Peptide or salt thereof, and nucleic acid

Abstract

To provide a technique for separating a novel lipase from a methane fermentation system and further identifying the microbial species responsible for the production of the lipase.SOLUTION: The present invention adopts a peptide selected from the group consisting of (a), (b), and (c) below, or a salt thereof: (a) a peptide comprising a specific amino acid sequence; (b) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the specific amino acid sequence; and (c) a peptide comprising an amino acid sequence having at least 90% sequence identity to the specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a peptide or a salt thereof, and a nucleic acid. [Background technology]

[0002] Anaerobic microbial ecosystems, mainly consisting of bacteria and archaea, exist in anaerobic environments and play an essential role in material circulation on Earth. However, since most anaerobic microorganisms cannot be isolated and cultured, the role that individual microorganisms play in material circulation in anaerobic microbial ecosystems remains largely unknown.

[0003] Lipase is an enzyme that plays an important role in the carbon cycle. Lipase has a wide range of substrate specificity and is used in various fields such as environmental purification and organic synthesis (e.g., Non-Patent Document 1). Most of the knowledge about lipase that has been revealed so far has been obtained from enzymes isolated from aerobic microorganisms. Although it is expected that various lipase-secreting microorganisms exist in anaerobic environments, only a few anaerobic microorganisms have been confirmed to produce lipase so far. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Khan, FI; 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. Summary of the Invention [Problem to be solved by the invention]

[0005] Anaerobic microorganisms may have novel lipases that exhibit reaction characteristics completely different from those of aerobic microorganisms. Therefore, the present invention aims to provide a technique for isolating novel lipases from methane fermentation systems and identifying the microbial species involved in the production of the lipases. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A peptide or a salt thereof selected from the group consisting of the following (a), (b), and (c): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO:1 in which one or more amino acids have been deleted, inserted, substituted and / or added; (c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:1. [2] A nucleic acid encoding the peptide described in [1]. Effect of the Invention

[0007] According to the present invention, a technique can be provided for isolating a novel lipase from a methane fermentation system and further identifying the microbial species involved in the production of the lipase. [Brief description of the drawings]

[0008] [Figure 1A] FIG. 2 shows the results of SDS-PAGE analysis of a crude protein extract from Escherichia coli expressing recombinant lipase X. [Figure 1B] FIG. 1 shows the results of zymography of crude protein extracts from E. coli expressing recombinant lipase X. [Figure 1C] FIG. 1C shows the results of detection of recombinant lipase X by Western blotting. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [peptide] In one embodiment, the present invention provides a peptide or a salt thereof selected from the group consisting of: (a), (b) and (c) below: (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a peptide comprising the amino acid sequence set forth in SEQ ID NO:1 in which one or more amino acids have been deleted, inserted, substituted and / or added; (c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:1.

[0010] As described later in the Examples, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is a peptide that has been detected in a microbial flora living in a methane fermentation environment.

[0011] The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 has lipase activity. Lipase is a group of enzymes that hydrolyze ester bonds constituting lipids. Lipase activity means the activity of the lipase to hydrolyze ester bonds. In this specification, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 may be referred to as "lipase X."

[0012] The peptide selected from the group consisting of (a), (b) and (c) preferably has lipase activity. The presence of lipase activity of a peptide can be confirmed, for example, by using 4-methylumbelliferyl oleate, which is a fluorescent substrate.

[0013] The lipase activity of a peptide selected from the group consisting of (a), (b), and (c) may be 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, or 70% or more of the lipase activity of a peptide consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0014] <Peptide (a)> The above peptide (a) may contain an amino acid sequence other than the amino acid sequence set forth in SEQ ID NO: 1. The above peptide (a) may be a peptide in which an amino acid sequence is added to the amino acid sequence set forth in SEQ ID NO: 1. When the peptide (a) above is a peptide in which amino acids have been added to the amino acid sequence set forth in SEQ ID NO: 1, the amino acids may be added to either the N-terminus or the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1, or to both the N-terminus and the C-terminus.

[0015] The peptide (a) may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, or 200 residues or less.

[0016] When the peptide (a) is a peptide in which one or more amino acids are added to the amino acid sequence set forth in SEQ ID NO: 1, the number of amino acids added to the amino acid sequence set forth in SEQ ID NO: 1 may be, for example, 1 to 5,000 residues, 1 to 3,000 residues, 1 to 2,000 residues, 1 to 1,000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0017] <Peptide (b)> The peptide (b) includes an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and / or added in the amino acid sequence set forth in SEQ ID NO: 1. Hereinafter, "one or more amino acids that have been deleted, inserted, substituted and / or added" will also be referred to as "mutated amino acids".

