WO2022176960A1 - Utilization of novel highly functional lipase derived from basidiomycetes - Google Patents
Utilization of novel highly functional lipase derived from basidiomycetes Download PDFInfo
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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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- Prior art keywords
- lipase
- enzyme composition
- enzyme
- amino acid
- acid sequence
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Images
Classifications
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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
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Provided is an enzyme composition, etc., that includes a lipase including an amino acid sequence having at least 90% sequence identity with an amino acid sequence shown by SEQ ID NO: 1.
Description
本発明は、担子菌由来の新規な高機能性リパーゼの利用に関する。
The present invention relates to the use of novel highly functional lipases derived from basidiomycetes.
リパーゼは、脂質のエステル結合を加水分解する酵素の総称であり、その反応は可逆性であるため、反応条件により、エステル化反応およびエステル交換反応の触媒としても作用することが知られている。リパーゼは、農薬製造、医薬品製造、化粧品製造、油脂製造、食品加工、食品製造、食品添加物の製造等において産業利用されており、また、消化薬、臨床検査薬、フレーバー剤、洗剤用酵素、ラセミ体分割の触媒等としても利用されている。基質特異性や分解特異性等の性質が異なる多くの種類のリパーゼが存在する。
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.
地球上で最も豊富な炭素源は木本植物資源(バイオマス)である。植物細胞壁の主成分は、セルロース、ヘミセルロースおよびリグニンであり、他の生物による資化や寄生を妨げる難分解性の植物細胞壁を形成している。また、これらの成分の他に、溶媒可溶性画分(一般に「抽出物」または「抽出成分」と呼ばれる)が全植物バイオマスの約10w/w%を構成する。抽出物の化学組成は、樹種や樹齢によって異なる。この抽出物もまた、植物の防御機能を担い、特に、伐採直後の針葉樹における菌のコロニー形成を妨害または排除する因子であると考えられている。一方で、担子菌の一種である木材腐朽菌は、セルラーゼ、ヘミセルラーゼ、リグニン分解酵素を含む様々な菌体外酵素を分泌することにより植物細胞壁を栄養源として生きている。木材腐朽菌のなかでも、リグニン分解性担子菌プレビオピシス・ギガンテア(Phlebiopsis gigantea、以下、P. gigantea)は、抽出成分を豊富に含む伐採したばかりの針葉樹に迅速に侵入できることが知られている。P. giganteaがどのように針葉樹由来の抽出物を解毒または利用するのか、そのメカニズムはほとんど分かっていないが、針葉樹を分解する際にリパーゼを分泌することが知られている(非特許文献1)。
The most abundant carbon source on earth is woody plant resources (biomass). 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. In addition to these components, the solvent-soluble fraction (commonly called "extract" or "extractables") 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. On the other hand, 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. Among wood-rotting fungi, 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).
本発明は、新規な高機能性リパーゼを見出し、その利用方法を提供することを目的とする。
The purpose of the present invention is to discover a novel highly functional lipase and to provide a method for using it.
本発明者は鋭意研究の結果、P. giganteaが分泌する複数のリパーゼのなかで、従来のリパーゼと比べて酸性条件かつ低温条件で高いリパーゼ活性を有するという特に有用な性質を有する酵素を同定し、リコンビナント酵素の製造に成功した。かくして、本発明を完成させた。
As a result of intensive research, the inventor of the present invention found that P. Among the multiple lipases secreted by gigantea, we identified an enzyme with a particularly useful property of having higher lipase activity under acidic and low-temperature conditions than conventional lipases, and succeeded in producing a recombinant enzyme. Thus, the present invention was completed.
本発明は、例えば、以下の態様を提供する。
[1]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼを含む酵素組成物、
[2]リパーゼの至適温度が10℃~35℃の範囲にある、[1]記載の酵素組成物、
[3]リパーゼの至適pHがpH3~pH5の範囲にある、[1]または[2]記載の酵素組成物、
[4]担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあるリパーゼを含む酵素組成物、
[5]担子菌由来のリパーゼであって、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物、
[6]担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあり、かつ、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物、
[7]担子菌がプレビオピシス・ギガンテアである、[4]~[6]のいずれか1項記載の酵素組成物、
[8]食品用酵素組成物、医薬品用酵素組成物、化粧品用酵素組成物、産業用酵素組成物、または洗浄組成物である、[1]~[7]のいずれか1項記載の酵素組成物、
[9]紙パルプの製造またはバイオリファイナリープロセスにおいて使用するための、[1]~[7]のいずれか1項記載の酵素組成物、
[10]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼのリコンビナント酵素、
[11]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼをコードする核酸を含む、ベクター、
[12]前記リパーゼをコードする核酸がコドン最適化された配列を含む、[11]記載のベクター、
[13][11]または[12]に記載したベクターが導入された宿主、
[14]酵母である、[13]記載の宿主、および
[15][13]または[14]記載の宿主を培養することを含む、リコンビナント酵素の製造方法。 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;
[2] The enzyme composition according to [1], wherein the optimum temperature of the lipase is in the range of 10°C to 35°C.
[3] The enzyme composition according to [1] or [2], wherein the optimum pH of the lipase is in the range ofpH 3 to pH 5;
[4] an enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10°C to 35°C;
[5] an enzyme composition containing a basidiomycete-derived lipase having an optimum pH in the range ofpH 3 to pH 5;
[6] An enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10° C. to 35° C. and an optimum pH in the range ofpH 3 to pH 5,
[7] The enzyme composition according to any one of [4] to [6], wherein the basidiomycete is Prebiopisis gigantea;
[8] The enzyme composition according to any one of [1] to [7], which is an enzyme composition for foods, an enzyme composition for pharmaceuticals, an enzyme composition for cosmetics, an enzyme composition for industrial use, or a cleaning composition. object,
[9] The enzyme composition of any one of [1] to [7] for use in paper pulp manufacturing or biorefinery processes,
[10] 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].
[1]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼを含む酵素組成物、
[2]リパーゼの至適温度が10℃~35℃の範囲にある、[1]記載の酵素組成物、
[3]リパーゼの至適pHがpH3~pH5の範囲にある、[1]または[2]記載の酵素組成物、
[4]担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあるリパーゼを含む酵素組成物、
[5]担子菌由来のリパーゼであって、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物、
[6]担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあり、かつ、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物、
[7]担子菌がプレビオピシス・ギガンテアである、[4]~[6]のいずれか1項記載の酵素組成物、
[8]食品用酵素組成物、医薬品用酵素組成物、化粧品用酵素組成物、産業用酵素組成物、または洗浄組成物である、[1]~[7]のいずれか1項記載の酵素組成物、
[9]紙パルプの製造またはバイオリファイナリープロセスにおいて使用するための、[1]~[7]のいずれか1項記載の酵素組成物、
[10]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼのリコンビナント酵素、
[11]配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼをコードする核酸を含む、ベクター、
[12]前記リパーゼをコードする核酸がコドン最適化された配列を含む、[11]記載のベクター、
[13][11]または[12]に記載したベクターが導入された宿主、
[14]酵母である、[13]記載の宿主、および
[15][13]または[14]記載の宿主を培養することを含む、リコンビナント酵素の製造方法。 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;
[2] The enzyme composition according to [1], wherein the optimum temperature of the lipase is in the range of 10°C to 35°C.
[3] The enzyme composition according to [1] or [2], wherein the optimum pH of the lipase is in the range of
[4] an enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10°C to 35°C;
[5] an enzyme composition containing a basidiomycete-derived lipase having an optimum pH in the range of
[6] An enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10° C. to 35° C. and an optimum pH in the range of
[7] The enzyme composition according to any one of [4] to [6], wherein the basidiomycete is Prebiopisis gigantea;
[8] The enzyme composition according to any one of [1] to [7], which is an enzyme composition for foods, an enzyme composition for pharmaceuticals, an enzyme composition for cosmetics, an enzyme composition for industrial use, or a cleaning composition. object,
[9] The enzyme composition of any one of [1] to [7] for use in paper pulp manufacturing or biorefinery processes,
[10] 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].
本発明によれば、従来のリパーゼとは異なり、酸性の至適pHおよび常温乃至低温での至適温度を有する、担子菌由来のリパーゼを含む酵素組成物が提供される。該組成物は、常温かつ酸性条件下で高い活性があるので、食品、医薬品、洗剤等の様々な用途に用いることができる。また、該リパーゼは天然物である担子菌由来なので、本発明の酵素組成物は安全性にも優れている。
According to the present invention, unlike conventional lipases, 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.
