Utilization of novel highly functional lipases derived from basidiomycetes

Abstract

To provide novel highly functional lipase derived from a basidiomycete Phlebiopsis gigantea and use methods thereof.SOLUTION: Provided herein is an enzyme composition comprising a lipase derived from Phlebiopsis gigantea, having an optimum temperature in a range of 10°C-35°C and an optimum pH in a range of pH3-pH5. Also provided are lipase-containing enzyme compositions for use in food, drugs, cosmetics, industry or washing, where the lipase has 90% or more sequence identity to a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of a novel highly functional lipase derived from a basidiomycete. [Background technology]

[0002] Lipase is a general term for enzymes that hydrolyze the ester bonds of lipids. Because this reaction is reversible, it is known that lipases also act as catalysts for esterification and transesterification reactions depending on the reaction conditions. Lipases are used industrially in agricultural chemical production, pharmaceutical production, cosmetic production, oil and fat production, food processing, food production, and food additive production, and are also used as digestive aids, clinical diagnostic reagents, flavoring agents, detergent enzymes, and catalysts for racemic separation. There are many types of lipases with different properties such as substrate specificity and degradation specificity.

[0003] Woody plant biomass is the most abundant carbon source on Earth. The main components of plant cell walls are cellulose, hemicellulose, and lignin, forming a highly degradable cell wall that prevents other organisms from assimilating or parasitizing them. In addition to these components, solvent-soluble fractions (commonly referred to as "extracts" or "extractives") comprise approximately 10 w / w% of total plant biomass. The chemical composition of extracts varies depending on the tree species and age. These extracts also play a role in plant defense, particularly in preventing or eliminating fungal colonization of newly felled coniferous trees. Meanwhile, wood-rotting fungi, a type of basidiomycete, survive by secreting various extracellular enzymes, including cellulases, hemicellulases, and ligninolytic enzymes, which allow them to utilize plant cell walls as a nutrient source. Among wood-decaying fungi, the ligninolytic basidiomycete Phlebiopsis gigantea (P. gigantea) is known to rapidly invade freshly felled coniferous trees, which are rich in extractives. Although the mechanism by which P. gigantea detoxifies or utilizes the extractives from conifers is largely unknown, it is known that it secretes lipases during coniferous wood decomposition (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] PLoS Genetics 2014; Volume 10, Issue 12: e1004759 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to discover a novel highly functional lipase and to provide a method for using the same. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have identified, among the multiple lipases secreted by P. gigantea, an enzyme that has particularly useful properties, namely, higher lipase activity under acidic and low-temperature conditions than conventional lipases, and have succeeded in producing a recombinant enzyme, thus completing the present invention.

[0007] The present invention provides, for example, the following aspects. [1] An enzyme composition comprising a lipase having 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 pH 3 to pH 5. [4] An enzyme composition containing a lipase derived from a basidiomycete fungus, the optimum temperature of which is in the range of 10°C to 35°C. [5] An enzyme composition containing a lipase derived from a basidiomycete fungus, the optimum pH of which is in the range of pH 3 to pH 5. [6] An enzyme composition comprising a lipase derived from a basidiomycete fungus, the lipase having an optimum temperature in the range of 10°C to 35°C and an optimum pH in the range of pH 3 to pH 5. [7] The enzyme composition according to any one of [4] to [6], wherein the Basidiomycete is Prebiopsis gigantea. [8] The enzyme composition according to any one of [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. [9] The enzyme composition according to any one of [1] to [7], for use in paper pulp production or a biorefinery process.

[10] A recombinant lipase 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 according to

[11] , wherein the nucleic acid encoding the lipase comprises a codon-optimized sequence.

[13] A host into which the vector described in

[11] or

[12] has been introduced.

