WO2016111368A1 - Procédé de production d'une protéine ayant une activité de dégradation de la lignine faisant appel à des thraustochytrides - Google Patents

Procédé de production d'une protéine ayant une activité de dégradation de la lignine faisant appel à des thraustochytrides Download PDF

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WO2016111368A1
WO2016111368A1 PCT/JP2016/050571 JP2016050571W WO2016111368A1 WO 2016111368 A1 WO2016111368 A1 WO 2016111368A1 JP 2016050571 W JP2016050571 W JP 2016050571W WO 2016111368 A1 WO2016111368 A1 WO 2016111368A1
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lignin
genus
aurantiochytrium
thraustochytrium
microorganism
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PCT/JP2016/050571
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English (en)
Japanese (ja)
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洋介 田岡
雅弘 林
大輔 本多
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国立大学法人宮崎大学
学校法人甲南学園
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Priority to JP2016568765A priority Critical patent/JP6682109B2/ja
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a method for producing a protein having lignin-degrading activity derived from microorganisms, a lignin-decomposing method using microorganisms, and the like.
  • Lignin is a constituent of woody biomass and is one of the substances that are most difficult 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).
  • Non-Patent Documents 1 to 4 Non-Patent Documents 1 to 4.
  • 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.
  • the jellyfish fungi which are marine eukaryotic microorganisms, accumulate significant amounts of oils and fats 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).
  • Patent Document 1 the lignin decomposing activity ability of the jellyfish fungi has not been studied.
  • An object of the present invention is to identify a novel organism having lignin resolution, to produce a lignin-degrading enzyme using the organism, and to provide a method for degrading lignin.
  • the present inventors screened thraustochytrids and examined conditions for inducing the degradation enzyme.
  • 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.
  • FIG. 1 shows an example of a plate assay of the present invention.
  • the left shows the results when RBBR-containing medium (medium 6 in Table 3) is used, and the right shows the results when lignin-containing medium (medium 8 in Table 3) is used.
  • FIG. 2 shows RBBR degradation activity of 7 strains having particularly excellent RBBR degradation activity among mutant strains of Aurantiochytriumchylimacinum mh0186.
  • the vertical axis represents the absorbance at 595 nm, which is the maximum absorption wavelength of RBBR.
  • 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.
  • 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 lignin-degrading activity of microorganisms can be measured by, for example, a plate assay.
  • 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.
  • 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.
  • 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.
  • 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.
  • Microorganisms classified as a kind of mold are marine eukaryotic microorganisms that produce highly unsaturated fatty acids as described 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.
  • a microorganism that is classified as a jellyfish and has the ability to produce a protein having lignin-degrading activity may be used.
  • a microorganism having capacity is preferred.
  • microorganisms that belong to the genus Aplanochytrium and have the ability to produce proteins having lignin-degrading activity include at least one species belonging to Aplanochytrium kerguelense.
  • the strain Aplanochytrium kerguelense SEK535 (Aplanochytrium kerguelense SEK535) is preferably used.
  • microorganism having the ability to produce a protein belonging to the genus Aurantiochytrium and having 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 SENB ATCC26185 or Aurantiochytrium sp. ATCC20888 is preferably used.
  • Examples of the microorganism having the ability to produce a protein belonging to the genus Botryochytrium and having a lignin-degrading activity include at least one species belonging to Botryochytrium radiatum moth.
  • Botryochytrium radiatum SEK353 accession number NBRC 104107) or Botryochytrium sp.
  • SEK598 accession number NBRC 110811 is preferably used.
  • Oblongichytrium sp. SEK347 (accession number NBRC 102618) or Oblongichytrium sp. SEK600 (accession number NBRC 110833) is preferably used.
  • 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 moth.
  • Parietichytrium sarkarianum SEK351 accession number NBRC 104108
  • Parietichytrium sarkarianum SEK364 accession number FERM BP-11298
  • SEK358 accesable. Used.
  • microorganism having the ability to produce a protein belonging to the genus Schizochytrium and having a lignin-degrading activity include at least one species belonging to Schizochytrium aggregatum or Schizochytrium limacinum.
  • Schizochytrium aggregatum ATCC28209, Schizochytrium sp. SEK210 (accession number NBRC 102615), or Schizochytrium sp. SEK345 (accession number NBRC 102616) is preferably used.
  • Microorganisms that belong to the genus Thraustochytrium and have the ability to produce a protein having lignin-degrading activity include Thraustochytrium aureum, Thraustochytrium kinnei, Thraustochytrium kinnei, Thraustochytrium kinnei, Or at least 1 sort which belongs to Thraustochytrium striatum (Thraustochytrium striatum) is mentioned.
  • Thraustochytrium aureum ATCC34304 Thraustochytrium aureum SEK621 (accession number NBRC 110820), Thraustochytrium kinnei SEK617 (accession number NBRC 110825), Thraustochytrium kinc ⁇ Thraustochytrium striatum ATCC24473, Thraustochytrium striatum NE03, or Thraustochytrium aff.
  • Caudivorum SEK 616 (identical to Schizochytrium sp. SEK616) (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 lignin-degrading activity include at least one species belonging to Ulkenia amoeboidea.
  • Ulkenia amoeboidea SEK214 accession number NBRC 104106
  • 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.
  • Aurantiochytrium limacinum mh0186 (FERM BP-11311) is disclosed in JP-A-2005-287380, and is an independent administrative institution National Institute of Advanced Industrial Science and Technology Patent, which is an international depositary organization under the Budapest Treaty Biological Deposit Center (1st, 1st, Higashi 1-chome, Tsukuba City, Ibaraki, Japan, 305-8566, Japan) (Currently, NITE Patent Biological Deposit Center (IPOD), 292-0818 Japan) Schizochytrium sp.
  • 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) MycoscienceTaxonomic rearrangement of the genus sen totolato morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov.
  • Aurantiochytrium limacinum mh0186 strain was obtained from the method of acquiring Schizophytrium Ssp. M-8 strain disclosed in JP-A-2005-287380 (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.
