WO2022176960A1 - Utilisation d'une nouvelle lipase hautement fonctionnelle dérivée de basidiomycètes - Google Patents

Utilisation d'une nouvelle lipase hautement fonctionnelle dérivée de basidiomycètes Download PDF

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WO2022176960A1
WO2022176960A1 PCT/JP2022/006467 JP2022006467W WO2022176960A1 WO 2022176960 A1 WO2022176960 A1 WO 2022176960A1 JP 2022006467 W JP2022006467 W JP 2022006467W WO 2022176960 A1 WO2022176960 A1 WO 2022176960A1
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lipase
enzyme composition
enzyme
amino acid
acid sequence
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千明 ▲高▼須賀
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N9/14—Hydrolases (3)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention relates to the use of novel highly functional lipases derived from basidiomycetes.
  • Lipase is a general term for enzymes that hydrolyze the ester bonds of lipids, and because the reaction is reversible, it is known to act as a catalyst for esterification and transesterification depending on the reaction conditions.
  • Lipase is industrially used in the production of agricultural chemicals, pharmaceuticals, cosmetics, fats and oils, food processing, food manufacturing, and food additive manufacturing. It is also used as a catalyst for splitting racemates. There are many types of lipases with different properties such as substrate specificity and degradation specificity.
  • biomass woody plant resources
  • the main components of plant cell walls are cellulose, hemicellulose and lignin, which form persistent plant cell walls that prevent assimilation and parasitism by other organisms.
  • solvent-soluble fraction commonly called “extract” or “extractables”
  • extract also constitutes about 10 w/w% of total plant biomass.
  • the chemical composition of the extract varies with tree species and age. This extract is also believed to be responsible for the plant's defense function, especially as a factor that prevents or eliminates fungal colonization in freshly felled conifers.
  • wood-rotting fungi which are a kind of basidiomycete, live on plant cell walls as a source of nutrition by secreting various extracellular enzymes including cellulase, hemicellulase, and lignin-degrading enzymes.
  • the lignin-degrading basidiomycete Phlebiopsis gigantea (hereinafter, P. gigantea) is known to be able to rapidly invade freshly felled conifers rich in extractives.
  • P. The mechanism of how gigantea detoxifies or utilizes extracts derived from conifers is largely unknown, but it is known that gigantea secretes lipase when decomposing conifers (Non-Patent Document 1).
  • the purpose of the present invention is to discover a novel highly functional lipase and to provide a method for using it.
  • the present invention provides, for example, the following aspects.
  • [1] an enzyme composition comprising a lipase comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1;
  • a lipase recombinant enzyme comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; [11] a vector comprising a nucleic acid encoding a lipase comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; [12] The vector of [11], wherein the lipase-encoding nucleic acid comprises a codon-optimized sequence; [13] a host into which the vector described in [11] or [12] has been introduced; [14] A method for producing a recombinant enzyme, which comprises culturing the host according to [13], which is yeast, and the host according to [15][13] or [14].
  • an enzyme composition containing a basidiomycete-derived lipase is provided, which has an optimal acidic pH and an optimal temperature between room temperature and low temperature. Since the composition has high activity at room temperature and under acidic conditions, it can be used in various applications such as foods, pharmaceuticals, and detergents. In addition, since the lipase is derived from a naturally occurring basidiomycete, the enzyme composition of the present invention is also excellent in safety.
  • P. spp. cultured on AV0X, AV1X, AV2X, or AV4X It shows the time course of lipase activity in the secretome of gigantea.
  • the transcription expression levels (RPKM) and ratios (Ratio) of the nine lipases encoded by the gigantea genome are shown.
  • Bold indicates p-value ⁇ 0.05.
  • 2 shows the amino acid sequence (SEQ ID NO: 2) of the secretory lipase PgLip19028. In the figure, the signal sequence is underlined.
  • FIG. 2 shows electrophoresis results of recombinant enzyme PgLip19028.
  • FIG. The results of a lipase activity test of recombinant PgLip19028 are shown. Relative activity of PgLip19028 under various pH conditions. Relative activity of PgLip19028 under various temperature conditions is shown.