[0018] The peptide (b) may be a peptide consisting of an amino acid sequence in which one or more amino acids have been mutated in the amino acid sequence set forth in SEQ ID NO: 1 (hereinafter also referred to as "mutant lipase X"), or may be a peptide in which one or more amino acid residues have been added to either or both of the N-terminus and C-terminus of mutant lipase X. The mutation may be any of deletion, insertion, substitution, and addition, or a combination of these.

[0019] In the peptide (b) above, the number of mutated amino acids is not particularly limited. The term "multiple" in (b) is not particularly limited, and examples thereof include 2 to 150, 2 to 120, 2 to 100, 2 to 80, 2 to 60, 2 to 40, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 3, or 2, etc.

[0020] The mutant lipase X may have a mutation in the amino acid sequence of lipase X that does not affect the lipase activity. Such mutations include, for example, substitutions called conservative substitutions. Conservative substitutions are substitutions between amino acids having functionally similar side chains. Classifications of functionally similar amino acid side chains include 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), aromatic side chains (H, F, Y, W), etc. Moreover, the amino acids described in the following groups 1) to 8) are known to be conservatively substituted for each other 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)

[0021] When the above peptide (b) is a peptide in which amino acids have been added to mutant lipase X, the amino acids may be added to either the N-terminus or the C-terminus of the amino acid sequence of mutant lipase X, or may be added to both the N-terminus and the C-terminus.

[0022] The peptide (b) is not particularly limited, and may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, or 200 residues or less.

[0023] When the peptide (b) is a peptide in which one or more amino acids have been added to a mutant lipase X, the number of amino acid residues in the amino acid sequence added to the mutant lipase X may be 1 to 5,000 residues, 1 to 3,000 residues, 1 to 2,000 residues, 1 to 1,000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0024] <Peptide (c)> The peptide (c) contains an amino acid sequence having 90% or more sequence identity with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 (i.e., the amino acid sequence of lipase X) (hereinafter, this sequence is referred to as a "lipase X homologous sequence"). In the amino acid sequence of the peptide (c), the lipase X homologous sequence has an amino acid sequence identity of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 98% or more, to the amino acid sequence set forth in SEQ ID NO:1.

[0025] Here, the sequence identity of an amino acid sequence is a value indicating the percentage of identity between a target amino acid sequence (target amino acid sequence) and a reference amino acid sequence (reference amino acid sequence). The reference amino acid sequence is the amino acid sequence set forth in SEQ ID NO: 1. The target amino acid sequence is a lipase X-homologous sequence in the peptide (c). The sequence identity of a subject amino acid sequence to a reference amino acid sequence can be determined, for example, as follows. First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, each amino acid sequence may contain gaps so as to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject 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)

[0026] When the peptide (c) above is a peptide in which one or more amino acids have been added to a lipase X homologous sequence, the amino acids may be added to either the N-terminus or the C-terminus of the lipase X homologous sequence, or to both the N-terminus and the C-terminus.

[0027] The peptide (c) may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, or 200 residues or less.

[0028] When the peptide (c) is a peptide in which one or more amino acids are added to a lipase X homologous sequence, the number of amino acids added to the lipase X homologous sequence may be 1 to 5,000 residues, 1 to 3,000 residues, 1 to 2,000 residues, 1 to 1,000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 200 residues, 1 to 100 residues, or 1 to 50 residues.

[0029] The peptide selected from the group consisting of (a), (b) and (c) may be a fusion peptide in which another protein or peptide tag is fused. For example, another protein or peptide tag may be bound to either or both of the N-terminus and C-terminus of a peptide (e.g., the peptides exemplified above) comprising the amino acid sequence of lipase X, mutant lipase X or a lipase X homologous sequence. Examples of the other protein or peptide include a marker enzyme protein such as alkaline phosphatase or a partial peptide thereof; a fluorescent protein such as GFP or a partial peptide thereof; and tag peptides such as His tag and FLAG tag.

[0030] The salt of the peptide according to this embodiment is not particularly limited as long as it is a physiologically acceptable salt, and may be an acid addition salt or a basic salt. Examples of the acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid; and salts with organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid. Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide; and salts with organic bases such as caffeine, piperidine, trimethylamine, and pyridine.

[0031] According to this embodiment, the peptide or salt thereof selected from the group consisting of (a), (b), and (c) above may be of one type or of two or more types.

[0032] Hereinafter, when the peptides according to this embodiment are collectively referred to, they may be referred to as "lipase X-like peptides."

[0033] The peptide according to the present embodiment described above is a novel protein having lipase activity. The peptide according to the present embodiment is derived from a microorganism present in a methane fermentation system. Since the peptide according to the present embodiment does not have a motif or domain sequence common to conventional lipases, it may have reaction characteristics completely different from those of conventional lipases.

