusuriniumu sp (Arthrinium sp.) F5518 strain (FERM P-17253), Humicola sp. (Humicola sp.) F5342 strain (FERM P- 17252), a filamentous fungus f5520 strain (FERM P-17254) and a filamentous fungus 2BW-1 strain (strain 2BW-1) (FERM P-17266).

Hereinafter, the present invention will be described in detail.
(1) Lignin-degrading filamentous fungi The inventors of the present invention have been widely used for cultivating wood-degrading basidiomycetous fungi collected from soil, decaying materials and plant remains throughout Japan. Is used to screen for lignin model compound molecules, which will be described later, using a method for measuring lignin degradation activity that uses the fluorescence intensity of a fluorescent substance (umbelliferone) released by cleavage of the β-allyl ether bond as an index. 15 strains that were recognized were selected. As a lignin model compound for screening, two types of molecular structures described later, benzyl type and phenol type, were used. Among the selected filamentous fungi, two strains (f5053 strain and f5518 strain) that showed strong degradation activity against benzyl type lignin model compounds and one strain (2BW) that showed strong degradation activity against phenol type lignin model compounds. -1 strain) and 2 strains (f5342 strain and f5520 strain) having both properties were further selected. These are incomplete fungi (Deuteromycotina) classified as so-called molds, and are different from fungi. Was identified species for these fungi, Trichoderma (Trichoderma), Arusuriniumu (Arthrinium), that is a filamentous fungi belonging to Humicola (Humicola), etc. were found.

The present inventors have discovered for the first time that filamentous fungi other than basidiomycetes have lignin-degrading activity. The lignin-degrading function that specifically cleaves β-allyl ether bonds is a function that is not found in basidiomycetes such as Phanerochaete chrysosporium and Coriolus versicolor that degrade lignin. From these strains, Trichoderma sp. F5053 (FERM P-17251), Arsolinium sp. F5518 ( Arthrinium sp. F5518, FERM P-17253), Humicola sp. F5342 ( Humicola sp. F5342 ). , FERM P-17252), filamentous fungus f5520 strain (Strain f5520, FERM P-17254), filamentous fungus 2BW-1 strain (Strain 2BW-1, FERM P-17266), dated February 25, 1999 Deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.

The filamentous fungi used in the lignin degradation method of the present invention are filamentous fungi other than basidiomycetes having lignin degradation activity.
For example, bacteria belonging to incomplete fungi (Deuteromycotina), Trichoderma ( Trichoderma ), Arthrinium ( Arthrinium ), Humicola ( Humicola ) and the like can be mentioned. Preferably, Trichoderma sp. F5053 strain ( Trichoderma sp. F5053, FERM P-17251), Arsolinium sp. F5518 strain ( Arthrinium sp. F5518, FERM P-17253), Humicola sp. F5342 strain ( Humicola sp. F5342, FERM P- 17252), filamentous fungus f5520 strain (Strain f5520, FERM P-17254), and filamentous fungus 2BW-1 strain (Strain 2BW-1, FERM P-17266), but are not limited thereto. In the present invention, these bacteria can be used alone or in combination of two or more.

(2) Culture conditions In the present invention, the lignin-degrading filamentous fungus isolated as described above can be cultured and grown under aerobic or anaerobic conditions, preferably aerobic conditions.
The aerobic culture may be a stationary culture or a shaking culture according to the culture of a normal mesophilic bacterium. The pH of the culture solution is 2-8, preferably 5-8.
The culture temperature is 10 to 40 ° C, preferably 20 to 30 ° C. The duration of culturing may be a time sufficient to decompose the lignin in the target lignin-containing substance, and is usually 1 to 20 days, preferably about 2 weeks. When zinc ions are added to the medium, the degradation activity is increased three-fold, and therefore 30 minutes to 1 week is preferable.
Anaerobic culture is in accordance with the above-described aerobic culture, but static culture is performed.

