JPWO2016111368A1 - Method for producing protein having lignin-degrading activity using jellyfish - Google Patents

Abstract

An object of the present invention is to provide a method for producing a protein having lignin degrading activity or a method for degrading lignin. The present invention provides a method for producing a protein having lignin-degrading activity and a method for degrading lignin by culturing at least one type of microorganism classified into the genus Cerebrum.

Description

  The present invention relates to a method for producing a protein having lignin-degrading activity derived from a microorganism, a lignin-decomposing method using the microorganism, and the like.

  Lignin is a constituent of woody biomass and is one of the most difficult materials to decompose. White rot fungi and the like are known as organisms that degrade lignin, and laccase, lignin peroxidase, manganese peroxidase, and the like are known as lignin-degrading enzymes produced by these organisms (Non-Patent Documents 1 to 4). Although the complete mechanism of lignin degradation has not been clarified, it has been suggested in nature that partial oxidative degradation of lignin by white-rot fungi and further reduction in molecular weight by bacterial groups are performed. The lignin-degrading enzyme group is expected to be widely applied to industrial applications such as recycling of wood waste, increasing efficiency of pulp bleaching from the paper industry, decomposition of dyes, and purification of environmental pollutants such as dioxins.

  On the other hand, it is known that the jellyfish fungi, which are marine eukaryotic microorganisms, accumulate a significant amount of fats and oils in the cells. The fats and oils accumulated by the jellyfish fungi contain highly unsaturated fatty acids such as docosahexaenoic acid at a high concentration (Patent Document 1). Conventionally, the lignin decomposing activity ability of the jellyfish fungi has not been studied.

JP 2005-287380 A

THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1991, Vol. 266, pp. 14464-14469 Wood Preservation, 1995, Vol. 21-4, pp.163-170 Microbiology, 2000, Vol. 146, pp. 759-765 Appl. Biochem. Biotechnol., 2011, Vol. 165, pp. 700-718

  Until now, most of the lignin-degrading bacteria are known to be fungi such as white rot fungi, and the lignin-degrading enzyme has not been put into practical use.

  An object of the present invention is to identify a novel organism having lignin-decomposing ability, to produce a lignin-degrading enzyme using the organism, and to provide a method for degrading lignin.

  In order to solve the above-mentioned problems, the present inventors screened thraustochytrids and examined conditions for inducing the degradation enzyme. As a result, microorganisms that are classified into the genus Germulus, namely, the genus Altornia, the genus Aplanochytrium, the genus Aurantiochytrium, the genus Botryochytrium, the Japonochytrium (Japonochytrium) ), Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium, or Ulkenia It has been found that a protein having lignin degrading activity can be produced by culturing at least one selected from the microorganisms to which it belongs.

  This specification includes the disclosure of Japanese Patent Application No. 2015-003488, which is the basis of the priority of the present application.

  According to the method of the present invention, it is possible to efficiently produce a protein having lignin degrading activity and to efficiently degrade lignin.

FIG. 1 shows an example of a plate assay of the present invention. The left shows the results when using the RBBR-containing medium (medium 6 in Table 3), and the right shows the lignin-containing medium (medium 8 in Table 3). FIG. 2 shows RBBR degradation activity of 7 strains having particularly excellent RBBR degradation activity among mutant strains of Aurantiochytrium limacinum mh0186. The vertical axis represents the absorbance at 595 nm, which is the maximum absorption wavelength of RBBR.

In one embodiment, the present invention relates to a method for producing a protein having lignin-degrading activity, comprising culturing at least one microorganism classified as a genus cerebrum and causing the microorganism to produce a protein having lignin-degrading activity. About.
(Lignin degradation activity)
As used herein, “lignin degrading activity” refers to an activity of degrading lignin or a similar substance. Examples of lignin-like substances include Remazol Brilliant Blue R (RBBR), Evans Blue, Azure B, and Orange II.

  The presence / absence and intensity of the lignin-degrading activity of the microorganism can be measured, for example, by a plate assay. In other words, microorganisms are seeded in a medium containing lignin or a lignin-like substance, the microorganisms are cultured for several days, and the presence and size of zona pellucida (decomposition zone) or browning (Berbendam reaction) around the grown colonies are measured. To do. A wider zona pellucida or browning range suggests higher lignin degradation activity. In addition, the presence or absence and strength of lignin-degrading activity can also be measured by measuring the absorbance of lignin or similar substances (for example, the absorbance of RBBR at 595 nm) in a culture solution from which microorganisms have been cultured in a liquid culture. it can.

The presence / absence and strength of lignin-degrading activity of a protein having lignin-degrading activity produced by a microorganism can be measured by a known method. For example, you may measure by the same plate assay as said microorganisms. Alternatively, the protein may be added to a lignin-containing solution and incubated, and the decolorization of lignin may be measured from the absorbance around 480 nm, or the degradation of the aromatic ring of lignin may be measured from the absorbance around 275 nm.
(Microorganism used in the present invention)
The method for producing a protein having lignin-degrading activity according to the present invention includes culturing at least one kind of microorganism classified as Thraustochytrids and causing the microorganism to produce a protein having lignin-degrading activity. It is characterized by.