[0034] <Method of producing peptides> The lipase X-like peptide may be produced as a recombinant protein by applying various known gene recombination techniques to the nucleic acid encoding the lipase X-like peptide. Chemical synthesis of the nucleic acid encoding the lipase X-like peptide and production of the recombinant protein are within the scope of ordinary skill in the art.

[0035] Alternatively, the lipase X-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, cultured mammalian cells, rabbit reticulocytes, Escherichia coli, etc.; synthesis systems in which factors necessary for translation are reconstituted, etc.

[0036] Alternatively, the lipase X-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), etc. Also, the lipase X-like peptide may be produced using a commercially available device generally called a peptide synthesizer.

[0037] The lipase X-like peptide may be produced in a form in which a tag peptide such as Flag tag, polyhistidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, etc. is added to its N-terminus and / or C-terminus, or in the form of a fusion protein with other proteins such as fluorescent protein, glutathione transferase, alkaline phosphatase, etc. The lipase X-like peptide may be labeled with a fluorescent substance, a luminescent substance, biotin, or other suitable labeling agent.

[0038] [Nucleic acid] In one embodiment, the present invention provides a nucleic acid encoding the above-mentioned peptide. The nucleic acid according to this embodiment can be used to produce the above-mentioned peptide. An example of the nucleic acid according to this embodiment is the DNA sequence shown in SEQ ID NO:2. EXAMPLES

[0039] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0040] [Experimental Example 1] In order to search for lipases contained in methanogens, proteomic analysis was performed on methane fermentation sludge using the following procedure.

[0041] Protein extraction from methane fermentation sludge Methane fermentation sludge was collected from a food waste treatment plant in Tokyo. 450 μl each of the methane fermentation sludge and RIPA Buffer (Nacalai Tesque) were added to a Lysing Matrix E tube (MP Biomedicals). Two sets of bead crushing at 3000 rpm for 120 seconds were then performed using a MicroSmash (Tomy). After centrifugation at 12,000 g for 4 minutes, the supernatant was collected as a crude protein extract. Next, the crude protein extract was subjected to TCA precipitation and acetone washing, and 200 μl of Solution 2 included in the EzApply2DKit (ATTO) was added to the protein pellet. The pellet was suspended by sonication (HANDY Sonic model UR-21P), 40 μl of Solution 2-2 was added, and the mixture was incubated at room temperature for 10 minutes. All of the above operations were performed on ice unless otherwise stated.

[0042] Two-dimensional electrophoresis and zymography 15 μl of the extracted protein solution was developed by two-dimensional electrophoresis. The first-dimension isoelectric focusing and subsequent SDS treatment were performed using agarGEL mini size 75 mm, pH 3-8 (ATTO) according to the manual for the 2D mini-slab system. The second-dimension SDS-PAGE was performed by mixing 4-MethylumbelliferylOleate as a zymography substrate at a final concentration of 100 μM with the separation gel in advance. After electrophoresis, the gel was immersed in 2.5% Tritonx-100 solution and gently shaken at room temperature for 1 hour to refold the protein. It was then thoroughly washed with millQ water, immersed in 50 mM phosphate buffer, and gently shaken at 35°C for 4 hours. Lipase activity was observed with a UV transilluminator.

[0043] mass spectrometry The active spots obtained by zymography were excised with a scalpel and treated with trypsin, and the partial amino acid sequences of the peptides contained in the active spots were then identified by nanoLC-MS / MS.

[0044] A Mascot search was performed on the obtained amino acid partial sequence with reference to the NCBInr database, but no protein was identified, suggesting that the obtained peptide sequence is a novel sequence.

[0045] [Experimental Example 2] A novel protein having lipase activity was identified by the following procedure.

[0046] Metagenomic sequencing DNA was extracted from the above-mentioned methane fermentation sludge using FastDNA SPINKit for Soil (MP Biomedicals). 250 μl of sludge was used, and the extraction procedure was performed according to the manual. Extracted DNA was purified using ProNex Beads (Promega). Quality check was performed using 5200 Fragment Analyzer System and Agilent HSGenomic DNA50kb Kit (Agilent Technologies). Library preparation was performed using SMRT bell Express Template Prep Kit 2.0 (PacBio) according to the manual Procedure & Checklist-Preparing HiFi Libraries from Low DNA Input Using SMRT bell Express Template PrepKit2.0. A polymerase complex was formed from the library created using Binding kit22 (PacBio), and sequencing was performed using SequelIIe (PacBio).

