Degrading enzymes and methods for degrading polyester resins

Abstract

To provide a technique for decomposing polyester plastic, using a marine microorganism-derived enzyme.SOLUTION: An enzyme comprises any amino acid sequence selected from specific 10 amino acid sequences, or an amino acid sequence having at least 80% sequence identity to any one selected from these amino acid sequences, the enzyme having hydrolysis activity to polyester resin.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a polyester resin-degrading enzyme, a recombinant microorganism expressing the enzyme, a method for degrading polyester resin using these, and a resin composition containing the enzyme. More specifically, it relates to a polyester resin-degrading enzyme derived from marine microorganisms, etc. [Background technology]

[0002] In light of concerns about marine pollution caused by microplastics, the importance of addressing environmental pollution problems caused by plastic waste has been re-emphasized, and the development of technologies for the decomposition and recycling of plastic waste is desired.

[0003] In relation to the present invention, for example, Patent Document 1 proposes a method for decomposing polyester plastics using enzymes derived from microorganisms of the genus Leptostrix. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2006-197883 [Overview of the project] [Problems that the invention aims to solve]

[0005] The main objective of this invention is to provide a technology for the decomposition of polyester-based plastics using enzymes derived from marine microorganisms. [Means for solving the problem]

[0006] To solve the above problems, the present invention provides the following [1]-

[22] . [1] Consists of any amino acid sequence of sequence numbers 1-10, or an amino acid sequence having at least 80% sequence identity to any amino acid sequence of sequence numbers 1-10, Having hydrolytic activity against polyester resins, enzyme. [2] The enzyme of [1] wherein the polyester resin is a biodegradable polyester resin. [2-2] The polyester resin is a biodegradable polyester resin or a non-biodegradable polyester resin, An enzyme according to [1], comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10. [3] The enzyme of [2] wherein the biodegradable polyester resin is an aliphatic polyester resin. [4] The enzyme of [3] wherein the aliphatic polyester resin is a polymer of a C2-C20 diol compound and a C2-C20 dicarboxylic acid. [5] A nucleic acid encoding the enzyme of [1], an expression vector containing the nucleic acid, or a recombinant microorganism expressing the enzyme.

[0007] A resin composition containing the enzyme [6] [1] and a polyester resin. [7] The resin composition of [6], wherein the polyester resin is a biodegradable polyester resin. [7-2] The polyester resin is a biodegradable polyester resin or a non-biodegradable polyester resin, The enzyme comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 10. [6] Resin composition. [8] The resin composition of [7], wherein the biodegradable polyester resin is an aliphatic polyester resin. [9] The resin composition of [8] wherein the aliphatic polyester resin is a polymer of a C2-C20 diol compound and a C2-C20 dicarboxylic acid.

[0008]

[10] A method for decomposing polyester resin, comprising the step of contacting the enzyme of [1] or the recombinant microorganism of [5] with the polyester resin.

[11] The decomposition method of

[10] , wherein the process is carried out in seawater.

[12] The decomposition method of

[10] , wherein the process is carried out in the presence of 3% by weight or more of NaCl.

[13] The decomposition method according to any one of

[10] -

[12] , wherein the polyester resin is a biodegradable polyester resin. [13-2] The polyester resin is a biodegradable polyester resin or a non-biodegradable polyester resin, The decomposition method according to any one of

[10] -

[12] , wherein the enzyme consists of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[14] The decomposition method according to any one of

[10] -

[13] , wherein the biodegradable polyester resin is an aliphatic polyester.

[15] The decomposition method of

[14] , wherein the aliphatic polyester resin is a polymer of a diol compound having 2 - 20 carbon atoms and a dicarboxylic acid having 2 - 20 carbon atoms.

[0009]

[16] A method for producing a polyester resin degradation product, comprising the step of contacting the enzyme of [1] or the recombinant microorganism of [5] with a polyester resin.

[17] The production method of

[16] , wherein the process is carried out in seawater.

[18] The production method of

[16] , wherein the process is carried out in the presence of 3% by weight or more of NaCl.

[19] The production method according to any one of

[16] -

[18] , wherein the polyester resin is a biodegradable polyester resin. [19-2] The polyester resin is a biodegradable polyester resin or a non-biodegradable polyester resin, The production method according to any one of

[16] -

[18] , wherein the enzyme consists of the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[20] The production method according to any one of

[16] -

[19] , wherein the biodegradable polyester resin is an aliphatic polyester resin.

[21] The production method according to

[20] , wherein the aliphatic polyester resin is a polymer of a diol compound having 2 to 20 carbon atoms and a dicarboxylic acid having 2 to 20 carbon atoms.

[22] The production method according to

[20] , wherein the polyester resin decomposition product is a diol compound having 2 to 20 carbon atoms, a dicarboxylic acid having 2 to 20 carbon atoms, and / or an oligomer thereof.

Advantages of the Invention

[0010] The present invention provides a technique for decomposing polyester-based plastics using an enzyme derived from marine microorganisms.

Brief Description of the Drawings

[0011] [Figure 1] The results of measuring the decomposition activity of ester bonds using p-nitrophenyl butyrate as a substrate are shown. [Figure 2] The results of measuring the decomposition activity of PBS are shown. In the figure, the vertical axis represents the decrease value of turbidity, and the horizontal axis represents time. [Figure 3] The results of evaluating the decomposition of PBS film and PBSA film are shown. [Figure 4] The results of evaluating the decomposition of PEF film and PET film are shown.

Modes for Carrying Out the Invention

[0012] Hereinafter, preferred embodiments for carrying out the present invention will be described. The embodiments described below show an example of typical embodiments of the present invention, and the scope of the present invention should not be construed narrowly thereby.

