Alginate lyase

Abstract

Provided is a novel alginate lyase. The present technology provides an alginate lyase containing a polypeptide described by any one of (1) to (3). (1) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2. (2) A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2 where at least one amino acid residue is substituted, added, inserted or deleted, the polypeptide having an alginate lyase activity. (3) With respect to the amino acid sequence represented by SEQ ID NO: 1 or 2, a polypeptide having at least 70% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having an alginate lyase activity.

Description

Alginate lyase

[0001] The present technology relates to an alginate lyase. More specifically, the present technology relates to a novel alginate lyase, DNA encoding the alginate lyase, an expression cassette or recombinant vector containing the DNA, a transformant, a method for producing alginate lyase, a method for producing alginate oligosaccharides, alginate oligosaccharides, and an enzyme preparation for producing alginate oligosaccharides.

[0002] Alginate lyase is an enzyme belonging to the polysaccharide lyase family that catalyzes the elimination reaction of cleaving the glycosidic bond of the substrate. The target substrate of alginate lyase is alginic acid, which is used as a thickener and gelling agent, and it is expected to be used in many industries, such as adjusting the texture of food and producing functional alginate oligosaccharides. Alginate lyase is known to exist in microorganisms and marine mollusks.

[0003] In recent years, alginate lyases with various characteristics have been developed. For example, Patent Document 1 discloses a novel alginate-degrading enzyme derived from Agarivorans sp. JAM-A1m strain, which has an optimum reaction pH in the alkaline range. Patent Document 2 discloses an alginate lyase derived from Nitratiruptor sp. SB155-2 strain, which exhibits high enzymatic activity and stability under a wide range of conditions. Patent Document 3 discloses a technique for easily producing alginate lyase on an industrial scale from a microorganism of the genus Flavobacterium.

[0004] Furthermore, Non-Patent Document 1 discloses a thermostable alginate lyase derived from Paenibacillus ehimensis, Non-Patent Document 2 discloses a thermostable alginate lyase derived from Rhodothermus marinus, and Non-Patent Document 3 discloses an alginate lyase derived from Paenibacillus sp. str. FPU-7 strain.

[0005] JP 2009-195222 A International Publication No. 2011 / 118582 JP 04-141090 A

[0006] Enzyme and Microbial Technology 166 (2023) 110221Frontiers in Plant Science Volume 13, 2022, 10.3389 / fpls.2022.981602SCIENTIFIC REPORTS (2019) 9:14870

[0007] As mentioned above, alginate lyase is expected to be widely used in various fields, and the discovery of a wide variety of alginate lyases will lead to development in industry.

[0008] Therefore, the main object of the present technology is to provide a novel alginate lyase.

[0009] The present technology first provides an alginate lyase comprising a polypeptide shown in any one of (1) to (3) below: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, (2) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2 in which one or more amino acid residues have been substituted, added, inserted, or deleted, and which has alginate lyase activity, and (3) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 2 that has 70% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 and which has alginate lyase activity. The alginate lyase according to the present technology may be derived from Paenibacillus elgii.

[0010] The present technology next provides DNA comprising a polynucleotide shown in any one of (1) to (3) below: (1) a polynucleotide encoding the alginate lyase according to claim 1, (2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 6, (3) a polynucleotide comprising a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 5 or 6 and encoding a polypeptide having alginate lyase activity. The present technology also provides an expression cassette or recombinant vector comprising the DNA of the present technology. The present technology also provides a transformant obtained by transforming a host with the expression cassette or recombinant vector of the present technology.

[0011] The present technology also provides a method for producing alginate lyase, the method comprising: a culture step of culturing Paenibacillus elgii or a transformant of the present technology; and a recovery step of recovering the alginate lyase of the present technology from the culture obtained in the culture step.

[0012] The present technology further provides a method for producing alginate oligosaccharides, which includes a step of reacting alginic acid and / or a salt thereof with the alginate lyase of the present technology. The present technology also provides alginate oligosaccharides obtained using the alginate lyase of the present technology. The present technology also provides an enzyme preparation for producing alginate oligosaccharides, which contains the alginate lyase of the present technology.

[0013] The technical terms used in this technology are explained below. <Amino acid notation> The 20 types of amino acid residues in an amino acid sequence may be expressed by single-letter abbreviations. That is, glycine (Gly) is sometimes represented as G, alanine (Ala) as A, valine (Val) as V, leucine (Leu) as L, isoleucine (Ile) as I, phenylalanine (Phe) as F, tyrosine (Tyr) as Y, tryptophan (Trp) as W, serine (Ser) as S, threonine (Thr) as T, cysteine ​​(Cys) as C, methionine (Met) as M, aspartic acid (Asp) as D, glutamic acid (Glu) as E, asparagine (Asn) as N, glutamine (Gln) as Q, lysine (Lys) as K, arginine (Arg) as R, histidine (His) as H, and proline (Pro) as P.

[0014] <Amino Acid Sequence> In the present technology, the amino acid sequence displayed has the N-terminus at the left end and the C-terminus at the right end.

[0015] <Types of Amino Acids> In the present technology, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0016] <Sequence Modifications (Substitution, Addition, Insertion, Deletion, Inversion, etc.)> In the present technology, modifications of amino acid sequences and / or nucleotide sequences include not only artificial modifications (substitution, addition, insertion, deletion, inversion, etc.), but also naturally occurring modifications (substitution, addition, insertion, deletion, inversion, etc.), i.e., cases where the sequences were originally different.