  • PgLip19028 is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, characterized by having a lipase conserved motif sequence GXSXG (SEQ ID NO: 3: GHSLG) and a catalytic triad (Ser180, Asp237, and His251) ( Figure 3). It is also predicted to have a secretory signal, three N-glycosylation sites and one O-glycosylation site.
  • the amino acid sequence of PgLip19028 without a secretion signal is shown in SEQ ID NO:1, and the amino acid sequence of PgLip19028 with a secretion signal is shown in SEQ ID NO:2.
  • nucleic acid sequence of PgLip19028 without a secretion signal is shown in SEQ ID NO:4, and the nucleic acid sequence of PgLip19028 with a secretion signal is shown in SEQ ID NO:5.
  • PgLip19028 was deduced to encode a lipase in triacylglycerol hydrolase (EC 3.1.1.3). It has now been shown in the present invention that PgLip19028 has a different molecular weight, pH and temperature optima, and substrate specificity than other previously characterized basidiomycete lipases. Biochemical characterization in the present invention has shown that PgLip19028 can liberate various unsaturated fatty acids under acidic conditions at ambient temperature. Furthermore, P.I. It was found that basidiomycetes other than gigantea also have lipases having amino acid sequences similar to PgLip19028 and having the same conserved motif sequence GHSLG.
  • the lipase used (hereinafter also referred to as “the lipase of the present invention”) is a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1.
  • the polypeptide may contain one to several amino acid mutations (substitutions, deletions, additions or insertions, or combinations thereof) in the amino acid sequence shown in SEQ ID NO: 1, as long as it has lipase activity, Alternatively, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85%, preferably at least 90%, at least the amino acid sequence shown in SEQ ID NO: 1, as long as it has lipase activity It may comprise amino acid sequences having 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the lipase of the present invention is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, or at least 45%, at least 50%, at least 60%, at least 70%, at least 80% with the amino acid sequence shown in SEQ ID NO: 1. %, or at least 85%, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and having lipase activity.
  • Peptides and polypeptides consisting of an amino acid sequence having at least 45% to at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 preferably retain the motif sequence "GHSLG".
  • “several” refers to about 2 to 10, and may be, for example, 3, 4, 5, 6, 7, 8, or 9.
  • amino acid substitutions may be conservative substitutions or non-conservative substitutions. Conservative substitutions are preferred.
  • the lipase of the present invention has an optimum temperature in the range of 10°C to 35°C, preferably 15°C to 30°C, more preferably 20°C to 30°C, and even more preferably 25°C. good too.
  • the lipase of the present invention may have an optimum pH in the range of pH 3 to pH 5, preferably in the range of pH 3.5 to pH 5, more preferably in the range of pH 4 to pH 5, still more preferably in the range of pH 4.5. good.
  • the optimum temperature and optimum pH for lipase are temperature conditions and conditions under which the relative activity of the enzyme is 40% or higher, preferably 60% or higher, more preferably 70% or higher, and even more preferably 80% or higher. Refers to pH conditions.
  • the relative activity is the relative value of the lipase activity under each condition when the lipase activity at the temperature or pH condition showing the highest lipase activity is defined as 100%.
  • lipase activity is based on hydrolytic activity on substrates. Measurement of lipase activity may be performed using a conventional method. It can be done by measuring the amount of nitrophenol.
  • the lipase of the present invention may have a molecular weight of about 30 kDa.
  • the lipase of the present invention can be obtained from a basidiomycete culture or culture supernatant.
  • a basidiomycete culture or culture supernatant may be the lysate of the bacterial cells or the culture supernatant itself, or it may be crudely purified or purified from the lysate of the bacterial cells or the culture supernatant.
  • the lipase of the present invention is usually a secretory type having an amino acid sequence with a secretory signal sequence added to the N-terminus, it is preferably obtained from the culture supernatant.
  • Basal media include, but are not limited to, potato dextrose agar (PDA) medium, malt extract agar (MYA) medium, Highley's basal medium, and the like.
  • PDA potato dextrose agar
  • MYA malt extract agar
  • Glucose, cellulose, or the like may be added to the medium as a carbon source.