1.リパーゼ
本発明者らの以前の研究において、針葉樹分解性担子菌P. giganteaのゲノム解析を行い、針葉樹を分解する際にリパーゼが分泌されることが液体クロマトグラフィータンデム型質量分析器(LC-MS/MS)を用いたプロテオーム解析で分かった(非特許文献1)。今回、本発明者は、前回の論文で予測された9つのリパーゼのうち、4つのリパーゼをコードする転写物が針葉樹抽出物の存在下で有意にアップレギュレートされることを見出した。本発明においては、4つのリパーゼのうち、針葉樹抽出物の存在下で特に転写量の多い分泌型リパーゼ(PgLip19028と称する)を同定した。PgLip19028は、配列番号1に示されるアミノ酸配列を含むポリペプチドであり、リパーゼの保存モチーフ配列GXSXG(配列番号3:GHSLG)および触媒トライアド(Ser180、Asp237、およびHis251)を有することを特徴とする(図3)。また、分泌シグナル、3つのN-グリコシル化部位、および1つのO-グリコシル化部位を有することが推定される。分泌シグナルを含まないPgLip19028のアミノ酸配列を配列番号1に、分泌シグナルを含むPgLip19028のアミノ酸配列を配列番号2に示す。また、分泌シグナルを含まないPgLip19028の核酸配列を配列番号4に、分泌シグナルを含むPgLip19028の核酸配列を配列番号5に示す。 1. lipase In our previous work, the conifer-degrading basidiomycete P. Genome analysis of gigantea was performed, and proteome analysis using a liquid chromatography tandem mass spectrometer (LC-MS/MS) revealed that lipase is secreted when decomposing conifers (Non-Patent Document 1). We have now found that transcripts encoding four of the nine lipases predicted in the previous paper are significantly upregulated in the presence of conifer extract. In the present invention, among the four lipases, a secretory lipase (referred to as PgLip19028) having a particularly high transcription amount in the presence of conifer extract was identified. 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. In addition, the 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.
本発明者らの以前の研究において、針葉樹分解性担子菌P. giganteaのゲノム解析を行い、針葉樹を分解する際にリパーゼが分泌されることが液体クロマトグラフィータンデム型質量分析器(LC-MS/MS)を用いたプロテオーム解析で分かった(非特許文献1)。今回、本発明者は、前回の論文で予測された9つのリパーゼのうち、4つのリパーゼをコードする転写物が針葉樹抽出物の存在下で有意にアップレギュレートされることを見出した。本発明においては、4つのリパーゼのうち、針葉樹抽出物の存在下で特に転写量の多い分泌型リパーゼ(PgLip19028と称する)を同定した。PgLip19028は、配列番号1に示されるアミノ酸配列を含むポリペプチドであり、リパーゼの保存モチーフ配列GXSXG(配列番号3:GHSLG)および触媒トライアド(Ser180、Asp237、およびHis251)を有することを特徴とする(図3)。また、分泌シグナル、3つのN-グリコシル化部位、および1つのO-グリコシル化部位を有することが推定される。分泌シグナルを含まないPgLip19028のアミノ酸配列を配列番号1に、分泌シグナルを含むPgLip19028のアミノ酸配列を配列番号2に示す。また、分泌シグナルを含まないPgLip19028の核酸配列を配列番号4に、分泌シグナルを含むPgLip19028の核酸配列を配列番号5に示す。 1. lipase In our previous work, the conifer-degrading basidiomycete P. Genome analysis of gigantea was performed, and proteome analysis using a liquid chromatography tandem mass spectrometer (LC-MS/MS) revealed that lipase is secreted when decomposing conifers (Non-Patent Document 1). We have now found that transcripts encoding four of the nine lipases predicted in the previous paper are significantly upregulated in the presence of conifer extract. In the present invention, among the four lipases, a secretory lipase (referred to as PgLip19028) having a particularly high transcription amount in the presence of conifer extract was identified. 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. In addition, the 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.
配列アノテーションに基づいて、PgLip19028は、トリアシルグリセロールヒドロラーゼ(E.C.3.1.1.3)におけるリパーゼをコードすることが推定された。今回、本発明において、PgLip19028は、以前に特徴付けられた他の担子菌由来リパーゼとは異なる分子量、至適pHおよび至適温度、および基質特異性を有することが示された。本発明における生物化学的特徴付けから、PgLip19028は、酸性条件下、周囲温度で、種々の不飽和脂肪酸を遊離できることが分かった。さらに、P. gigantea以外の担子菌にも、PgLip19028と類似のアミノ酸配列を有し、かつ、同一の保存モチーフ配列GHSLGを有するリパーゼが存在することが分かった。
Based on sequence annotation, 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.
したがって、本発明の一態様において、使用されるリパーゼ(以下、「本発明のリパーゼ」ともいう)は、配列番号1に示されるアミノ酸配列を含むポリペプチドである。該ポリペプチドは、リパーゼ活性を有する限りにおいて、配列番号1に示されるアミノ酸配列において1~数個のアミノ酸変異(置換、欠失、付加または挿入、またはこれらの組み合わせ)を含んでいてもよく、あるいはリパーゼ活性を有する限りにおいて、配列番号1に示されるアミノ酸配列と少なくとも45%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または少なくとも85%、好ましくは、少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%の配列同一性を有するアミノ酸配列を含んでいてもよい。好ましくは、本発明のリパーゼは、配列番号1に示されるアミノ酸配列からなるポリペプチド、あるいは配列番号1に示されるアミノ酸配列と少なくとも45%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または少なくとも85%、好ましくは、少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%の配列同一性を有するアミノ酸配列からなるポリペプチドであって、リパーゼ活性を有するポリペプチドである。上記の配列番号1に示されるアミノ酸配列において1~数個のアミノ酸変異を含むポリペプチド、配列番号1に示されるアミノ酸配列と少なくとも45%~少なくとも99%の配列同一性を有するアミノ酸配列を含むポリペプチド、および配列番号1に示されるアミノ酸配列と少なくとも45%~少なくとも99%の配列同一性を有するアミノ酸配列からなるポリペプチドは、好ましくは、モチーフ配列「GHSLG」を保持する。本明細書において、「数個」とは、2~10個程度をいい、例えば、3個、4個、5個、6個、7個、8個、または9個であってもよい。本明細書において、アミノ酸置換は、保存的置換または非保存的置換であってもよい。好ましくは保存的置換である。
Therefore, in one aspect of the present invention, 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. Preferably, 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. A polypeptide containing one to several amino acid mutations in the amino acid sequence shown in SEQ ID NO: 1 above, a polypeptide containing 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 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". As used herein, "several" refers to about 2 to 10, and may be, for example, 3, 4, 5, 6, 7, 8, or 9. As used herein, amino acid substitutions may be conservative substitutions or non-conservative substitutions. Conservative substitutions are preferred.
本発明のリパーゼは、10℃~35℃の範囲、好ましくは15℃~30℃の範囲、さらに好ましくは20℃~30℃の範囲、またさらに好ましくは25℃の至適温度を有していてもよい。また、本発明のリパーゼは、pH3~pH5の範囲、好ましくはpH3.5~pH5の範囲、さらに好ましくはpH4~pH5の範囲、またさらに好ましくはpH4.5の至適pHを有していてもよい。本明細書において、リパーゼの至適温度および至適pHは、酵素の相対活性が40%以上、好ましくは60%以上、さらに好ましくは70%以上、またさらに好ましくは80%以上である温度条件およびpH条件をいう。相対活性は、最も高いリパーゼ活性を示した温度またはpH条件でのリパーゼ活性を100%とした場合の、各条件におけるリパーゼ活性の相対値である。
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. Moreover, 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. As used herein, 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%.
本明細書において、リパーゼ活性は、基質に対する加水分解活性に基づく。リパーゼ活性の測定は常法を用いて行えばよく、例えば、ドデカン酸p-ニトロフェニル、パルミチン酸p-ニトロフェニル、酪酸p-ニトロフェニル等のリパーゼモデル基質を用い、酵素反応によって生成したp-ニトロフェノールの量を測定することによって行うことができる。
As used herein, 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.
本発明のリパーゼは、約30kDaの分子量を有していてもよい。
The lipase of the present invention may have a molecular weight of about 30 kDa.
本発明のリパーゼは、担子菌の培養物または培養上清から得ることができる。例えば、菌体の破砕液または培養上清そのものであってもよく、あるいは菌体の破砕液または培養上清からの粗精製または精製したものであってもよい。本発明のリパーゼは、通常、N末端に分泌シグナル配列が付加されたアミノ酸配列を有する分泌型であるため、好ましくは培養上清から得られる。粗精製または精製は、例えば、塩析、遠心分離、限外ろ過、ゲルろ過、各種クロマトグラフィー(吸着クロマトグラフィー、疎水性クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、高速液体クロマトグラフィー等)、透析、電気泳動などの既知の方法によって行うことができる。
The lipase of the present invention can be obtained from a basidiomycete culture or culture supernatant. For example, it 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. Since 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. 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.