[0017] <Isolation / Purification> In this technology, the term "isolated" is used interchangeably with "purified." The term "isolated" is used to distinguish it from the natural state, i.e., the state that exists in nature. The artificial procedure of "isolating" results in an "isolated state" that is different from the natural state. An isolated product is clearly and decisively different from the natural product itself.

[0018] <Equivalent Sequence> In the present technology, an "equivalent sequence" refers to an amino acid sequence or a nucleotide sequence that differs in part from a reference amino acid sequence (in the present technology, SEQ ID NO: 1 or SEQ ID NO: 2) or a reference nucleotide sequence (in the present technology, SEQ ID NO: 5 or SEQ ID NO: 6), but the difference does not substantially affect the function of the protein (in the present technology, alginate lyase activity). Therefore, in the present technology, an enzyme having a polypeptide chain consisting of an equivalent amino acid sequence, or an enzyme encoded by a polynucleotide consisting of an equivalent nucleotide sequence, exhibits alginate lyase activity.

[0019] 1 is a graph showing the pH stability of alginate lyase obtained from a strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and a strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. 2 is a graph showing the optimum pH of alginate lyase obtained from a strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and a strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. 3 is a graph showing the temperature stability of alginate lyase obtained from a strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and a strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. 4 is a graph showing the optimum pH of alginate lyase obtained from a strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and a strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. 5 is a graph showing the optimum pH of alginate lyase obtained from a strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and a strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. 1 is a graph showing the optimum temperature of alginate lyase obtained from a strain heterologously expressing an alginate lyase derived from Paenibacillus elgii No. 1 and a strain heterologously expressing an alginate lyase derived from Paenibacillus elgii No. 2. FIG. 2 is a graph showing the optimum salt concentration of alginate lyase obtained from a strain heterologously expressing an alginate lyase derived from Paenibacillus elgii No. 1 and a strain heterologously expressing an alginate lyase derived from Paenibacillus elgii No. 2.

[0020] Preferred embodiments for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0021] 1. Alginate lyase The alginate lyase according to the present technology will be described in detail below.

[0022] [Amino acid sequence] The alginate lyase according to the present technology includes a polypeptide shown in any one of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, (2) a polypeptide having alginate lyase activity, in which one or several amino acid residues have been substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 or 2, and (3) a polypeptide having alginate lyase activity, in which the amino acid sequence shown in SEQ ID NO: 1 or 2 has 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0023] In the polypeptide of (2), the amino acid modification may include only one type of modification (e.g., substitution only) from substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (2), the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1, 2, or 1.

[0024] In the polypeptide of (3) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2 may be 70% or more, preferably 75% or more, more preferably 80% or more, even more preferably 85% or more, and even more preferably 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0025] Here, in the polypeptide (3), the sequence identity to each amino acid sequence shown in SEQ ID NO: 1 or 2 is the sequence identity calculated by comparing with the amino acid sequence shown in SEQ ID NO: 1 or 2. Furthermore, the term "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0026] In the polypeptides (2) and (3), the amino acid residues corresponding to positions 178 (tyrosine residue), 215 (lysine residue), and 246 (lysine residue) in the amino acid sequence shown in SEQ ID NO: 1 are presumed to be highly likely to be active centers, and therefore it is desirable not to introduce substitutions or deletions into these positions.

[0027] The polypeptides of the present invention may be part of a larger protein (e.g., a fusion protein), in which additional sequences may be added to aid in purification, such as multiple histidine residues, or to ensure stability during recombinant production.

[0028] In the polypeptides of (2) and (3), when an amino acid substitution is introduced into SEQ ID NO: 1 or 2, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of substitutions in the polypeptides of (2) and (3) include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another uncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0029] The alginate lyase containing the polypeptides (2) and (3) preferably has alginate lyase activity equal to or greater than that of the alginate lyase containing the polypeptide (1). Specifically, the relative activity of the alginate lyase containing the polypeptides (2) and (3) is 0.7-fold or more, 0.8-fold or more, 0.9-fold or more, 1.0-fold or more, 1.1-fold or more, preferably 1.15-fold or more, more preferably 1.2-fold or more, and even more preferably 1.25-fold or more of the relative activity of the alginate lyase containing the polypeptide (1).

[0030] [Origin] The origin of the alginate lyase according to the present technology is not particularly limited as long as it contains the aforementioned polypeptide. For example, alginate lyase derived from Paenibacillus elgii can be used. The Paenibacillus elgii strain used is not particularly limited as long as it does not impair the action and effect of the present technology, and strains generally available from biological resource organizations can be used. In the present technology, as shown in the Examples below, an alginate lyase containing the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2 was purified from Paenibacillus elgii.

[0031] More specifically, the alginate lyase containing the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is an alginate lyase derived from Paenibacillus elgii strain No. 1. The amino acid sequence including the signal sequence and pro-sequence is shown in SEQ ID NO: 3.

[0032] The alginate lyase containing the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is derived from Paenibacillus elgii strain No. 2. The amino acid sequence including the signal sequence and pro-sequence is shown in SEQ ID NO: 4.

[0033] The alginate lyase according to the present technology may be an artificial protein artificially designed based on the amino acid sequences (1) to (3) described above.

[0034] [Enzyme Chemical Properties] (1) Action The alginate lyase according to the present technology produces alginate oligosaccharides using alginic acid as a substrate.

[0035] (2) Optimum pH The alginate lyase according to the present technology has an optimum pH in the range of pH 7.5 to 9.0. The optimum pH of the alginate lyase according to the present technology varies depending on the amino acid sequence, origin, etc., as described above, but is in the range of pH 7.5 to 9.0. As a specific example, the optimum pH of an alginate lyase consisting of the amino acid sequence of SEQ ID NO: 1 is 8.5. The optimum pH of an alginate lyase consisting of the amino acid sequence of SEQ ID NO: 2 is 8.0.