[0013] [Polyester Resin] In the present invention, the polyester resin may be an aliphatic polyester resin, an aromatic polyester resin, or an aliphatic-aromatic polyester resin. The polyester resin may include biodegradable and non-biodegradable ones. Examples of non-biodegradable polyester resins include polyethylene terephthalate, polyethylene furanoate, and polybutylene terephthalate. The polyester resin may be a biodegradable polyester resin, and from the viewpoint of high biodegradability, it is preferably an aliphatic polyester resin, and more preferably an aliphatic polyester resin having 2-20 C12 diol units and 2-20 C12 dicarboxylic acid units. Here, "biodegradability" refers to the property of a resin being broken down into low molecular weight molecules such as oligomers and monomers through hydrolysis by microorganisms, and then further broken down into water and carbon dioxide. Furthermore, each repeating unit in polyester resin is also called a compound unit for the compound from which the repeating unit originates. For example, a repeating unit derived from an aliphatic diol is called an "aliphatic diol unit," a repeating unit derived from an aliphatic dicarboxylic acid is called an "aliphatic dicarboxylic acid unit," and a repeating unit derived from an aromatic dicarboxylic acid is called an "aromatic dicarboxylic acid unit." In addition, the "main constituent unit" in polyester resin is usually a constituent unit that is present in the polyester resin at a concentration of 80% by weight or more, and there may be cases where no constituent units other than the main constituent unit are present. Polyester resins may contain each constituent unit individually or in any combination and ratio. Furthermore, the diol units, dicarboxylic acid units, and aliphatic oxycarboxylic acid units may be derived from compounds derived from petroleum or from plant-based materials; however, those derived from plant-based materials are preferable due to their environmental benefits.

[0014] The diol units contained in the polyester resin may be aliphatic or aromatic, but aliphatic units are preferred because they are more easily biodegradable, and diol units represented by the following general formula (1) are particularly preferred. -OR 1 -O- (1) In formula (1), R 1 This represents an aliphatic hydrocarbon group with 2 to 20 carbon atoms.

[0015] R 1 The number of carbon atoms in the aliphatic hydrocarbon group represented by is usually 2 or more, preferably 4 or more, and usually 20 or less, preferably 16 or less, more preferably 12 or less, and even more preferably 6 or less. A particularly preferred aliphatic hydrocarbon group is an aliphatic hydrocarbon group with 4 carbon atoms. Examples of aliphatic diols that give the aliphatic diol unit represented by formula (1) are ethylene glycol, 1,3-propanediol, 1,4-butanediol, and 1,4-cyclohexanedimethanol, with 1,4-butanediol, 1,3-propanediol, and ethylene glycol being more preferred, and 1,4-butanediol being particularly preferred. The diol units contained in the polyester resin may be of one type or may consist of two or more types of units in any combination and ratio. When the polyester resin contains multiple types of diol units, it is preferable that it contains 30 mol% or more of aliphatic diol units, and more preferably 50 mol% or more.

[0016] The diol units contained in the polyester resin may include aromatic diol units. Specific examples of aromatic diol components that provide aromatic diol units include xylylene glycol, 4,4'-dihydroxybiphenyl, 2,2-bis(4'-hydroxyphenyl)propane, 2,2-bis(4'-β-hydroxyethoxyphenyl)propane, bis(4-hydroxyphenyl)sulfone, and bis(4-β-hydroxyethoxyphenyl)sulfonic acid. The aromatic diol component may also be a derivative of an aromatic diol compound. Furthermore, it may be a compound having a structure in which multiple aliphatic diol compounds and / or aromatic diol compounds are dehydrated and condensed with each other.

[0017] The dicarboxylic acid units contained in the polyester resin may be aliphatic or aromatic. Furthermore, the polyester resin may contain only one type of dicarboxylic acid unit, or two or more types of units in any combination and ratio, and may contain both aliphatic and aromatic dicarboxylic acid units. However, from the viewpoint of biodegradability, the dicarboxylic acid units are preferably aliphatic. When the polyester resin contains multiple types of dicarboxylic acid units, it is preferable that the aliphatic dicarboxylic acid units are present in 30 mol% or more, and more preferably 40 mol% or more. On the other hand, there is no particular lower limit for the aliphatic dicarboxylic acid units; they may not be present at all. Furthermore, when the polyester resin contains aromatic dicarboxylic acid units, it is preferable that the aromatic dicarboxylic acid units are present in 70 mol% or less, and more preferably 60 mol% or less. The number of carbon atoms in the dicarboxylic acid unit is preferably 2 to 22. The dicarboxylic acid units contained in the polyester resin are preferably dicarboxylic acid units represented by the following general formula (2), or oxalic acid. -OC-R 2 -CO- (2) In formula (2), R 2 The symbols represent a single bond, an aliphatic hydrocarbon group having 1 to 22 carbon atoms, or an aromatic hydrocarbon group or heteroaromatic group having 4 to 8 carbon atoms.

[0018] R 2 The number of carbon atoms in the hydrocarbon group represented is preferably 2 or more and 22 or less. R 2 The number of carbon atoms in the aliphatic hydrocarbon group represented by formula (2) is usually 1 or more, preferably 2 or more, and on the other hand, preferably 22 or less, more preferably 16 or less, even more preferably 12 or less, and particularly preferably 8 or less. When the polyester resin contains two or more types of aliphatic dicarboxylic acid units represented by formula (2), a combination of an aliphatic hydrocarbon group having 2 carbon atoms and an aliphatic hydrocarbon group having 4 to 10 carbon atoms is preferred.

[0019] The aliphatic dicarboxylic acid component that provides the aliphatic dicarboxylic acid unit represented by formula (2) is not particularly limited, but the number of carbon atoms is preferably 2 or more, more preferably 4 or more, and on the other hand, preferably 22 or less, more preferably 10 or less. That is, an aliphatic dicarboxylic acid having 2 to 22 carbon atoms or a derivative such as its alkyl ester is preferable, and an aliphatic carboxylic acid having 4 to 10 carbon atoms or a derivative such as its alkyl ester is more preferable. Preferred aliphatic dicarboxylic acids or derivatives thereof include, for example, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, dodecanedioic acid or derivatives thereof. Among these, adipic acid, succinic acid and sebacic acid are preferable, succinic acid and sebacic acid are more preferable, and succinic acid is particularly preferable.