担子菌の培養には、通常の培養装置および基礎培地を用いることができ、液体培養または固体培養のいずれの方法で行ってもよい。基礎培地としては、例えば、限定するものではないが、ポテトデキストロース寒天(PDA)培地、麦芽エキス寒天(MYA)培地、Highley無機塩培地(Highley's basal medium)等が挙げられる。培地には、炭素源として、グルコースやセルロース等を追加してもよい。培地にはさらに、リパーゼ誘導を促すために、好ましくは針葉樹抽出物が添加される。針葉樹としては、例えば、限定するものではないが、マツ(例えば、テーダマツなど)、スギ、ヒノキ、ツガ、モミ、トウヒ、イチイ等が挙げられる。針葉樹抽出物は、例えば、針葉樹の樹皮を剥いだ木質部分を破砕して得た木粉を、アセトン等の有機溶媒を用いて抽出することによって得てもよい。抽出方法として、有機溶媒に浸漬し、加温・攪拌し調製する方法や、ソックスレー抽出法を用いてもよい。抽出物は、例えばロータリーエバポレーターや凍結乾燥法等により、濃縮してもよい。培養温度、培養期間等の培養条件は、当業者により適宜設定することができる。例えば、20~30℃で3~14日間培養してもよい。
A normal culture apparatus and basal medium can be used for culturing basidiomycetes, and either liquid culture or solid culture can be used. 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. 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. As 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.
担子菌として、好ましくはP. giganteaが用いられるが、他の担子菌、例えば、Shizophilum commune、Coniophora puteana、Serpula lacrymans、Heterobasidion annosum、Stereum hirsutum、Postia planceta、Wolfiporia cocos、Fomitopsis pinicola、Ceriporiopsis suvermispora、Dichomitus squalens、Trametes versicolor、Phanerochaete、Phanerochaete carnosa、Phlebia breviospora等を用いてもよい。これらの担子菌は、いずれの菌株であってもよく、自然界から採取されたもの、またはATCC(American Type Culture Collection)等の保存機関から入手したものであってもよい。
As a basidiomycete, preferably P. giganteaが用いられるが、他の担子菌、例えば、Shizophilum commune、Coniophora puteana、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).
また、本発明の一態様において使用されるリパーゼは、そのアミノ酸配列を基に化学合成(ペプチド合成)によって製造してもよい。
In addition, the lipase used in one aspect of the present invention may be produced by chemical synthesis (peptide synthesis) based on its amino acid sequence.
2.リコンビナント酵素
本発明で使用されるリパーゼは、リコンビナント酵素であってもよい。PgLip19028のリコンビナント酵素は、本発明で初めて製造された。また、本発明において、上記リパーゼのリコンビナント酵素を産生する発現系が初めて構築された。 2. Recombinant Enzyme 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. In addition, in the present invention, an expression system for producing the recombinant lipase was constructed for the first time.
本発明で使用されるリパーゼは、リコンビナント酵素であってもよい。PgLip19028のリコンビナント酵素は、本発明で初めて製造された。また、本発明において、上記リパーゼのリコンビナント酵素を産生する発現系が初めて構築された。 2. Recombinant Enzyme 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. In addition, in the present invention, an expression system for producing the recombinant lipase was constructed for the first time.
リコンビナント酵素の製造には、無細胞タンパク質合成系(大腸菌由来、小麦胚芽由来など)または細胞タンパク質発現系を用いてもよい。細胞タンパク質発現系のための宿主は、原核細胞または真核細胞のいずれであってもよく、例えば、真菌(酵母、糸状菌等)、動物細胞(脊椎動物細胞、哺乳動物細胞等)、昆虫細胞、細菌(大腸菌、枯草菌、放線菌等)等が挙げられる。好ましくは酵母が用いられ、例えば、限定するものではないが、サッカロマイセス属酵母、カンジダ属酵母、ピキア属酵母などが挙げられる。例えば、Pichia pastorisを使用してもよい。
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.
リコンビナント酵素の生産のために、本発明のリパーゼをコードする核酸を宿主細胞中に導入する。本発明のリパーゼをコードする核酸は、P. giganteaに関する既知の配列情報に基づき合成することができる。本発明のリパーゼをコードする核酸配列は、宿主細胞における発現のためにコドン最適化されていてもよい。各宿主に最適なコドンおよびコドン最適化の方法は当業者によく知られている。本発明のリパーゼをコードする核酸は、PgLip19028のアミノ酸配列、すなわち、配列番号1に示されるアミノ酸配列と少なくとも45%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または少なくとも85%、好ましくは、少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%配列同一性を有するアミノ酸配列をコードするヌクレオチド配列を含む。好ましくは、本発明のリパーゼをコードする核酸は、配列番号1に示されるアミノ酸配列と少なくとも45%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または少なくとも85%、好ましくは、少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%配列同一性を有するアミノ酸配列をコードするヌクレオチド配列からなる核酸であってもよい。本発明のリパーゼをコードする核酸としては、例えば、配列番号4で示されるヌクレオチド配列を含む核酸が挙げられる。本発明のリパーゼをコードする核酸のさらなる例としては、配列番号4で示されるヌクレオチド配列に対して少なくとも45%、少なくとも50%、少なくとも60%、少なくとも70%、少なくとも80%、または少なくとも85%、好ましくは、少なくとも90%、少なくとも91%、少なくとも92%、少なくとも93%、少なくとも94%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%配列同一性を有するヌクレオチド配列を含む核酸が挙げられる。また、宿主としてPichia pastorisにおける発現のためにコドン最適化された核酸配列の一例として、配列番号6に示される配列が挙げられる。
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. Preferably, 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. Further examples of 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.
本発明のリパーゼをコードする核酸配列には、分泌シグナル配列が付加されていてもよい。分泌シグナル配列としては、例えば、PgLip19028の分泌シグナル配列、またはα-ファクタの分泌シグナル配列などを使用してもよい。また、本発明のリパーゼをコードする核酸配列は、プロモーター配列および/または核酸の発現に関わるいずれかの配列、例えばエンハンサー配列等、さらにタンパク質安定化や精製・発現量測定など種々の分析や解析に関わる配列、例えば、マルトースバインディングプロテイン(MBP)、ポリヒスチジンタグ(His-tag)、ヒトc-Mycタグ等と作動可能に連結されていてもよい。プロモーター配列としては、例えば、限定するものではないが、メタノール添加によってタンパク質生産を誘導できるアルコールオキシダーゼ(AOX)のプロモーター配列や、恒常的なタンパク質生産のためのプロモーターとして、グリセルアルデヒド-3-リン酸デヒドロゲナーゼGAPのプロモーター配列を使用することができる。
A secretory signal sequence may be added to the nucleic acid sequence encoding the lipase of the present invention. As the secretory signal sequence, for example, the secretory signal sequence of PgLip19028 or the secretory signal sequence of α-factor may be used. In addition, 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. Examples of 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. The promoter sequence for acid dehydrogenase GAP can be used.
本発明のリパーゼをコードする核酸は、適当なベクター中に挿入されていてもよく、該ベクターを宿主細胞中に導入してもよい。本明細書において、「ベクター」とは、目的の核酸を宿主細胞中に輸送する核酸分子をいう。ベクターとしては、プラスミドベクター、コスミド、ラムダファージ、人工染色体、ウイルスベクター(例えば、バキュロウイルスベクター、レトロウイルスベクター、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、単純ヘルペスウイルス等)、リポソームなど当該分野で既知の種々のベクターを使用することができる。宿主細胞に適当なベクターを選択すればよい。ベクターは、好ましくは発現ベクターであり、プロモーター配列および/または挿入核酸の発現に関わるいずれかの配列、例えばエンハンサー配列等を含んでいてもよい。種々のプロモーターが知られており、宿主細胞に適当なプロモーターを選択すればよい。ベクターはさらに、宿主細胞への導入後、目的遺伝子が導入された細胞(形質転換細胞)の選抜のための選択マーカー遺伝子、例えば、抗生物質または薬物耐性遺伝子等を含んでいてもよい。
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. As used herein, "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. 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.
ベクターの宿主細胞への導入は、当該分野で既知の方法によって行うことができる。例えば、カチオン性脂質媒介性導入法、ジエチルアミノエチル(DEAE)-デキストラン法、リン酸カルシウム共沈殿法、カチオン性ポリマーによる導入法、エレクトロポレーション法、マイクロインジェクション法、ソノポレーション法、レーザー照射法、ウイルスベクターによる導入方法などが挙げられる。
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.
得られた遺伝子導入細胞を培養することにより、目的の本発明のリパーゼが生産される。好ましくは、本発明のリパーゼは細胞外に(培地中に)分泌される。また、大腸菌などの細胞系で生産した場合、目的の酵素が不溶化形態(沈殿物)として得られることもある。このような場合は、可溶化作業またはホールディング作業を行うことにより、活性形態の酵素を得ることができる。可溶化作業またはホールディング作業は、当該分野で既知の方法により行えばよい。
By culturing the obtained gene-introduced cells, the desired lipase of the present invention is produced. Preferably, the lipase of the invention is secreted extracellularly (into the medium). Also, when produced in a cell line such as E. coli, the desired enzyme may be obtained in an insolubilized form (precipitate). In such cases, 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.