[0036] Furthermore, the alginate lyase according to the present technology has 70% or more of the activity at the optimum pH at pH 7.5 to 9.5.

[0037] The optimum pH is a value measured using 50 mM acetate buffer (pH 4.0 to 5.5), 50 mM phosphate buffer (pH 5.5 to 8.0), 50 mM Tris-HCl buffer (pH 8.0 to 9.5), and 50 mM CAPS buffer (pH 9.5 to 11.0).

[0038] (3) pH Stability The alginate lyase according to the present technology is stable in the pH range of 4.0 to 10.5, and even in this range, 70% or more of its activity remains after treatment at 37°C for 30 minutes. The residual activity at pH 4.0 is 85% or more, preferably 90% or more, when the activity at the optimal pH is taken as 100%. Furthermore, the residual activity at pH 5.0 is 90% or more, preferably 95% or more, when the activity at the optimal pH is taken as 100%.

[0039] (4) Optimum Temperature The alginate lyase according to the present technology has an optimum temperature in the range of 55 to 65°C. The optimum temperature of the alginate lyase according to the present technology varies depending on the amino acid sequence, origin, etc., as described above, but is in the range of 55 to 65°C. As a specific example, the optimum temperature of an alginate lyase consisting of the amino acid sequence of SEQ ID NO: 1 is 65°C, and it exhibits 70% or more of its activity at the optimum temperature between 55 and 70°C. The optimum temperature of an alginate lyase consisting of the amino acid sequence of SEQ ID NO: 2 is 60°C, and it exhibits 75% or more of its activity at the optimum temperature between 50 and 65°C.

[0040] The optimum temperature is a value measured using 50 mM Tris-HCl buffer, pH 8.0.

[0041] As mentioned above, alginate lyase is expected to be widely used in industry. This application includes catalyzing decomposition reactions under high-temperature conditions. However, the active temperature of alginate lyase currently produced on an industrial scale and put into practical use is between 30°C and 40°C, and it cannot be used at temperatures above 50°C. In contrast, the optimal temperature of the alginate lyase of the present technology is in the range of 55-65°C, and it can function sufficiently even under high-temperature conditions.

[0042] (5) Thermostability: When treated for 30 minutes at a high temperature of 40°C or higher, the alginate lyase according to the present technology has a residual activity of 80% or more at 40°C and 40% or more at 50°C. As a specific example, when treated for 30 minutes at a high temperature of 40°C or higher, the alginate lyase derived from Paenibacillus elgii No. 1 has a residual activity of approximately 100% at 40°C and 65% at 50°C. When treated for 30 minutes at a high temperature of 40°C or higher, the alginate lyase derived from Paenibacillus elgii No. 2 has a residual activity of approximately 95% at 40°C and 45% or more at 50°C. While conventional alginate lyases cannot be used at temperatures above 50°C, the alginate lyase according to the present technology is stable to a certain extent even at temperatures above 50°C, and therefore can be expected to be usable at temperatures above 50°C.

[0043] (6) Molecular Weight The molecular weight of the alginate lyase according to the present technology is 32 to 36 kDa. Specific examples include the molecular weight of the alginate lyase derived from Paenibacillus elgii No. 1 strain and the alginate lyase derived from Paenibacillus elgii No. 2 strain, both of which have a molecular weight of 34 kDa.

[0044] The molecular weight is a value measured by SDS-PAGE.

[0045] (7) Other Features The alginate lyase according to the present technology exhibits activity even without the addition of salt, and when comparing the activity at final salt concentrations of 0 (no addition), 50 mM, 100 mM, and 150 mM, it exhibits the maximum activity without the addition of salt.

[0046] Many common alginate lyases exhibit maximum activity when salts such as sodium chloride are added. The use of salts can place a burden on factory equipment, and conventional alginate lyases have not always been able to fully meet the expected application needs of industry. In contrast, the alginate lyase of the present technology exhibits high activity even without added salt and exhibits maximum activity without added salt, thereby reducing the burden on factory equipment and enabling application in a variety of fields with sufficient activity.

[0047] 2. DNA encoding alginate lyase The DNA according to the present technology includes any of the polynucleotides (1) to (3) below: (1) a polynucleotide encoding the alginate lyase according to the present technology, (2) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 or 6, and (3) a polynucleotide comprising a sequence equivalent to the nucleotide sequence shown in SEQ ID NO: 5 or 6 and encoding a polypeptide having alginate lyase activity.

[0048] The "polynucleotide encoding the alginate lyase of the present technology" in (1) above refers to a polynucleotide that, when expressed, gives the alginate lyase of the present technology, and includes not only a polynucleotide having a base sequence corresponding to the amino acid sequence of the alginate lyase of the present technology, but also a polynucleotide in which a sequence that does not encode an amino acid sequence has been added to such a polynucleotide. It also includes a polynucleotide with degenerated codons.

[0049] Specific examples of polynucleotides encoding the alginate lyase of the present technology are shown in SEQ ID NO: 5 (a genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 1), SEQ ID NO: 7 (a cDNA sequence encoding the amino acid sequence of SEQ ID NO: 3), SEQ ID NO: 6 (a genomic DNA sequence encoding the amino acid sequence of SEQ ID NO: 2), and SEQ ID NO: 8 (a cDNA sequence encoding the amino acid sequence of SEQ ID NO: 4).

