Lipase, composition for lipid degradation, lipid degradation method, and nucleic acid

Abstract

Provided are a novel lipase obtained from a methane fermentation system, a composition for lipid degradation containing the lipase, a lipid degradation method using the lipase, and a nucleic acid encoding the lipase.　A lipase composed of a peptide selected from (a), (b), and (c).　(a) A peptide having an amino acid sequence set forth in SEQ ID NO: 5; (b) a peptide having the amino acid sequence set forth in SEQ ID NO: 5 with one or multiple amino acids deleted, inserted, substituted, and / or added; and (c) a peptide having an amino acid sequence having a sequence identity of 90% or greater with the amino acid sequence set forth in SEQ ID NO: 5.

Description

Lipase, lipid-degrading composition, lipid degradation method and nucleic acid

[0001] The present invention relates to a lipase, a lipid-degrading composition, a lipid degradation method, and a nucleic acid. The present application claims priority based on Japanese Patent Application No. 2025-032055 filed with Japan on February 28, 2025, the content of which is incorporated herein by reference.

[0002] An anaerobic microbial ecosystem dominated by bacteria and archaea is established in anaerobic environments, and plays an indispensable role in material circulation on Earth. Since most anaerobic microorganisms cannot be isolated and cultured, the role that individual microorganisms play in material circulation within anaerobic microbial ecosystems remains largely unclear.

[0003] Lipase is an enzyme that plays an important role in the carbon cycle. Lipases have broad substrate specificity and are applied in various fields such as environmental remediation and organic synthesis (for example, Non-Patent Document 1). Most of the knowledge about lipases that has been clarified so far is obtained from enzymes isolated from aerobic microorganisms. Although it is expected that various lipase-secreting microorganisms exist even in anaerobic environments, very few anaerobic microorganisms have been confirmed to produce lipases so far.

[0004] Khan, F. I.; Lan, D.; Durrani, R.; Huan, W.; Zhao, Z.; Wang, Y. The Lid Domain in Lipases: Structural and Functional Determinant of Enzymatic Properties. Front. Bioeng. Biotechnol. 2017, 5.

[0005] Anaerobic microorganisms may have novel lipases exhibiting completely different reaction characteristics from those of aerobic microorganisms. Accordingly, an object of the present invention is to provide a novel lipase obtained from a methane fermentation system, a lipid-degrading composition containing the lipase, a lipid degradation method using the lipase, and a nucleic acid encoding the lipase.

[0006] The present invention includes the following embodiments: [1] A lipase comprising a peptide selected from (a), (b), and (c) below: (a) A peptide comprising the amino acid sequence described in SEQ ID NO: 5; (b) A peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence described in SEQ ID NO: 5; (c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 5. [2] The lipase according to claim 1, wherein the peptide of (b) or (c) is such that the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is serine or threonine, and the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is serine or threonine. [3] The lipase according to claim 1, which is a high-temperature resistant lipase.

[0007] A lipid-degrading composition containing the lipase described in any one of [4] [1] to [3].

[0008] [5] A lipid decomposition method comprising the step of contacting a lipid with a lipase described in any one of [1] to [3] to hydrolyze the ester bonds of the lipid. [6] The lipid decomposition method according to [5], wherein the step is performed at 60°C or higher.

[0009] [7] A nucleic acid encoding a lipase as described in any one of [1] to [3].

[0010] According to the present invention, it is possible to isolate a novel lipase obtained from a methane fermentation system, provide a lipid-degrading composition containing the lipase, a lipid-degrading method using the lipase, and a nucleic acid encoding the lipase.

[0011] Figure 1 is a schematic diagram showing the method used to identify a novel lipase from anaerobic sludge in Experimental Example 1. Figure 2A shows the results of developing recombinant lipase A and purified lipase A on SDS-PAGE and staining with CBB in Experimental Example 2. Figure 2B shows the results of detecting fluorescent substances produced by hydrolysis by recombinant lipase A and purified lipase A after SDS-PAGE in Experimental Example 2. Figure 3 shows the results of analyzing the enzymatic reaction of lipase A based on the Michaelis-Menten model in Experimental Example 3. Figure 4 shows the results of analyzing the optimal temperature for the enzymatic reaction by lipase A in Experimental Example 4. Figure 5 shows the results of analyzing the substrate characteristics of lipase A in Experimental Example 5. Figure 6 shows the results of analyzing the inhibition of lipase A's enzymatic activity by PMSF in Experimental Example 6. Figure 7 shows the results of analyzing the binding of lipase A to PMSF by simulation in Experimental Example 7. Figure 8A shows the results of analyzing the enzymatic reaction of the mutant lipase in Experimental Example 8 at 50°C for 3 hours. Figure 8B shows the results of analyzing the enzymatic reaction of the mutant lipase in Experimental Example 8 at 90°C for 1.5 hours.

[0012] (Lipase) In one embodiment, the present invention provides a lipase comprising a peptide selected from (a), (b), and (c) below: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 5; (b) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence described in SEQ ID NO: 5; (c) a peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 5.

[0013] The peptides selected from (a), (b), and (c) above have lipase activity. Lipases are a group of enzymes that hydrolyze ester bonds that make up lipids. Lipase activity refers to the activity of the lipase in hydrolyzing ester bonds.