遺伝子導入細胞の培養は常法にしたがって行えばよく、培養培地は宿主に適当なものを選択すればよい。例えば、限定するものではないが、YPD培地、YPG培地等が挙げられる。培養温度、時間等の培養条件は、適宜選択することができる。例えば、約20~30℃で1日~1週間程度培養してもよい。
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.
宿主細胞によって生産された本発明のリパーゼは、培養細胞の破砕液または培養上清から、好ましくは培養上清から、粗精製または精製してもよい。粗精製、および精製操作は、当該分野で既知の方法によって行うことができる。粗精製または精製は、例えば、塩析、遠心分離、限外ろ過、ゲルろ過、各種クロマトグラフィー(吸着クロマトグラフィー、疎水性クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、高速液体クロマトグラフィー等)、透析、電気泳動などの既知の方法によって行うことができる。
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.
3.酵素組成物
本発明のリパーゼは、酵素組成物の形態で提供される。本発明のリパーゼを含む酵素組成物(以下、「本発明の酵素組成物」ともいう)は、本発明のリパーゼそのものであってもよく、または有効成分である本発明のリパーゼの他に付加成分を含んでいてもよい。該付加成分の例としては、本発明の酵素組成物の用途にもよるが、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤などが挙げられる。賦形剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、デンプン、デキストリン、糖類、糖アルコール、グリセロールなどが挙げられる。緩衝剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、リン酸塩、クエン酸塩、酢酸塩などが挙げられる。安定剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、プロピレングリコール、アスコルビン酸などが挙げられる。保存剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、フェノール、塩化ベンザルコニウム、メチルパラベンなどが挙げられる。防腐剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、エタノール、塩化ベンザルコニウム、パラオキシ安息香酸などが挙げられる。酵素組成物中における本発明のリパーゼの量および付加成分の量は、当業者により適宜決定することができる。 3. Enzyme Composition 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. Examples of buffering agents include, but are not limited to, phosphates, citrates, acetates, and the like, depending on the application of the enzyme composition. Examples of stabilizers include, but are not limited to, propylene glycol, ascorbic acid, and the like, depending on the application of the enzyme composition. Examples of preservatives include, but are not limited to, phenol, benzalkonium chloride, methylparaben, and the like, depending on the use of the enzyme composition. Examples of 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.
本発明のリパーゼは、酵素組成物の形態で提供される。本発明のリパーゼを含む酵素組成物(以下、「本発明の酵素組成物」ともいう)は、本発明のリパーゼそのものであってもよく、または有効成分である本発明のリパーゼの他に付加成分を含んでいてもよい。該付加成分の例としては、本発明の酵素組成物の用途にもよるが、賦形剤、緩衝剤、懸濁剤、安定剤、保存剤、防腐剤などが挙げられる。賦形剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、デンプン、デキストリン、糖類、糖アルコール、グリセロールなどが挙げられる。緩衝剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、リン酸塩、クエン酸塩、酢酸塩などが挙げられる。安定剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、プロピレングリコール、アスコルビン酸などが挙げられる。保存剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、フェノール、塩化ベンザルコニウム、メチルパラベンなどが挙げられる。防腐剤としては、酵素組成物の用途にもよるが、例えば、限定するものではないが、エタノール、塩化ベンザルコニウム、パラオキシ安息香酸などが挙げられる。酵素組成物中における本発明のリパーゼの量および付加成分の量は、当業者により適宜決定することができる。 3. Enzyme Composition 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. Examples of buffering agents include, but are not limited to, phosphates, citrates, acetates, and the like, depending on the application of the enzyme composition. Examples of stabilizers include, but are not limited to, propylene glycol, ascorbic acid, and the like, depending on the application of the enzyme composition. Examples of preservatives include, but are not limited to, phenol, benzalkonium chloride, methylparaben, and the like, depending on the use of the enzyme composition. Examples of 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.
本発明の酵素組成物はまた、tert-ブチルメチルエーテルを含んでいてもよい。当該組成物中におけるtert-ブチルメチルエーテルの配合量は、当業者により適宜決定することができる。例えば、組成物中5v/v%~30v/v%、好ましくは15v/v%が挙げられる。
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.
本発明の酵素組成物は、食品用酵素組成物であってもよい。例えば、本発明の酵素組成物は、食品または食品原料の風味改善剤(フレーバー剤)として使用することができる。上記食品または食品原料としては、例えば、限定するものではないが、各種乳製品(例えば、チーズ、バター、ヨーグルト等)、マーガリン、ショートニング、各種植物油(大豆油、菜種油、コーン油、パーム油、パーム核油、ヤシ油、ひまわり油、綿実油等)、ドレッシングなどが挙げられる。したがって、本発明のさらなる態様においては、本発明の酵素組成物を食品または食品原料に添加する、または作用させることを特徴とする、食品または食品原料の風味改善方法が提供される。
The enzyme composition of the present invention may be a food enzyme composition. For example, the enzyme composition of the present invention can be used as a flavor improving agent (flavor agent) for food or food ingredients. Examples of 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.
食品用の本発明の酵素組成物は、また、食品添加物(例えば、フレーバー剤等)の製造、食品加工(例えば、清酒の製造等)、食用油脂(例えば、クッキングオイル、ショートニング等)の製造に用いてもよい。したがって、本発明のさらなる態様においては、本発明の酵素組成物を食品原料または中間生成物に添加する、または作用させることを特徴とする、食品の製造方法が提供される。
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.
本発明の酵素組成物は、医薬品用酵素組成物であってもよい。医薬品用の本発明の酵素組成物は、例えば、限定するものではないが、医薬品(例えば、消化酵素剤、代替酵素、例えば、ライソゾーム酸性リパーゼ欠損症治療薬等)、臨床検査薬(例えば、血中コレステロールの測定用検査薬等)等であってもよい。
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.
本発明の酵素組成物は、また、洗浄組成物として使用できる。したがって、本発明のさらなる態様においては、本発明の酵素組成物を含む洗浄組成物が提供される。例えば、本発明の酵素組成物を含む洗浄組成物は、被洗浄物(洗濯物等)に付着した脂質を分解除去して洗浄効果を向上させ、さらに、低温(水温)で高い活性を有するため、さらに洗浄効果が向上する。
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. For example, 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.
本発明の酵素組成物は、さらに、化粧品用酵素組成物であってもよく、例えば化粧品(例えば、洗顔剤など)の添加物としても使用できる。
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.).
さらに、本発明の酵素組成物は、脂肪酸、石鹸の製造、排水処理、光学活性医薬・農薬原料の製造、光学活性化合物の合成等に利用でき、産業用酵素として有用である。
Furthermore, 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.
本発明の酵素組成物は、さらに、紙パルプ製造において使用することができる。本発明の酵素組成物を使用することにより、針葉樹の樹木からの紙パルプ生産工程で抽出物を取り除くことができ、それにより製造される紙の性質が向上する。本発明の酵素組成物は、さらに、バイオリファイナリープロセスにおいて使用することができる。
The enzyme composition of the present invention can also be used in paper pulp production. By using the enzyme composition of the present invention, 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.
さらに、本発明の酵素組成物は、樹木から、特に針葉樹の抽出物から、各種脂肪酸等の脂質成分を得るために使用できる。このようにして得られる樹木由来の脂質成分は、例えば、食品、化粧品、医薬などの分野において使用される。
Furthermore, 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.
以下、実施例を用いて本発明をより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
The present invention will be described in more detail below using examples, but the present invention is not limited to these examples.
実施例1:P. gigantea培養上清のリパーゼ活性
樹皮を剥ぎ、木質部分を粉砕したテーダマツ木粉(1mmメッシュ)に対して70w/v%アセトンを用いてソックスレー抽出法で得られた抽出成分を、微結晶セルロース(Avicel PH101, 50uM, Fluka Chemika, Switzerland)1.25gに加え、これらをロータリーエバポレーターで乾燥させて基質を調製した。セルロースへの抽出成分の添加量は、木粉重量あたりの天然のテーダマツ抽出物含量(AV1X)、天然濃度の2倍量(AV2X)および4倍量(AV4X)になるようにした。テーダマツ抽出物を含有しない微結晶セルロースのアセトン懸濁基質を「AV0X」とした。250mLの基礎培地(Highley培地)中に基質を加え、P. gigantea単離株11061-1の単一担子胞子株5-6をPDA培地で培養した菌糸プラグ(mycelial plugs)を接種し、ロータリー振盪器(150rpm)において22℃で7日間培養した。培養上清を経時的(培養3日目、5日目、7日目)に収集し、培養上清中のタンパク質濃度をProtein Assay(Bio-Rad)によって測定した。セクレトーム(培養上清分泌物)中のリパーゼ活性を以下のように測定した。 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. 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.