The medium is not particularly limited as long as it is used for culturing ordinary microorganisms, preferably filamentous fungi. For example, a potato starch-dextrose medium, corn meal medium, oatmeal medium, a Vogel medium described later, or the like may be used. Preferred is a Vogel medium.
Woody components such as cellulose and lignin can be added to the medium. Furthermore, various carbon sources or nitrogen sources can be added as necessary. Examples of the carbon source include glucose, shochu, maltose, saccharose, sucrose, brown sugar, molasses, waste molasses, malt extract and the like. Examples of the nitrogen source include meat extract, peptone, gluten meal, soybean flour, dry yeast, yeast extract, ammonium sulfate, ammonium tartrate, urea and the like. In addition, if necessary, inorganic salts such as sodium salt, magnesium salt, manganese salt, iron salt, calcium salt, phosphate, zinc salt, vitamins such as inositol, vitamin B ₁ hydrochloride, L-asparagine, biotin, etc. Kinds may be added.

(3) Lignin Decomposing Method The lignin decomposing method of the present invention is carried out by treating lignin with the above-described culture of filamentous fungus having lignin decomposing activity or its treated product. For example, the lignin-containing substance is added to the above-described culture solution, or conversely, the above-described culture solution or an extract component thereof is added to the lignin-containing substance for treatment.
For the reaction, any of batch method, continuous method, semi-continuous method and the like can be used. Moreover, as long as it contains the lignin-degrading filamentous fungus of the present invention, it may be used together with other lignin-degrading bacteria. Moreover, in order to raise reaction efficiency, it is preferable to add zinc ion.
When the lignin-containing substance is added to the culture solution for treatment, it can be carried out according to the above-mentioned culture conditions for lignin-degrading bacteria.

(4) Lignin treatment agent The lignin treatment agent according to the present invention contains a lignin-degrading filamentous fungus other than the basidiomycete described above, and it does not matter whether it is a solid when it is liquid. That is, the culture solution itself having the above lignin degrading activity can be used as a lignin treatment agent. Moreover, what dried the culture solution and added the manufacturing aid and made it the solid agent can also be used by adding to the lignin containing substance itself to process, or a processing medium.

  EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention more concretely, the scope of the present invention is not limited by these examples.

Example 1: Isolation of filamentous fungus having β-allyl ether bond-cleaving activity (1) Collection and culture of fungus Microorganisms were collected from soil, decayed material, and plant remains samples from all over Japan for culturing wood-degradable basidiomycetes. 1 ml of the frequently used Vogel medium (liquid) was placed in a Wasselman, sterilized, inoculated with bacteria, and allowed to stand for 2 weeks.
The Vogel medium contains 10 g of glucose (made by Seikagaku) and 2.5 g of yeast extract (Yeast Extract, manufactured by Oriental Yeast Co., Ltd.), 20 ml of stock solution (Trace metal) with the following composition and trace metal per liter of medium. ) 100 μl, 0.01 μl Biotin Solution 100 μl.

(2) Measurement of cleavage activity of β-allyl ether bond using lignin model compound As a lignin model compound, umbelliferone represented by the following chemical formula (I) having 4-methylumbelliferone as a fluorescent substance in the B ring Two types of ether (phenol type; hereinafter referred to as substrate-1) and umbelliferone ether (benzyl type; hereinafter referred to as substrate-2) represented by the following chemical formula (II) were used. The benzyl type umbelliferone ether has a structure closer to that of natural lignin.
Since these compounds produce 4-methylumbelliferone which is a fluorescent substance when the β-allyl ether bond is cleaved as shown by the following chemical formula, the decomposition activity of the substrate can be measured from the intensity of the fluorescence.

A DMSO (dimethyl sulfoxide, 0.1%) solution containing umbelliferone ether as a substrate is added to the cultured cell suspension so that the final concentration becomes 0.01%, and after about 24 hours, an ether bond is formed. The fluorescence intensity of umbelliferone released by the cleavage of was measured with a fluorometer (Shimadzu RF1500). Measurement was performed at an excitation wavelength of 360 nm and a measurement wavelength of 450 nm.
As a result, the most active 2BW-1 strain was isolated from the three microbial strains that showed extremely strong degradation activity against substrate-1. Similarly, 4 strains f5053, f5518, f5342 and f5520 having strong activity were isolated from 6 types of microorganisms which showed strong degradation activity against substrate-2.