  • the medium is further preferably supplemented with a coniferous extract to facilitate lipase induction. Examples of conifers include, but are not limited to, pine (eg, locust pine), cedar, cypress, hemlock, fir, spruce, yew, and the like.
  • the coniferous tree extract may be obtained, for example, by extracting wood flour obtained by crushing the bark-stripped woody part of the coniferous tree with an organic solvent such as acetone.
  • an extraction method a method of preparing by immersing in an organic solvent, heating and stirring, or a Soxhlet extraction method may be used.
  • the extract may be concentrated, for example, by rotary evaporation, freeze-drying, or the like. Culture conditions such as culture temperature and culture period can be appropriately set by those skilled in the art. For example, it may be cultured at 20-30° C. for 3-14 days.
  • basidiomycete preferably P. gigantea ⁇ Shizophilum commune ⁇ Coniophora souna ⁇ Serpula lacrymans ⁇ Heterobasidion annosum ⁇ Stereum hirsutum ⁇ Postia planceta ⁇ Wolfiporia cocos ⁇ Fomitopsis pinicola ⁇ Ceriporiopsis suvermispora ⁇ Dichomitus squalens ⁇ Trametes versicolor ⁇ Phanerochaete ⁇ Phanerochaete carnosa , Phlebia breviospora and the like may be used.
  • These basidiomycetes may be of any strain, and may be collected from the natural world or obtained from preservation organizations such as ATCC (American Type Culture Collection).
  • the lipase used in one aspect of the present invention may be produced by chemical synthesis (peptide synthesis) based on its amino acid sequence.
  • the lipase used in the present invention may be a recombinant enzyme.
  • a recombinant enzyme of PgLip19028 was produced for the first time in the present invention.
  • an expression system for producing the recombinant lipase was constructed for the first time.
  • a cell-free protein synthesis system (derived from E. coli, wheat germ, etc.) or a cellular protein expression system may be used for the production of recombinant enzymes.
  • Hosts for cellular protein expression systems can be either prokaryotic or eukaryotic cells, such as fungi (yeast, filamentous fungi, etc.), animal cells (vertebrate cells, mammalian cells, etc.), insect cells. , bacteria (Escherichia coli, Bacillus subtilis, actinomycetes, etc.), and the like.
  • Yeast is preferably used, and examples thereof include, but are not limited to, yeast of the genus Saccharomyces, yeast of the genus Candida, and yeast of the genus Pichia. For example, Pichia pastoris may be used.
  • a nucleic acid encoding the lipase of the present invention is introduced into a host cell for the production of a recombinant enzyme.
  • Nucleic acids encoding the lipases of the present invention are obtained from P. gigantea can be synthesized based on known sequence information.
  • a nucleic acid sequence encoding a lipase of the invention may be codon-optimized for expression in a host cell. The optimal codons for each host and methods of codon optimization are well known to those skilled in the art.
  • the nucleic acid encoding the lipase of the present invention is at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% the amino acid sequence of PgLip19028, i.e., the amino acid sequence shown in SEQ ID NO: 1. , preferably amino acids having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity It includes a nucleotide sequence that encodes a sequence.
  • the nucleic acid encoding the lipase of the invention is at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% the amino acid sequence shown in SEQ ID NO: 1, preferably encode amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity It may be a nucleic acid consisting of a nucleotide sequence. Nucleic acids encoding the lipases of the present invention include, for example, nucleic acids comprising the nucleotide sequence shown in SEQ ID NO:4.
  • nucleic acids encoding lipases of the invention include at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% relative to the nucleotide sequence shown in SEQ ID NO:4, Preferably, nucleotide sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity
  • a nucleic acid containing An example of a nucleic acid sequence codon-optimized for expression in Pichia pastoris as a host is the sequence shown in SEQ ID NO:6.
  • a secretory signal sequence may be added to the nucleic acid sequence encoding the lipase of the present invention.
  • the secretory signal sequence for example, the secretory signal sequence of PgLip19028 or the secretory signal sequence of ⁇ -factor may be used.