樹皮を剥ぎ、木質部分を粉砕したテーダマツ木粉(1mmメッシュ)に対して70w/v%アセトンを用いてソックスレー抽出法で得られた抽出成分を、微結晶セルロース(Avicel PH101, 50uM, Fluka Chemika, Switzerland)1.25gに加え、これらをロータリーエバポレーターで乾燥させて基質を調製した。セルロースへの抽出成分の添加量は、木粉重量あたりの天然のテーダマツ抽出物含量(AV1X)、天然濃度の2倍量(AV2X)および4倍量(AV4X)になるようにした。テーダマツ抽出物を含有しない微結晶セルロースのアセトン懸濁基質を「AV0X」とした。250mLの基礎培地(Highley培地)中に基質を加え、P. gigantea単離株11061-1の単一担子胞子株5-6をPDA培地で培養した菌糸プラグ(mycelial plugs)を接種し、ロータリー振盪器(150rpm)において22℃で7日間培養した。培養上清を経時的(培養3日目、5日目、7日目)に収集し、培養上清中のタンパク質濃度をProtein Assay(Bio-Rad)によって測定した。セクレトーム(培養上清分泌物)中のリパーゼ活性を以下のように測定した。 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. 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.
リパーゼ活性の測定
リパーゼ活性は、ドデカン酸p-ニトロフェニル(pNPD、Sigma-Aldrich社製)を基質として用いて測定した。ジメチルスルホキシド(DMSO)中の75mM pNPD(2μL)を50μLの培養上清および25μLの100mM酢酸バッファー(pH5.0)と混合し、全量100μLにした。37℃で30分間インキュベーション後、25μLの100mM Na2CO3を加えて反応を止めた。遊離されたp-ニトロフェノール(pNP)を405nmで検出し、pNP(Sigma-Aldrich社製)の標準曲線を用いてpNP遊離量を決定した。1単位のリパーゼ活性は、1分間の反応当たり1μモルのpNPを遊離させる酵素量として定義した。 Measurement of Lipase Activity Lipase activity was measured using p-nitrophenyl dodecanoate (pNPD, Sigma-Aldrich) as a substrate. 75 mM pNPD (2 μL) in dimethylsulfoxide (DMSO) was mixed with 50 μL of culture supernatant and 25 μL of 100 mM acetate buffer (pH 5.0) to a total volume of 100 μL. After 30 min incubation at 37° C., the reaction was stopped by adding 25 μL 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 μmole of pNP per minute of reaction.
リパーゼ活性は、ドデカン酸p-ニトロフェニル(pNPD、Sigma-Aldrich社製)を基質として用いて測定した。ジメチルスルホキシド(DMSO)中の75mM pNPD(2μL)を50μLの培養上清および25μLの100mM酢酸バッファー(pH5.0)と混合し、全量100μLにした。37℃で30分間インキュベーション後、25μLの100mM Na2CO3を加えて反応を止めた。遊離されたp-ニトロフェノール(pNP)を405nmで検出し、pNP(Sigma-Aldrich社製)の標準曲線を用いてpNP遊離量を決定した。1単位のリパーゼ活性は、1分間の反応当たり1μモルのpNPを遊離させる酵素量として定義した。 Measurement of Lipase Activity Lipase activity was measured using p-nitrophenyl dodecanoate (pNPD, Sigma-Aldrich) as a substrate. 75 mM pNPD (2 μL) in dimethylsulfoxide (DMSO) was mixed with 50 μL of culture supernatant and 25 μL of 100 mM acetate buffer (pH 5.0) to a total volume of 100 μL. After 30 min incubation at 37° C., the reaction was stopped by adding 25 μL 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 μmole of pNP per minute of reaction.
結果を図1に示す。いずれの時点においても、AV1X、AV2XおよびAV4Xの培養上清で増加したリパーゼ活性が観察された。一方、AV0Xでは比較的低い活性が検出された。これらの結果から、P. giganteaの培養上清中にリパーゼが分泌されており、そのリパーゼ活性は抽出成分を添加することで誘導されることが分かった。
The results are shown in Figure 1. Increased lipase activity was observed in AV1X, AV2X and AV4X culture supernatants at all time points. On the other hand, relatively low activity was detected with AV0X. From these results, P. It was found that lipase is secreted in the culture supernatant of gigantea, and its lipase activity can be induced by adding an extract component.
実施例2:針葉樹抽出物の存在下で誘導されるリパーゼ
実施例1と同様にしてP. giganteaを5日間培養し、得られた菌体から全RNAを常法によって精製した。各サンプルから得られた100ngの全RNAを対象に、イルミナRNAシーケンス(イルミナ社、アメリカ)のプロトコール通りに、mRNAを精製し、PCRおよびアダプター付加作業によりRNAシーケンス用の2X150bpペアエンドライブラリを作成し、NovaSeqシーケンサーでトランスクリプトーム解析した。得られたデータをゲノム配列にマッピングし、遺伝子の発現量をサンプルの総リード数および遺伝子長で補正したRPKM値を算出した。 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.
実施例1と同様にしてP. giganteaを5日間培養し、得られた菌体から全RNAを常法によって精製した。各サンプルから得られた100ngの全RNAを対象に、イルミナRNAシーケンス(イルミナ社、アメリカ)のプロトコール通りに、mRNAを精製し、PCRおよびアダプター付加作業によりRNAシーケンス用の2X150bpペアエンドライブラリを作成し、NovaSeqシーケンサーでトランスクリプトーム解析した。得られたデータをゲノム配列にマッピングし、遺伝子の発現量をサンプルの総リード数および遺伝子長で補正したRPKM値を算出した。 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.
さらに、この際の培養上清を液体クロマトグラフィータンデム型質量分析器(LC-MS/MS)を用いたプロテオーム解析した。培養上清中のタンパク質を10%(w/v)トリクロロ酢酸で沈殿させ、冷アセトンで3回洗浄後、空気乾燥させた。得られたペレットにクロロホルムおよびメタノールを加え、次いで水を加えることにより、ペレット由来の全タンパク質をメタノール/クロロホルム/水分配によって精製した。タンパク質は中間相に分配された。メタノールで複数回洗浄後、該精製タンパク質を最終的に8M尿素/50mM NH4HCO3(pH8.5)/1mM TrisHCl中に再溶解した。各サンプル中のタンパク質総量が等量になるよう調整し、トリプシン/LysC消化し、OMIX C18 SPE(Agilent Technologies)で精製して、最終的に2μgのタンパク質を、EASY-SprayTM(商標)エレクトロスプレーソースを備えたハイブリッド・リニア・イオントラップ-オービトラップ質量分析計(LTQ-Orbitrap Elite TM, ThermoFisher Scientific)に連結したAgilent 1100 ナノフローシステム(Agilent Technologies)を用いるLC-MS/MS分析に付した。質量スペクトル分析前のペプチドのクロマトグラフィーは、キャピラリーエミッターカラム(PepMap(登録商標)C18、3μM、100オングストローム、150x0.075mm、ThermoFisher Scientific)に2μlの精製ペプチドが自動的に負荷されることによって達成された。HPLCシステムには、溶媒A:0.1%(v/v)ギ酸を流し、溶媒B:99.9%(v/v)アセトニトリルおよび0.1%(v/v)ギ酸を0.50μL/分で流してペプチドを負荷し(30分間)、0.3μL/分の流速で3%(v/v)B~20%(v/v)Bの勾配で154分かけてペプチドをエレクトロスプレー中に直接溶出させ、最後に12分間の20%(v/v)B~50%(v/v)勾配で溶出し、50-95%(v/v)Bで5分間のフラッシュアウトを行った。HPLCカラム/エレクトロスプレーソースから溶出されたペプチドとして、オービトラップにおいて解像度120,000でsurvey MSスキャンを獲得し、次いで、380~1800m/zのMS1スキャンにおいて検出された20個の最も高い強度のペプチドのMS2フラグメンテーションを行い、ダイナミックエクスクルージョン(dynamic exclusion)によって重複(redundancy)を制限した。下流分析のために、MSConvert(Proteo Wizard:迅速なプロテオミクスツール開発のためのオープンソースソフトウェア)MS/MS生データをmgfファイルフォーマットに変換した。得られたmgfファイルを用いて、JGIポータル(https://genome.jgi.doe.gov/portal/Phlgi1/Phlgi1.download.html)を介してP. giganteaタンパク質データベースをサーチし、MS/MSペプチドを同定した。
Furthermore, 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. The total amount of protein in each sample was adjusted to equal amounts, trypsin/LysC digested and purified by OMIX C18 SPE (Agilent Technologies) to give a final 2 μg of protein by EASY-Spray ™ electrospray. 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 ™ , ThermoFisher Scientific) with source. Chromatography of peptides prior to mass spectral analysis was accomplished by automatically loading 2 μl of purified peptide onto a capillary emitter column (PepMap® C18, 3 μM, 100 Å, 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 μL/ Peptides were loaded (30 min) at a flow rate of 0.3 μL/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 μL/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. . As peptides eluted from the HPLC column/electrospray source, a survey MS scan was acquired at 120,000 resolution on the Orbitrap, followed by the 20 highest intensity peptides detected in the MS1 scan from 380-1800 m/z. was subjected to MS2 fragmentation and redundancy was limited by dynamic exclusion. MSConvert (Proteo Wizard: open source software for rapid proteomics tool development) MS/MS raw data were converted to mgf file format for downstream analysis. Using the resulting mgf file, P.G. The gigantea protein database was searched to identify MS/MS peptides.