[0014] In this specification, the peptide consisting of the amino acid sequence described in Sequence ID No. 5 may be referred to as "lipase A". When peptides selected from the group consisting of (a), (b), and (c) above are referred to collectively as "lipase A-like peptides".

[0015] The ratio of the lipase activity of the lipase A-like peptide to the lipase activity of lipase A (the ratio of lipase activities) may be 5% to 500%, 10% or more, 20% or more, 30% or more, 50% or more, or 70% or more. The ratio of the lipase activity of the lipase A-like peptide to the lipase activity of lipase A (the ratio of lipase activities) may be 300% or less, 200% or less, or 100% or less.

[0016] The ratio of lipase activity to lipase activity can be measured and calculated as follows: As the enzyme solution, purified lipase A-like peptide is added to a buffer (50 mM sodium phosphate, pH 7.0, 62.5 mM NaCl) (enzyme concentration 0.3 mg / mL). As the enzyme reaction buffer, 50 mM sodium phosphate buffer (pH 7.0) is prepared. As the substrate stock solution, 20 mM p-nitrophenylpalmitic acid (99% ethanol) is prepared. 10 μL of the enzyme solution and 10 μL of the substrate stock solution are added to 180 μL of the enzyme reaction buffer and reacted for 1.5 hours. Then, the absorbance at a wavelength of 410 nm is measured. Based on the absorbance, the lipase activity is calculated. The reaction temperature is not particularly limited, but may be 20°C, 50°C, 80°C, 90°C, or 97.5°C.

[0017] <Peptide (a)> The peptide (a) above may include an amino acid sequence other than the amino acid sequence described in SEQ ID NO: 5. The peptide (a) above may also be a peptide in which an amino acid sequence is added to the amino acid sequence described in SEQ ID NO: 5. If the peptide (a) above is a peptide in which an amino acid is added to the amino acid sequence described in SEQ ID NO: 5, the addition of the amino acid may be performed at either the N-terminus or the C-terminus of the amino acid sequence described in SEQ ID NO: 5, or at both the N-terminus and the C-terminus.

[0018] The peptide in (a) may be, for example, 5000 residues or less, 3000 residues or less, 2000 residues or less, 1000 residues or less, 500 residues or less, 400 residues or less, or 350 residues or less.

[0019] If the peptide in (a) is a peptide in which one or more amino acids are added to the amino acid sequence described in SEQ ID NO: 5, the number of amino acid sequences added to the amino acid sequence described in SEQ ID NO: 5 may be, for example, 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0020] <Peptide (b)> Peptide (b) includes an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence described in Sequence ID No. 5. Hereinafter, "one or more amino acids that are deleted, inserted, substituted and / or added" will also be referred to as "mutated amino acids".

[0021] The peptide in (b) may be a peptide consisting of an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence described in Sequence ID No. 5 (hereinafter also referred to as "mutant lipase A"), or it may be a peptide in which one or more amino acid residues are added to either the N-terminus and / or C-terminus of mutant lipase A. The mutation may be a deletion, insertion, substitution, or addition, or a combination thereof.

[0022] In the peptide described in (b) above, the number of mutated amino acids is not particularly limited. The term "multiple" in (b) is not particularly limited, but examples include 2 to 50, 2 to 40, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 3, or 2.

[0023] Mutant lipase A may have mutations in the amino acid sequence of lipase A. Examples of such mutations include those called conservative substitutions. Conservative substitutions are substitutions between amino acids that have functionally similar side chains. Classification of functionally similar amino acid side chains includes hydrophobic side chains (A, I, L, M, F, P, W, Y, V), hydrophilic side chains (R, D, N, C, E, Q, G, H, K, S, T), aliphatic side chains (G, A, V, L, I, P), hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), carboxylic acid and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), and fragrant group-containing side chains (H, F, Y, W). Furthermore, the amino acids listed in groups 1) to 8) below are known to be mutually conserved substitutions within each group. 1) Alanine (A), Glycine (G) 2) Aspartic acid (D), Glutamic acid (E) 3) Asparagine (N), Glutamine (Q) 4) Arginine (R), Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) 7) Serine (S), Threonine (T) 8) Cysteine ​​(C), Methionine (M)

[0024] The peptide in (b) is not particularly limited and may have, for example, 5000 residues or less, 3000 residues or less, 2000 residues or less, 1000 residues or less, 500 residues or less, 400 residues or less, or 350 residues or less.

[0025] If the peptide in (b) is a peptide to which one or more amino acids have been added in mutant lipase A, the number of amino acid sequences added to mutant lipase A may be 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0026] <Peptide (c)> Peptide (c) contains an amino acid sequence having 90% or more sequence identity with the peptide consisting of the amino acid sequence described in Sequence ID No. 5 (i.e., the amino acid sequence of lipase A) (hereinafter, this sequence is referred to as the "lipase A homologous sequence"). In the amino acid sequence of peptide (c), the lipase A homologous sequence may have a sequence identity of 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence described in Sequence ID No. 5.