[0020] The aromatic dicarboxylic acid component that provides the aromatic dicarboxylic acid unit represented by formula (2) is not particularly limited, but the number of carbon atoms is usually 4 or more and 8 or less, preferably 6 or more. Preferred aromatic dicarboxylic acids include phthalic acid, isophthalic acid, terephthalic acid, 2,5-furandicarboxylic acid, etc. Among them, terephthalic acid and 2,5-furandicarboxylic acid are preferable, and 2,5-furandicarboxylic acid is more preferable.

[0021] The polyester resin may be a resin containing an oxycarboxylic acid unit. The oxycarboxylic acid unit contained in the polyester resin may be a polyester resin containing an aliphatic oxycarboxylic acid unit represented by the following general formula (3). -O-R 3 -CO- (3) In formula (3), R 3 represents an aliphatic hydrocarbon group having 1 to 20 carbon atoms.

[0022] R 3 The number of carbon atoms of the aliphatic hydrocarbon group represented by is preferably 1 or more, and on the other hand, preferably 16 or less, more preferably 12 or less, and still more preferably 8 or less.

[0023] The aliphatic oxycarboxylic acid component that gives the aliphatic oxycarboxylic acid unit represented by formula (3) is not particularly limited and includes, for example, lactic acid, glycolic acid, 3-hydroxybutyric acid, 2-hydroxy-n-butyric acid, 2-hydroxycaproic acid, 6-hydroxycaproic acid, 2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxyisocaproic acid, 3-hydroxypropionic acid, 4-hydroxybutyric acid, 5-hydroxyvaleric acid, 6-hydroxycaproic acid, and other hydroxy acids, or derivatives thereof such as lower alkyl esters or intramolecular esters. If optical isomers exist for these, either the D-form or the L-form may be used. Among these, lactic acid and glycolic acid are preferred.

[0024] The oxycarboxylic acid units contained in the polyester resin may also include aromatic oxycarboxylic acid units. Specific examples of aromatic oxycarboxylic acid components that yield aromatic oxycarboxylic acid units include, for example, p-hydroxybenzoic acid and p-β-hydroxyethoxybenzoic acid. The aromatic oxycarboxylic acid component may also be a derivative of an aromatic oxycarboxylic acid compound. Alternatively, it may be a compound (oligomer) having a structure in which multiple aromatic oxycarboxylic acid compounds and / or aromatic oxycarboxylic acid compounds are dehydrated and condensed with each other. In other words, oligomers may be used as raw materials. If optical isomers exist for the aromatic compound components that give rise to these aromatic compound units, they may be D-isomers, L-isomers, or racemic mixtures.

[0025] Examples of polyester resins include polyester resins containing a diol unit represented by the above general formula (1) and a dicarboxylic acid unit represented by the above general formula (2); polyester resins containing a diol unit represented by the above general formula (1), a dicarboxylic acid unit represented by the above general formula (2), and an oxycarboxylic acid unit represented by the above general formula (3); and polyester resins containing an oxycarboxylic acid unit represented by the above general formula (3). Specifically, polyester resins include aliphatic polyester resins containing aliphatic diol units and aliphatic dicarboxylic acid units as the main constituent units (hereinafter sometimes referred to as "aliphatic polyester resin (A)"), aliphatic-aromatic polyester resins (B) in which at least a portion of the repeating units of aliphatic polyester resin (A) are replaced with aromatic compound units, and aromatic polyester resins (polyarylate) (C) in which the repeating units of aliphatic polyester resin (A) are replaced with aromatic compound units.

[0026] (Aliphatic polyester resin (A)) The aliphatic polyester resin (A) consists of aliphatic diol units represented by the above formula (1) and R 2 Aliphatic polyester resin containing an aliphatic dicarboxylic acid unit represented by the above formula (2), where is an aliphatic hydrocarbon group; and an aliphatic diol unit represented by the above formula (1) and R 2 An aliphatic polyester resin containing an aliphatic dicarboxylic acid unit represented by the above formula (2), in which the aliphatic hydrocarbon group is, and an aliphatic oxycarboxylic acid unit represented by the above formula (3) is preferred. Note that the aliphatic diol unit represented by formula (1), R 2 The aliphatic dicarboxylic acid unit represented by formula (2) and the aliphatic oxycarboxylic acid unit represented by formula (3), where is an aliphatic hydrocarbon group, are as described above. Furthermore, the preferred aliphatic polyester resin as aliphatic polyester (A) is also as described above, including the case in which the aliphatic polyester resin (A) is copolymerized with a trifunctional or higher aliphatic polyhydric alcohol and a trifunctional or higher aliphatic polyhydric carboxylic acid or its acid anhydride or a trifunctional or higher aliphatic polyhydric oxycarboxylic acid component.

[0027] As the aliphatic polyester resin (A), polybutylene succinate-based resins such as polybutylene succinate (PBS), polybutylene succinate adipate (PBSA), and polybutylene succinate sebacate (PBSSe) are particularly preferred.

[0028] (Aliphatic-aromatic polyester resin (B)) Aliphatic-aromatic polyester resin (B) is a resin in which at least some of the repeating units of the aliphatic polyester resin (A) described above are replaced with aromatic compound units. Aliphatic-aromatic polyester resin (B) is an aliphatic diol unit represented by formula (1) described above and R 2 The aromatic dicarboxylic acid unit represented by formula (2) above, in which is an aromatic group, Aliphatic-aromatic polyester resin; aliphatic diol unit represented by formula (1), R 2 Aliphatic-aromatic polyester resins are preferred that contain aromatic dicarboxylic acid units represented by formula (2) and aliphatic oxycarboxylic acid units represented by formula (3), where the aromatic hydrocarbon group is the aromatic hydrocarbon group.