トランスクリプトーム解析の結果を図2に示す。9つのリパーゼのなかで、4つのリパーゼをコードする転写産物が抽出物の存在によって有意にアップレギュレートされた。なかでも、PgLip19028転写産物が最も豊富であり(AV4X中でRPKM=1767)、AV0Xの4.64倍蓄積した。また、プロテオーム解析により、9つのリパーゼのうちPgLip19028を同定し(配列番号1)、PgLip19028が培養上清中に分泌されていることを確認した。
The results of transcriptome analysis are shown in Figure 2. Among the nine lipases, transcripts encoding four lipases were significantly upregulated by the presence of the extract. Among them, the PgLip19028 transcript was the most abundant (RPKM=1767 in AV4X) and accumulated 4.64-fold that of AV0X. Furthermore, by proteome analysis, PgLip19028 was identified among the nine lipases (SEQ ID NO: 1), and it was confirmed that PgLip19028 was secreted into the culture supernatant.
実施例3:リコンビナントPgLip19028の製造
PgLip19028をコードするDNAから天然分泌シグナルを除いたものを合成した(JGIサービス)。その遺伝子(配列番号4)を、PIPE(Polymerase Incomplete Primer Extension)クローニングのためのプライマーを用いて発現ベクターpPICZα(Invitrogen)中にサブクローン化した。分泌シグナルとして、pPICZα上のαファクタの分泌シグナルを利用した。プロモーター配列として、AOXのプロモーター配列を用いた。使用したプライマー配列を表1に示す。製造者(Invitrogen)の指示書にしたがって、発現ベクターを酵母Pichia pastorisに形質転換した。形質転換したPichia pastorisをYPD液体培地中30℃で1~3日間振盪培養し、得られた菌体をよく洗浄してYPG液体培地中に移した。該培地にメタノールを添加後、1~3日間培養してタンパク質発現を誘導し、PgLip19028を過剰発現する該形質転換酵母からリコンビナントリパーゼが生産された。形質転換したPichia pastorisの培養上清を飽和硫酸アンモニウム沈殿に付し、得られたタンパク質沈殿物を100mM酢酸バッファー(pH5.0)中に溶解し、これを粗酵素として用いた。10μgの粗酵素を変性させ、タンパク質脱グリコシル化のためにエンドグリコシダーゼH(Endo H、New England Biolab社製)で消化し、次いで粗酵素サンプルをSDS-PAGE分析(Bio-Rad)に付し、ゲルをクーマシーブリリアントブルー(CBB)R-250(FUJIFILM Wako Pure Chemical Corporation, Osaka Japan)で染色した。コントロールとして、空の(上記遺伝子をクローニングしていない)ベクターpPICZαを導入したPichia pastorisの培養上清を用い、上記と同様にして粗タンパク質を得た(以下、「ベクターコントロール」という)。リパーゼ活性測定のために、上記粗酵素のタンパク質濃度を10μg/mLに調整した。1μg/mLの粗酵素またはベクターコントロールを用いて、以下の方法でリパーゼ活性を測定した。 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 pPICZα (Invitrogen) using primers for PIPE (Polymerase Incomplete Primer Extension) cloning. As a secretion signal, the α-factor secretion signal on pPICZα 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. 10 μg of crude enzyme was denatured and digested with endoglycosidase H (Endo H, New England Biolab) for protein deglycosylation, then crude enzyme samples were subjected to SDS-PAGE analysis (Bio-Rad), Gels were stained with Coomassie Brilliant Blue (CBB) R-250 (FUJIFILM Wako Pure Chemical Corporation, Osaka Japan). As a control, the culture supernatant of Pichia pastoris into which the empty vector pPICZα was introduced was used to obtain crude protein in the same manner as described above (hereinafter referred to as "vector control"). For lipase activity measurement, the protein concentration of the crude enzyme was adjusted to 10 μg/mL. Using 1 μg/mL crude enzyme or vector control, lipase activity was measured by the following method.
PgLip19028をコードするDNAから天然分泌シグナルを除いたものを合成した(JGIサービス)。その遺伝子(配列番号4)を、PIPE(Polymerase Incomplete Primer Extension)クローニングのためのプライマーを用いて発現ベクターpPICZα(Invitrogen)中にサブクローン化した。分泌シグナルとして、pPICZα上のαファクタの分泌シグナルを利用した。プロモーター配列として、AOXのプロモーター配列を用いた。使用したプライマー配列を表1に示す。製造者(Invitrogen)の指示書にしたがって、発現ベクターを酵母Pichia pastorisに形質転換した。形質転換したPichia pastorisをYPD液体培地中30℃で1~3日間振盪培養し、得られた菌体をよく洗浄してYPG液体培地中に移した。該培地にメタノールを添加後、1~3日間培養してタンパク質発現を誘導し、PgLip19028を過剰発現する該形質転換酵母からリコンビナントリパーゼが生産された。形質転換したPichia pastorisの培養上清を飽和硫酸アンモニウム沈殿に付し、得られたタンパク質沈殿物を100mM酢酸バッファー(pH5.0)中に溶解し、これを粗酵素として用いた。10μgの粗酵素を変性させ、タンパク質脱グリコシル化のためにエンドグリコシダーゼH(Endo H、New England Biolab社製)で消化し、次いで粗酵素サンプルをSDS-PAGE分析(Bio-Rad)に付し、ゲルをクーマシーブリリアントブルー(CBB)R-250(FUJIFILM Wako Pure Chemical Corporation, Osaka Japan)で染色した。コントロールとして、空の(上記遺伝子をクローニングしていない)ベクターpPICZαを導入したPichia pastorisの培養上清を用い、上記と同様にして粗タンパク質を得た(以下、「ベクターコントロール」という)。リパーゼ活性測定のために、上記粗酵素のタンパク質濃度を10μg/mLに調整した。1μg/mLの粗酵素またはベクターコントロールを用いて、以下の方法でリパーゼ活性を測定した。 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 pPICZα (Invitrogen) using primers for PIPE (Polymerase Incomplete Primer Extension) cloning. As a secretion signal, the α-factor secretion signal on pPICZα 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. 10 μg of crude enzyme was denatured and digested with endoglycosidase H (Endo H, New England Biolab) for protein deglycosylation, then crude enzyme samples were subjected to SDS-PAGE analysis (Bio-Rad), Gels were stained with Coomassie Brilliant Blue (CBB) R-250 (FUJIFILM Wako Pure Chemical Corporation, Osaka Japan). As a control, the culture supernatant of Pichia pastoris into which the empty vector pPICZα was introduced was used to obtain crude protein in the same manner as described above (hereinafter referred to as "vector control"). For lipase activity measurement, the protein concentration of the crude enzyme was adjusted to 10 μg/mL. Using 1 μg/mL crude enzyme or vector control, lipase activity was measured by the following method.
リパーゼ活性の測定
ドデカン酸p-ニトロフェニル(pNPD、Sigma-Aldrich社製)を基質として用いた。5v/v%ジメチルスルホキシド(DMSO)中の1.5mM pNPDを1μg/mLの酵素サンプル、25μLの25mM酢酸バッファー(pH4.5)およびtert-ブチルメチルエーテル(tBE)と混合し、全量160μLにした。tBEの最終濃度は15v/v%に維持した。反応混合物を25℃で10分間インキュベーション後、40μLの100mM Na2CO3を加えて反応を止めた。405nmでの吸光度を測定して遊離されたp-ニトロフェノール(pNP)を検出し、pNP(Sigma-Aldrich社製)の検量線を用いてpNP遊離量を決定した。1単位のリパーゼ活性は、1分間の反応当たり1μモルのpNPを遊離させる酵素量として定義した。なお、予備試験において、15v/v%tBEは、5v/v%、10v/v%、15v/v%、20v/v%、25v/v%および30v/v%tBEと比べて最も高い活性を示した。 Measurement of lipase activity p-Nitrophenyl dodecanoate (pNPD, manufactured by Sigma-Aldrich) was used as a substrate. 1.5 mM pNPD in 5 v/v% dimethylsulfoxide (DMSO) was mixed with 1 μg/mL enzyme sample, 25 μL of 25 mM acetate buffer (pH 4.5) and tert-butyl methyl ether (tBE) to a total volume of 160 μL. . The final concentration of tBE was maintained at 15 v/v%. After incubating the reaction mixture at 25° C. for 10 minutes, the reaction was stopped by adding 40 μL of 100 mM Na 2 CO 3 . Released p-nitrophenol (pNP) was detected by measuring absorbance at 405 nm, and 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 μmole of pNP per minute of reaction. In preliminary studies, 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.