[14] The host according to

[13] , which is a yeast; and

[15] A method for producing a recombinant enzyme, comprising culturing the host according to

[13] or

[14] . [Effects of the Invention]

[0008] According to the present invention, an enzyme composition containing a lipase derived from a basidiomycete fungus is provided, which, unlike conventional lipases, has an acidic optimum pH and an optimum temperature at room to low temperatures. Because the composition has high activity at room temperature and under acidic conditions, it can be used in a variety of applications, including food, pharmaceuticals, and detergents. Furthermore, because the lipase is derived from a natural basidiomycete fungus, the enzyme composition of the present invention is also highly safe. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the time course of lipase activity in the secretome of P. gigantea cultured on AV0X, AV1X, AV2X, or AV4X. [Figure 2]Figure 1 shows the transcript expression levels (RPKM) and ratios (Ratios) of nine lipases encoded by the genome of P. gigantea cultivated on AV0X, AV1X, AV2X, or AV4X. Bold indicates p-value < 0.05. [Figure 3] The amino acid sequence of secretory lipase PgLip19028 (SEQ ID NO: 2) is shown. In the figure, the signal sequence is underlined. [Figure 4] The electrophoresis results of the recombinant enzyme PgLip19028 are shown. [Figure 5] 1 shows the results of a lipase activity test of recombinant PgLip19028. [Figure 6] The relative activity of PgLip19028 under various pH conditions is shown. [Figure 7] The relative activity of PgLip19028 under various temperature conditions is shown. [Figure 8] GC-MS analysis of triolein (A) and released products from conifer extract (B) after overnight incubation at 25°C in acetate buffer (pH 4.5) with recombinant PgLip19028 expressed by P. pastoris or a vector control. [Figure 9] The activity of PgLip19028 is compared with that of commercially available lipases. DETAILED DESCRIPTION OF THE INVENTION

[0010] 1. Lipase In our previous study, we performed genome analysis of the conifer-degrading basidiomycete P. gigantea and found that lipases are secreted during conifer decomposition by liquid chromatography-tandem mass spectrometry (LC-MS / MS) proteome analysis (Non-Patent Document 1). In this study, we found that transcripts encoding four of the nine lipases predicted in the previous paper were significantly upregulated in the presence of conifer extract. In this study, we focused on a secreted lipase (designated PgLip19028) that was particularly highly transcribed in the presence of conifer extract. PgLip19028 is a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 and is characterized by a conserved lipase 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 secretion signal, three N-glycosylation sites, and one O-glycosylation site. The amino acid sequence of PgLip19028 without the secretion signal is shown in SEQ ID NO: 1, and the amino acid sequence of PgLip19028 with the secretion signal is shown in SEQ ID NO: 2. The nucleic acid sequence of PgLip19028 without the secretion signal is shown in SEQ ID NO: 4, and the nucleic acid sequence of PgLip19028 with the secretion signal is shown in SEQ ID NO: 5. include The nucleic acid sequence of PgLip19028 is shown in SEQ ID NO:5.

[0011] Based on sequence annotation, PgLip19028 was predicted to encode a lipase in the triacylglycerol hydrolase (EC 3.1.1.3) family. In the present invention, PgLip19028 was shown to have a molecular weight, pH and temperature optimum, and substrate specificity distinct from those of other previously characterized lipases from basidiomycetes. Biochemical characterization in the present invention revealed that PgLip19028 can liberate various unsaturated fatty acids under acidic conditions at ambient temperature. Furthermore, lipases with amino acid sequences similar to those of PgLip19028 and the same conserved motif sequence, GHSLG, were found in basidiomycetes other than P. gigantea.

[0012] Therefore, the lipase used in the present invention (hereinafter also referred to as "lipase of the present invention") is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1. As long as the polypeptide has lipase activity, it may contain one to several amino acid mutations (substitutions, deletions, additions, or insertions, or a combination thereof) in the amino acid sequence shown in SEQ ID NO: 1, or it may comprise an amino acid sequence having 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 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 to the amino acid sequence shown in SEQ ID NO: 1, as long as it has lipase activity. Preferably, the lipase used in the present invention is a polypeptide having lipase activity, which is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence having 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 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 to the amino acid sequence set forth in SEQ ID NO: 1. The polypeptides containing one to several amino acid mutations in the amino acid sequence set forth in SEQ ID NO: 1, polypeptides having an amino acid sequence having at least 45% to at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and polypeptides consisting of an amino acid sequence having at least 45% to at least 99% sequence identity to the amino acid sequence set forth 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 or non-conservative substitutions. Conservative substitutions are preferred.

[0013] The lipase used in 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. The lipase used in the present invention also has an optimum pH in the range of pH 3 to pH 5, preferably pH 3.5 to pH 5, more preferably pH 4 to pH 5, and even more preferably pH 4.5. As used herein, the optimum temperature and optimum pH of a lipase refer to the temperature and pH 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. The relative activity is the relative value of the lipase activity under each condition, with the lipase activity at the temperature or pH condition showing the highest lipase activity being taken as 100%.