  • the nucleic acid sequence encoding the lipase of the present invention can be a promoter sequence and/or any sequence involved in nucleic acid expression, such as an enhancer sequence, and can be used for various analyzes such as protein stabilization, purification, and expression level measurement. It may be operably linked to sequences of interest, eg, maltose binding protein (MBP), polyhistidine tag (His-tag), human c-Myc tag, and the like.
  • MBP maltose binding protein
  • His-tag polyhistidine tag
  • human c-Myc tag and the like.
  • promoter sequences include, but are not limited to, alcohol oxidase (AOX) promoter sequences that can induce protein production by addition of methanol, and glyceraldehyde-3-phosphorus as a promoter for constant protein production.
  • AOX alcohol oxidase
  • the promoter sequence for acid dehydrogenase GAP can be used.
  • a nucleic acid encoding the lipase of the present invention may be inserted into an appropriate vector, and the vector may be introduced into host cells.
  • vector refers to a nucleic acid molecule that transports a nucleic acid of interest into a host cell.
  • Vectors known in the art include plasmid vectors, cosmids, lambda phages, artificial chromosomes, viral vectors (e.g., baculovirus vectors, retroviral vectors, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, etc.), liposomes, and the like.
  • a variety of vectors can be used.
  • a vector suitable for the host cell may be selected.
  • the vector is preferably an expression vector and may contain a promoter sequence and/or any sequence involved in the expression of the inserted nucleic acid, such as an enhancer sequence.
  • a promoter sequence and/or any sequence involved in the expression of the inserted nucleic acid, such as an enhancer sequence.
  • Various promoters are known, and a suitable promoter for the host cell may be selected.
  • the vector may further contain a selection marker gene, such as an antibiotic or drug resistance gene, for selection of cells into which the gene of interest has been introduced (transformed cells) after introduction into host cells.
  • Introduction of vectors into host cells can be performed by methods known in the art. For example, cationic lipid-mediated introduction method, diethylaminoethyl (DEAE)-dextran method, calcium phosphate co-precipitation method, cationic polymer introduction method, electroporation method, microinjection method, sonoporation method, laser irradiation method, virus A vector-based introduction method and the like can be mentioned.
  • cationic lipid-mediated introduction method diethylaminoethyl (DEAE)-dextran method
  • calcium phosphate co-precipitation method calcium phosphate co-precipitation method
  • cationic polymer introduction method electroporation method
  • microinjection method microinjection method
  • sonoporation method laser irradiation method
  • virus A vector-based introduction method and the like can be mentioned.
  • the desired lipase of the present invention is produced.
  • the lipase of the invention is secreted extracellularly (into the medium).
  • the desired enzyme when produced in a cell line such as E. coli, the desired enzyme may be obtained in an insolubilized form (precipitate).
  • the active form of the enzyme can be obtained by carrying out a solubilization or a folding operation. Solubilization or holding may be performed by methods known in the art.
  • Gene-introduced cells can be cultured according to a conventional method, and a culture medium suitable for the host can be selected. Examples include, but are not limited to, YPD medium, YPG medium, and the like. Culture conditions such as culture temperature and time can be appropriately selected. For example, it may be cultured at about 20 to 30° C. for about 1 day to 1 week.
  • the lipase of the present invention produced by host cells may be crudely purified or purified from the lysate or culture supernatant of cultured cells, preferably from the culture supernatant.
  • Crude purification and purification operations can be performed by methods known in the art. For crude purification or purification, for example, salting out, centrifugation, ultrafiltration, gel filtration, various chromatography (adsorption chromatography, hydrophobic chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, etc.), It can be carried out by known methods such as dialysis and electrophoresis.
  • the lipase of the present invention is provided in the form of an enzyme composition.
  • the enzyme composition containing the lipase of the present invention (hereinafter also referred to as the "enzyme composition of the present invention") may be the lipase of the present invention itself, or an additional component in addition to the lipase of the present invention as an active ingredient. may contain Examples of such additional components include excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, etc., depending on the application of the enzyme composition of the present invention. Excipients may include, but are not limited to, starch, dextrin, sugars, sugar alcohols, glycerol, and the like, depending on the use of the enzyme composition.