[0050] The DNA according to the present technology also includes DNA containing a polynucleotide that, when compared with the base sequence of the polynucleotide encoding the alginate lyase according to the present technology described in (1) above, has a sequence that differs in part from the base sequence of the polynucleotide (hereinafter also referred to as an "equivalent sequence"), although the function of the protein it encodes is equivalent.

[0051] Specific examples of equivalent sequences include polynucleotides that contain modifications such as substitution, addition, insertion, deletion, or inversion of one or more bases based on the base sequence of a polynucleotide encoding the alginate lyase of the present technology (e.g., SEQ ID NO: 5, 6, 7, or 8), and that encode a polypeptide having alginate lyase activity.

[0052] The base modifications may occur at multiple sites in the sequence. Here, "multiple" refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, and more preferably 2 to 10 bases, although this varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA.

[0053] Specific examples of equivalent sequences include sequences that have, for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably about 90% or more, even more preferably 95% or more, and particularly preferably 99% or more sequence identity to the base sequence of the polynucleotide encoding the alginate lyase of the present technology (e.g., SEQ ID NO: 5, 6, 7, or 8).

[0054] Here, "sequence identity" can be calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (Software Development Co., Ltd.) can be used, and these may be used with default parameters.

[0055] In the present technology, DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of the base sequence shown in SEQ ID NO: 5, 6, 7, or 8 is also included in the DNA related to the present technology, as long as it encodes a polypeptide having alginate lyase activity.

[0056] Here, "under stringent conditions" refers to conditions in which the sample is incubated at 50°C to 65°C for 4 hours to overnight in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5x Denhartz's (0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400), and 100 μg / ml salmon sperm DNA.

[0057] Hybridization under stringent conditions is specifically performed by the following method. Specifically, a nylon membrane onto which a DNA library or cDNA library is immobilized is prepared, and the nylon membrane is blocked at 65°C in a prehybridization solution containing 6xSSC, 0.5% SDS, 5x Denhardt's buffer, and 100 μg / mL salmon sperm DNA. Subsequently, each 32P-labeled probe is added, and the membrane is incubated overnight at 65°C. The nylon membrane is washed in 6xSSC at room temperature for 10 minutes, in 2xSSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2xSSC containing 0.1% SDS at 45°C for 30 minutes, and then autoradiography is performed to detect DNA that specifically hybridizes with the probe.

[0058] [Method for Obtaining DNA According to the Present Technology] The DNA according to the present technology can be prepared in an isolated state by standard genetic engineering techniques, molecular biological techniques, biochemical techniques, chemical synthesis, PCR (e.g., overlap PCR), or a combination thereof, with reference to the sequence information disclosed in this specification or the attached sequence listing.

[0059] Furthermore, the DNA according to the present technology can also be isolated from a microorganism that produces a polypeptide having alginate lyase activity. For example, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using primers or probes designed from known amino acid sequence information taking into account gene degeneracy, or primers or probes designed based on known nucleotide sequence information.

[0060] The DNA of the present technology includes various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be easily artificially produced using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are infrequently used in the host, the protein expression level may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0061] The sum of the host-optimal codon usage frequencies for each codon can be used as an index of codon usage frequency. An optimal codon is defined as the codon with the highest usage frequency among codons corresponding to the same amino acid. The codon usage frequency is not particularly limited as long as it is optimized for the host. For example, the following is an example of an optimal codon for E. coli: F: phenylalanine (ttt), L: leucine (ctg), I: isoleucine (att), M: methionine (atg), V: valine (gtg), Y: tyrosine (tat), stop codon (taa), H: histidine (cat), Q: glutamine (cag), N: asparagine (aat), K: lysine (aaa), D: aspartic acid (gat), E: glutamic acid (gaa), S: serine (agc), P: proline (ccg), T: threonine (acc), A: alanine (gcg), C: cysteine ​​(tgc), W: tryptophan (tgg), R: arginine (cgc), G: glycine (ggc).

[0062] Methods for introducing a mutation into a gene and artificially modifying the amino acid sequence include known techniques such as the Kunkel method and the Gapped duplex method, and mutagenesis kits using site-directed mutagenesis, such as QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio Inc.).

[0063] The DNA base sequence can be confirmed by conventional sequencing, such as the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463). Alternatively, the sequence can be analyzed using an appropriate DNA sequencer.

[0064] Whether the obtained DNA encodes the polypeptide of interest can be confirmed by comparing the determined nucleotide sequence with any of the nucleotide sequences set forth in SEQ ID NOs: 5 to 8. Alternatively, the amino acid sequence deduced from the determined nucleotide sequence can be compared with any of the amino acid sequences set forth in SEQ ID NOs: 1 to 4.

[0065] [Uses of DNA Relating to the Present Technology] The DNA relating to the present technology can be used as a gene encoding the alginate lyase relating to the present technology described above, a probe for identifying the DNA encoding the alginate lyase relating to the present technology, or a primer for amplifying or mutating the DNA encoding the alginate lyase relating to the present technology.

[0066] The DNA of the present technology can be used to prepare the alginate lyase of the present technology. A genetic engineering preparation method using the DNA of the present technology makes it possible to obtain a more homogeneous alginate lyase of the present technology. This method is also suitable for preparing large quantities of the alginate lyase of the present technology. When the DNA of the present technology is a DNA having a polynucleotide that does not contain an initiation codon, the alginate lyase of the present technology can be obtained by adding an initiation codon or a signal peptide containing an initiation codon and then expressing it.

[0067] The DNA according to the present technology can also be used as an experimental tool for the purpose of elucidating the mechanism of action of the alginate lyase according to the present technology, or as a tool for designing or producing mutants (modified forms) of the alginate lyase according to the present technology.