[0014] In this specification, lipase activity is based on the hydrolysis activity of a substrate. Lipase activity can be measured by a conventional method, for example, by measuring the amount of p-nitrophenol produced by an enzymatic reaction using lipase model substrates such as p-nitrophenyl dodecanoate, p-nitrophenyl palmitate, and p-nitrophenyl butyrate.

[0015] The lipase used in the present invention has a molecular weight of about 30 kDa.

[0016] The lipase used in the present invention can be obtained from a culture or culture supernatant of a basidiomycete fungus. For example, it may be the cell lysate or culture supernatant itself, or it may be a crude or purified product obtained from the cell lysate or culture supernatant. The lipase used in the present invention is typically a secretory lipase having an amino acid sequence with a secretory signal sequence added to the N-terminus, and is therefore preferably obtained from the culture supernatant. Crude or purified lipase can be achieved by known methods, such as salting out, centrifugation, ultrafiltration, gel filtration, various types of chromatography (such as adsorption chromatography, hydrophobic chromatography, ion exchange chromatography, affinity chromatography, and high-performance liquid chromatography), dialysis, and electrophoresis.

[0017] Basidiomycete culture can be performed using conventional culture equipment and basal media, either liquid or solid. Examples of basal media include, but are not limited to, potato dextrose agar (PDA), malt extract agar (MYA), and Highley's basal medium. Carbon sources such as glucose and cellulose may be added to the medium. To promote lipase induction, a coniferous tree extract is preferably added. Examples of coniferous trees include, but are not limited to, pine (e.g., loblolly pine), cedar, cypress, hemlock, fir, spruce, and yew. Coniferous tree extracts may be obtained by, for example, extracting wood flour obtained by crushing the woody parts of conifers after removing the bark with an organic solvent such as acetone. Extraction methods include immersing the material in an organic solvent, heating, and stirring, or Soxhlet extraction. The extract may be concentrated, for example, using a rotary evaporator or freeze-drying. Culture conditions such as culture temperature and culture period can be appropriately determined by those skilled in the art. For example, culture may be performed at 20 to 30°C for 3 to 14 days.

[0018] Although P. gigantea is preferably used as the basidiomycete, other basidiomycetes may also be used, such as Shizophilum commune, Coniophora puteana, Serpula lacrymans, Heterobasidion annosum, Stereum hirsutum, Postia planceta, Wolfiporia cocos, Fomitopsis pinicola, Ceriporiopsis subvermispora, Dichomitus squalens, Trametes versicolor, Phanerochaete, Phanerochaete carnosa, and Phlebia breviospora. These basidiomycetes may be any strain, and may be collected from nature or obtained from a collection institution such as the American Type Culture Collection (ATCC).

[0019] The lipase used in the present invention may also be produced by chemical synthesis (peptide synthesis) based on its amino acid sequence.

[0020] 2. Recombinant enzymes The lipase used in the present invention may be a recombinant enzyme. The recombinant enzyme of PgLip19028 was produced for the first time in the present invention. Furthermore, an expression system for producing the recombinant lipase was constructed for the first time in the present invention.

[0021] Recombinant enzymes may be produced using a cell-free protein synthesis system (Escherichia coli-derived, wheat germ-derived, etc.) or a cellular protein expression system. Hosts for cellular protein expression systems may be either prokaryotic or eukaryotic cells, including, for example, fungi (yeast, filamentous fungi, etc.), animal cells (vertebrate cells, mammalian cells, etc.), insect cells, and bacteria (Escherichia coli, Bacillus subtilis, actinomycetes, etc.). Yeasts are preferably used, including, but not limited to, yeasts of the genus Saccharomyces, Candida, and Pichia. For example, Pichia pastoris may be used.