[0027] Here, amino acid sequence identity is a value that indicates the percentage of the target amino acid sequence that matches a reference amino acid sequence. The reference amino acid sequence is the amino acid sequence described in Sequence ID No. 5. The target amino acid sequence is the lipase A homologous sequence in peptide (c). The sequence identity of the target amino acid sequence with respect to the reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the target amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (F1). Sequence identity (%) = Number of matching amino acids / Total number of amino acids in the target amino acid sequence × 100 … (F1) The value of amino acid sequence identity can be obtained by calculation based on alignment obtained by known homology search software such as BLASTP.

[0028] If the peptide in (c) above is a peptide to which one or more amino acids have been added to a lipase A homologous sequence, the addition of amino acids may be performed at either the N-terminus or the C-terminus of the lipase A homologous sequence, or at both the N-terminus and the C-terminus.

[0029] The peptide in (c) may be, for example, 5000 residues or less, 3000 residues or less, 2000 residues or less, 1000 residues or less, 500 residues or less, 400 residues or less, or 350 residues or less.

[0030] If the peptide in (c) is a peptide in which one or more amino acids are added to a lipase A homologous sequence, the number of amino acid sequences added to the lipase A homologous sequence may be 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0031] In peptide (b) or (c), the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5, and the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 are important for the enzymatic activity of the lipase A-like peptide.

[0032] In peptide (b) or (c), the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5, and the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5, can be identified, for example, as follows:

[0033] The amino acid in the peptide of (b) or (c) (lipase A-like peptide) that corresponds to the m-th amino acid from the N-terminus of the amino acid sequence of lipase A described in Sequence ID No. 5 can be identified by aligning the amino acid sequences of lipase A and the lipase A-like peptide. Here, gaps may be included in each amino acid sequence to maximize sequence identity. Known homology search software such as BLASTP may be used for alignment, or gaps may be included in each amino acid sequence to maximize sequence identity.

[0034] Preferably, the peptide of (b) or (c) has serine or threonine as the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5, and serine or threonine as the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5.

[0035] In the peptide described in (b) or (c), it is preferable that the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is serine. In the peptide described in (b) or (c), it is preferable that the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is threonine.

[0036] The peptide selected from the group consisting of (a), (b), and (c) above may be a fusion peptide formed by fusing another protein or peptide tag. For example, another protein or peptide tag may be bound to either or both of the N-terminus and C-terminus of a peptide containing the amino acid sequence of lipase A, mutant lipase A, or lipase A homologous sequence (for example, the peptides exemplified above). Examples of other proteins or peptides include signal peptides that regulate intracellular localization, extracellular secretion, etc.; marker enzyme proteins such as alkaline phosphatase or partial peptides thereof; fluorescent proteins such as GFP or partial peptides thereof; tag peptides such as His tags and FLAG tags.

[0037] The lipase A-like peptide is preferably a high-temperature resistant lipase. A "high-temperature resistant lipase" means a lipase that retains lipase activity even under high-temperature reaction conditions.

[0038] More specifically, a high-temperature resistant lipase may be a lipase A-like peptide as described below. The ratio of the lipase activity of the lipase A-like peptide to the lipase activity of lipase A (the ratio of lipase activities) may be 5% to 500%, 10% or more, 20% or more, 30% or more, 50% or more, or 70% or more. The ratio of the lipase activity of the lipase A-like peptide to the lipase activity of lipase A (the ratio of lipase activities) may be 300% or less, 200% or less, or 100% or less. The above ratio of lipase activities is measured at a high reaction temperature. The high reaction temperature may be 60°C, 80°C, 90°C, or 97.5°C.

[0039] In this embodiment, the peptide selected from the group consisting of (a), (b), and (c) above may be one type or two or more types.

[0040] The lipase according to the present embodiment described above is derived from a microorganism present in a methane fermentation system. This lipase is a novel protein that does not have motifs or domain sequences common to conventional lipases. This lipase may have reaction characteristics completely different from those of conventional lipases. Conventionally known lipases are denatured and inactivated at high temperatures (for example, 60°C). The lipase according to the present embodiment has high lipase activity at high temperatures (for example, 60°C).

[0041] Lipids, which are substrates of lipase, may solidify at normal temperature (for example, 10 to 25°C), and solid lipids adhering to fibers, containers, drains, and the like are difficult to wash. The lipase according to the present embodiment has high activity at high temperatures, and solid lipids become liquid at high temperatures. Therefore, by bringing this lipase into contact with lipids that have turned from solid to liquid at high temperatures, the lipids can be efficiently washed.

[0042] <Method for producing peptide> The lipase A-like peptide may be produced as a recombinant protein by applying various known genetic recombination techniques to a nucleic acid encoding the same. Chemical synthesis of a nucleic acid encoding a lipase A-like peptide and production of a recombinant protein can be performed within the scope of the ordinary working ability of those skilled in the art.

[0043] Alternatively, the lipase A-like peptide may be obtained by a cell-free protein synthesis system. The cell-free protein synthesis system is not particularly limited, and examples thereof include synthesis systems using cell extracts obtained from wheat germ, yeast, insect cells, mammalian cultured cells, rabbit reticulocytes, Escherichia coli, etc.; and synthesis systems reconstituted with factors necessary for translation.

[0044] Alternatively, the lipase A-like peptide may be produced by a method commonly used in the art, for example, an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). It may also be produced using a commercially available instrument generally called a peptide synthesizer.