[0068] 3. Expression Cassette, Recombinant Vector The expression cassette and the recombinant vector according to the present technology contain the above-described DNA according to the present technology.

[0069] The expression cassettes and recombinant vectors according to the present technology may contain, as control elements, transcription elements such as promoters and terminators, as well as enhancers, CCAAT boxes, TATA boxes, and SPI sites, as necessary. These control elements may be operably linked to the DNA of the present invention. "Operably linked" means that the DNA of the present invention is linked to various control elements that regulate the DNA of the present invention in a state that allows it to operate in a host cell.

[0070] Regarding the recombinant vector according to the present technology, an expression vector constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host is suitable. Such expression vectors are known, and examples of commercially available expression vectors include pQE-based vectors (Qiagen, Inc.), pDR540, pRIT2T (GE Healthcare Biosciences, Inc.), pET-based vectors (Merck Ltd.), and pBE-S (Takara Bio Inc.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α E. coli strain, a combination of a pET-based vector and a BL21(DE3) E. coli strain, or a combination of a pDR540 vector and a JM109 E. coli strain.

[0071] 4. Transformant The transformant according to the present technology can be obtained by transforming a host with the above-described expression cassette according to the present technology or the recombinant vector according to the present technology.

[0072] The host used to produce the transformant according to the present technology is not particularly limited as long as it has the following characteristics (1) to (4): (1) an expression cassette or recombinant vector can be introduced, (2) the expression cassette or recombinant vector is stable, (3) it is capable of autonomous replication, and (4) it is capable of expressing the traits of the gene in the introduced expression cassette or recombinant vector. Suitable examples of such hosts include bacteria belonging to the genus Escherichia, such as Escherichia coli, the genus Bacillus, such as Bacillus subtilis, and the genus Pseudomonas, such as Pseudomonas putida; filamentous fungi; and yeast. Animal cells, insect cells, plants, etc. may also be used.

[0073] The transformant of the present invention can be obtained by introducing the DNA according to the present technology, the expression cassette according to the present technology, or the recombinant vector according to the present technology into a host. The location where the DNA according to the present technology is introduced is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or on the genome.

[0074] Specific methods for introducing the expression cassette or recombinant vector according to the present technology include, for example, recombinant vector methods and genome editing methods. Conditions for introducing the expression cassette or recombinant vector into the host may be appropriately set depending on the type of host, etc. When the host is a bacterium, examples include a method using competent cells treated with calcium ions and electroporation. When the host is a yeast, examples include electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples include electroporation, the calcium phosphate method, and lipofection. When the host is an insect cell, examples include the calcium phosphate method, the lipofection method, and electroporation. When the host is a plant cell, examples include electroporation, the Agrobacterium method, the particle gun method, and the PEG method.

[0075] Whether or not the DNA according to the present technology, the expression cassette according to the present technology, or the recombinant vector according to the present technology has been incorporated into a host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like.

[0076] When PCR is used to confirm whether the DNA, expression cassette, or recombinant vector of the present technology has been incorporated into a host, for example, the genomic DNA, expression cassette, or recombinant vector may be isolated and purified from a transformant. For example, when the host is a bacterium, the isolation and purification of the genomic DNA, expression cassette, or recombinant vector is performed using a lysate obtained by lysing the bacterium. Lysis may be achieved by treatment with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS).

[0077] Furthermore, physical disruption methods such as freeze-thawing and French press treatment may be combined. DNA can be separated and purified from the lysate by, for example, an appropriate combination of deproteinization treatments using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation treatment, and commercially available kits.

[0078] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, a type II restriction enzyme that acts on a specific nucleotide sequence can be used. DNA can be ligated to an expression cassette or expression vector using, for example, DNA ligase.

[0079] Then, PCR is performed using the isolated and purified DNA as a template and primers specific to the DNA of the present technology. The PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product is detected as a band, thereby confirming transformation.

[0080] Alternatively, PCR may be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzyme reaction, or the like.

[0081] 5. Method for Producing Alginate Lyase The method for producing alginate lyase according to the present technology involves at least a culture step and a recovery step. If necessary, various other steps commonly used in the production of enzymes can be freely combined. Each step will be described in detail below.

[0082] (1) Culturing step The culturing step is a step of culturing a microorganism that produces alginate lyase or a transformant according to the present technology. Examples of microorganisms that produce alginate lyase include microorganisms of the genus Paenibacillus, such as Paenibacillus elgii. The microorganisms used may be wild-type strains or mutant strains (e.g., mutant strains induced by ultraviolet irradiation, etc.).

[0083] The culture conditions in the culture step can be appropriately set taking into consideration the properties of the microorganism to be cultured and the nutritional and physiological properties of the transformant to be cultured. The microorganism or transformant can be cultured in a solid or liquid medium, with liquid culture being preferred. For industrial production, aeration and agitation culture is preferred.

[0084] As nutrient sources for the medium, substances required for the growth of microorganisms or transformants can be used as appropriate. Carbon sources may be any assimilable carbon compounds, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. Nitrogen sources may be any assimilable nitrogen compounds, such as peptone, meat extract, yeast extract, casein hydrolysate, and alkaline extract of soybean meal. In addition to carbon and nitrogen sources, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, and alginates (e.g., sodium alginate), as well as specific amino acids and specific vitamins, may also be used as needed.

[0085] The culture temperature may be appropriately set within a range in which the microorganism or transformant to be cultured can grow and the microorganism or transformant can produce alginate lyase. For example, the culture temperature can be set at 15 to 37°C.