[0022] To produce a recombinant enzyme, a nucleic acid encoding the lipase of the present invention is introduced into a host cell. The nucleic acid encoding the lipase of the present invention can be synthesized based on known sequence information for P. gigantea. The nucleic acid sequence encoding the lipase of the present invention may be codon-optimized for expression in a host cell. The optimal codons for each host and methods for codon optimization are well known to those skilled in the art. The nucleic acid encoding the lipase of the present invention includes a nucleotide sequence encoding an amino acid sequence having 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 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 to the amino acid sequence of PgLip19028, i.e., the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the nucleic acid encoding the lipase of the present invention may be a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 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 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 to the amino acid sequence set forth in SEQ ID NO: 1. An example of a nucleic acid encoding a lipase of the present invention is a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 4. Further examples of nucleic acids encoding the lipase of the present invention include nucleic acids comprising a nucleotide sequence having 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 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 to the nucleotide sequence set forth in SEQ ID NO: 4.Furthermore, an example of a nucleic acid sequence that has been codon-optimized for expression in Pichia pastoris as a host is the sequence shown in SEQ ID NO:6.

[0023] A secretory signal sequence may be added to the nucleic acid sequence encoding the lipase of the present invention. Examples of secretory signal sequences that may be used include the secretory signal sequence of PgLip19028 or the secretory signal sequence of α-factor. Furthermore, the nucleic acid sequence encoding the lipase of the present invention may be operably linked to a promoter sequence and / or any sequence involved in nucleic acid expression, such as an enhancer sequence, as well as sequences involved in various analyses such as protein stabilization, purification, and expression level measurement, such as maltose binding protein (MBP), polyhistidine tag (His-tag), and human c-Myc tag. Examples of promoter sequences that can be used include, but are not limited to, the alcohol oxidase (AOX) promoter, which can induce protein production by the addition of methanol, and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, which is used as a promoter for constitutive protein production.

[0024] The nucleic acid encoding the lipase of the present invention may be inserted into an appropriate vector, and the vector may be introduced into a host cell. As used herein, the term "vector" refers to a nucleic acid molecule that transports a nucleic acid of interest into a host cell. Various vectors known in the art can be used, including plasmid vectors, cosmids, lambda phage, artificial chromosomes, viral vectors (e.g., baculovirus vectors, retrovirus vectors, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, etc.), and liposomes. A vector appropriate 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 promoter appropriate 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 (transformed cells) into which the gene of interest has been introduced after introduction into the host cell.

[0025] Vectors can be introduced into host cells by methods known in the art, such as cationic lipid-mediated introduction, diethylaminoethyl (DEAE)-dextran, calcium phosphate coprecipitation, cationic polymer introduction, electroporation, microinjection, sonoporation, laser irradiation, and viral vector introduction.

[0026] The desired lipase of the present invention is produced by culturing the resulting gene-transfected cells. Preferably, the lipase of the present invention is secreted extracellularly (into the medium). When produced in a cell system such as E. coli, the desired enzyme may be obtained in an insoluble form (precipitate). In such cases, the enzyme can be obtained in an active form by solubilizing or folding. The solubilizing or folding may be performed by a method known in the art.

[0027] The transfected cells may be cultured according to standard methods, and a culture medium appropriate for the host may be selected. Examples include, but are not limited to, YPD medium and YPG medium. Culture conditions such as culture temperature and time may be selected appropriately. For example, the cells may be cultured at about 20 to 30°C for about one day to one week.

[0028] The lipase of the present invention produced by host cells may be crudely or purified from the culture cell lysate or culture supernatant, preferably from the culture supernatant. Crude purification and purification procedures can be performed by methods known in the art. Crude purification and purification can be performed by known methods such as salting out, centrifugation, ultrafiltration, gel filtration, various types of chromatography (adsorption chromatography, hydrophobic chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography, etc.), dialysis, electrophoresis, etc.

[0029] 3. Enzyme Composition The lipase of the present invention is provided in the form of an enzyme composition. An enzyme composition containing the lipase of the present invention (hereinafter also referred to as the "enzyme composition of the present invention") may contain the lipase of the present invention itself, or may contain additional components in addition to the lipase of the present invention as an active ingredient. Examples of such additional components, depending on the intended use of the enzyme composition of the present invention, include excipients, buffers, suspending agents, stabilizers, preservatives, and antiseptics. Excipients, depending on the intended use of the enzyme composition, include, but are not limited to, starch, dextrin, sugars, sugar alcohols, and glycerol. Buffers, depending on the intended use of the enzyme composition, include, but are not limited to, phosphates, citrates, and acetates. Stabilizers, depending on the intended use of the enzyme composition, include, but are not limited to, propylene glycol and ascorbic acid. Preservatives, depending on the intended use of the enzyme composition, include, but are not limited to, phenol, benzalkonium chloride, and methylparaben. Preservatives vary depending on the intended use of the enzyme composition, but include, but are not limited to, ethanol, benzalkonium chloride, parahydroxybenzoic acid, etc. The amounts of the lipase of the present invention and the additional components in the enzyme composition can be appropriately determined by those skilled in the art.