ドデカン酸p-ニトロフェニル(pNPD、Sigma-Aldrich社製)を基質として用いた。5v/v%ジメチルスルホキシド(DMSO)中の1.5mM pNPDを1μg/mLの酵素サンプル、25μLの25mM酢酸バッファー(pH4.5)およびtert-ブチルメチルエーテル(tBE)と混合し、全量160μLにした。tBEの最終濃度は15v/v%に維持した。反応混合物を25℃で10分間インキュベーション後、40μLの100mM Na2CO3を加えて反応を止めた。405nmでの吸光度を測定して遊離されたp-ニトロフェノール(pNP)を検出し、pNP(Sigma-Aldrich社製)の検量線を用いてpNP遊離量を決定した。1単位のリパーゼ活性は、1分間の反応当たり1μモルのpNPを遊離させる酵素量として定義した。なお、予備試験において、15v/v%tBEは、5v/v%、10v/v%、15v/v%、20v/v%、25v/v%および30v/v%tBEと比べて最も高い活性を示した。 Measurement of lipase activity p-Nitrophenyl dodecanoate (pNPD, manufactured by Sigma-Aldrich) was used as a substrate. 1.5 mM pNPD in 5 v/v% dimethylsulfoxide (DMSO) was mixed with 1 μg/mL enzyme sample, 25 μL of 25 mM acetate buffer (pH 4.5) and tert-butyl methyl ether (tBE) to a total volume of 160 μL. . The final concentration of tBE was maintained at 15 v/v%. After incubating the reaction mixture at 25° C. for 10 minutes, the reaction was stopped by adding 40 μL of 100 mM Na 2 CO 3 . Released p-nitrophenol (pNP) was detected by measuring absorbance at 405 nm, and 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 μmole of pNP per minute of reaction. In preliminary studies, 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.
電気泳動結果を図4に、リパーゼ活性測定結果を図5に示す。図4から明らかなように、リコンビナント酵素PgLip19028は、Pichia pastoris培養上清における主要タンパク質として生産された(図4中、レーン1:PgLip19028、レーン3:ベクターコントロール)。Endo Hによる脱グリコシル化後、該リパーゼは、SDS-PAGEによって概算される約30.0kDaの算出分子量(グリコシル化およびシグナルペプチドを減算した)と合致した(図4のレーン2)。実際、PgLip19028では、ベクターコントロール(2.55±0.06U/mg)と比べて、モデル基質pNPDに対する有意に高いリパーゼ活性(85.41±3.75U/mg)が検出された(図5)。上記の酵母発現系において、主要なタンパク質として分子量約30kDaの粗酵素を約60mg/Lを得ることができた。
Fig. 4 shows the results of electrophoresis, and Fig. 5 shows the results of lipase activity measurement. As is clear from FIG. 4, 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). After deglycosylation with Endo H, 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). Indeed, significantly higher lipase activity (85.41±3.75 U/mg) against the model substrate pNPD was detected with PgLip19028 compared to the vector control (2.55±0.06 U/mg) (FIG. 5). . About 60 mg/L of crude enzyme with a molecular weight of about 30 kDa could be obtained as the main protein in the yeast expression system described above.
実施例4:PgLip19028の至適pHおよび至適温度の決定
PgLip19028の至適pH条件を決定するために、pH3.0~pH3.5の酒石酸バッファー、pH3.5~pH5.5の酢酸バッファー、およびpH6.0~pH6.5の酢酸バッファーを用いて、実施例3で得られた粗酵素のリパーゼ活性を測定した。pH条件以外は、実施例3に記載のとおりである。ベクターコントロールのリパーゼ活性を測定値から引き算した。結果を図6に示す。図中、最も高い活性を100%とし、各条件下での相対値を示した。 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.
PgLip19028の至適pH条件を決定するために、pH3.0~pH3.5の酒石酸バッファー、pH3.5~pH5.5の酢酸バッファー、およびpH6.0~pH6.5の酢酸バッファーを用いて、実施例3で得られた粗酵素のリパーゼ活性を測定した。pH条件以外は、実施例3に記載のとおりである。ベクターコントロールのリパーゼ活性を測定値から引き算した。結果を図6に示す。図中、最も高い活性を100%とし、各条件下での相対値を示した。 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.
PgLip19028の至適温度条件を決定するために、10℃、15℃、20℃、25℃、30℃および35℃の温度条件下で反応混合物を10分間インキュベーションし、実施例3で得られた粗酵素のリパーゼ活性を測定した。温度条件以外は、実施例3に記載のとおりである。ベクターコントロールのリパーゼ活性を測定値から引き算した。結果を図7に示す。図中、最も高い活性を100%とし、各条件下での相対値を示した。
To determine the optimum temperature conditions for PgLip19028, 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.
その結果、PgLip19028の最適な反応温度およびpHをそれぞれ、25℃およびpH4.5であった。また、pH3~pH5の範囲および10℃~35℃の範囲で相対活性40%以上を示し、これらのpH条件または温度条件がPgLip19028を用いた反応に適することが分かった。最適pH4.5は、明らかに他の従来のリパーゼよりも低い。このような最適pHは、シュウ酸などの分泌有機酸のために通常酸性であるP. giganteaの外的環境に合致する。
As a result, the optimum reaction temperature and pH for PgLip19028 were 25°C and pH 4.5, respectively. In addition, 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.
実施例5:PgLip19028のグリセリド分解アッセイ
グリセリド分解アッセイのために、実施例3で得られた1μg/mLの粗酵素またはベクターコントロールを、10mg/mLのトリオレインまたはマツ抽出物、最終濃度15v/v%のtBE、5v/v%DMSOおよび25mM酢酸バッファー(pH4.5)と混合して、全量を1000μLにした。マツ抽出物は、樹皮を剥ぎ、木質部分を粉砕したテーダマツ木粉(1mmメッシュ)を70w/v%アセトンに浸漬し一晩攪拌して得られた。反応混合物を25℃で24時間、120rpmで振盪させながらインキュベーションし、凍結乾燥させ、クロロホルム中に溶解させた。サンプルを薄層クロマトグラフィー(TLC)プレートにアプライし、残りのサンプルをBSTFAによりトリメチルシリル化後、GC-MS分析に付した。GC-MS分析は以下の方法で行った。 Example 5 Glyceride Degradation Assay of PgLip19028 For the glyceride degradation assay , 1 μg/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 μL. 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. for 24 hours with shaking at 120 rpm, lyophilized and dissolved in chloroform. Samples were applied to thin layer chromatography (TLC) plates and the remaining samples were subjected to GC-MS analysis after trimethylsilylation with BSTFA. GC-MS analysis was performed by the following method.
グリセリド分解アッセイのために、実施例3で得られた1μg/mLの粗酵素またはベクターコントロールを、10mg/mLのトリオレインまたはマツ抽出物、最終濃度15v/v%のtBE、5v/v%DMSOおよび25mM酢酸バッファー(pH4.5)と混合して、全量を1000μLにした。マツ抽出物は、樹皮を剥ぎ、木質部分を粉砕したテーダマツ木粉(1mmメッシュ)を70w/v%アセトンに浸漬し一晩攪拌して得られた。反応混合物を25℃で24時間、120rpmで振盪させながらインキュベーションし、凍結乾燥させ、クロロホルム中に溶解させた。サンプルを薄層クロマトグラフィー(TLC)プレートにアプライし、残りのサンプルをBSTFAによりトリメチルシリル化後、GC-MS分析に付した。GC-MS分析は以下の方法で行った。 Example 5 Glyceride Degradation Assay of PgLip19028 For the glyceride degradation assay , 1 μg/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 μL. 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. for 24 hours with shaking at 120 rpm, lyophilized and dissolved in chloroform. Samples were applied to thin layer chromatography (TLC) plates and the remaining samples were subjected to GC-MS analysis after trimethylsilylation with BSTFA. GC-MS analysis was performed by the following method.