FIG. 1 shows the degradation activity (fluorescence intensity) for substrate-1 and FIG. 2 shows the degradation activity for substrate-2 of these separated strains. As a control, Phanerochaete chrysosporium ME446 (P in the figure) and Kawaratake ( Coriolus versicolor (C in the figure)) belonging to the basidiomycete known to degrade lignin are used in the same manner. The degradation activity was examined. The horizontal axis in the figure represents the fluorescence intensity, and the numerical value 2000 represents the decomposition intensity of almost 100%.

  The 2BW-1 strain showed extremely strong degrading activity against substrate-1 having a phenolic hydroxyl group, but almost all β-allyl ether in a substrate having no phenolic hydroxyl group such as substrate-2 showed degrading activity. There wasn't. The f5053 strain and f5518 strain also showed strong degradation activity against substrate-2. The f5342 and f5520 strains showed both properties. On the other hand, Fanelocate chrysosporium and Kawaratake, which are known to produce lignin-degrading enzymes, completely decomposed the fluorescent substance generated from the substrate, and the fluorescence intensity was zero.

  Therefore, it was revealed that the filamentous fungus isolated according to the present invention has a function of specifically decomposing β-allyl ether bond. In addition, the cleavage activity of substrate-1 by 2BW-1 strain, f5342 strain and f5520 strain is presumed to be due to phenolic hydroxyl group-dependent laccase. It is considered different. On the other hand, the f5053 and f5518 strains are presumably due to the action different from that of laccase, which decomposes non-phenolic substrates.

(3) Measurement of cleavage activity of β-allyl ether bond using high molecular weight lignin model compound As described above, except that a high molecular weight substrate (DHP) in which substrate-1 is copolymerized with a lignin high molecular structure is used as the lignin model compound ( Performed in the same manner as 2). The results of examining the activity of 2BW-1 strain and f5053 strain are shown in FIG. When the fluorescence intensity is measured only with DHP as a control, there is about 100 fluorescence intensity (DHP Control in the figure). As is clear from FIG. 3, these two strains also showed strong degradation activity against the β ether bond contained in the polymer substrate (DHP). In particular, the 2BW-1 strain showed strong degradation activity.

Example 2: Form and identification of filamentous fungi having β-allyl ether bond cleavage activity
(i) Strain 2BW-1: This strain is entirely colorless on cornmeal agar, without any distinction between the front and the true, and lacks aerial hyphae. The surface is partly white powder. On oatmeal agar, the flora is almost uniform, gray and tan, and the surface is partially covered with white powdery fluffy mycelium. The back side is dark green. On malt agar, it is yellowish brown and the center is covered with a white mycelium. A radial brown line is formed. The back side is tan.

(ii) f5053 strain: The conidial pattern of this strain is upright and branched, and the branches are mainly rotated at the tip, forming a spore soul at the tip. The conidia are phyarotypes (endospores come from phialide), colorless single vesicles that are oval or subspherical (3-6 × 2-4 μm). Thick membrane spores are light brown and subspherical (diameter 8-12 μm) (form in PDA medium). Therefore, this strain was identified as a filamentous fungus belonging to Trichoderma .

(iii) f5518 strain: The conidia of this strain stands upright, does not branch, and produces 1 to several budding type conidia at the tip. Conidia are brown, spherical, elliptical, or disk-shaped with a thin slit-like line in the center, with a major axis of 8-11 μm and a minor axis of 4-5 μm (form in PDA medium) ). From these characteristics, this strain was identified as a filamentous fungus belonging to Arthrinium .

(iv) f5342 strain: This strain is formed by unilaterally or chain-forming contiguous spores of allelo-type (thick spore-like spores) directly on the mycelium or on the petite. Allelo-type conidia are light brown, spherical or subspherical (diameter 8-11 μm) (form spores on elementary agar, cornmeal agar, oatmeal agar). From these characteristics, this strain was identified as a filamentous fungus belonging to Humicola .