[0045] It should be noted that the lipase A-like peptide may be produced in a form in which a tag peptide such as a Flag tag, polyhistidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, or MBP tag is added to its N-terminus and / or C-terminus, and may also be produced in the form of a fusion protein with another protein such as a fluorescent protein, glutathione transferase, or alkaline phosphatase. Further, the lipase A-like peptide may be labeled with an appropriate labeling agent such as a fluorescent substance, a luminescent substance, biotin, or the like.

[0046] (Composition for Lipolysis) In one embodiment, the present invention provides a composition for lipolysis comprising the lipase according to the above embodiment. Said composition for lipolysis is suitable for use in lipolysis.

[0047] The lipid to be degraded by the composition for lipolysis is not particularly limited as long as it is a lipid that can be degraded by lipase activity. The lipid to be degraded preferably has a structure in which a hydroxy group and a carboxy group of a fatty acid form an ester bond.

[0048] Regarding the lipid to be degraded, the number of carbon atoms of the fatty acid, which may be a component derived from the lipid, is not particularly limited as long as the effects of the present invention are exhibited, and may be 1 to 50, 2 to 30, 3 to 20, 3 to 8, or 3 to 6.

[0049] The composition for lipolysis may contain components other than the lipase according to the above embodiment (other components). The other components are not particularly limited as long as the effects of the present invention are achieved, and examples thereof include solvents, surfactants, acids, alkalis, salts, chelating agents, buffer solutions, foaming enhancers, foam inhibitors, fluorescent whitening agents, fragrances, bactericides, fungicides, enzyme inhibitors, enzyme stabilizers, enzyme activators, antioxidants, and solubilizers.

[0050] The lipase in the lipid-degrading composition may be one type or two or more types. The content of the lipase according to the above embodiment in the lipid-degrading composition is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 0.000001 to 1% by mass, 0.0001 to 1% by mass, 0.001 to 1% by mass, or 0.1 to 1% by mass, relative to the total amount (100% by mass) of the lipid-degrading composition.

[0051] The lipid-degrading composition according to this embodiment contains the lipase according to the embodiment, and therefore can efficiently degrade lipids. Since this lipase retains its lipase activity even at high temperatures, the lipid-degrading composition can efficiently degrade lipids at high temperatures.

[0052] (Lipid Decomposition Method) In one embodiment, the present invention includes the step of contacting the lipase according to the above embodiment with a lipid to hydrolyze the ester bonds of the lipid.

[0053] In this embodiment, the lipids to be brought into contact with the lipase are the same as the lipids that the lipid-degrading composition is degraded in the above embodiment.

[0054] The hydrolysis step may be carried out at temperatures of 10 to 200°C, or at temperatures of 20°C or higher, 40°C or higher, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, or 95°C or higher. The upper limit of the temperature for the hydrolysis step is not particularly limited, but for example, it may be 180°C or lower, 150°C or lower, 130°C or lower, 120°C or lower, 100°C or lower, or 97.5°C or lower. Animal lipids, which may be the lipids to be decomposed, have high melting points and are solid at room temperature, but by carrying out the hydrolysis step at a higher temperature, the lipids to be decomposed become liquid. Furthermore, it is possible to increase the reaction rate of hydrolysis catalyzed by lipase. This makes it possible to proceed with the hydrolysis of lipids more efficiently.

[0055] The lipid decomposition method according to this embodiment uses the lipase according to the embodiment, and therefore can decompose lipids with high efficiency even at high temperatures.

[0056] (Nucleic Acid) In one embodiment, the present invention provides a nucleic acid encoding the above-described peptide. The above-described peptide can be produced using the nucleic acid according to this embodiment. Examples of nucleic acids according to this embodiment include the base sequence described in Sequence ID No. 6.

[0057] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0058] (Materials and Methods) <Extraction of Genomic DNA and Proteins> Sludge was collected from an anaerobic sludge treatment tank at a large-scale mesothermal food waste treatment facility (Tokyo, Japan). Genomic DNA was extracted using Fast DNA® SPIN Kit for Soil (MP Biomedicals). DNA concentration was measured using QuantiFluor dsDNA System and Quantus Fluorometer (Promega). DNA quality was evaluated using 5200 Fragment Analyzer System and Agilent HS Genomic DNA 50kb Kit (Agilent Technologies).

[0059] Total protein was extracted from the collected sludge by bead beating. Equal volumes (450 μl each) of sludge and RIPA buffer (Cayman Chemical) were placed in Lysing Matrix E tubes (MP Biomedicals) and homogenized for 120 seconds at 3,000 rpm using a Micro Smash (Tomy). After repeating the homogenization process, the mixture was centrifuged at 12,000 g for 10 minutes. The resulting supernatant (500 μl) was mixed with 50 μl of ice-cold trichloroacetic acid and incubated for 30 minutes. After incubation, the mixture was again centrifuged at 14,000 g for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 1 ml of acetone at -20°C, followed by centrifugation at 14,000 g for 10 minutes, and the supernatant was removed. This acetone washing step was performed twice, and then the pellets were air-dried at room temperature for 5 minutes.

[0060] For 2D gel electrophoresis, the protein solution was prepared using the Ez Apply 2D Kit (ATTO) as follows: First, the pellet was resuspended in 450 μL of Solution 2 using a Handy Sonic model UR-21P (Tomy). Next, 40 μL of Solution 2-2 was added to the suspension and incubated at room temperature for 10 minutes. Unless otherwise noted, these steps were performed at -4°C. The final protein solution was stored at -80°C until the next step.