  The “microorganisms classified into the genus Amaranthus” are marine eukaryotic microorganisms that produce highly unsaturated fatty acids as explained in the background art. The microorganisms that are classified into the genus Germulus are the genus Altornia, the genus Aplanochytrium, the genus Aurantiochytrium, the genus Botryochytrium, the genus Japonochytrium, the ob Examples include microorganisms belonging to the genus Oblongichytrium, the genus Parietichytrium, the genus Schizochytrium, the genus Sicyoidochytrium, the genus Thraustochytrium, or the genus Ulkenia. It is done. In the present invention, a microorganism that is classified as a jellyfish and has the ability to produce a protein having lignin-degrading activity may be used.

  In particular, it produces at least one protein belonging to the genus Aplanochytrium, Aurantiochytrium, Botryochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium, or Ulkenia. A microorganism having capacity is preferred.

  Examples of the microorganism having the ability to produce a protein belonging to the genus Aplanochytrium and having a lignin-degrading activity include at least one species belonging to Aplanochytrium kerguelense. As a specific strain of a microorganism belonging to the genus Aplanochytrium, the strain Aplanochytrium kerguelense SEK535 (Aplanochytrium kerguelense SEK535) is preferably used.

  Examples of the microorganism having the ability to produce a protein belonging to the genus Aurantiochytrium and having a lignin degrading activity include at least one species belonging to Aurantiochytrium limacinum or Aurantiochytrium mangrovei. Specific strains of microorganisms belonging to the genus Aurantiochytrium include Aurantiochytrium limacinum SR21 ATCC MYA-1381, Aurantiochytrium limacinum mh0186 (accession number FERM BP-11311), Aurantiochytrium mangrovei SEK243, Aurantiochytrium mangrovei SEK218 (accession number NBRC 103269), Aurantiochytrium sp. ATCC26185 or Aurantiochytrium sp. ATCC20888 is preferably used.

  Examples of microorganisms belonging to the genus Botryochytrium and capable of producing a protein having lignin-degrading activity include at least one species belonging to Botryochytrium radiatum. As specific strains of microorganisms belonging to the genus Botryochytrium, Botryochytrium radiatum SEK353 (accession number NBRC 104107) or Botryochytrium sp. SEK598 (accession number NBRC 110811) is preferably used.

  Oblongichytrium sp. SEK347 (Accession No. NBRC 102618) or Oblongichytrium sp. SEK600 (Accession No. NBRC 110833) is preferably used as a specific strain of a microorganism belonging to the genus Oblongichytrium and capable of producing a protein having lignin-degrading activity.

  Examples of the microorganism having the ability to produce a protein belonging to the genus Parietichytrium and having a lignin-degrading activity include at least one species belonging to Parietichytrium sarkarianum. Parietichytrium sarkarianum SEK351 (Accession No. NBRC 104108), Parietichytrium sarkarianum SEK364 (Accession No. FERM BP-11298), or Parietichytrium sp. SEK358 (Accession No. FERM BP-11405) is preferable as a specific strain of a microorganism belonging to the genus Parietichytrium. Used.

  Examples of the microorganism belonging to the genus Schizochytrium and having the ability to produce a protein having lignin-degrading activity include at least one species belonging to Schizochytrium aggregatum or Schizochytrium limacinum. As specific strains of microorganisms belonging to the genus Schizochytrium, Schizochytrium aggregatum ATCC28209, Schizochytrium sp. SEK210 (accession number NBRC 102615), or Schizochytrium sp. SEK345 (accession number NBRC 102616) is preferably used.

  Microorganisms belonging to the genus Thraustochytrium and having the ability to produce a protein having lignin-degrading activity include Thraustochytrium aureum, Thraustochytrium kinnei, Thraustochytrium kinnei, Thraustochytrium roseum, Or at least 1 sort which belongs to Thraustochytrium striatum (Thraustochytrium striatum) is mentioned. Specific strains of microorganisms belonging to the genus Thraustochytrium include Thraustochytrium aureum ATCC34304, Thraustochytrium aureum SEK621 (Accession number NBRC 110820), Thraustochytrium kinnei SEK617 (Accession number NBRC 110825), Thraustochytrium kinnei SEK618 (Accession number NBRC ATCC282 Thraustochytrium striatum ATCC 24473, Thraustochytrium striatum NE03, or Thraustochytrium aff. Caudivorum SEK 616 (identical to Schizophytrium sp. SEK 616) (accession number NBRC 110822) is preferably used.