  • buffering agents include, but are not limited to, phosphates, citrates, acetates, and the like, depending on the application of the enzyme composition.
  • stabilizers include, but are not limited to, propylene glycol, ascorbic acid, and the like, depending on the application of the enzyme composition.
  • preservatives include, but are not limited to, phenol, benzalkonium chloride, methylparaben, and the like, depending on the use of the enzyme composition.
  • preservatives include, but are not limited to, ethanol, benzalkonium chloride, paraoxybenzoic acid, and the like, depending on the application of the enzyme composition.
  • the amount of the lipase of the present invention and the amount of additional components in the enzyme composition can be appropriately determined by those skilled in the art.
  • the enzyme composition of the present invention may also contain tert-butyl methyl ether.
  • the blending amount of tert-butyl methyl ether in the composition can be appropriately determined by those skilled in the art. For example, 5 v/v % to 30 v/v %, preferably 15 v/v % in the composition.
  • the enzyme composition of the present invention may be a food enzyme composition.
  • the enzyme composition of the present invention can be used as a flavor improving agent (flavor agent) for food or food ingredients.
  • the food or food ingredients include, but are not limited to, various dairy products (eg, cheese, butter, yogurt, etc.), margarine, shortening, various vegetable oils (soybean oil, rapeseed oil, corn oil, palm oil, palm kernel oil, coconut oil, sunflower oil, cottonseed oil, etc.), dressings, and the like. Therefore, in a further aspect of the present invention, there is provided a method for improving the flavor of food or food ingredients, characterized by adding to or acting on the food or food ingredients the enzyme composition of the present invention.
  • the enzyme composition of the present invention for foods can also be used in the production of food additives (e.g., flavoring agents, etc.), food processing (e.g., sake production, etc.), and production of edible fats and oils (e.g., cooking oil, shortening, etc.). may be used for Therefore, in a further aspect of the present invention there is provided a method for producing food, characterized in that the enzyme composition of the present invention is added to or allowed to act on a food raw material or intermediate product.
  • food additives e.g., flavoring agents, etc.
  • food processing e.g., sake production, etc.
  • edible fats and oils e.g., cooking oil, shortening, etc.
  • the enzyme composition of the present invention may be a pharmaceutical enzyme composition.
  • the enzyme composition of the present invention for pharmaceuticals includes, but is not limited to, pharmaceuticals (e.g., digestive enzyme agents, alternative enzymes, e.g., therapeutic agents for lysosomal acid lipase deficiency, etc.), laboratory test agents (e.g., blood A test drug for measuring middle cholesterol, etc.) may also be used.
  • the enzyme composition of the present invention can also be used as a cleaning composition. Accordingly, in a further aspect of the invention there is provided a cleaning composition comprising the enzymatic composition of the invention.
  • the cleaning composition containing the enzyme composition of the present invention decomposes and removes lipids adhering to the items to be cleaned (laundry, etc.) to improve the cleaning effect, and has high activity at low temperatures (water temperature). , further improving the cleaning effect.
  • the enzyme composition of the present invention may further be an enzyme composition for cosmetics, and can be used, for example, as an additive for cosmetics (eg, facial cleanser, etc.).
  • the enzyme composition of the present invention can be used for the production of fatty acids and soaps, wastewater treatment, the production of raw materials for optically active pharmaceuticals and agricultural chemicals, the synthesis of optically active compounds, and is useful as an industrial enzyme.
  • the enzyme composition of the present invention can also be used in paper pulp production.
  • the extractables can be removed in the paper pulp production process from coniferous trees, thereby improving the properties of the paper produced.
  • the enzyme compositions of the invention can also be used in biorefinery processes.
  • the enzyme composition of the present invention can be used to obtain lipid components such as various fatty acids from trees, particularly from conifer extracts.
  • the tree-derived lipid component thus obtained is used in fields such as foods, cosmetics, and pharmaceuticals.
  • Example 1 P.I.