[0029] Note that the aliphatic diol unit represented by formula (1), R 2 The aromatic dicarboxylic acid unit represented by formula (2) and the aliphatic oxycarboxylic acid unit represented by formula (3), where is an aromatic hydrocarbon group, are as described above. Furthermore, the preferred aliphatic polyester as the aliphatic-aromatic polyester (B) is also as described above, including the case in which the aliphatic-aromatic polyester resin (B) is copolymerized with a trifunctional or higher aliphatic polyhydric alcohol and a trifunctional or higher aliphatic polyhydric carboxylic acid or its acid anhydride or a trifunctional or higher aliphatic polyhydric oxycarboxylic acid component.

[0030] Aliphatic-aromatic polyester resin (B) may contain aromatic diol units. That is, aliphatic-aromatic polyester resin (B) may be a polyester resin having aromatic diol units and aliphatic dicarboxylic acid units; aromatic diol units and aliphatic dicarboxylic acid units and aromatic dicarboxylic acid units; aliphatic diol units and aromatic diol units and aromatic dicarboxylic acid units; aliphatic diol units and aromatic diol units and aliphatic dicarboxylic acid units and aromatic dicarboxylic acid units. Specific examples of aromatic diol components are as described above.

[0031] Aliphatic-aromatic polyester resin (B) may contain aromatic oxycarboxylic acid units. Specific examples of aromatic oxycarboxylic acid components that provide aromatic oxycarboxylic acid units are as described above.

[0032] As the aliphatic-aromatic polyester resin (B), it is preferable to use an aromatic dicarboxylic acid component as the component that provides aromatic compound units. In this case, the content of aromatic dicarboxylic acid units is preferably 10 mol% or more and 80 mol% or less, based on the total amount of aliphatic dicarboxylic acid units and aromatic dicarboxylic acid units (100 mol%).

[0033] As the aromatic dicarboxylic acid component, terephthalic acid or 2,5-franglionic acid is preferred. Specifically, as the aliphatic-aromatic polyester resin (B), polybutylene terephthalate resins such as polybutylene adipate terephthalate (PBAT), polybutylene succinate terephthalate (PBST), and polybutylene sebacate terephthalate (PBSeT), and polyfranglionic carboxylate resins such as polybutylene adipate furanoate (PBAF), polybutylene succinate furanoate (PBSF), polybutylene sebacate furanoate (PBSeF), and polybutylene succinate sebacate furanoate (PBSSeF) are preferred.

[0034] As the aliphatic-aromatic polyester resin (B), resins having succinic acid, adipic acid, and sebacic acid as dicarboxylic acid units are preferred. Therefore, as the aliphatic-aromatic polyester resin (B), polybutylene succinate resins such as PBST, PBSF, and PBSSeF; polybutylene adivate resins such as PBAT, PBAF, and PBASeF; and polybutylene sebacate resins such as PBSeT and PBSeF are preferred, and polybutylene succinate-aromatic dicarboxylic acid resins such as PBST, PBSF, and PBSSeF are even more preferred.

[0035] (Aromatic polyester resin (C)) Aromatic polyester resin (polyarylate) (C) is a resin in which the repeating units of the aliphatic polyester resin (A) described above are replaced with aromatic compound units. The aromatic polyester resin (C) may contain aromatic diol units and R 2 Aromatic polyester resin containing aromatic dicarboxylic acid units represented by the above formula (2), where is an aromatic hydrocarbon group; aliphatic-aromatic polyester resin (B) may contain aromatic diol units, R 2 is an aromatic hydrocarbon group Examples include aromatic polyester resins containing aromatic dicarboxylic acid units represented by the above formula (2) and aromatic oxycarboxylic acid units that may be contained in the aliphatic-aromatic polyester resin (B).

[0036] The units and other components contained in aromatic polyester resin (C) are as described above.

[0037] [enzyme] The enzyme according to the present invention can be a protein comprising any of the amino acid sequences of SEQ ID NOs: 1-10 or a protein consisting of said amino acid sequence. Furthermore, the enzyme according to the present invention is a protein comprising an amino acid sequence in which one or several amino acids are deleted, inserted, substituted and / or added in any of the amino acid sequences of SEQ ID NOs: 1-10, and / or an amino acid sequence having at least 70%, 75%, preferably 80%, 85%, more preferably 90%, 95%, even more preferably 96%, 97%, and particularly preferably 98%, 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 1-10, and which has hydrolytic activity against polyester resins. Here, "several" refers to 2-40, 2-30, preferably 2-20, 2-10, more preferably 2-5, 2-4, and particularly preferably 3 or 2. To introduce deletions or other changes into the amino acid sequence, site-directed mutagenesis kits, such as the QuikChange™ Site-Directed Mutagenesis Kit (Stratagene), GeneTailor™ Site-Directed Mutagenesis System (Invitrogen), or TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.: Takara Bio), can be used by known methods such as the Kunkel method or the Gapped duplex method. Alternatively, the entire gene containing the deletion or other changes may be artificially synthesized. "Sequence identity" is calculated by aligning two amino acid sequences to be compared so that as many residues as possible match, and then dividing the number of matching residues by the total number of residues, expressed as a percentage. During the alignment process, gaps are inserted into one or both of the two sequences being compared as needed. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, or CLUSTALW. When gaps are inserted, the total number of residues is calculated by counting each gap as one residue. If the total number of residues counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of matching residues by the total number of residues in the longer sequence.