  Examples of the microorganism having the ability to produce a protein belonging to the genus Ulkenia and having a lignin-degrading activity include at least one species belonging to Ulkenia amoeboidea. As specific strains of microorganisms belonging to the genus Ulkenia, Ulkenia amoeboidea SEK214 (Accession No. NBRC 104106), Ulkenia sp. ATCC28207, or Ulkenia sp. SEK615 (Accession No. NBRC 110830) is preferably used.

  The range of microorganisms used in the present invention is not limited to microorganisms that are currently classified into the above genera or species, but are currently classified into other genera or species, or the classification is not clear However, in terms of molecular evolution, microorganisms to be classified into any of the above genera or species are also included.

  Among the above-mentioned specific strains, Aurantiochytrium limacinum mh0186 strain (FERM BP-11311) is disclosed in Japanese Patent Application Laid-Open No. 2005-287380, and is an independent administrative agency, National Institute of Advanced Industrial Science and Technology that is an international depositary organization under the Budapest Treaty. Biological Deposit Center (1st, 1st, 1st East, 1-chome, Tsukuba City, Ibaraki, Japan 305-8566) (currently National Institute of Technology and Evaluation (NITE) Patent Biological Deposit Center (IPOD), 292-0818 Japan Schizochytrium sp. M-8, which was deposited domestically on March 29, 2004, with the accession number FERM P-19755 in Kisarazu City, Kazusa-Kamazu 2-5-8, Room 120), was transferred to the international deposit. It is what. This strain can be obtained from the depository institution and can also be sold from the applicant, Miyazaki University. The genus that was considered to be the genus Schizophytrium at the time of deposit of FERM P-19755 was reorganized into the genus Schizophytrium, Aurantiochytrium, and Oblongichytrium (Rinka Yokoyama, Daiske Honda (2007) Mycoscience Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience, 48, 199-21). The Aurantiochytrium limacinum mh0186 strain was obtained from the method of obtaining the Schizothytrium sp. M-8 strain disclosed in JP-A-2005-287380 (the Mabletaceae M-8 strain was obtained as follows. First, a mangrove forest on Ishigaki Island Place the seawater and deciduous leaves collected in step 3 into a 300 ml Erlenmeyer flask, and add about 0.05 g of pine pollen (here, collected from the coast around Miyazaki city). Seawater was collected so as to contain, and 0.1 ml was applied on a potato dextrose agar medium prepared in a petri dish, cultured at 28 ° C. for 5 days, picked up a creamy, non-glossy colony and applied on a new agar medium Three days later, the grown microorganisms were observed under a microscope and stored in a slant medium, judging from the size and form of the cells as being Labyrinthula.

  Aurantiochytrium limacinum SR21 ATCC MYA-1381, Aurantiochytrium sp. ATCC26185, Aurantiochytrium sp. It is generally available.

  Aurantiochytrium limacinum SR21 ATCC MYA-1381 is Schizophytrium limacinum Honda et Yokochi (ATCC MYA-1381), Aurantiochytrium sp. ATCC26185 is Thraustochytrium sp. (ATCC 26185), Aurantiochytrium sp. Schizochytrium aggregatum ATCC28209 is Schizochytrium aggregatum Goldstein et Belsky (ATCC 28209), Thraustochytrium aureum ATCC34304 is Thraustochytrium aureum Goldstein (ATCC 34304), Thraustochytrium roseum ATCC28210 is Thraustochytrium Thruustotritrium roseCC ATCC 24473) and Ulkenia sp. ATCC28207 are the same as Japonochytrium sp. (ATCC 28207), respectively, and the genus or species classification is reconstructed by the present inventors.

  Aurantiochytrium mangrovei SEK218 (Accession number NBRC 103269), Botryochytrium radiatum SEK353 (Accession number NBRC 104107), Botryochytrium sp. SEK598 (Accession number NBRC 110811), Oblongichytrium sp. SEK347 (Accession number NBRC 102618), Oblongichytrium sp. 110833), Parietichytrium sarkarianum SEK351 (Accession No. NBRC 104108), Schizochchytrium sp. SEK210 (Accession No. NBRC 102615), Schizophytrium sp. SEK345 (Accession No. NBRC 102616), Thraustochytrium aff. Caudivorum SEK616 (Accession No. NBRC 110822), Thureustochy (Accession number NBRC 110820), Thraustochytrium kinnei SEK617 (Accession number NBRC 110825), Thraustochytrium kinnei SEK618 (Accession number NBRC 110826), Ulkenia amoeboidea SEK214 (Accession number NBRC 104106), and Ulkenia sp. SEK615 (Accession number NBRC 110830) It is deposited with the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818). These strains are generally available from the preservation institution and can also be sold from the applicant, Miyazaki University.

  Parietichytrium sp. SEK358 (Accession No. FERM BP-11405) and Parietichytrium sarkarianum SEK364 (Accession No. FERM BP-11298) are under the Treaty of Budapest with the date of accession as of August 11, 2011 and September 24, 2010, respectively. National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, an international depositary organization (1st, 1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan, 305-8566) (currently NITE) ) Deposited at the Patent Biological Deposit Center (IPOD), Room 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan). These strains can be obtained from the depository institution and can also be sold from the applicant, Miyazaki University.