GC-MS分析
GC-MS分析は、異なる脂質クラスの同時溶出を可能にする、ミディアム長フューズドシリカDB-5HTキャピラリーカラム(12m×0.25mm内径、0.1μmフィルム厚)を用いるイオントラップ検出器(Varian 4000)と結合させたVarian 3800クロマトグラフ(いずれもJ&W Scientific社製)で行った。温度プログラムは100℃(1分)で開始し、1分毎に10℃ずつ380℃まで昇温し、5分間維持した。トランスファーラインを300℃で維持し、インジェクターを120℃(0.1分)から200℃/分で380℃にプログラムし、ヘリウムをキャリアガスとして2mL/分の速度で用いた。マスフラグメントグラフィーによって、およびそれらのマススペクトルをWileyおよびNISTライブラリーおよび標準のマススペクトルと比較することによって、化合物を同定した。N,O-ビス(トリメチルシリル)トリフルオロアセトアミド(BSTFA)をピリジンの存在下で用いて、トリメチルシリル誘導体を調製した。 GC-MS analysis 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 μm 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. Compounds were identified by mass fragmentography and by comparing their mass spectra with those of Wiley and NIST libraries and standards. Trimethylsilyl derivatives were prepared using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine.
GC-MS分析は、異なる脂質クラスの同時溶出を可能にする、ミディアム長フューズドシリカDB-5HTキャピラリーカラム(12m×0.25mm内径、0.1μmフィルム厚)を用いるイオントラップ検出器(Varian 4000)と結合させたVarian 3800クロマトグラフ(いずれもJ&W Scientific社製)で行った。温度プログラムは100℃(1分)で開始し、1分毎に10℃ずつ380℃まで昇温し、5分間維持した。トランスファーラインを300℃で維持し、インジェクターを120℃(0.1分)から200℃/分で380℃にプログラムし、ヘリウムをキャリアガスとして2mL/分の速度で用いた。マスフラグメントグラフィーによって、およびそれらのマススペクトルをWileyおよびNISTライブラリーおよび標準のマススペクトルと比較することによって、化合物を同定した。N,O-ビス(トリメチルシリル)トリフルオロアセトアミド(BSTFA)をピリジンの存在下で用いて、トリメチルシリル誘導体を調製した。 GC-MS analysis 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 μm 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. Compounds were identified by mass fragmentography and by comparing their mass spectra with those of Wiley and NIST libraries and standards. Trimethylsilyl derivatives were prepared using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine.
GC-MS分析の結果を図8に示す。トリグリセリドはリパーゼ反応後、有意に減少した。一方、ジオレイン、モノオレインおよびオレイン酸が最終産物として同定された(図8A)。さらに、PgLip19028は、針葉樹抽出物中のトリグリセリドおよびジグリセリドから、オレイン酸、リノール酸、リノレン酸およびパルミチン酸を遊離させた(図8B)。これらの結果は、該リパーゼが、種々の炭素鎖長および不飽和結合を有する広い種類の脂肪酸を生産することを示す。
The results of GC-MS analysis are shown in FIG. Triglycerides decreased significantly after lipase reaction. On the other hand, diolein, monoolein and oleic acid were identified as end products (Fig. 8A). Furthermore, PgLip19028 liberated oleic, linoleic, linolenic and palmitic acids from triglycerides and diglycerides in conifer extracts (Fig. 8B). These results indicate that the lipase produces a wide variety of fatty acids with varying carbon chain lengths and unsaturation.
実施例5:市販のリパーゼとの活性比較試験
市販の商業用糸状菌由来リパーゼ4種とPgLip19028のリパーゼ活性を比較した。市販のリパーゼとして、リパーゼM1アマノ(Rhizomucor miehei由来;天野エンザイム株式会社)、リパーゼA6アマノ(Asperigillus niger由来;天野エンザイム株式会社)、リパーゼF-AP15アマノ(Rhizopus oryzae由来;天野エンザイム株式会社)、およびNovozym 51032(Asperigillus 由来;Novozymes)を用いた。実施例3で得られた粗酵素またはベクターコントロールあるいは市販のリパーゼ1μg/mLを用いて、実施例3に記載にしたがってリパーゼ活性を測定した。結果を図9に示す。PgLip19028は粗酵素の状態で、市販のリパーゼよりも高い活性を示した。 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 activity was measured as described in Example 3 using the crude enzyme or vector control obtained in Example 3 or 1 μg/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.
市販の商業用糸状菌由来リパーゼ4種とPgLip19028のリパーゼ活性を比較した。市販のリパーゼとして、リパーゼM1アマノ(Rhizomucor miehei由来;天野エンザイム株式会社)、リパーゼA6アマノ(Asperigillus niger由来;天野エンザイム株式会社)、リパーゼF-AP15アマノ(Rhizopus oryzae由来;天野エンザイム株式会社)、およびNovozym 51032(Asperigillus 由来;Novozymes)を用いた。実施例3で得られた粗酵素またはベクターコントロールあるいは市販のリパーゼ1μg/mLを用いて、実施例3に記載にしたがってリパーゼ活性を測定した。結果を図9に示す。PgLip19028は粗酵素の状態で、市販のリパーゼよりも高い活性を示した。 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 activity was measured as described in Example 3 using the crude enzyme or vector control obtained in Example 3 or 1 μg/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; Primer
SEQ ID NO: 8; Primer
SEQ ID NO: 9; Primer
SEQ ID NO: 10; Primer 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;
SEQ ID NO: 7; Primer
SEQ ID NO: 8; Primer
SEQ ID NO: 9; Primer
SEQ ID NO: 10; Primer 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;
Claims (15)
- 配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼを含む酵素組成物。 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.
- リパーゼの至適温度が10℃~35℃の範囲にある、請求項1記載の酵素組成物。 The enzyme composition according to claim 1, wherein the optimum temperature of lipase is in the range of 10°C to 35°C.
- リパーゼの至適pHがpH3~pH5の範囲にある、請求項1または2記載の酵素組成物。 The enzyme composition according to claim 1 or 2, wherein the optimum pH of the lipase is in the range of pH3-pH5.
- 担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあるリパーゼを含む酵素組成物。 An enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10°C to 35°C.
- 担子菌由来のリパーゼであって、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物。 An enzyme composition containing a basidiomycete-derived lipase having an optimum pH in the range of pH3 to pH5.
- 担子菌由来のリパーゼであって、至適温度が10℃~35℃の範囲にあり、かつ、至適pHがpH3~pH5の範囲にあるリパーゼを含む酵素組成物。 An enzyme composition containing a basidiomycete-derived lipase having an optimum temperature in the range of 10°C to 35°C and an optimum pH in the range of pH3 to pH5.
- 担子菌がプレビオピシス・ギガンテアである、請求項4~6のいずれか1項記載の酵素組成物。 The enzyme composition according to any one of claims 4 to 6, wherein the basidiomycete is Prebiopisis gigantea.
- 食品用酵素組成物、医薬品用酵素組成物、化粧品用酵素組成物、産業用酵素組成物、または洗浄組成物である、請求項1~7のいずれか1項記載の酵素組成物。 The enzyme composition according to any one of claims 1 to 7, which is an enzyme composition for food, an enzyme composition for pharmaceuticals, an enzyme composition for cosmetics, an enzyme composition for industrial use, or a cleaning composition.
- 紙パルプの製造またはバイオリファイナリープロセスにおいて使用するための、請求項1~7のいずれか1項記載の酵素組成物。 The enzyme composition according to any one of claims 1 to 7, for use in paper pulp manufacturing or biorefinery processes.
- 配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼのリコンビナント酵素。 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.
- 配列番号1に示されるアミノ酸配列と少なくとも90%の配列同一性を有するアミノ酸配列を含むリパーゼをコードする核酸を含む、ベクター。 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.
- 前記リパーゼをコードする核酸がコドン最適化された配列を含む、請求項11記載のベクター。 The vector according to claim 11, wherein the lipase-encoding nucleic acid comprises a codon-optimized sequence.
- 請求項11または12に記載したベクターが導入された宿主。 A host into which the vector according to claim 11 or 12 has been introduced.
- 酵母である、請求項13記載の宿主。 The host according to claim 13, which is yeast.
- 請求項13または14記載の宿主を培養することを含む、リコンビナント酵素の製造方法。 A method for producing a recombinant enzyme, comprising culturing the host according to claim 13 or 14.
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DATABASE UNIPROTKB 1 April 2015 (2015-04-01), ANONYMOUS: "UNIPROT", XP055960564, Database accession no. A0A0C3PMX9 * |
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 * |
PAUL CHRISTAKOPOULOS; CONSTANTINA TZIA; DIMITRIS KEKOS; BASIL J. MACRIS: "Production and characterization of extracellular lipase from", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 38, no. 2, 1 November 1992 (1992-11-01), Berlin, DE , pages 194 - 197, XP035172588, ISSN: 1432-0614, DOI: 10.1007/BF00174467 * |
REHMAN SAIMA, BHATTI HAQ NAWAZ, BILAL MUHAMMAD, ASGHER MUHAMMAD: "Optimization of process variables for enhanced production of extracellular lipase by Pleurotus ostreatus IBL-02 in solid-state fermentation", PAKISTAN JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 32, no. 2, 1 March 2019 (2019-03-01), PK , pages 617 - 624, XP055960566, ISSN: 1011-601X * |
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