[0030] The enzyme composition of the present invention may also contain tert-butyl methyl ether. The amount of tert-butyl methyl ether in the composition can be appropriately determined by those skilled in the art. For example, the amount is 5 v / v % to 30 v / v %, preferably 15 v / v %, of the composition.

[0031] 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 improver (flavoring agent) for foods or food ingredients. Examples of such foods or food ingredients include, but are not limited to, various dairy products (e.g., 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, etc. Therefore, in a further aspect of the present invention, there is provided a method for improving the flavor of foods or food ingredients, which comprises adding or allowing the enzyme composition of the present invention to act on the foods or food ingredients.

[0032] The enzyme composition of the present invention for food use may also be used in the production of food additives (e.g., flavoring agents, etc.), food processing (e.g., sake production, etc.), and the production of edible oils and fats (e.g., cooking oil, shortening, etc.). Thus, in a further aspect of the present invention, there is provided a method for producing food, which comprises adding or allowing the enzyme composition of the present invention to act on a food raw material or intermediate product.

[0033] The enzyme composition of the present invention may be an enzyme composition for pharmaceutical use. For example, the enzyme composition of the present invention for pharmaceutical use may be, but is not limited to, pharmaceuticals (e.g., digestive enzyme preparations, alternative enzymes, e.g., drugs for treating lysosomal acid lipase deficiency, etc.), clinical diagnostic reagents (e.g., test reagents for measuring blood cholesterol, etc.), etc.

[0034] The enzyme composition of the present invention can also be used as a cleaning composition. Accordingly, in a further aspect of the present invention, a cleaning composition containing the enzyme composition of the present invention is provided. For example, a cleaning composition containing the enzyme composition of the present invention decomposes and removes lipids attached to items to be cleaned (such as laundry), thereby improving cleaning effectiveness, and furthermore, has high activity at low temperatures (water temperatures), thereby further improving cleaning effectiveness.

[0035] The enzyme composition of the present invention may also be an enzyme composition for cosmetics, and can be used, for example, as an additive for cosmetics (for example, facial cleansers, etc.).

[0036] Furthermore, the enzyme composition of the present invention can be used in the production of fatty acids and soap, wastewater treatment, the production of optically active pharmaceutical and agricultural chemical raw materials, the synthesis of optically active compounds, and the like, and is therefore useful as an industrial enzyme.

[0037] The enzyme composition of the present invention can also be used in paper pulp production. By using the enzyme composition of the present invention, extractives can be removed during the paper pulp production process from coniferous trees, thereby improving the properties of the paper produced. The enzyme composition of the present invention can also be used in biorefinery processes.

[0038] Furthermore, the enzyme composition of the present invention can be used to obtain lipid components such as various fatty acids from extracts of trees, particularly coniferous trees, and the tree-derived lipid components thus obtained are used in the fields of food, cosmetics, medicine, etc.

[0039] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. [Example]

[0040] Example 1: Lipase activity of P. gigantea culture supernatant Loblolly pine wood flour (1 mm mesh) was stripped of bark and ground to a fine powder. The extract was then subjected to Soxhlet extraction using 70 w / v% acetone. The extract was added to 1.25 g of microcrystalline cellulose (Avicel PH101, 50 μM, Fluka Chemika, Switzerland) and dried on a rotary evaporator to prepare a matrix. The amount of extract added to the cellulose was determined to be the natural loblolly pine extract content per weight of wood flour (AV1X), twice the natural concentration (AV2X), or four times the natural concentration (AV4X). The acetone-suspended matrix of microcrystalline cellulose without loblolly pine extract was designated "AV0X." Substrate was added to 250 mL of basal medium (Highley medium), and mycelial plugs of P. gigantea isolate 11061-1 (single basidiospore strain 5-6) cultured in PDA medium were inoculated and cultured on a rotary shaker (150 rpm) at 22°C for 7 days. The culture supernatant was collected over time (on days 3, 5, and 7 of culture), and the protein concentration in the culture supernatant was measured by Protein Assay (Bio-Rad). Lipase activity in the secretome (culture supernatant secretions) was measured as follows.