  • Aurantiochytrium limacinum SR21 ATCC MYA-1381 is Schizophytrium limacinum Hyundai et Yokochi (ATCC MYA-1381), Aurantiochytrium sp. ATCC26185 is Thraustochytrium sp.
  • ATCC 26185 AurantiochyAT20 , Schizochytrium aggregatum ATCC28209 the Schizochytrium aggregatum Goldstein et Belsky (ATCC 28209), Thraustochytrium aureum ATCC34304 and Thraustochytrium aureum Goldstein (ATCC 34304), Thraustochytrium roseum ATCC28210 and Thraustochytrium roseum Goldstein (ATCC 28210_TT), Thraustochytrium striatum ATCC24473 is Thraustochytrium striatum Schneider ( ATCC 24473) and Ulkenia sp. ATCC 28207 are the same as Japonochytrium sp. (ATCC 28207), respectively, and the present inventors have reconstructed the genus or species classification.
  • 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 102SEK) 110833), Parietichytrium sarkarianum SEK351 (Accession number NBRC 104108), Schizophytrium sp. SEK210 (Accession number NBRC 102615), Schizochytrium sp.
  • SEK345 (Accession number NBRC 102616), Thraustochytrium aff. Caudivorum SERC616 (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. National Institute of Technology and Evaluation (2-5 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818) -8). 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 on 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 the National Institute for Product Evaluation Technology (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: “a novel astaxanthin-producing ravirinthura 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 lignin-degrading activity of Aurantiochytrium limacinum mh0186 (Accession No. FERM BP-11311).
  • 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.
  • radiation treatment include UV irradiation, gamma irradiation, X-ray irradiation, and heavy ion beam irradiation.
  • mutagens include ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, bromouracil. And nucleotide base analogs, and acridines.
  • 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.
  • 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.
  • a fixed medium, a liquid medium, or the like suitable for cultivating the crucible is used as the medium.
  • the medium is not particularly limited. Examples of commonly used mediums 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)), etc. are known, and these 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, and 25 to 35. Incubate at °C. 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 amount of protein having lignin-degrading activity by the microorganism is significantly increased by culturing the microorganism with 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 TM (polysorbate 20, polyoxyethylene sorbitan monolaurate), Tween® 40 TM (polysorbate 40, polyoxyethylene sorbitan).
  • Tween 60 TM polysorbate 60, polyoxyethylene sorbitan monostearate
  • Tween 80 TM polysorbate 80, polyoxyethylene sorbitan monooleate
  • Brij 35 TM polyoxyethylene (23) lauryl ether
  • Triton® X-100 TM poly (oxyethylene) octylphenyl ether
  • 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.
  • a carbon source such as glucose, a nitrogen source such as polypeptone, and a growth promoting substance such as yeast extract.
  • 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.
  • the nitrogen source examples 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.
  • the growth promoting substance examples include meat extract and malt extract in addition to yeast extract.
  • 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.
  • 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.
  • 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.
  • 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, 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.
  • the microorganism may be cultured using a rich medium.
  • 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.
  • 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.
  • 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, 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
  • 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 at a concentration of, for example, 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.
  • 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.
  • 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.
  • 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.
  • the culture supernatant fraction can be used in the method of the present invention.
  • a culture supernatant fraction 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.
  • 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.
  • 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.
  • 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.
  • the lignin-containing material preferably contains a buffer solution or the like in order to maintain conditions suitable for lignin degradation.
  • the conditions of 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.
  • it can be incubated under appropriate temperature and pH conditions for several tens of minutes to two weeks, preferably several hours to several days, or about 1 to 3 days.
  • temperature conditions include 10 ° C. to 40 ° C., 15 ° C. to 35 ° C., preferably 20 ° C. to 30 ° C.
  • examples of pH conditions include pH 5 to 9, 6 to 8, preferably 6.5 to 7.5.
  • 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.
  • 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. .
  • composition for use in protein production or for degradation of lignin 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.
  • 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.
  • 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.
  • the concentration of the composition of the present invention depends on the dilution ratio.
  • the concentration of the above component may be, for example, several times to several hundred times, preferably several times to several tens of times the above concentration).
  • 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.
  • the concentration of the above 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 above concentration, depending on the dilution ratio. May be).
  • 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.
  • 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) ( Medium 1 to 8 were prepared according to Table 3 below.
  • RBBR Remazol Brilliant Blue R
  • lignin Nacalai Tesque
  • Tween 80 Nacalai Tesque
  • polypeptone yeast extract
  • 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
  • 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]
  • 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.
  • 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.
  • 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 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.
  • 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 100 ⁇ l of RBBR-containing liquid medium (composition of medium 3 shown in Table 3 above, excluding agar, vitamin liquid, and trace elements) was aseptically placed in each well of 96-well microplate Was 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 for 5 seconds 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.