  Thraustochytrium striatum NE03 was isolated by the present inventors in the coastal area of Kagoshima Prefecture (Reference: “New Astaxanthin-Producing Labyrinthura Thraustochytrium sp. NE03” Authors: Naoki Nagano, Yosuke Taoka, Tomoyo Kuramura, Masahiro Hayashi (Miyazaki Univ.) Material ： Abstracts of the Annual Meeting of the Marine Biotechnology Society Volume ： 12th Page ： 109, Publication year ： May 30, 2009).

  The range of microorganisms used in the present invention includes the above-mentioned specific strains, preferably mutant strains having the ability to produce a protein having a lignin-degrading activity of Aurantiochytrium limacinum mh0186 (Accession No. FERM BP-11311). Preferably, the mutant strain of the above-mentioned specific strain has the ability to produce a protein having lignin-degrading activity that is comparable to or higher than that of the wild-type strain, and has the ability to produce a protein having lignin-degrading activity than the wild-type strain. It is particularly preferable that the height is high.

Those skilled in the art can easily produce mutants based on known mutagenesis methods. Examples of such a method include radiation treatment and mutagen treatment. Examples of radiation treatment include UV irradiation, gamma irradiation, X-ray irradiation, and heavy ion beam irradiation. Examples of mutagens include ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, bromouracil. And nucleotide base analogs, and acridines. After performing these treatments, mutants may be obtained by randomly isolating colonies, or screened based on the ability to produce a protein having a desired trait such as lignin-degrading activity to obtain a mutant. May be.
(Microbial culture)
The method of the present invention includes a culturing step of culturing the microorganism and causing the microorganism to produce a protein having lignin-degrading activity.

  In the culturing step, a fixed medium, a liquid medium, or the like suitable for cultivating the jarlet is used as the medium. The medium is not particularly limited, and examples of the medium that is usually used include GY medium (Journal of Fisheries Science, Vol. 68, No. 5, 674-678 (2002)), B1 agar plate medium (Appl. Microbiol. Biotechnol., 72 , 1161-1169 (2006)), and the like can be used as appropriate.

  The culture conditions in the culture step are not particularly limited, but typically, the microorganism is used in an appropriate medium for about 1 day to 2 weeks, preferably 2 to 12 days, more preferably 3 to 10 days, 25 to 35. Incubate at ℃. In the case of a liquid medium, culture is performed under shaking conditions as necessary. The light / dark conditions during the culture are not particularly limited, but are usually performed under dark conditions.

  The present inventors have found that the production of a protein having lignin-degrading activity by the microorganism is remarkably increased by culturing the microorganism using a medium containing a surfactant. The surfactant can be used, for example, at a concentration of 0.1 g / L medium to 10 g / L medium, particularly 0.5 g / L medium to 5 g / L medium. Preferred surfactants used in the present invention include nonionic surfactants such as Tween 20 ™ (polysorbate 20, polyoxyethylene sorbitan monolaurate), Tween 40 ™ (polysorbate 40, polyoxyethylene sorbitan). Monopalmitate), Tween 60 ™ (polysorbate 60, polyoxyethylene sorbitan monostearate), Tween 80 ™ (polysorbate 80, polyoxyethylene sorbitan monooleate), Brij 35 ™ (polyoxyethylene) (23) lauryl ether), and Triton X-100 ™ (poly (oxyethylene) octylphenyl ether), especially Tween 80.

  The present inventors have also found that the production of a protein having lignin-degrading activity by the microorganism is remarkably increased by culturing the microorganism using an oligotrophic medium. In the present specification, the “poor nutrient medium” means a medium having a low content of a carbon source such as glucose, a nitrogen source such as polypeptone, and a growth promoting substance such as yeast extract. Examples of the carbon source include glucose, natural extracts such as potato, malt, and carrot, and sugars such as maltose, sucrose, lactose, and amylose, and alcohols such as ethanol and glycerol. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, organic acid ammonium salts such as ammonium citrate, and nitrates such as sodium nitrate in addition to polypeptone. Examples of the growth promoting substance include meat extract and malt extract in addition to yeast extract. For example, the “oligotrophic medium” in the present specification is at least one of a nitrogen source concentration such as polypeptone, a carbon source concentration such as glucose, and a growth promoting substance concentration such as yeast extract, preferably a nitrogen source or a carbon source. , And all concentrations of growth promoters are low. Preferable concentrations of the nitrogen source, the carbon source, and the growth promoting substance are, for example, 1.0 g / L medium or less, preferably 0.5 g / L medium or less, particularly preferably 0.1 g / L medium or less. On the other hand, it is preferable that the nitrogen source, the carbon source, and the growth promoting substance are contained in the medium to the extent necessary to promote the growth of microorganisms. Preferable concentrations of the nitrogen source, the carbon source, and the growth promoting substance are, for example, 0.001 g / L medium or more, preferably 0.005 g / L medium or more, particularly preferably 0.01 g / L medium or more. Therefore, the nitrogen source concentration is, for example, 0.001 g / L to 1.0 g / L medium, preferably 0.005 g / L to 0.5 g / L medium, and particularly preferably 0.01 g / L to 0.1 g / L medium. The concentration is, for example, 0.001 g / L to 1.0 g / L medium, preferably 0.005 g / L to 0.5 g / L medium, particularly preferably 0.01 g / L to 0.1 g / L medium, and the growth promoting substance concentration is For example, 0.001 g / L to 1.0 g / L medium, preferably 0.005 g / L to 0.5 g / L medium, and particularly preferably 0.01 g / L to 0.1 g / L medium.