[0041] Measurement of lipase activity Lipase activity was measured using p-nitrophenyl dodecanoate (pNPD, Sigma-Aldrich) as a substrate. 2 μL of 75 mM pNPD in dimethyl sulfoxide (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 of incubation at 37°C, the reaction was stopped by adding 25 μL of 100 mM Na2CO3. The liberated p-nitrophenol (pNP) was detected at 405 nm, and the amount of pNP released was determined using a standard curve of pNP (Sigma-Aldrich). One unit of lipase activity was defined as the amount of enzyme liberating 1 μmole of pNP per minute of reaction.

[0042] The results are shown in Figure 1. At all time points, increased lipase activity was observed in the culture supernatants of AV1X, AV2X, and AV4X. In contrast, relatively low activity was detected in AV0X. These results indicate that lipase is secreted into the culture supernatant of P. gigantea and that its lipase activity is induced by the addition of the extract.

[0043] Example 2: Lipase induced in the presence of coniferous tree extract P. gigantea was cultured for 5 days as in Example 1, and total RNA was purified from the resulting cells using standard methods. mRNA was purified from 100 ng of total RNA obtained from each sample according to the Illumina RNA Sequencing (Illumina, USA) protocol. A 2x150 bp paired-end library for RNA sequencing was then prepared by PCR and adapter addition, and the transcriptome was analyzed using a NovaSeq sequencer. The resulting data was mapped to the genome sequence, and RPKM values were calculated by correcting gene expression levels for the total number of reads and gene length in the sample.

[0044] Furthermore, the culture supernatant 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. Chloroform and methanol were added to the resulting pellet, followed by water, to purify the total protein from the pellet by methanol / chloroform / water partitioning. The protein partitioned into the interphase. After multiple washes with methanol, the purified protein was finally redissolved in 8 M urea / 50 mM NH4HCO3 (pH 8.5) / 1 mM TrisHCl. The total amount of protein in each sample was adjusted to be equal, digested with trypsin / LysC, and purified using an OMIX C18 SPE (Agilent Technologies). Finally, 2 μg of protein was purified using an EASY-Spray TM Hybrid Linear Ion Trap-Orbitrap Mass Spectrometer (LTQ-Orbitrap Elite™) with an Electrospray SourceTM The peptides were subjected to LC-MS / MS analysis using an Agilent 1100 nanoflow system (Agilent Technologies) coupled to a 1000 sieve capillary (ThermoFisher Scientific). Chromatography of the peptides prior to mass spectrometry analysis was achieved 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). The HPLC system was loaded 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 / min for 30 min. Peptides were then directly eluted into the electrospray column 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. Finally, peptides were eluted with a gradient of 20% (v / v) B to 50% (v / v) B over 12 min, followed by a 5 min flush with 50-95% (v / v) B. Survey MS scans were acquired at 120,000 resolution on the Orbitrap as peptides eluted from the HPLC column / electrospray source. MS2 fragmentation of the 20 most intense peptides detected in the MS1 scan from 380 to 1800 m / z was then performed, with redundancy limited by dynamic exclusion. For downstream analysis, MS / MS raw data were converted to mgf file format using MSConvert (Proteo Wizard: open-source software for rapid proteomics tool development). The resulting mgf files were used to search the P. gigantea protein database via the JGI portal (https: / / genome.jgi.doe.gov / portal / Phlgi1 / Phlgi1.download.html) to identify MS / MS peptides.

[0045] The results of the transcriptome analysis are shown in Figure 2. Of 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), 4.64-fold more abundant than in AV0X. Furthermore, proteome analysis identified only PgLip19028 among the nine lipases, confirming that it was secreted into the culture supernatant.