(iv) f5520 strain: This strain has a generally homogeneous flora on cornmeal agar medium, grayish green, entirely covered with white aerial hyphae, and the back surface is grayish green. On the oatmeal agar medium, the whole is yellowish brown and the central part is particularly dark and covered with yellowish brown mycelia, the back is dark brown in the central part and the surrounding is yellowish brown. It is yellowish brown on malt agar and is covered with a thin aerial mycelium of white to tan. The back side is yellowish brown and has almost uniform color.

These strains include Trichoderma sp. F5053 strain (FERM P-17251), Arthrinium sp. F5518 strain (FERM P-17253), Humicola sp. F5342 strain (FERM) P-17252), strain f5520 strain (FERM P-17254) and filamentous strain 2BW-1 strain (Strain 2BW-1) (FERM P-17266) deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology Has been.

Comparative Example 1: Comparison of Bacteria and Filamentous Fungi in Lignin Degradation Reaction Substrate-1 was used as a lignin model compound, and Trichoderma sp. F5053 was known as a degrading bacterium and a bacterium that cleaves β-allyl ether bond . Pseudomonas paucimobilis bacterium (Journal of Bacteriology (J. Bact., 173 , 7950-7955 (1991))) is used in the same manner as in Example 1 (2). The degradation activity was examined. As a result, both showed almost the same level of degradation activity, but it was found that Pseudomonas takes much longer time for the membrane permeation reaction in which the substrate enters the cells, and the degradation reaction time is much faster for filamentous fungi.

The present invention provides a lignin decomposition method and a lignin treatment agent that specifically cleaves β-allyl ether bond, which is the main intramolecular bond of lignin, using a culture of lignin-degrading filamentous fungi or a processed product thereof. It is.
In particular, the inventors found Trichoderma sp. F5053 strain (FERM P-17251), Arthrinium sp. F5518 strain (FERM P-17253), Humicola sp. The f5342 strain (FERM P-17252), the filamentous fungus f5520 strain (Strain f5520) (FERM P-17254), or the filamentous fungus 2BW-1 strain (strain 2BW-1) (FERM P-17266) is β- in the lignin molecule. Since only the allyl ether bond is cleaved and its activity is high, lignin can be used as a biomass resource by using this strain. Furthermore, the lignin decomposition method and lignin treatment agent of the present invention can be effectively used in fields such as the paper industry, the industrial waste treatment industry, and soil improvement.

The result of evaluating the cleavage activity of the lignin intramolecular ether bond by a lignin-degrading filamentous fungus by fluorescence intensity measurement using umbelliferone ether (phenol type) (substrate-1) is shown. The result of evaluating the cleavage activity of the lignin intramolecular ether bond by a lignin-degrading filamentous fungus by fluorescence intensity measurement using umbelliferone ether (benzyl type) (substrate-2) is shown. Using lignin-degrading filamentous fungi 2BW-1 and f5053 strains as fluorescent polymer lignin model compounds, the lignin-degrading filamentous fungus 2BW-1 and f5053 strains were used to evaluate the lignin intramolecular ether bond cleavage activity. Results are shown.

Claims (4)

Humicola sp. F5342 strain (FERM P-17252), Arthrinium sp. F5518 strain (FERM P-17253), filamentous fungus f5520 strain (Strain f5520) (FERM P-17254) or filamentous A lignin decomposition method comprising treating lignin using a strain 2BW-1 strain (Strain2BW-1) (FERM P-17266) .   The lignin decomposition method according to claim 1, wherein the β-allyl ether bond in the lignin molecule is cleaved. Humicola sp. F5342 strain (FERM P-17252), Arthrinium sp. F5518 strain (FERM P-17253), filamentous fungus f5520 strain (Strain f5520) (FERM P-17254) or filamentous A lignin treating agent comprising a strain 2BW-1 strain (strain2BW-1) (FERM P-17266). Humicola sp. F5342 strain (FERM P-17252), Arthrinium sp. F5518 strain (FERM P-17253), filamentous fungus f5520 strain (Strain f5520) (FERM P-17254) and filamentous A lignin-degrading bacterium selected from the group consisting of strain 2BW-1 (strain2BW-1) (FERM P-17266).

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