  • the lipase-activated bark of the gigantea culture supernatant was peeled off, and the woody part was pulverized to lobe pine wood powder (1 mm mesh) using 70 w/v% acetone for Soxhlet extraction.
  • PH101, 50 uM, Fluka Chemika, Switzerland and dried on a rotary evaporator to prepare the substrates.
  • the amount of extractives added to the cellulose was such that the amount of natural Lodzia pine extract per weight of wood flour (AV1X), twice the natural concentration (AV2X) and four times the amount (AV4X).
  • An acetone suspension substrate of microcrystalline cellulose without loblolly pine extract was labeled "AV0X”.
  • Substrate was added in 250 mL of basal medium (Highley medium) and P.
  • Basal medium Highley medium
  • Single basidiospore strains 5-6 of gigantea isolate 11061-1 were inoculated with mycelial plugs cultured in PDA medium and cultured for 7 days at 22° C. on a rotary shaker (150 rpm).
  • Culture supernatants were collected over time (3rd, 5th, and 7th days of culture), and the protein concentration in the culture supernatant was measured by Protein Assay (Bio-Rad). Lipase activity in the secretome (culture supernatant secretion) was measured as follows.
  • Lipase activity was measured using p-nitrophenyl dodecanoate (pNPD, Sigma-Aldrich) as a substrate.
  • pNPD p-nitrophenyl dodecanoate
  • DMSO dimethylsulfoxide
  • 25 ⁇ L of 100 mM acetate buffer (pH 5.0) was mixed with 50 ⁇ L of culture supernatant and 25 ⁇ L of 100 mM acetate buffer (pH 5.0) to a total volume of 100 ⁇ L. After 30 min incubation at 37° C., the reaction was stopped by adding 25 ⁇ L of 100 mM Na 2 CO 3 . Released p-nitrophenol (pNP) was detected at 405 nm, and pNP release was determined using a standard curve of pNP (Sigma-Aldrich).
  • One unit of lipase activity was defined as the amount of enzyme that liberates 1 ⁇ mole of pNP per minute of reaction.
  • Example 2 Lipase Induced in the Presence of Conifer Extract Analogously to Example 1, P. gigantea was cultured for 5 days, and total RNA was purified from the obtained cells by a conventional method. Using 100 ng of total RNA obtained from each sample, mRNA was purified according to the protocol of Illumina RNA Sequencing (Illumina Inc., USA), and a 2X150 bp paired-end library for RNA sequencing was created by PCR and adapter addition, Transcriptome analysis was performed with NovaSeq sequencer. The obtained data were mapped to the genome sequence, and the RPKM value was calculated by correcting the expression level of the gene by the total number of reads of the sample and the gene length.
  • Illumina RNA Sequencing Illumina Inc., USA
  • 2X150 bp paired-end library for RNA sequencing was created by PCR and adapter addition
  • Transcriptome analysis was performed with NovaSeq sequencer. The obtained data were mapped to the genome sequence, and the RPKM value was
  • the culture supernatant at this time was subjected to proteome analysis using a liquid chromatography tandem mass spectrometer (LC-MS/MS). Proteins in the culture supernatant were precipitated with 10% (w/v) trichloroacetic acid, washed three times with cold acetone, and air-dried. Total protein from the pellet was purified by methanol/chloroform/water partitioning by adding chloroform and methanol to the resulting pellet followed by water. Proteins partitioned into the interphase. After multiple washes with methanol, the purified protein was finally redissolved in 8 M urea/50 mM NH 4 HCO 3 (pH 8.5)/1 mM TrisHCl.
  • LC-MS/MS liquid chromatography tandem mass spectrometer
  • LC-MS/MS analysis was performed using an Agilent 1100 nanoflow system (Agilent Technologies) coupled to a hybrid linear ion trap-orbitrap mass spectrometer (LTQ-Orbitrap Elite TM , ThermoFisher Scientific) with source.
  • Chromatography of peptides prior to mass spectral analysis was accomplished by automatically loading 2 ⁇ l of purified peptide onto a capillary emitter column (PepMap® C18, 3 ⁇ M, 100 ⁇ , 150 ⁇ 0.075 mm, ThermoFisher Scientific). rice field.