[0047] Data analysis and database construction Subreads were generated by removing adapter sequences from the sequences obtained by sequencing using SMRTLink (v.10.1.0.119528). Subreads were aligned to generate consensus sequences (CCS), and CCS reads with an average quality score of less than 20 per read were excluded to define HiFi reads. The average read length of the obtained sequences was 6,957 bp, and a total of 1.1 Gb of sequence was obtained (Q>=30: 98.4%). As a result of assembly, 2076 contigs with an average length of 40,590 bp were obtained. The assembly was performed using hifiasm_meta. Annotation was performed using Prokka, and the resulting protein sequences were used as a database for Mascot searches.

[0048] A Mascot search was performed with reference to a protein database constructed from annotations, and a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 was identified (hereinafter, this protein may be referred to as lipase X). In addition, the base sequence set forth in SEQ ID NO: 2, which codes for lipase X, was identified.

[0049] [Experimental Example 3] The protein (lipase X) consisting of the amino acid sequence set forth in SEQ ID NO: 1, identified in Experimental Example 2, was expressed in Escherichia coli, and the lipase activity of the resulting recombinant protein was examined.

[0050] Primers were designed to amplify the region excluding the start codon and stop codon of the gene encoding lipase X. Next, PCR was performed using DNA extracted from methane fermentation sludge as a template to obtain the target PCR amplified fragment. This was introduced into a protein expression vector (pET-22b(+) plasmid). Sanger sequencing was used to confirm that there were no mutations in the base sequence of the lipase gene introduced into the expression vector, and the lipase expression vector was introduced into Escherichia coli BL21 using the electroporation method. Due to the structure of the expression vector, the resulting recombinant protein has a signal peptide attached to the N-terminus of the amino acid sequence described in SEQ ID NO:1 and a 6xHis tag sequence attached to the C-terminus. The transformed E. coli was cultured at 100 μg·ml -1 After overnight preculture in LB liquid medium containing ampicillin, IPTG induction was performed. The culture was then centrifuged at 4000g for 15 minutes at 4°C, and the cell pellet was suspended in SDS-PAGE sample buffer and sonicated. This was used as a protein extract, and after development by SDS-PAGE, the expression of the recombinant protein was confirmed by CBB staining and Western blotting. In Western blotting, anti-His6tag rabbit antibody was used as the primary antibody, and HRP-conjugated anti-rabbit donkey antibody was used as the secondary antibody. Furthermore, the crude protein extract was subjected to zymography using 4-methylumbelliferyloleate as a substrate to verify the lipase activity of the recombinant protein. The results are shown in Figures 1A, 1B, and 1C.

[0051] Figure 1A shows the results of SDS-PAGE of E. coli crude protein extracts. Figure 1B shows the results of zymography. Figure 1C shows the results of Western blotting. The results of zymography revealed that the recombinant protein had lipase activity.

[0052] [Experimental Example 4] The lineage of the protein (lipase X) consisting of the amino acid sequence set forth in SEQ ID NO: 1, identified in Experimental Example 2, and the microbial species having the base sequence set forth in SEQ ID NO: 2 encoding lipase X, was analyzed.

[0053] Metabat2 was used to bin the metagenomic sequences obtained in Experimental Example 2. For quality confirmation and phylogenetic classification of metagenomic assembled genomes (MAGs), MDMcleaner was used, which is considered more suitable than CheckM for evaluating the genome quality of difficult-to-culture microorganisms.

[0054] Lipase X was found to be encoded by MAGmetabat2.19. The quality of this MAG was evaluated by MDMcleaner. No sequences were found to be contaminating. This MAG consisted of 17 contigs (shortest contig length: 15,032 bp, longest contig length: 91,220 bp, N50: 53,784 bp) with a total base length of 758,414 bp. The completeness of MAG was 75%. MAG contained two 16S rRNA genes, and the number of genes predicted to code for proteins was 859. Phylogenetic analysis of MAG revealed that it was classified into the genus Candidatus Scatomorpha in the family Oscillospiraceae.

[0055] Furthermore, a domain / motif search for lipase X using InterPro revealed no domains or motifs in the amino acid sequence of lipase X that were common to known lipases.

[0056] Lipase X does not share any motifs or domain sequences in common with conventional lipases, suggesting that it may have reaction properties completely different from those of conventional lipases. [Industrial Applicability]

[0057] The peptide (lipase) of the present invention can be used to decompose waste oil contained in food wastewater and the like.

Claims

1. A peptide or a salt thereof selected from the group consisting of the following (a), (b) and (c): (a) a peptide comprising the amino acid sequence set forth in SEQ ID NO:1; (b) a peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and / or added in the amino acid sequence set forth in SEQ ID NO:1; (c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

1.

2. A nucleic acid encoding the peptide of claim 1.

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