[0038] As an example of the above-mentioned amino acid substitutions, amino acids near the active site residue may be substituted with hydrophobic amino acids (F, Y, W, L, I, V, A, P) or basic amino acids (K, R, H) to improve interaction with the polyester chain. For example, an enzyme obtained by introducing such amino acid substitutions into an enzyme with the amino acid sequence of SEQ ID NO: 9 is the enzyme with the amino acid sequence of SEQ ID NO: 10. Here, the active site residue refers to the serine in the conserved region -Gly-X-Ser-Gly-X- (where X is any amino acid). In the enzyme of SEQ ID NO: 9, the serine at position 154 is the active site residue. Furthermore, amino acids near the active site residue refer to residues located around the active residue in the three-dimensional structure of the enzyme and which are expected to interact with the substrate. For example, in the enzyme of sequence 9, amino acids near the active site include glycine at position 80 to serine at position 83, glutamic acid at position 113, tryptophan at position 153, methionine at position 155, tyrosine at position 178, isoleucine at position 200, asparagine at position 205, and threonine at position 231. Furthermore, to enhance the structural stability of the enzyme, disulfide bonds may be introduced at appropriate positions, or amino acid substitutions may be made to narrow the space between peptide backchains. The location for introducing mutations can be determined from the enzyme's three-dimensional structural information by identifying areas where voids exist between adjacent secondary structures or backchains. For example, in the enzyme of sequence 9, a void is observed at the intersection of the α-helix consisting of amino acid residues from methionine at position 155 to asparagine at position 166 and the α-helix consisting of amino acid residues from proline at position 114 to serine at position 133. The three-dimensional structure of the enzyme can be determined using coordinate information obtained by X-ray crystallography or coordinate information derived from simulations.

[0039] The enzyme according to the present invention may have modifications as long as it has the above-described amino acid sequence and possesses hydrolytic activity towards polyester resins. Modifications include intramolecular crosslinking to improve enzyme stability, modification of amino acid side chain groups, and addition of purification tags at the ends of the amino acid sequence.

[0040] Furthermore, the enzyme according to the present invention can also be defined as a protein comprising (comprising of) an amino acid sequence encoded by DNA consisting of any of the base sequences of SEQ ID NOs. 11-20, or a protein consisting of (consisting of) said amino acid sequence. Furthermore, the enzyme according to the present invention may be a protein comprising an amino acid sequence encoded in DNA that hybridizes under stringent conditions to the complementary strand of any of the base sequences of SEQ ID NOs: 11-20, and which has hydrolytic activity against polyester resins. Stringent conditions include, for example, a condition in which a nylon membrane immobilized with DNA is incubated with a probe at 65°C for 20 hours in a solution containing 6×SSC (1×SSC is made by dissolving 8.76g of sodium chloride and 4.41g of sodium citrate in 1 liter of water), 1% SDS, 100 μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficol, and hybridization is performed, but is not limited to this. Those skilled in the art can set hybridization conditions by taking into account not only the salt concentration and temperature of such buffers, but also other conditions such as probe concentration, probe length, and reaction time. Examples of washing conditions after hybridization include "2×SSC, 0.1%SDS, 42°C" and "1×SSC, 0.1%SDS, 37°C," while more stringent conditions include "1×SSC, 0.1%SDS, 65°C" and "0.5×SSC, 0.1%SDS, 50°C." For detailed procedures regarding hybridization, refer to Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Laboratory Press (1989)), Current Protocols in Molecular Biology (John Wiley & Sons (1987-1997)), etc.

[0041] The enzyme may be derived from marine microorganisms, for example, from microorganisms belonging to the class Gammaproteobacteria. Microorganisms belonging to the class Gammaproteobacteria include those belonging to the genera Pseudomonas, Vibrio, Alcanivorax, Alteromonas, and Pseudoalteromonas.

[0042] Microorganisms of the genus Pseudomonas include P. salina, P. oceani, P. bauzanensis, P. saudimassiliensis, P. litoralis, P. pachastrellae, P. salegens, P. jilinensis, P. aestusnigri, P. abyssi, P. oceani, P. formosensis, P. gallaeciensis, P. pelagia, P. xinjiangensis, P. sabulinigri, and P. yangmingensis.

[0043] Examples of microorganisms belonging to the genus Vibrio include V. gazogenes, V. spartinae, V. ruber, V. palustris, V. aerogenes, and V. rhizosphirae.

[0044] Examples of microorganisms belonging to the genus Alcanivorax include A. balearicus, A. borkumensis, A. dieselolei, A. gelatiniphagus, A. hongdengensis, A. jadensis, A. marinus, A. mobilis, A. pacificus, A. venustensis, A. xenomutans, A. profundi, A. indicus, and A. nanhaiticus.

[0045] Structure of Alteromonas and A. addita, A. genovensis, A. hispanica, A. macleodii, A. litorea, A. marina, A. simiduii, A. stellipolaris, A. tagae, A. mediterranea, A. naphthalenivorans, A. australica, A. ponticola.

[0046] The structure of pseudoalteromonas and P.S. agarivorans, P. aliena, P. antarctica,P. arctica, P. atlantica, P. aurantia, P. bacteriolytica, P. byunsanensis, P. carrageenovora, P. citrea, P. denitrificans, P. distincta, P. donghaensis, P. elyakovii, P. spejiana, P. flavipulchra, P. haloplanktis, P. issachenkonii, luteoviolacea, P. lipolytica, P. maricaloris, P. marina, P. mariniglutinosa, P. nigrifaciens, P. paragorgicola, P. peptidolytica, P. phenolica, P. piscicida, P. prydzensis, P. rubra, P. ruthenica, P. sagamiensis, P. spongiae, P. tetraodonis, translucida, P. tunicate, P. ulvae, P. undina, P. xiamenensis.

[0047] [Current and architectural] The enzyme according to the present invention may be isolated and purified from the marine microorganisms described above, or it may be expressed in recombinant microorganisms using conventionally known molecular biological methods and then purified. Recombinant microorganisms are produced by introducing nucleic acids encoding the enzyme according to the present invention into a general host vector system and transforming the microorganisms with the vector system. In addition to the marine microorganisms mentioned above, suitable hosts include bacteria such as Escherichia coli, Rhodococcus, Pseudomonas, Corynebacterium, Bacillus, Streptococcus, and Streptomyces; yeasts such as Saccharomyces, Candida, Shizosaccharomyces, and Pichia; and filamentous fungi such as Aspergillus. Among these, Escherichia coli is particularly convenient, efficient, and preferable.