  In another embodiment, the microorganism may be cultured using a rich medium. In the present specification, the “rich medium” means a medium having a high content of a carbon source such as glucose, a nitrogen source such as polypeptone, and a growth promoting substance such as yeast extract. For example, the “enriched medium” in the present specification includes at least one of a nitrogen source concentration, a carbon source concentration, and a growth promoting substance concentration, preferably all concentrations of the nitrogen source, the carbon source, and the growth promoting substance. High medium. The preferable concentration of the nitrogen source, carbon source and growth promoting substance in the rich medium is, for example, 1.0 g / L medium or more, preferably 1.5 g / L medium or more, particularly preferably 2.0 g / L medium or more. The upper limit of the nitrogen source, carbon source, and growth promoting substance in the eutrophic medium is not particularly limited, for example, 50.0 g / L medium or less, 20.0 g / L medium or less, preferably 10.0 g / L medium or less, 9.0 g / L Below medium, below 8.0 g / L medium, below 7.0 g / L medium, below 6.0 g / L medium, particularly preferably below 5.0 g / L medium. Therefore, the nitrogen source concentration is, for example, 1.0 g / L to 20.0 g / L medium, preferably 1.5 g / L to 10.0 g / L medium, and particularly preferably 2.0 g / L to 5.0 g / L medium. The concentration is, for example, 1.0 g / L to 20.0 g / L medium, preferably 1.5 g / L to 10.0 g / L medium, particularly preferably 2.0 g / L to 5.0 g / L medium, and the growth promoting substance concentration is For example, 1.0 g / L to 20.0 g / L medium, preferably 1.5 g / L to 10.0 g / L medium, and particularly preferably 2.0 g / L to 5.0 g / L medium.

  The medium preferably further contains lignin or a lignin-like substance. A lignin-like substance such as lignin or RBBR can be used, for example, at a concentration of 0.1 g / L medium to 10 g / L medium, particularly 0.3 g / L medium to 1 g / L medium.

The medium may contain a vitamin mixed solution and trace elements as necessary. The vitamin mixed solution and the trace element can be used at a concentration of, for example, 0.1 ml / L medium to 10 ml / L medium, particularly 0.5 ml / L medium to 5 ml / L medium.
(Protein recovery)
The method for producing a protein having lignin-degrading activity of the present invention may optionally include a step of recovering or isolating the protein having lignin-degrading activity. The method of the present invention may not include the step of recovering or isolating protein. In this case, the microorganism culture, the culture supernatant fraction, the cell-containing fraction, the cell disruption product, the cell extract A protein having lignin-degrading activity contained in (crude enzyme extract) or the like can be used as it is. When a protein having lignin-degrading activity produced by a cultured microorganism accumulates in the body of the microorganism, a microorganism culture, a cell-containing fraction, a cell fragment, a cell extract, etc. are used in the method of the present invention. When the microorganism is secreted outside the body of the microorganism, the culture supernatant fraction can be used in the method of the present invention.

  From a microorganism culture, a culture supernatant fraction, a cell-containing fraction, a cell disruption product, a cell extract (crude enzyme extract), etc. Separation means include gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography and other chromatography, ammonium sulfate fractionation, sodium sulfate fractionation, sodium chloride fractionation salting out, dialysis, etc. One or more general means such as electric point electrophoresis can be used in combination. At this time, the protein having lignin degradation activity can be increased in concentration using the presence of lignin degradation activity as an index.

Proteins having lignin decomposing activity obtained by the present invention are used in industries that require lignin decomposing treatment, for example, recycling of wood waste, efficiency of pulp bleaching from the paper industry, degradation of dyes, and environmental pollution such as dioxins. It can be used for purification of substances.
(Lignin degradation method and use of the microorganism of the present invention in degradation of lignin)
In one embodiment, the present invention includes a step of degrading lignin by allowing lignin to act on at least one kind of microorganism classified into the genus Aedes, or a protein having lignin-degrading activity produced by the microorganism. It relates to a decomposition method. The method for degrading lignin according to the present invention comprises culturing at least one kind of microorganism classified as a jellyfish with a lignin-containing material, causing the microorganism to produce a protein having lignin-degrading activity, and causing the lignin in the material to produce the protein. May be performed by directly acting. The lignin decomposition method of the present invention may be carried out by mixing a protein having lignin decomposition activity obtained by the above-described production method with a lignin-containing material.