[0086] The culture time may be, for example, about 12 to 48 hours. The culture step can be completed when the yield of alginate lyase reaches its maximum.

[0087] (2) Recovery step The recovery step is a step of recovering the alginate lyase according to the present technology from the culture obtained in the culture step. The recovery method in the recovery step is not particularly limited, and one or more recovery methods commonly used in enzyme production can be used in any combination.

[0088] Specifically, for example, when alginate lyase expressed using a transformant is present within the transformant, the transformant is separated from the culture supernatant using a common separation method such as centrifugation. The culture supernatant is removed, and the separated transformant is recovered. The separated transformant is treated using mechanical methods such as ultrasound or a French press, or enzymatic methods such as lysozyme, and, if necessary, solubilized using an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), thereby obtaining a water-soluble fraction containing alginate lyase. Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed alginate lyase can be secreted into the culture medium.

[0089] The water-soluble fraction containing alginate lyase thus obtained may be subjected to purification treatment as is, or the desired alginate lyase in the water-soluble fraction may be concentrated and then subjected to purification treatment. Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.). Purification of alginate lyase can be carried out, for example, by an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

[0090] The purified alginate lyase can be powdered by freeze-drying, vacuum drying, spray drying, etc., as needed, and then distributed on the market.

[0091] 6. Method for Producing Alginate Oligosaccharides Alginate oligosaccharides are disaccharide-hexasaccharide oligosaccharides whose constituent monosaccharides are mannuronic acid and guluronic acid. They are used in plant growth promoters and cosmetics, and have recently attracted attention as prebiotics. The method for producing alginate oligosaccharides according to the present technology includes a step of reacting alginic acid and / or a salt thereof with the alginate lyase according to the present technology (hereinafter also referred to as the "enzyme action step"). Furthermore, as necessary, a raw material preparation step, an enzyme inactivation step, a recovery step, etc. can also be performed. Each step is described in detail below.

[0092] (1) Raw Material Preparation Step The raw material preparation step is a step of preparing alginic acid and / or a salt thereof as a raw material. The alginic acid and / or a salt thereof may be a mixture of two or more selected from alginic acid and its salts, or may be in the form of a composition mixed with other components as long as the action and effect of the present technology are not impaired.

[0093] Examples of alginic acid salts that can be used in the present technology include sodium alginate.

[0094] (2) Enzyme action step The enzyme action step is a step in which the alginate lyase according to the present technology is allowed to act on alginic acid and / or a salt thereof. The alginate lyase according to the present technology may be used alone or in combination with two or more types. Furthermore, as long as the action and effect of the present technology are not impaired, it may also be used in combination with enzymes other than the alginate lyase according to the present technology depending on the purpose. When two or more types of enzymes are used in combination, the two or more types of enzymes may be added to alginic acid and / or a salt thereof simultaneously or separately. When two or more types of enzymes are added separately, the order in which they are added is not particularly limited. Note that the details of the alginate lyase according to the present technology are as described above, and therefore will not be described here.

[0095] Various conditions for the enzyme reaction step can be freely set as long as they do not impair the effects of the present technology. For example, pH, temperature, reaction time, etc. can be set depending on the physicochemical properties of the enzyme used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. Optimal reaction conditions can be determined through preliminary experiments. Below are examples of conditions for the reaction of each enzyme.

[0096] When the alginate lyase according to the present technology is allowed to act on alginic acid and / or a salt thereof, the pH can be set to, for example, pH 4.0 to 11.5, preferably pH 4.0 to 10.5, more preferably pH 5.0 to 10.0, even more preferably pH 6.0 to 10.0, still more preferably pH 7.0 to 9.5, and particularly preferably pH 7.5 to 9.0.

[0097] When the alginate lyase according to the present technology is allowed to act on alginic acid and / or a salt thereof, the temperature can be set to, for example, 35°C to 70°C, preferably 40°C to 70°C, more preferably 45°C to 70°C, even more preferably 50°C to 70°C, and particularly preferably 55°C to 65°C.

[0098] When the alginate lyase according to the present technology is allowed to act on alginic acid and / or a salt thereof, the lower limit of the duration of the action may be, for example, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more, and the upper limit may be, for example, 48 hours or less, 24 hours or less, 10 hours or less, 2 hours or less, or 1 hour or less.

[0099] (3) Enzyme deactivation step The enzyme deactivation step is a step of deactivating an enzyme after the above-described enzyme action step. The enzyme deactivation step is not an essential step and can be performed as needed. Furthermore, when multiple enzymes are used in the above-described enzyme action step, enzyme deactivation can also be performed between the actions of the enzymes. For example, when multiple enzymes are acted on in the enzyme action step, the enzyme deactivation step can be appropriately performed during the enzyme action step.

[0100] The method for inactivating the enzyme is not particularly limited, and one or more general inactivation methods can be freely combined depending on the properties of the enzyme to be inactivated. For example, an enzyme can be inactivated by heating it to a temperature equal to or higher than its inactivation temperature.

[0101] (4) Recovery Step The recovery step is a step of recovering the produced alginate oligosaccharide. Specific recovery methods can be selected from one or a combination of two or more common recovery methods used in the production of alginate oligosaccharide, depending on the type of alginate oligosaccharide to be produced.

[0102] The recovered alginate oligosaccharides can be further processed depending on the form of the final product, as long as the action and effect of the present technology are not impaired.

[0103] 7. Enzyme preparation for producing alginate oligosaccharides The enzymatic preparation for producing alginate oligosaccharides according to the present technology is an enzymatic preparation containing the alginate lyase according to the present technology. Details of the alginate lyase according to the present technology are as described above, and therefore will not be described here.