[0048] The marine microorganisms or recombinant microorganisms expressing the enzymes described above according to the present invention can be obtained by using the culture supernatant obtained from the culture medium by a collection operation such as centrifugation, or by using the cells or their processed products. Examples of processed cell products include cells treated with acetone and toluene, freeze-dried cells, cell lysates, cell-free extracts obtained by lysing cells, and crude or purified enzymes extracted from these.

[0049] [Method for decomposing polyester resin] By bringing the enzyme or recombinant microorganism according to the present invention into contact with the polyester resin, the polyester resin can be decomposed. The decomposition of polyester resin can be confirmed, for example, by a decrease in the weight of the resin subjected to decomposition or a decrease in the average molecular weight. Furthermore, when used as an emulsion, it can be confirmed by the formation of a clear zone due to the decomposition of the resin.

[0050] The step of contacting the enzyme or recombinant microorganism with the polyester resin may be carried out in a suitable solvent. Typically, an aqueous solvent such as a buffer solution is used as the solvent. The enzyme according to the present invention is derived from marine microorganisms and exhibits hydrolytic activity even under high salt concentrations, so the enzymatic reaction can be carried out even in solvents with high salt concentrations (for example, 3% by weight or more of NaCl). Therefore, seawater, which is inexpensive, readily available, and can be used in large quantities as a solvent, can be used, contributing to the effective use of water resources. Furthermore, the step of bringing the enzyme or recombinant microorganism into contact with the polyester resin may be carried out by directly applying or spraying the solution containing the enzyme or recombinant microorganism onto the polyester resin. Furthermore, the step of contacting the enzyme or recombinant microorganism with the polyester resin may be carried out in a compost. The compost may be in a device with stirring and heating, or it may be open-system fermented compost. The decomposition of compost resin in a compost is usually slow. The enzyme according to the present invention is active even in compost with a high salt concentration and shows an effect of promoting the decomposition of polyester resin products in the compost. Therefore, it reduces the amount of polyester resin to be incinerated and contributes to the reduction of carbon dioxide emissions.

[0051] The duration, temperature, pH, and amount of enzyme added to the enzymatic reaction are not particularly limited and can be adjusted as appropriate. The reaction temperature and duration are usually 1 hour to 1 week at 10-60°C, preferably 1 day or more at 20-50°C, and more preferably 3 days or more at 30-40°C. The pH conditions for the enzymatic reaction are, for example, in the range of pH 4 to 10, preferably pH 5.0 to 9.0. The amount of enzyme added is, for example, 0.001-20% (w / w), preferably 0.01-10% (w / w), and more preferably 0.1-5% (w / w) relative to the polyester resin.

[0052] The decomposition products of the polyester resin produced by the reaction of the enzyme according to the present invention with the polyester resin may be recovered and used for the resynthesis of the polyester resin. The decomposition products of polyester resins are determined by the type of polyester resin, but may be C2-C20 diol compounds and C2-C20 dicarboxylic acids and / or oligomers (approximately 10-mers or less) thereof, such as succinic acid, adipic acid, 6-hydroxyhexanoic acid, 1,4-butanediol, ethylene glycol, and oligoesters consisting of combinations thereof.

[0053] [Resin composition] The resin composition containing the enzyme and polyester resin according to the present invention exhibits excellent biodegradability. The resin composition according to the present invention is normally biodegradable in at least one of the following environments: seawater, freshwater, brackish water, soil, or compost. In particular, high biodegradability in seawater (marine biodegradable resin composition) is preferred because the amount of microorganisms in seawater is low. The resin composition according to the present invention can be expected to decompose in seawater even if it is dumped into the ocean, for example, because the enzyme exhibits hydrolytic activity even under high salt concentrations. Embodiments of the decomposition of polyester resin according to the present invention include not only a method in which the resin is intentionally exposed to seawater to decompose, but also a method in which the polyester resin according to the present invention is dumped into seawater and unintentionally subjected to decomposition in seawater.

[0054] In the resin composition according to the present invention, the polyester resin may be used alone, or two or more resins may be used in any combination and ratio.

[0055] The amount of enzyme blended with the polyester resin in the resin composition is not particularly limited, but is, for example, 0.001-20% (w / w), preferably 0.01-10% (w / w), and more preferably 0.1-5% (w / w).

[0056] Enzymes can be incorporated directly into the resin composition. Enzymes can also be incorporated into the resin composition attached to or fixed to the resin. Furthermore, enzymes can be incorporated into the resin composition attached to or fixed to a carrier of any shape, encapsulated within the lattice space of a three-dimensional lattice-shaped carrier, or encapsulated within a water-soluble capsule-shaped carrier. Examples of carriers include synthetic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyacrylamide, and photocurable resins; gel carriers made of natural polymers such as cellulose, carrageenan, and sodium alginate; and carriers made of polyethylene, polyurethane, polypropylene, polyester, polyolefin, and rayon. Inorganic carriers such as activated carbon and anthracite can also be used. These carriers can also include those known as microspheres. [Examples]

[0057] 1. Preparation of PBS / PBSA emulsified agar medium 2 g of polybutylene succinate (PBS) or polybutylene succinate adipate (PBSA) was dissolved in 40 mL of chloroform, and 40 mg of surfactant (Plysurf A210G) and 250 mL of water were added. The solution was emulsified using a process homogenizer. The emulsion was kept warm and stirred in a hot stirrer for 3 hours to evaporate the chloroform. After diluting with water to make up 250 mL, the solution was sterilized in an autoclave to obtain the resin solution. To 750 mL of filtered seawater, 1 g NH4Cl, 0.05 g K2HPO4, 0.1 g BD Yeast Extract, and 15 g agar were added, and the mixture was sterilized by autoclaving to obtain an agar solution. The agar solution and emulsion were mixed in a volume ratio of 3:1, and then solidified to form a plate medium (PBS(A) emulsified agar medium).