  The lignin-containing material is not particularly limited as long as it contains lignin, and may be solid or liquid. When microorganisms are cultured and acted on or in a lignin-containing material, the lignin-containing material includes the components described in the above “microorganism culture”, such as a surfactant, a carbon source, a nitrogen source, and a growth promoting substance. It is preferable to contain. When the lignin-containing material is allowed to act on the recovered or isolated protein, the lignin-containing material preferably contains a buffer solution or the like in order to maintain conditions suitable for lignin degradation.

  The conditions for the degradation step of the method of the present invention are not particularly limited as long as a protein having lignin degradation activity acts on lignin. For example, it can be incubated for about several tens of minutes to two weeks, preferably several hours to several days, or about one to three days under appropriate temperature and pH conditions. Examples of the temperature condition include 10 ° C. to 40 ° C., 15 ° C. to 35 ° C., preferably 20 ° C. to 30 ° C., and examples of the pH condition include pH 5 to 9, 6 to 8, and preferably 6.5 to 7.5. When the lignin-containing material is a liquid, incubation is performed under shaking conditions as necessary. The light / dark conditions during the incubation are not particularly limited, but are usually performed under dark conditions.

  In another aspect, the present invention relates to the use of at least one of the microorganisms classified into the genus Aedes, or the protein having lignin-degrading activity produced in the microorganism, in the degradation of lignin or in the degradation method of lignin. .

The types of microorganisms used in the method for degrading lignin and the method for degrading lignin or the method for degrading lignin of the present invention are as described above.
(Composition for use in protein production or for degradation of lignin)
In one aspect, the present invention is for use in the production of a protein having lignin degrading activity, comprising at least one microorganism classified in the genus Aspergillus as described herein, or for use in degrading lignin. Of the composition. The composition of the present invention is preferably a surfactant, and / or a carbon source such as glucose, a nitrogen source such as polypeptone, and a yeast extract, in addition to at least one microorganism classified as a fungus. Contains growth-promoting substances. The types and amounts of surfactants that can be included in the composition of the present invention, and the amounts of glucose, polypeptone, and yeast extract are as described in the above-mentioned "Microbial culture", so description is omitted here (however, The above concentration is the final concentration after the composition is added to another liquid.When the composition of the present invention is used after being diluted, the concentration of the composition of the present invention depends on the dilution ratio. The concentration of the component may be several times to several hundred times, preferably several times to several tens times the concentration, for example. The composition of the present invention may further contain lignin or a lignin-like substance, a vitamin mixed solution, and trace elements as necessary. Since the amounts of these components are also as described above, the description is omitted here (however, the above concentration is the final concentration after the composition is added to another liquid. When used after being diluted, the concentration of the component in the composition of the present invention is, for example, several times to several hundred times, preferably several times to several tens of times the concentration according to the dilution ratio. May be).

  The composition of the present invention may be used directly on a solid containing lignin or added to a liquid, and if necessary, a solid containing lignin suspended and / or diluted in a medium such as water or a buffer. It may be added to the top or in the liquid.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

<Example 1: Lignin or RBBR-degrading activity of jellyfish in various media>
1) Medium preparation and plate assay method Remazol Brilliant Blue R (RBBR) or lignin (Nacalai Tesque), Tween 80 (Nacalai Tesque), polypeptone, yeast extract (Nacalai Tesque), glucose (Nacalai Tesque), agar powder (Nacalai Tesque), vitamin mixed solution (composition shown in Table 1 below), trace elements (composition shown in Table 2 below), 1L of 50% artificial seawater (ASW) ( Media 1-8 were prepared according to Table 3 below.

Grow on B1 agar medium (yeast extract 0.2g, polypeptone 0.2g, glucose 0.5g, vitamin mixed solution 0.1ml, agar 1.5g, 50% ASW 100mL [pH 6.8]) (28 ° C, 4-7 days) Each colony of 28 strains of the fungus was collected with a platinum wire and inoculated on media 1 to 8 prepared as described above. Thereafter, the medium was incubated at 28 ° C. for 7 days, and the zona pellucida (decomposition zone) and browning (Berbendam reaction) around the grown colonies were observed. Degradation zone and browning were not observed-, unclear but degradation zone or blackening was identified as +, and those with clear degradation zone or browning were identified as size ( mm, average value ± standard deviation (n = 3)) were measured.
2) Results An example of the plate assay is shown in FIG. The results for all the test strains are shown in Table 4 below.

  In medium 1 and 2, all 28 strains did not show RBBR degradation activity. In medium 3, zona pellucida was confirmed around the colony in 24 out of 28 strains, and RBBR degradation activity was confirmed. Although the medium 4 was weak, RBBR degradation activity was confirmed in 19 strains. In medium 5, RBBR degradation activity was confirmed in all 28 strains. In medium 6, RBBR degradation activity was confirmed in 19 strains.