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Abstract

Le problème abordé dans la présente invention est de pourvoir à un procédé de production d'une protéine ayant une activité de dégradation de la lignine ou à un procédé de dégradation de la lignine. La solution selon l'invention porte sur un procédé de production d'une protéine ayant une activité de dégradation de la lignine et sur un procédé de dégradation de la lignine, chaque procédé comprenant la culture d'au moins un type de micro-organisme appartenant à la famille des thraustochytrides.
PCT/JP2016/050571 2015-01-09 2016-01-08 Procédé de production d'une protéine ayant une activité de dégradation de la lignine faisant appel à des thraustochytrides WO2016111368A1 (fr)

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Title
ATALLA, M. MABROUK. ET AL.: "Screening of some marine-derived fungal isolates for lignin degrading enzymes (LDEs) production", AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA, vol. 1, 2010, pages 591 - 599 *
KAZUTO FUKAYA ET AL.: "Remazol Brilliant Blue R o Shihyo to shita Kaiyosei Shinkaku Biseibutsu Yaburetsubokabi-rui ni yoru Lignin Shori Noryoku no Hyoka", JAPANESE SOCIETY FOR MARINE BIOTECHNOLOGY TAIKAI KOEN YOSHISHU, vol. 15th, 1 June 2013 (2013-06-01), pages 108 *

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