[0058] 2. Search for polyester-degrading microorganisms in the ocean Seawater was concentrated to 1 / 100 of its original volume using an ultrafiltration membrane (molecular weight cutoff 200,000 Da). Concentrated seawater was seeded onto a culture medium (Marine Broth 2216). PBS or PBSA powder was added to the medium at a concentration of 10 mg / mL, and the cultures were incubated at 25°C and 200 rpm for several weeks. Once microbial growth was visually confirmed, a small amount of culture medium was taken and subcultured. The culture was incubated at 25°C and 200 rpm for several weeks, and the culture medium was collected as isolation source 1.

[0059] Biodegradation tests were conducted using a solution prepared by adding trace amounts of minerals (8.5 mg / L KH2PO4, 22 mg / L K2HPO4, 33 mg / L Na2HPO4·2H2O, 1.7 mg / L NH4Cl, 0.25 mg / L FeCl3·6H2O) and 30 mg of PBS powder to 100 mL of concentrated seawater. The solution was incubated at 25°C, and the solution was collected when an increase in biodegradation was observed, and this was used as isolation source 2.

[0060] After spreading isolation source 1 or isolation source 2 onto PBS(A) emulsified agar medium, the cultures were incubated statically at 30°C. Polyester-degrading microorganisms were isolated using the clear zone formed around the colonies as an indicator.

[0061] 3. Identification of polyester-degrading enzyme genes Polyester-degrading microorganisms were collected using a platinum loop and inoculated onto culture medium (Marine Broth 2216). After incubation at 30°C and 200 rpm for several days, the cells were collected by centrifugation. Genomic DNA was extracted from extracts obtained by disrupting the bacterial cells, and its sequence was determined. Hydrolytic enzyme genes were searched within the genome sequence, and enzymes 1-9 (amino acid sequences are shown in SEQ ID NOs: 1-9, and nucleic acid sequences in SEQ ID NOs: 11-19) were identified. Enzyme 3 (SEQ ID NOs: 3,13) was found by homology search with sequences 5 and 9 from the Vibrio gazogenes genome sequence. The genes for enzymes 1-9 were cloned into vectors (pET26b or pET22b) to obtain expression vectors.

[0062] 4. Introducing mutations into enzymes Using an expression vector containing the gene for Sequence ID No. 19 (enzyme 9) (5-10 ng) as a template, amino acid substitutions were introduced by PCR using 50 pmol of primers and KODOne polymerase (Toyobo Co., Ltd.). The primer sequences are shown in Table 1. After the PCR reaction, 1 μL of DpnI enzyme solution was added to the reaction mixture and incubated at 37°C for 1 hour. 3 μL of the reaction mixture was added to competent E. coli JM109 cells, and transformation was performed using a standard method. Plasmids were extracted from the resulting transformants and used as an expression vector for enzyme 10. Enzyme 10 is a modified enzyme in which the amino acid sequence of enzyme 9 is modified by substituting Gln at position 113 with Tyr, Gly at position 159 with Ala, Tyr at position 178 with Trp, and Asn at position 205 with Lys.

[0063] [Table 1]

[0064] 5. Expression and purification of hydrolytic enzymes Escherichia coli (BL21(DE3) strain or Rosetta2(DE3) strain) was transformed with an expression vector. Recombinant strains were inoculated into 10 mL of LB medium (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) using one loop of platinum. The cultures were incubated overnight at 37°C and 200 rpm to obtain the pre-culture medium. The pre-culture medium was added to 250 mL of 2X YT medium. The culture was incubated at 30°C at 150 rpm, and when the turbidity (OD600) exceeded 1, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the culture was incubated for another night at 150 rpm to obtain the main culture medium.

[0065] The enzymes were purified from the extract of the recovered bacterial cells or from the periplasm fraction. (1) Preparation of the periplasm fraction The culture medium was centrifuged to collect the bacterial cells. The bacterial cells were suspended in 1 / 10 the volume of buffer (20 mM Tris-HCl (pH 8.0), 20% (w / v) sucrose, 1 mM EDTA) of the culture medium. The suspension was centrifuged, the supernatant was removed, and the bacterial cells were resuspended in the same volume of water. The suspension was centrifuged again, and the supernatant (periplasm fraction) was collected. Tris-HCl buffer (pH 8.0) was added to the periplasm fraction to a final concentration of 20 mM, and the mixture was filtered through a membrane filter to obtain the periplasm fraction. (2) Preparation of bacterial cell extract To 10 mL of culture medium containing bacterial cells, 8 mL of Merck BugBuster was added and the mixture was stirred at room temperature for 30 minutes to 2 hours. Then, the mixture was centrifuged at 8,000 g for 10 minutes, and the supernatant was sterilized through a 0.22 μm filter to obtain a bacterial cell extract.

[0066] The periplasm fraction or bacterial cell extract was fractionated using an affinity column (HisTrap HP) to obtain purified enzymes.

[0067] 6. Measurement of enzyme activity (1) Measurement 1 p-nitrophenyl butyrate was reacted with purified enzyme, and the amount of p-nitrophenol produced by the decomposition of the ester bond was quantified based on absorbance (405 nm), and the enzyme activity was measured. One unit of enzyme was defined as the amount of enzyme producing 1 μmol of p-nitrophenol per minute. The reaction was carried out in 100 mM Tris-HCl buffer (pH 8.0), with a reaction system volume of 200 μL. p-nitrophenyl butyrate was dissolved in DMSO and added to the reaction system at a final concentration of 1 mM. Absorbance at 405 nm was measured over time, and the initial velocity was calculated from the linear region.