[0061] <Two-dimensional electrophoresis and zymography of proteins> 15 μl of protein solution and 10 μl of overlay solution from Ez Apply 2D Kit (ATTO) were loaded onto Agar GEL A-M38 (ATTO). Isoelectric focusing was performed for 210 minutes at 300 V using WSE-1510 DiscRun Ace (ATTO). The agar gel was then shaken with 2.5% trichloroacetic acid solution and washed with sterile water.

[0062] Prior to SDS-PAGE, the agar gel was equilibrated for 10 minutes in a buffer consisting of 50 mM Tris (pH 6.8), 2% SDS, and 0.01% bromophenol blue (BPB). The agar gel was then placed on an 8% SDS-polyacrylamide gel containing 100 μM paramethylumbelliferyl oleate dissolved in 25 μl of DMSO. Electrophoresis was then performed at 200 V for 60 minutes using an electrophoresis buffer consisting of 1% SDS, 1.92 M glycine, and 250 mM Tris.

[0063] After electrophoresis, the gel was stirred with 2.5% Triton X-100 solution for 60 minutes to regenerate the proteins. Next, the gel was washed with distilled water and incubated in 50 mM phosphate buffer (pH 7) at 35°C. Lipase activity in the gel was detected by hydrolysis of paramethylumbelliferyl oleate, generating visible fluorescent paramethylumbelliferone under UV light. Protein content was estimated by staining the gel with Silver Stain MS Kit (APRO Science Group).

[0064] <Analysis of Protein Amino Acid Sequences> Protein spots exhibiting lipase activity were excised from the gel and sent to Japan Proteomics Co., Ltd. (Tokyo, Japan) for analysis using mass spectrometry. In this analysis, the obtained peptides were analyzed by nanoLC-MS / MS after digestion within the gel. The generated mass spectrometry data were searched against metagenomics databases using the Mascot program (Matrix Science).

[0065] <Metagenomic Analysis> Sequencing was performed using PacBio Sequel IIe (Pacific Biosciences of California) and DNBSEC-G400 (MGI Tech) installed at the Bioengineering Laboratory (Kanagawa, Japan).

[0066] For PacBio sequencing, the library was prepared using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences). Next, the sequencing polymerase was bound to the SMRTbell library using Binding Kit 2.2 (Pacific Biosciences). After sequencing, adapter sequences were trimmed from the raw data using SMRT Link (version 10.1.0.119528). HiFi reads were generated by aligning the trimmed reads with Pancake with KSW2 at an average quality of 20 or higher, resulting in 157,984 reads (average: 6,957 bp).

[0067] For DNBSEC sequencing, libraries were constructed using the MGIEasy FS DNA Library Preparation Kit (MGI Tech). Library quality was verified using a fragment analyzer and the dsDNA 915 reagent kit (Agilent Technologies).

[0068] Circular DNA was constructed using the MGIEasy Circularization Kit, and DNA nanoballs (DNBs) were prepared using the DNBSEC-G400RS High-throughput Sequencing Kit (MGI Tech). 2×200bp paired-end sequencing was performed on the DNBSEC-G400 platform. Adapter trimming and quality assessment were performed using Trimmomatic v. 0.39 and Fastqc v. 0.11.9, respectively. Raw sequencing data was stored under BioSample accession number SAMD00662589.

[0069] Hybrid assembly was performed using metaSPADE v. 3.10.1, and HiFi reads were assembled separately using hifiasm_meta v. 0.3.1. They were then merged using quickmerge v. 0.3. Assembly scaffolding was performed using ntLink. Coverage was calculated using Bowtie2 v. 2.4.1 by mapping DNBseq reads to the resulting assemblies.

[0070] Binning was performed using SemiBin2 v. 1.5.1 with the built-in model "wastewater". Potential contamination within the metagenomic assembly genome (MAG) was detected and removed using MDMcleaner pipeline v. 0.8.7, and completeness was estimated by CheckM2. The coding sequence of each MAG was predicted by prokka. The resulting protein database was used for the Mascot search described above. These analyses were performed using default parameters unless otherwise specified.

[0071] The taxonomic assignment of MAGs was determined using GTDB-tk v. 2.3.2. The proportion of conserved proteins (POCPs) was calculated using the POCP-nf pipeline. Pathway analysis was performed using BlastKOALA with annotation files created by prokka. The habitability and distribution of target microorganisms were analyzed using the IMNGS platform and ProkAtlas tools, with queries set to the full-length 16S rRNA gene sequence and a sequence similarity threshold of 99%.

[0072] <Hexomorphic Expression and Purification of Lipase> The base sequence encoding lipase A, consisting of the amino acid sequence described in Sequence ID No. 5, was amplified by PCR using DNA obtained from sludge as a template. The primer sets used here were WT Fw and WT Re, as shown in Table 1. These primers were designed so that the BamHI and NotI sequences were positioned on both sides of the target gene. PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (Takara Bio). The PCR product was purified using Monarch DNA Gel Extraction Kit (New England Biolabs). The amplification products and the pET22(b)+ vector were digested in CutSmart Buffer (New England Biolabs) with BamHI HF and NotI HF restriction enzymes, respectively, and ligated using a DNA Ligation Kit (Takara Bio) to obtain the expression construct. The sequence of the expression construct was confirmed using a DS3000 compact CE sequencer (Hitachi, Tokyo, Japan).