In medium 7, all 28 strains did not show lignin degrading activity. In medium 8, browning was confirmed around the colonies in 27 strains, and lignin degradation activity was confirmed.
<Example 2: RBBR degradation activity of mutant strain of Aurantiochytrium limacinum mh0186>
1) Method
UV irradiation treatment Aurantiochytrium limacinum mh0186 (Aurantiochytrium limacinum mh0186) strain was precultured at 28 ° C. for 72 hours in a 100 ml baffled Erlenmeyer flask containing 20 ml of GY medium. The pre-cultured strain was transferred to GY medium and cultured for 72 hours under the same conditions. All the culture broth was collected, and the cultured cells were washed twice by centrifugation (5,000 g × 5 min) with sterile 50% ASW. The washed cultured cells were diluted to 20 ml with sterile 50% ASW and then cultured overnight at 28 ° C. in a 100 ml baffled Erlenmeyer flask. 1.0 ml of the culture solution was collected in a 1.5 ml microtube, and allowed to stand for 10 minutes after low-speed centrifugation at 500 g for 5 minutes. After recovering the supernatant, 10 ² to 10 ⁶ bacteria suspended in sterile 50% ASW were applied to a B1 agar medium. UV irradiation to plate using AS ONE Handy UV lamp SLUV-6 (As One Co., Osaka, Japan) in combination of irradiation distance 5, 10 cm and irradiation time 0, 5, 10, 15, 30 seconds (254 nm )did. After UV irradiation, the plate was cultured for 72 hours at 28 ° C. in the dark, and colonies grown on the plate were counted. The death rate was calculated by comparison with UV non-irradiation treatment (control group). The resulting colonies were streaked three times on B1 agar medium and purified as UV-irradiated strains.
RBBR degradation screening test using 96-well microplate
To each well of the 96-well microplate, 100 μl of RBBR-containing liquid medium (the composition of medium 3 shown in Table 3 above, excluding agar, vitamin liquid, and trace elements) was aseptically added. The above UV-irradiated strain was cultured in B1 agar medium at 28 ° C. for 72 hours, and colonies formed on the plate were scraped with a 1-ase platinum ear and suspended in each well. The microplate was cultured at 28 ° C. for 48 hours with stirring at 500 rpm in a microplate shaker. After culturing, the culture solution in each well was collected and centrifuged. The degradation level of RBBR was evaluated by measuring absorbance at 595 nm, which is the maximum absorption wavelength of RBBR in the supernatant, using a microplate reader.
2) Results
In the section treated with UV at 5 cm for 10 seconds, the death rate was 99.8%. The colonies grown in this test area were purified, and the 78 strains obtained were designated as UV-irradiated strains.

  The results of measuring the absorbance at 595 nm which is the RBBR maximum absorption wavelength of the supernatant after culturing the UV-irradiated strain are shown in Table 5 below, and the results of 7 strains in which a significant decrease in absorbance was observed are shown in FIG. .

  Absorbance decreased in many of the mutant strains, especially in 7 irradiated strains (# 25, # 29, # 39, # 47, # 64, # 71, # 74) Decrease was observed. As described above, a mutant having RBBR degradation activity could be obtained.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (13)