[0068] The results are shown in Figure 1. For enzymes 1-10, ester bond degrading activity of p-nitrophenyl butyrate was confirmed.

[0069] (2) Measurement 2 The emulsified PBS solution was diluted with 20 mM Tris-HCl buffer (pH 8.0) to an OD660 of 0.5. 100 μL of purified enzyme solution was added to 900 μL of the emulsified PBS solution. The decrease in turbidity at 660 nm was measured over time using an absorbance meter. The amount of enzyme added was as follows:

[0070] [Table 2]

[0071] The results are shown in Figure 2. In the figure, the vertical axis represents the decrease in turbidity, and the horizontal axis represents time. The degradation activity of PBS was confirmed for enzymes 1-10.

[0072] (3) Measurement 3 Add 1 cm to 1 mL of 20 mM Tris-HCl buffer (pH 8.0) containing purified enzyme. 2 The PBS or PBSA film was immersed and allowed to react at 30°C for approximately 1-3 days. After the reaction was complete, the film surface was washed with 70% ethanol, air-dried, and the weight loss of the film was measured. The amount of enzyme added was as follows:

[0073] [Table 3]

[0074] The results are shown in Figure 3. For enzymes 1-10, the progression of degradation of PBS film and PBSA film was confirmed.

[0075] (4) Measurement 4 Add 1 cm to 5 mL of 200 mM Tris-HCl buffer (pH 8.0) containing enzyme 10. 2 PEF film and PET film were immersed in the solution and reacted at 40°C and 120 rpm for 8 days. After the reaction was complete, the film surface was washed with 70% ethanol, air-dried, and the weight loss of the film was measured. The amount of enzyme added was 50 μg.

[0076] The results are shown in Figure 4. Degradation was observed in both the PEF film and the PET film polyester. [Sequence Listing Free Text]

[0077] Sequence ID 1: Amino acid sequence of enzyme 1 Sequence ID 2: Amino acid sequence of enzyme 2 Sequence ID 3: Amino acid sequence of enzyme 3 Sequence ID 4: Amino acid sequence of enzyme 4 Sequence ID 5: Amino acid sequence of enzyme 5 Sequence ID 6: Amino acid sequence of enzyme 6 Sequence ID 7: Amino acid sequence of enzyme 7 Sequence ID 8: Amino acid sequence of enzyme 8 Sequence ID 9: Amino acid sequence of enzyme 9 Sequence ID 10: Amino acid sequence of enzyme 10 Sequence ID 11: Gene sequence of enzyme 1 Sequence ID 12: Gene sequence of enzyme 2 Sequence ID 13: Gene sequence of enzyme 3 Sequence ID No. 14: Gene sequence of enzyme 4 Sequence ID 15: Gene sequence of enzyme 5 Sequence ID 16: Gene sequence of enzyme 6 Sequence ID 17: Gene sequence of enzyme 7 Sequence ID 18: Gene sequence of enzyme 8 Sequence ID 19: Gene sequence of enzyme 9 Sequence ID 20: Gene sequence of enzyme 10 Sequence ID 21: Base sequence of primer PS2-3_Q113Y_F Sequence ID 22: Base sequence of primer PS2-3_Q113Y_R Sequence ID 23: Base sequence of primer PS2-3_G159A_F Sequence ID 24: Base sequence of primer PS2-3_G159A_R Sequence ID 25: Base sequence of primer PS2-3_Y178W_F Sequence ID 26: Base sequence of primer PS2-3_Y178W_R Sequence ID 27: Base sequence of primer PS2-3_N205K_F Sequence ID 28: Base sequence of primer PS2-3_N205K_R

Claims

1. It consists of an amino acid sequence of any of SEQ ID NOs: 1, 2, 4, 6, 7, 9, or 10, or an amino acid sequence having at least 95% sequence identity to any of SEQ ID NOs: 1, 2, 4, 6, 7, 9, or 10. Having hydrolytic activity against polyester resins, enzyme.

2. The amino acid sequence is one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 7, 9, or 10, or an amino acid sequence having at least 95% sequence identity with one of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 7, 9, or 10. Having hydrolytic activity against polyester resins, enzyme.

3. The enzyme according to claim 1 or 2, wherein the polyester resin is a biodegradable polyester resin.

4. The enzyme according to claim 3, wherein the biodegradable polyester resin is an aliphatic polyester resin.

5. The enzyme according to claim 4, wherein the aliphatic polyester resin is a polymer of a C2-C20 diol compound and a C2-C20 dicarboxylic acid.

6. A recombinant microorganism expressing the enzyme described in claim 1 or 2.

7. The amino acid sequence is one of the amino acid sequences of SEQ ID NOs: 1-10, or an amino acid sequence having at least 95% sequence identity with one of the amino acid sequences of SEQ ID NOs: 1-10. An enzyme having hydrolytic activity against aliphatic polyester resins, Aliphatic polyester resin, A resin composition containing the following:

8. The resin composition according to claim 7, wherein the aliphatic polyester resin is a polymer of a C2-C20 diol compound and a C2-C20 dicarboxylic acid.

9. The amino acid sequence is one of the amino acid sequences of SEQ ID NOs: 1-10, or an amino acid sequence having at least 95% sequence identity with one of the amino acid sequences of SEQ ID NOs: 1-10. An enzyme having hydrolytic activity against polyester resin, or a recombinant microorganism expressing said enzyme, Aliphatic polyester resin, A method for decomposing polyester resin, including a step of bringing it into contact with something.

10. The decomposition method according to claim 9, wherein the aliphatic polyester resin is a polymer of a C2-C20 diol compound and a C2-C20 dicarboxylic acid.

11. The amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10, An enzyme having hydrolytic activity against polyester resin, or a recombinant microorganism expressing said enzyme, A method for decomposing a polyester resin, comprising the step of contacting it with polyethylene terephthalate or polyethylene furanoate.

12. The decomposition method according to claim 9 or 11, wherein the above step is performed in seawater.

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