  • the HPLC system was run with solvent A: 0.1% (v/v) formic acid and solvent B: 99.9% (v/v) acetonitrile and 0.1% (v/v) formic acid at 0.50 ⁇ L/ Peptides were loaded (30 min) at a flow rate of 0.3 ⁇ L/min and peptides were electrosprayed with a gradient of 3% (v/v) B to 20% (v/v) B over 154 min at a flow rate of 0.3 ⁇ L/min. followed by a final 12 min gradient of 20% (v/v) B to 50% (v/v) followed by a 5 min flashout of 50-95% (v/v) B. .
  • MSConvert Proteo Wizard: open source software for rapid proteomics tool development
  • transcripts encoding four lipases were significantly upregulated by the presence of the extract.
  • PgLip19028 was identified among the nine lipases (SEQ ID NO: 1), and it was confirmed that PgLip19028 was secreted into the culture supernatant.
  • Example 3 Production of Recombinant PgLip19028 A DNA encoding PgLip19028 from which the natural secretion signal was removed was synthesized (JGI Service). The gene (SEQ ID NO:4) was subcloned into the expression vector pPICZ ⁇ (Invitrogen) using primers for PIPE (Polymerase Incomplete Primer Extension) cloning. As a secretion signal, the ⁇ -factor secretion signal on pPICZ ⁇ was used. As the promoter sequence, the AOX promoter sequence was used. Table 1 shows the primer sequences used. The expression vector was transformed into the yeast Pichia pastoris according to the manufacturer's instructions (Invitrogen).
  • the transformed Pichia pastoris was shake-cultured in YPD liquid medium at 30° C. for 1 to 3 days, and the obtained cells were washed well and transferred to YPG liquid medium. After adding methanol to the medium, it was cultured for 1 to 3 days to induce protein expression, and recombinant lipase was produced from the transformed yeast overexpressing PgLip19028.
  • the culture supernatant of the transformed Pichia pastoris was subjected to saturated ammonium sulfate precipitation, and the resulting protein precipitate was dissolved in 100 mM acetate buffer (pH 5.0) and used as crude enzyme.
  • p-Nitrophenyl dodecanoate manufactured by Sigma-Aldrich
  • DMSO dimethylsulfoxide
  • tBE tert-butyl methyl ether
  • pNP release amount was determined using a standard curve of pNP (manufactured by Sigma-Aldrich).
  • One unit of lipase activity was defined as the amount of enzyme that liberates 1 ⁇ mole of pNP per minute of reaction.
  • 15 v/v% tBE exhibited the highest activity compared to 5 v/v%, 10 v/v%, 15 v/v%, 20 v/v%, 25 v/v% and 30 v/v% tBE. Indicated.
  • Fig. 4 shows the results of electrophoresis
  • Fig. 5 shows the results of lipase activity measurement.
  • the recombinant enzyme PgLip19028 was produced as a major protein in the Pichia pastoris culture supernatant (lane 1: PgLip19028, lane 3: vector control in FIG. 4).
  • the lipase was consistent with a calculated molecular weight (glycosylation and signal peptide subtracted) of approximately 30.0 kDa as estimated by SDS-PAGE (lane 2 in Figure 4).
  • Example 4 Determination of Optimum pH and Temperature of PgLip19028 To determine the optimum pH conditions for PgLip19028, pH 3.0-pH 3.5 tartrate buffer, pH 3.5-pH 5.5 acetate buffer, and The lipase activity of the crude enzyme obtained in Example 3 was measured using an acetate buffer of pH 6.0 to pH 6.5. As described in Example 3, except for pH conditions. Vector control lipase activity was subtracted from the measurements. The results are shown in FIG. In the figure, the highest activity was defined as 100%, and relative values under each condition were shown.
  • the reaction mixture was incubated for 10 minutes at temperatures of 10°C, 15°C, 20°C, 25°C, 30°C and 35°C.
  • the lipase activity of the enzyme was measured. It is as described in Example 3 except for the temperature conditions.