[0046] Example 3: Production of recombinant PgLip19028 DNA encoding PgLip19028 was synthesized (JGI Service) without the native secretion signal. The gene (SEQ ID NO: 4) was subcloned into the expression vector pPICZα (Invitrogen) using primers for PIPE (Polymerase Incomplete Primer Extension) cloning. The α-factor secretion signal on pPICZα was used as the secretion signal. The AOX promoter sequence was used as the promoter sequence. The primer sequences used are shown in Table 1. The expression vector was transformed into the yeast Pichia pastoris according to the manufacturer's instructions (Invitrogen). The transformed Pichia pastoris was cultured with shaking in YPD liquid medium at 30°C for 1 to 3 days, and the resulting cells were thoroughly washed and transferred to YPG liquid medium. Methanol was added to the medium, followed by culture for 1 to 3 days to induce protein expression. Recombinant lipase was produced from the transformed yeast overexpressing Lip19028. Pichia pastoris culture supernatant 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 the crude enzyme. 10 μg of crude enzyme was denatured and digested with endoglycosidase H (Endo H, New England Biolabs) for protein deglycosylation. The crude enzyme sample was then subjected to SDS-PAGE analysis (Bio-Rad), and the gel was stained with Coomassie Brilliant Blue (CBB) R-250 (FUJIFILM Wako Pure Chemical Corporation, Osaka Japan). As a control, Pichia pastoris culture supernatant containing the empty vector pPICZα (no cloned genes) was used to obtain crude protein in the same manner as above (hereinafter referred to as "vector control"). The protein concentration of the crude enzyme was adjusted to 10 μg / mL for lipase activity measurement. Lipase activity was measured using 1 μg / mL of crude enzyme or vector control as follows.

[0047] Measurement of lipase activity p-Nitrophenyl dodecanoate (pNPD, Sigma-Aldrich) was used as the substrate. 1.5 mM pNPD in 5% (v / v) dimethyl sulfoxide (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 tBE concentration was maintained at 15% (v / v). The reaction mixture was incubated at 25°C for 10 min, and then 40 μL of 100 mM Na2CO3 was added to stop the reaction. The absorbance at 405 nm was measured to detect liberated p-nitrophenol (pNP), and the amount of pNP released was determined using a standard curve for pNP (Sigma-Aldrich). One unit of lipase activity was defined as the amount of enzyme required to liberate 1 μmole of pNP per minute of reaction. In preliminary tests, 15 v / v% tBE showed 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.

[0048] The electrophoresis results are shown in Figure 4, and the lipase activity measurement results are shown in Figure 5. As can be seen from Figure 4, the recombinant enzyme PgLip19028 was produced as the major protein in the Pichia pastoris culture supernatant (Lane 1: PgLip19028, Lane 3: vector control). After deglycosylation with Endo H, the lipase matched the calculated molecular weight of approximately 30.0 kDa (after subtraction of glycosylation and signal peptide) estimated by SDS-PAGE (Lane 2, Figure 4). In fact, PgLip19028 exhibited significantly higher lipase activity (85.41 ± 3.75 U / mg) against the model substrate pNPD compared with the vector control (2.55 ± 0.06 U / mg) (Figure 5). In the yeast expression system described above, approximately 60 mg / L of crude enzyme with a molecular weight of approximately 30 kDa was obtained as the major protein.

[0049] [Table 1]

[0050] Example 4: Determination of the optimum pH and temperature of PgLip19028 To determine the optimal pH conditions for PgLip19028, the lipase activity of the crude enzyme obtained in Example 3 was measured using tartrate buffers of pH 3.0 to 3.5, acetate buffers of pH 3.5 to 5.5, and acetate buffers of pH 6.0 to 6.5. The conditions other than the pH were the same as those described in Example 3. The lipase activity of the vector control was subtracted from the measured values. The results are shown in Figure 6. In the figure, the highest activity is set to 100%, and the relative values under each condition are shown.

[0051] To determine the optimal 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, and the lipase activity of the crude enzyme obtained in Example 3 was measured. Other than the temperature conditions, the conditions were the same as those described in Example 3. The lipase activity of the vector control was subtracted from the measured values. The results are shown in Figure 7. In the figure, the highest activity is set to 100%, and the relative values under each condition are shown.

[0052] The optimal reaction temperature and pH for PgLip19028 were determined to be 25°C and 4.5, respectively. Furthermore, relative activity was over 40% in the pH range of 3 to 5 and 10 to 35°C, indicating that these pH and temperature conditions are suitable for reactions using PgLip19028. The optimal pH of 4.5 is clearly lower than that of other conventional lipases. This optimal pH matches the external environment of P. gigantea, which is usually acidic due to secreted organic acids such as oxalic acid.