[0104] (1) Content of alginate lyase The content of alginate lyase in the enzyme preparation for producing alginate oligosaccharides according to the present technology can be freely set as long as it does not impair the effects of the present technology. The content of alginate lyase can be set to, for example, 0.006 U or more, 0.03 U or more, 0.06 U or more, 0.5 U or more, 1 U or more, 10 U or more, 20 U or more, 30 U or more, 40 U or more, 50 U or more, or 100 U or more per 1 g of alginic acid and / or a salt thereof used in the production of alginate oligosaccharides.

[0105] The upper limit of the content of alginate lyase is not particularly limited as long as it does not impair the effects of the present technology, and can be set to, for example, 12,000 U or less, 10,000 U or less, 9,000 U or less, 8,000 U or less, 7,000 U or less, 6,000 U or less, 5,000 U or less, 4,000 U or less, 3,000 U or less, 2,400 U or less, 2,000 U or less, 1,500 U or less, 1,200 U or less, 1,000 U or less, 900 U or less, 800 U or less, 700 U or less, 600 U or less, 500 U or less, 400 U or less, 300 U or less, 200 U or less, 100 U or less, 50 U or less, 40 U or less, 30 U or less, 24 U or less, 20 U or less, 18 U or less, or 13 U or less per gram of alginic acid and / or a salt thereof used in the production of alginate oligosaccharides.

[0106] In the present technology, the alginate lyase activity is a value measured by the measurement method described in the Examples below.

[0107] (2) Other Components The enzyme preparation for producing alginic acid oligosaccharides according to the present technology can be used in combination with other components as long as the action and effect of the present technology are not impaired. Examples of other components that can be used include excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, stabilizers, enzymes, and other components commonly used in formulations. Furthermore, components with known or future functions can also be used in combination as appropriate depending on the purpose.

[0108] The present invention will be described in more detail below with reference to examples. Note that the examples described below are representative examples of the present invention and should not be construed as narrowing the scope of the present invention.

[0109] 1. Selection of Microorganisms In order to discover a novel alginate lyase, a library of over 10,000 strains was screened using alginate lyase activity as an indicator. As a result, two strains of microorganisms, namely, Paenibacillus elgii No. 1 and Paenibacillus elgii No. 2, were selected as promising candidates.

[0110] 2. Sequence Confirmation The amino acid sequences (SEQ ID NOs: 1 and 2) of alginate lyase derived from Paenibacillus elgii No. 1 strain and Paenibacillus elgii No. 2 strain were determined from genomic DNA information revealed by genomic DNA analysis using a next-generation sequencer.

[0111] 3. Construction of Expression System The target sequence was incorporated into a commercially available expression vector. PCR and infusion reactions were performed using primers. Expression plasmids incorporating the target sequence were constructed using Escherichia coli strains JM109 and BL21 (DE3), and transformants (Paenibacillus elgii No. 1-derived alginate lyase heterologous expression strains and Paenibacillus elgii No. 2-derived alginate lyase heterologous expression strains) were obtained using the heat shock method. The obtained transformants were subjected to sequence analysis to confirm the integration of the target sequence.

[0112] 4. Cultivation of Selected Microorganisms The components listed in Table 1 were dissolved in water to the concentrations listed in Table 1, and the mixture was autoclaved at 121°C for 30 minutes to prepare a medium. The selected Paenibacillus elgii No. 1 and Paenibacillus elgii No. 2 strains were inoculated into the medium, and cultured with shaking at 30°C for 48 hours. After culture, the medium was centrifuged and the supernatant was collected. Diatomaceous earth was added to the supernatant, which was then filtered. The culture filtrate was then concentrated using an ultrafiltration membrane.

[0113]

[0114] 5. Cultivation of Transformants The components listed in Table 2 below were dissolved in water to the concentrations listed in Table 2, and the mixture was autoclaved at 121°C for 30 minutes to prepare a medium. The above-obtained strains heterologously expressing alginate lyase derived from Paenibacillus elgii No. 1 and Paenibacillus elgii No. 2 were inoculated into the medium and cultured with shaking at 37°C for 48 hours. After culturing, the medium was centrifuged and the supernatant was collected. Diatomaceous earth was added to the supernatant, which was then filtered. The culture filtrate was then concentrated using an ultrafiltration membrane.

[0115]

[0116] 6. Purification of alginate lyase The crude enzyme solution was subjected to salting out with 40% ammonium sulfate to remove contaminating proteins, and the pH was adjusted with sodium hydroxide to obtain a crude purified solution. This crude purified solution was applied to a column ("Hitrap" column) equilibrated with 50 mM potassium phosphate buffer (pH 7.5) + 40% saturated ammonium sulfate. TM The column was loaded onto 5 mL of "Butyl FF" (Cytiva). After washing the column with 50 mL of the same buffer, the alginate lyase adsorbed to the column was eluted and fractionated using a stepwise gradient of saturated ammonium sulfate (40% to 0%, 50 mL). The alginate lyase activity of a portion of each obtained fraction was confirmed using the method described below, and fractions having alginate lyase activity were collected to obtain a purified alginate lyase solution. Purification was confirmed by SDS-PAGE. The molecular weight was 34 kDa. The purified enzyme solution was prepared by ultrafiltration (UF) and diafiltration (DF).