  A method for producing a protein having lignin-degrading activity, comprising a culturing step of culturing at least one kind of microorganism classified as a cerebral fungus and causing the microorganism to produce a protein having lignin-degrading activity.   The microorganism is genus Aurantiochytrium, genus Ulkenia, genus Thraustochytrium, genus Oblongichytrium, genus Schizothytrium, genus Aplanochytrium, 2. The bacterium of claim 1, which is at least one selected from the genus Botryochytrium, the genus Sicyoidochytrium, the genus Parietichytrium, and the genus Japonochytrium. Method.   Said microorganisms are Ulkenia amoeboidea, Thraustochytrium kinnei, Aurantiochytrium limacinum, Thraustochytrium strium Aurantiochytrium mangrovei, Schizochytrium aggregatum, Thraustochytrium aureum, Thraustochytrium roseum, Aranochetium kell At least one species selected from microorganisms belonging to Botryochytrium radiatum and Parietichytrium sarkarianum The method of claim 1.   The microorganisms are Aurantiochytrium sp. ATCC 20888 (Aurantiochytrium sp. ATCC 20888), Aurantiochytrium sp. SEK618 (Thraustochytrium kinnei SEK618), Urkenia sp.ATCC28207 (Ulkenia sp. ) Strain, Thraustochytrium striatum ATCC 24473 (Thraustochytrium striatum ATCC 24473) strain, Aurantiochytrium mangrovei SEK218 strain, Aurantiochytrium remacinum SR21 ATCC MYA-1381 (Aurantiochytrium limacinum SR21 Strain of Oblongichytrium sp. SEK347 (Oblongichytri um sp. SEK347), Urkenia sp. SEK615 (Ulkenia sp. SEK615), Schizochytrium aggregatum ATCC28209 (Schizochytrium aggregatum ATCC28209), Traustochytrium aureum ATCC34304 (Thraustochytrium aureum ATCC34304), 282 Thraustochytrium roseum ATCC28210) strain, Oblongichytrium sp SEK600 (Oblongichytrium sp. SEK600) strain, Botriochitrium sp. SEK598 (Botryochytrium sp. SEK598) strain, Schizochytrium sp. (Schizochytrium sp. SEK345), Traustochytrium aureum SEK621 (Thraustochytrium aureum SEK621), Thraustochytrium af Kaudyborum SEK616 (Thraustochytrium aff. Caudivorum SEK 616), Botriochitrium radiochy SEK353 Co., Ltd., Paris Etchichytrium Sarcalianum SEK351 (Par ietichytrium sarkarianum SEK351), Parietichytrium sarkarianum SEK364 (Parietichytrium sarkarianum SEK364), and Parietichytrium sp. SEK358 (Parietichytrium sp. SEK358), and the ability of these strains to produce proteins with lignin-degrading activity 2. The method of claim 1, wherein the method is at least one selected from mutant strains having.   The method according to any one of claims 1 to 4, wherein the culturing step is a step of culturing the microorganism using a medium containing a surfactant.   6. The method of claim 5, wherein the surfactant is Tween 80 ™.   The culturing step is a step of culturing the microorganism using a medium having a polypeptone concentration of 1.0 g / L or less, a glucose concentration of 1.0 g / L or less, and / or a yeast extract concentration of 1.0 g / L or less. The method as described in any one of -6.   The culturing step is a step of culturing the microorganism using a medium having a polypeptone concentration of 1.0 g / L or more, a glucose concentration of 1.0 g / L or more, and / or a yeast extract concentration of 1.0 g / L or more. The method as described in any one of -6.   A method for degrading lignin, comprising a step of degrading lignin by acting on lignin a protein having a lignin-degrading activity produced by at least one of microorganisms classified into the genus Cerebrum.   Use of at least one kind of microorganism classified into the genus Cerebrum, or a protein having lignin-degrading activity produced in the microorganism, in the degradation of lignin.   The microorganism is genus Aurantiochytrium, genus Ulkenia, genus Thraustochytrium, genus Oblongichytrium, genus Schizothytrium, genus Aplanochytrium, 10.At least one selected from microorganisms belonging to the genus Botryochytrium, the genus Sicyoidochytrium, the genus Parietichytrium, and the genus Japonochytrium. Use of the method or claim 10.   Said microorganisms are Ulkenia amoeboidea, Thraustochytrium kinnei, Aurantiochytrium limacinum, Thraustochytrium strium Aurantiochytrium mangrovei, Schizochytrium aggregatum, Thraustochytrium aureum, Thraustochytrium roseum, Aranochetium kell At least one species selected from microorganisms belonging to Botryochytrium radiatum and Parietichytrium sarkarianum Use of the method or claim 10 of claim 9.   The microorganisms are Aurantiochytrium sp. ATCC 20888 (Aurantiochytrium sp. ATCC 20888), Aurantiochytrium sp. SEK618 (Thraustochytrium kinnei SEK618), Urkenia sp.ATCC28207 (Ulkenia sp. ) Strain, Thraustochytrium striatum ATCC 24473 (Thraustochytrium striatum ATCC 24473) strain, Aurantiochytrium mangrovei SEK218 strain, Aurantiochytrium remacinum SR21 ATCC MYA-1381 (Aurantiochytrium limacinum SR21 Strain of Oblongichytrium sp. SEK347 (Oblongichytri um sp. SEK347), Urkenia sp. SEK615 (Ulkenia sp. SEK615), Schizochytrium aggregatum ATCC28209 (Schizochytrium aggregatum ATCC28209), Traustochytrium aureum ATCC34304 (Thraustochytrium aureum ATCC34304), 282 Thraustochytrium roseum ATCC28210) strain, Oblongichytrium sp SEK600 (Oblongichytrium sp. SEK600) strain, Botriochitrium sp. SEK598 (Botryochytrium sp. SEK598) strain, Schizochytrium sp. (Schizochytrium sp. SEK345), Traustochytrium aureum SEK621 (Thraustochytrium aureum SEK621), Thraustochytrium af Kaudyborum SEK616 (Thraustochytrium aff. Caudivorum SEK 616), Botriochitrium radiochy SEK353 Co., Ltd., Paris Etchichytrium Sarcalianum SEK351 (Par ietichytrium sarkarianum SEK351), Parietichytrium sarkarianum SEK364 (Parietichytrium sarkarianum SEK364), and Parietichytrium sp. SEK358 (Parietichytrium sp. SEK358), and the ability of these strains to produce proteins with lignin-degrading activity 11. The method of claim 9 or the use of claim 10, which is at least one selected from mutants having

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