[0053] Example 5: Glyceride degradation assay of PgLip19028 For the glyceride degradation assay, 1 μg / mL of crude enzyme or vector control obtained in Example 3 was mixed with 10 mg / mL triolein or pine extract, a final concentration of 15% (v / v) tBE, 5% (v / v) DMSO, and 25 mM acetate buffer (pH 4.5) to a total volume of 1000 μL. Pine extract was obtained by soaking loblolly pine wood flour (1 mm mesh) obtained by debarking and pulverizing the woody parts in 70% (v / 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 sample was trimethylsilylated with BSTFA and subjected to GC-MS analysis. GC-MS analysis was performed as follows.

[0054] GC-MS analysis GC-MS analysis was performed on a Varian 3800 chromatograph (both J&W Scientific) coupled to an ion trap detector (Varian 4000) using a medium-length fused silica DB-5HT capillary column (12 m × 0.25 mm i.d., 0.1 μm film thickness), which allows for the simultaneous elution of different lipid classes. The temperature program started at 100 °C (1 min) and increased by 10 °C every min to 380 °C, where it was maintained for 5 min. The transfer line was maintained at 300 °C, and the injector was programmed from 120 °C (0.1 min) to 380 °C at 200 °C / min, using helium 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. The trimethylsilyl derivative was prepared using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in the presence of pyridine.

[0055] The results of GC-MS analysis are shown in Figure 8. Triglycerides were significantly reduced after the lipase reaction, while diolein, monoolein, and oleic acid were identified as the end products (Figure 8A). Furthermore, PgLip19028 liberated oleic acid, linoleic acid, linolenic acid, and palmitic acid from triglycerides and diglycerides in the conifer extract (Figure 8B). These results indicate that the lipase produces a wide variety of fatty acids with various carbon chain lengths and unsaturated bonds.

[0056] Example 5: Activity comparison test with commercially available lipase The lipase activity of PgLip19028 was compared with that of four commercially available lipases derived from filamentous fungi. The commercially available lipases used were 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). Lipase activity was measured as described in Example 3 using the crude enzyme obtained in Example 3, a vector control, or 1 μg / mL of a commercially available lipase. The results are shown in Figure 9. PgLip19028, in its crude enzyme form, exhibited higher activity than the commercially available lipases. [Sequence List Free Text]

[0057] 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

Claims

1. An enzyme composition comprising a lipase comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and tert-butyl methyl ether.

2. 2. The enzyme composition according to claim 1, wherein the optimum temperature of the lipase is in the range of 10°C to 35°C.

3. 3. The enzyme composition according to claim 1, wherein the optimum pH of the lipase is in the range of pH 3 to pH 5.

4. A method for increasing the expression level of a lipase derived from Prebiopsis gigantea, the lipase having an optimum temperature range of 10°C to 35°C, the method comprising culturing Prebiopsis gigantea in the presence of a coniferous tree extract, wherein the coniferous tree is pine, hemlock, fir, or spruce.

5. A method for increasing the expression level of a lipase derived from Prebiopsis gigantea, the lipase having an optimum pH in the range of pH 3 to pH 5, the method comprising culturing Prebiopsis gigantea in the presence of a coniferous tree extract, wherein the coniferous tree is pine, hemlock, fir, or spruce.

6. A method for increasing the expression level of a lipase derived from Prebiopsis gigantea, the lipase having an optimum temperature in the range of 10°C to 35°C and an optimum pH in the range of pH 3 to pH 5, the method comprising culturing Prebiopsis gigantea in the presence of a coniferous tree extract, wherein the coniferous tree is pine, hemlock, fir, or spruce.

7. A method described in any one of claims 4 to 6, wherein the lipase comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

8. The enzyme composition according to any one of claims 1 to 3, which is an enzyme composition for food, pharmaceuticals, cosmetics, industrial use, or a cleaning composition.

9. The enzyme composition according to any one of claims 1 to 3 for use in paper pulp production or biorefinery processes.

10. An enzyme composition described in any one of claims 1 to 3, 8 and 9, which contains 5 v / v% to 30 v / v% tert-butyl methyl ether in the composition.

11. A method for producing lipase derived from Prebiopsis gigantea, the method comprising increasing the expression level of the lipase by the method described in any one of claims 4 to 7.

Images