[0073] Next, the expression construct was introduced into E. coli BL21 strain by electroporation. BL21 cells containing the expression construct were pre-incubated overnight at 37°C in 5 ml of LB liquid medium containing 100 μg / ml ampicillin. The culture was then transferred to 50 ml of fresh LB medium containing 100 μg / ml ampicillin and incubated until the OD600 reached 0.5. At this point, IPTG was added to a final concentration of 2 mM, and the culture was incubated for a further 2.5 hours. The cells were harvested by centrifugation at 4,000 g for 15 minutes, and the supernatant was discarded. The cell pellet was resuspended in 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 55 mM imidazole, and sonicated using a HANDY Sonic model UR-21P (Tomy). The target protein was purified using HisTrap® HP (Cytiva).

[0074] Expression constructs for mutant lipase A (T85A, S86A, S104A, T106A, S188A, T85A / T106A, T85A / S86A, and S104A / T106A) were prepared using the PrimeSTAR® Mutagenes Basal Kit.

[0075] For example, T85A refers to a mutant lipase in which the 85th threonine from the N-terminus of lipase A, which has the amino acid sequence described in Sequence ID No. 5, is replaced with alanine. T85A / T106A refers to a mutant lipase in which the 85th threonine from the N-terminus of lipase A is replaced with alanine, and the 106th threonine from the N-terminus of lipase A is replaced with alanine.

[0076] The primer sets used to introduce the mutations are shown in Table 1. Expression and purification of these mutants were performed according to the same protocol as described above. The expressed wild-type lipase A and mutant lipase A had their signal peptide sequences removed for heterologous expression.

[0077]

[0078] <In silico analysis> Lipase_reclassification was used to compare the amino acid sequence described in Sequence ID No. 4 with representative examples of known lipolytic enzyme families (35 lipase families and 11 true lipase subfamilies). Here, the true lipase subfamilies refer to a group of lipases that exhibit particularly high hydrolytic activity towards long-chain fatty acid esters. MAFFT v7.055b was used for multiple alignment. Functional prediction was performed using NCBI Conserved Domain Search (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) and InterPro (https: / / www.ebi.ac.uk / interpro / ) with default settings.

[0079] The signal peptide sequence and its cleavage site in the amino acid sequence described in Sequence ID No. 4 were predicted using the SignalP-6.0 server (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ).

[0080] Alphafold2 v. 2.3.0 was used to model the three-dimensional structure of lipase A from its amino acid sequence. Molecular docking simulations were performed using DynamicBind with the obtained model. The simulation results were visualized and analyzed using PyMOL.

[0081] <Optimal Temperature> Lipase activity was evaluated by mono-p-nitrophenyl palmitate assay at reaction temperatures ranging from 20°C to 97.5°C. The substrate was prepared as a 20 mM stock solution in 99% ethanol and stored in the dark at -20°C. Before use, 10 μl of the substrate stock solution was diluted in 180 μl of assay buffer (50 mM sodium phosphate buffer, pH 7.0) containing 1 mg / mL gum arabic and 0.4% Triton X-100.

[0082] 10 μL of purified protein solution (50 mM sodium phosphate, pH 7.0, 62.5 mM NaCl) was added to the mixture (final concentration 0.013 mg / mL). The reaction mixture was transferred to a 1.5 ml tube and incubated for 1.5 hours on a heating block set to the respective reaction temperature. The sample was then placed on an ice block and NaOH was added to a final concentration of 5 mM to terminate the reaction. The reaction mixture was transferred to a 96-well plate and absorbance was measured at λ = 410 nm using a DS PHARMA BIOMEDICAL (Bio-Tec) microplate reader. The assay buffer was preheated to the specified reaction temperature. All measurements were taken three times, and the blank (without enzyme) was subtracted to correct for autohydrolysis.

[0083] <Substrate Specificity> Enzyme specificity was evaluated by a p-nitrophenylacyl ester (pNP) assay using C4-C18 alkyl chains (C4, C6, C8, C10, C12, C14, C16, C18). Each substrate was prepared as a 20 mM stock solution in 99% ethanol and stored in the dark at -20°C. Before use, 10 μl of the nuclear substrate stock solution was diluted in 180 μl of assay buffer (50 mM sodium phosphate buffer, pH 7.0) containing 1 mg / mL gum arabic and 0.4% Triton X-100.

[0084] 10 μL of purified protein solution (50 mM sodium phosphate, pH 7.0, 62.5 mM NaCl) was added to the mixture (final concentration 0.016 mg / mL). The reaction mixture was transferred to a 96-well plate and incubated at 50°C for 1.5 hours. Absorbance was measured at λ = 410 nm using a DS PHARMA BIOMEDICAL (Bio-Tec) microplate reader with gentle shaking for 15 minutes at 50°C. All measurements were taken three times, and autohydrolysis was corrected by subtracting the blank (no enzyme).