  • Vector control lipase activity was subtracted from the measurements. The results are shown in FIG. In the figure, the highest activity was defined as 100%, and relative values under each condition were shown.
  • the optimum reaction temperature and pH for PgLip19028 were 25°C and pH 4.5, respectively.
  • it showed a relative activity of 40% or more in the range of pH 3 to pH 5 and in the range of 10°C to 35°C, and it was found that these pH conditions or temperature conditions are suitable for the reaction using PgLip19028.
  • the optimum pH 4.5 is clearly lower than other conventional lipases.
  • Such a pH optimum is for P. elegans, which is normally acidic due to secreted organic acids such as oxalic acid. It matches gigantea's external environment.
  • Example 5 Glyceride Degradation Assay of PgLip19028
  • 1 ⁇ g/mL crude enzyme obtained in Example 3 or vector control was added to 10 mg/mL triolein or pine extract, final concentration 15 v/v. % tBE, 5 v/v % DMSO and 25 mM acetate buffer (pH 4.5) to a total volume of 1000 ⁇ L.
  • the pine extract was obtained by stripping the bark and crushing the woody portion of Lodia pine wood powder (1 mm mesh), immersing it in 70 w/v % acetone and stirring overnight. The reaction mixture was incubated at 25° C.
  • GC-MS analysis was performed with an ion trap detector using a medium length fused silica DB-5HT capillary column (12 m x 0.25 mm id, 0.1 ⁇ m film thickness), which allows co-elution of different lipid classes. (Varian 4000) coupled with a Varian 3800 chromatograph (both manufactured by J&W Scientific). The temperature program started at 100° C. (1 minute), increased by 10° C. every minute up to 380° C. and held for 5 minutes. The transfer line was maintained at 300° C., the injector was programmed from 120° C. (0.1 min) to 380° C. at 200° C./min, and helium was used as the carrier gas at a rate of 2 mL/min.
  • Trimethylsilyl derivatives were prepared using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine.
  • Triglycerides decreased significantly after lipase reaction.
  • diolein, monoolein and oleic acid were identified as end products (Fig. 8A).
  • PgLip19028 liberated oleic, linoleic, linolenic and palmitic acids from triglycerides and diglycerides in conifer extracts (Fig. 8B).
  • Example 5 Activity comparison test with commercially available lipases The lipase activities of PgLip19028 and four commercially available lipases derived from filamentous fungi were compared.
  • Commercially available lipases include Lipase M1 Amano (derived from Rhizomucor miehei; Amano Enzyme Inc.), Lipase A6 Amano (derived from Asperigillus niger; Amano Enzyme Inc.), Lipase F-AP15 Amano (derived from Rhizopus oryzae; Amano Enzyme Inc.), and Novozym 51032 (derived from Asperigillus; Novozymes) was used.
  • Lipase M1 Amano derived from Rhizomucor miehei; Amano Enzyme Inc.
  • Lipase A6 Amano derived from Asperigillus niger; Amano Enzyme Inc.
  • Lipase F-AP15 Amano derived from Rhizopus
  • Lipase activity was measured as described in Example 3 using the crude enzyme or vector control obtained in Example 3 or 1 ⁇ g/mL of commercially available lipase. The results are shown in FIG. PgLip19028, in the state of crude enzyme, showed higher activity than commercially available lipase.
  • SEQ ID NO: 6 Nucleotide sequence coding PgiLip19028 (-signal), codon optimized for Pichia pastoris SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10;

Abstract

L'invention concerne une composition enzymatique, etc, qui comprend une lipase comprenant une séquence d'acides aminés ayant au moins 90 % d'identité de séquence avec une séquence d'acides aminés représentée par SEQ ID NO : 1.
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IWATA MANA, GUTIÉRREZ ANA, MARQUES GISELA, SABAT GRZEGORZ, KERSTEN PHILIP J., CULLEN DANIEL, BHATNAGAR JENNIFER M., YADAV JAGJIT, : "Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives", SCIENTIFIC REPORTS, vol. 11, no. 1, 1 December 2021 (2021-12-01), pages 1 - 14, XP055960572, DOI: 10.1038/s41598-021-91756-5 *
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