[0117] 7. Measurement of alginate lyase activity 150 μL of 2% sodium alginate solution as substrate and 50 mM potassium phosphate buffer (pH 7.5) were placed in a 2650 μL cuvette and incubated at 50°C for 5 minutes. 200 μL of enzyme solution was added and mixed, and the reaction was carried out at 50°C for 5 minutes. The increase in absorbance at 235 nm during the reaction was measured. The presence of alginate lyase activity was confirmed by confirming the production of alginate oligosaccharides. The amount of enzyme that increased the absorbance measurement at 235 nm by 1.0 per minute was defined as 1 unit (1 U).

[0118] 8. Characterization of Alginate Lyase (1) pH Stability Alginate lyase obtained from the strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and the strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase was dissolved in 50 mM Britton-Robinson buffers (pH 2-12) with different pH values. The resulting enzyme solutions were incubated at 37°C for 30 minutes for pH treatment. The enzyme activity of each enzyme solution was measured using the activity measurement method described above. The residual activity (%) was calculated as the relative value of the enzyme activity after pH treatment, with the enzyme activity at the treatment pH showing the highest residual activity taken as 100%. The results are shown in Figure 1. As is clear from Figure 1, the alginate lyase obtained from the strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase and the strain heterologously expressing Paenibacillus elgii No. 2-derived alginate lyase. The alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Bacillus subtilis 2 showed high stability at pH 4.0 to 10.5.

[0119] (2) Optimum pH The pH of the substrate solution was adjusted to pH 4, 5, 6, 7, 8, 9, 10, or 11 using 50 mM acetate buffer (pH 4.0-5.5), 50 mM phosphate buffer (pH 5.5-8.0), 50 mM Tris-HCl buffer (pH 8.0-9.5), and 50 mM CAPS buffer (pH 9.5-11.0). Activity was measured in the same manner as described above, except that the enzyme reaction was carried out at each pH. The activity at the pH condition at which the purified enzyme exhibited maximum activity was defined as 100%, and the relative activity at each pH was calculated as relative activity (%). The results are shown in Figure 2. As is clear from Figure 2, the optimal pH of the alginate lyase obtained from the strain heterologously expressing Paenibacillus elgii No. 1-derived alginate lyase was pH 8.5, whereas the activity at the pH condition at which the purified enzyme exhibited maximum activity was 100%. The optimum pH of the alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Bacillus subtilis No. 2 was pH 8.0.

[0120] (3) Temperature Stability The purified alginate lyase was dissolved in 50 mM potassium phosphate buffer (pH 7.5), and the resulting enzyme solution was incubated at 30°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 70°C for 30 minutes for temperature treatment. The enzyme activity of each enzyme solution before and after temperature treatment was measured using the activity measurement method described above. The relative enzyme activity after temperature treatment, defined as 100% enzyme activity before temperature treatment, was calculated as residual activity (%). The results are shown in Figure 3. As is clear from Figure 3, the alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Paenibacillus elgii No. 1, exhibited high stability below 50°C, while the alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Paenibacillus elgii No. 2, exhibited high stability below 45°C. Furthermore, all alginate lyases showed high stability, with residual activity of 80% or more up to 45°C, and also showed a certain level of stability, with residual activity of 40% or more even at 50°C.

[0121] (4) Optimum Temperature Activity was measured in the same manner as described above, except that the enzyme reaction temperature was changed to 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. The activity at the temperature condition where the purified enzyme showed maximum activity was defined as 100%, and the relative activity (%) was calculated as the relative amount of activity at each temperature condition. The results are shown in Figure 4. As shown in Figure 4, the optimum temperature of the alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Paenibacillus elgii No. 1 was 65°C, and the optimum temperature of the alginate lyase obtained from the strain heterologously expressing alginate lyase derived from Paenibacillus elgii No. 2 was 60°C.

[0122] (5) Optimal Salt Concentration When measuring activity, NaCl was added to final concentrations of 0, 50, 100, and 150 mM, and activity was measured in the same manner as in the activity measurement method described above. The activity under the salt concentration condition that showed the maximum activity of the purified enzyme was defined as 100%, and the relative amount of activity under each temperature condition was calculated as relative activity (%). The results are shown in Figure 5. As is clear from Figure 5, the activity of both strains decreased with the addition of NaCl. That is, both strains showed maximum activity under conditions without the addition of NaCl.

Claims

1. An alginate lyase comprising a polypeptide shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) a polypeptide in which one or more amino acid residues have been substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1 or 2, and which has alginate lyase activity; and (3) a polypeptide in which the amino acid sequence shown in SEQ ID NO: 1 or 2 has a sequence identity of 70% or more to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which has alginate lyase activity.

2. The alginate lyase according to claim 1, which is derived from Paenibacillus elgii.

3. DNA comprising a polynucleotide shown in any one of (1) to (3) below: (1) a polynucleotide encoding the alginate lyase described in claim 1; (2) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 5 or 6; (3) a polynucleotide comprising a sequence equivalent to the base sequence shown in SEQ ID NO: 5 or 6 and encoding a polypeptide having alginate lyase activity.

4. An expression cassette or recombinant vector containing the DNA of claim 3.

5. A transformant obtained by transforming a host with the expression cassette or recombinant vector according to claim 4.

6. A method for producing alginate lyase, comprising: a culturing step of culturing Paenibacillus elgii or the transformant according to claim 5; and a recovery step of recovering the alginate lyase according to claim 1 from the culture obtained in the culturing step.

7. A method for producing alginic acid oligosaccharides, comprising the step of reacting alginic acid and / or a salt thereof with the alginate lyase of claim 1.

8. Alginate oligosaccharides obtained using the alginate lyase of claim 1.

9. An enzyme preparation for producing alginate oligosaccharides, comprising the alginate lyase according to claim 1.

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