[0085] <Michaelis-Menten Reaction Kinetics> The Michaelis-Menten reaction kinetics of lipase A, consisting of the amino acid sequence described in Sequence ID No. 5, were evaluated using p-nitrophenylpalmitic acid at different substrate concentrations of 0.25, 0.5, 1, 1.5, 2, and 2.5 mM. The corresponding hydrolytic activity values ​​were fitted to the Michaelis-Menten model using the R package "drc" to determine the reaction kinetic parameters, including Vmax and Km.

[0086] <Enzyme Catalytic Mechanism> Lipase activity was evaluated by mono-p-nitrophenyl palmitate assay. The substrate was prepared as a 20 mM stock solution in 99% ethanol and stored in the dark at -20°C. Before use, 10 μl of the substrate stock solution was diluted with 160 μl of assay buffer (50 mM sodium phosphate buffer, pH 7.0) containing 1 mg / mL gum arabic and 0.4% Triton X-100.

[0087] 30 μL of 0.3 mg / mL purified protein solution (50 mM sodium phosphate, pH 7.0, 62.5 mM NaCl) was added to the mixture. Furthermore, 10 mM PMSF was added to the experimental group, and 10 μL of assay buffer was added to the control group. The reaction mixture was transferred to a 96-well plate and incubated at 50°C for 1.5 hours. Absorbance was then measured at λ = 410 nm using a DS PHARMA BIOMEDICAL (Bio-Tec) microplate reader.

[0088] The activity of mutant lipase A, prepared in <Hexagram Expression and Purification of Lipase>, was evaluated by mono-p-nitrophenyl palmitate assay at reaction temperatures of 50°C or 90°C. Each substrate was prepared as a 20 mM stock solution in 99% ethanol and stored in the dark at -20°C. Before use, 10 μL of the substrate stock solution was diluted with 180 μL of assay buffer (50 mM sodium phosphate buffer, pH 7.0) containing 1 mg / mL gum arabic and 0.4% Triton X-100.

[0089] 10 μL of purified protein solution (50 mM sodium phosphate, pH 7.0, 62.5 mM NaCl) was added to the mixture. The reaction mixture was then transferred to a 96-well plate and reacted at 50°C for 3 hours or at 90°C for 1.5 hours. Absorbance was measured at λ = 410 nm using a DS PHARMA BIOMEDICAL (Bio-Tec) microplate reader with gentle shaking at 50°C for 15 minutes.

[0090] [Experimental Example 1] Genomic DNA and proteins were extracted from an anaerobic sludge treatment tank at a large-scale medium-temperature food waste treatment facility in Tokyo using the following procedure. Figure 1 is a schematic diagram illustrating the method used to identify a novel lipase from anaerobic sludge.

[0091] Based on the results of metagenomic analysis and protein amino acid sequence analysis, a Mascot search was performed using a protein database constructed from annotations using Prokka. As a result, a protein consisting of the amino acid sequence described in SEQ ID NO: 1 was identified. The nucleotide sequence described in SEQ ID NO: 2, which encodes the protein consisting of the amino acid sequence described in SEQ ID NO: 1, was identified.

[0092] Furthermore, the contig sequence having the nucleotide sequence described in SEQ ID NO: 2 was analyzed. As a result, the nucleotide sequence described in SEQ ID NO: 3, which is longer than the nucleotide sequence described in SEQ ID NO: 2, was identified. The nucleotide sequence described in SEQ ID NO: 3 encoded the amino acid sequence described in SEQ ID NO: 4.

[0093] Phylogenetic analysis of the metagenomic assembly genome (MAG) revealed that the species possessing the nucleotide sequence described in Sequence ID No. 3 is classified into the genus Candidatus Scatomorpha of the family Oscillospiraceae.

[0094] When the amino acid sequence described in SEQ ID NO: 4 was analyzed using the method described above in silico, it was found that the 327-residue amino acid sequence described in SEQ ID NO: 4 had a 20-residue signal peptide sequence at its N-terminus. The amino acid sequence described in SEQ ID NO: 4, excluding the signal peptide sequence, is the 307-residue amino acid sequence described in SEQ ID NO: 5. Hereinafter, the protein consisting of the amino acid sequence described in SEQ ID NO: 5 may be referred to as "lipase A". The nucleotide sequence described in SEQ ID NO: 6 is the nucleotide sequence that encodes the amino acid sequence described in SEQ ID NO: 5.

[0095] A homology search for lipase A, consisting of the amino acid sequence described in Sequence ID No. 5, using NCBI BLASTp revealed that it is homologous to ABC transporter substrate binding protein and does not belong to any known lipase family.

[0096] [Experimental Example 2] Lipase A was expressed in E. coli BL21 using the method described in <Hexagram Expression and Purification of Lipase>, and its enzyme activity was confirmed.

[0097] Recombinant lipase A expressed and purified lipase A were subjected to SDS-PAGE using a separation gel containing paramethylumbelliferyl oleate as the substrate. After electrophoresis, the gel was immersed in 2.5% Triton X-100 solution and gently shaken at room temperature for 1 hour to refold the protein. Subsequently, the gel was thoroughly washed with distilled water, immersed in 50 mM phosphate buffer, and gently shaken at 35°C for 4 hours to react the substrate with lipase A. The fluorescent substance produced by hydrolysis was then detected under UV light.

[0098] Figure 2A shows the results of developing the expressed recombinant lipase A and the purified lipase A using SDS-PAGE and staining with CBB. Figure 2B shows the results of detecting hydrolysates with UV.

[0099] [Experimental Example 3] The enzyme activity of recombinant lipase A obtained in Experimental Example 2 was measured using the method described above in the Michaelis-Menten reaction kinetics.

[0100] Figure 3 shows the relationship between substrate concentration and enzyme reaction rate. The enzymatic reaction by lipase A was found to follow the Michaelis-Menten model. Vmax was 0.92 μmol / min, Km was 9.15 mM, and kcat was 2614 min. －１ That was the case.

[0101] [Experimental Example 4] Using the method described above, the optimal temperature for the lipid ester bond decomposition reaction of recombinant lipase A obtained in Experimental Example 2 was analyzed.

[0102] Figure 4 shows the relationship between reaction temperature and enzyme activity. Lipase A is extremely thermogenic, and it was found to exhibit the highest activity between 90 and 97.5°C within the range of 20 to 100°C. Furthermore, it was found that lipase A is active even at a low temperature of 20°C.

[0103] [Experimental Example 5] <Substrate Specificity> The substrate specificity of recombinant lipase A obtained in Experimental Example 2 was analyzed using the method described above.

[0104] Figure 5 shows the enzyme activity when using p-nitrophenylacyl esters with alkyl chain lengths of C4 to C18 as substrates. It has been revealed that lipase A catalyzes the decomposition reaction of lipid ester bonds for a wide range of lipids, from short-chain to long-chain fatty acid esters.

[0105] [Experimental Example 6] <Reaction Mechanism Analysis> The reaction mechanism of recombinant lipase A obtained in Experimental Example 2 was analyzed using PMSF by the method described above.

[0106] The analysis results are shown in Figure 6. It was revealed that the enzymatic activity of lipase A is inhibited by PMSF. This suggests that the catalytic mechanism of lipase A is similar to that of known lipases.

[0107] [Experimental Example 7] The structure of lipase A was analyzed using the method described above in <in silico analysis>.

[0108] Analysis using InterPro predicted that five sites—A84-T85-S86, S104-I105-H106, N161, D210, and E235—would bind to the ligand. The numbers in the five sites indicate the number of amino acid residues from the N-terminus in the amino acid sequence of Sequence ID No. 5, and the letters represent the amino acid residues. For example, A84 refers to the 84th alanine residue from the N-terminus in the amino acid sequence described in Sequence ID No. 5.

[0109] Alphafold2 v. 2.3.0 was used to model the three-dimensional structure of lipase A. Using the obtained model, molecular docking simulations with PMSF were performed using DynamicBind, and the simulation results were visualized using PyMOL. The results are shown in Figure 7. It was predicted that PMSF would bind to the oxygen atoms T85 and T106 of lipase A. This result suggests that T85 and T106 may be the active sites of lipase A.

[0110] [Experimental Example 8] In <Hexogenous Expression and Purification of Lipase> and <Mutant Lipase>, the enzymatic activity of wild-type lipase A and mutant lipase A was analyzed using the methods described above.

[0111] Wild-type lipase A is a lipase consisting of the amino acid sequence described in Sequence ID No. 5. There are eight types of mutant lipase A: T85A, S86A, S104A, T106A, S188A, T85A / T106A, T85A / S86A, and S104A / T106A. For example, T85A refers to mutant lipase A in which the 85th threonine from the N-terminus of lipase A, consisting of the amino acid sequence described in Sequence ID No. 5, is replaced with alanine. T85A / T106A refers to mutant lipase A in which the 85th serine from the N-terminus of lipase A is replaced with alanine, and the 106th threonine from the N-terminus of lipase A is replaced with alanine.

[0112] The absorbance was corrected to the value per 1 mg of mutant lipase A, and the activity relative to wild-type lipase A was calculated. The results are shown in Figures 8A and 8B. Figure 8A shows the results of the enzyme reaction at 50°C for 3 hours, and Figure 8B shows the results of the enzyme reaction at 90°C for 1.5 hours. It was revealed that S104 and T106 of lipase A are important for enzyme activity.

[0113] The peptide (lipase) of the present invention can be used to decompose waste oil contained in food wastewater, etc.

Claims

1. A lipase comprising a peptide selected from (a), (b), and (c) below: (a) a peptide containing the amino acid sequence described in SEQ ID NO: 5; (b) a peptide containing an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and / or added in the amino acid sequence described in SEQ ID NO: 5; (c) a peptide containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO:

5.

2. The lipase according to claim 1, wherein the peptide of (b) or (c) is such that the amino acid corresponding to the 104th serine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is serine or threonine, and the amino acid corresponding to the 106th threonine from the N-terminus of the amino acid sequence described in SEQ ID NO: 5 is serine or threonine.

3. The lipase according to claim 1, which is a high-temperature resistant lipase.

4. A lipid-degrading composition containing the lipase described in any one of claims 1 to 3.

5. A method for lipid decomposition comprising the step of contacting a lipid with a lipase according to any one of claims 1 to 3 to hydrolyze the ester bonds of the lipid.

6. The lipid decomposition method according to claim 5, wherein the above step is performed at a temperature of 60°C or higher.

7. A nucleic acid encoding a lipase according to any one of claims 1 to 3.

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