Proteins, polynucleotides, recombinant vectors, transformants, compositions, methods for detecting polyethylene terephthalate, and methods for degrading polyethylene terephthalate.

Abstract

The present invention provides a protein that can specifically bind to PET; a polynucleotide encoding the protein; a recombinant vector and transformant containing the polynucleotide; a composition containing the protein; and a method for detecting and degrading PET. [Solution] A modified protein having the ability to bind to polyethylene terephthalate; a polynucleotide encoding the protein; a recombinant vector and transformant containing the polynucleotide; a composition containing the protein; and a method for detecting and degrading PET.

Description

[Technical Field]

[0001] This disclosure relates to proteins, polynucleotides, recombinant vectors, transformants, compositions, methods for detecting polyethylene terephthalate, and methods for degrading polyethylene terephthalate. [Background technology]

[0002] Polyethylene terephthalate (PET) accounts for approximately 4% of the plastics produced in Japan and is used in everyday items such as beverage bottles and clothing. It is also used as an engineering plastic in composite materials with glass fibers and other materials. However, it is known that used plastics leak into the environment, break down into small particles, and float in water. Such plastics floating in the environment are called "microplastics." To address environmental pollution, technologies for removing or decomposing PET are desired, and various PET decomposition technologies aimed at the sustainable use of PET have been reported. For example, Patent Documents 1-3 and Non-Patent Document 1 report enzymes capable of decomposing PET. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2023-58381 [Patent Document 2] International Publication No. 2024 / 122636 [Patent Document 3] Japanese Patent Publication No. 2015-119670 [Non-patent literature]

[0004] [Non-Patent Document 1] Akihiko Nakamura et al., Positive Charge Introduction on the Surface of Thermostabilized PET Hydrolase Facilitates PET Binding and Degradation, ACS Catal. 2021, 11, p.8550-8564 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] For enzymatic decomposition of solid polymers such as PET, it is crucial that the enzyme adsorbs to the substrate surface. In fact, many enzymes that decompose natural solids like cellulose and chitin often possess both a decomposition domain and an adsorption domain. Furthermore, for PET decomposition, it is desirable that the enzyme specifically binds to PET.

[0006] In addition, while the detection of plastics floating in the environment is currently done visually, the analysis is complicated due to the small size and enormous number of particles. Therefore, in order to quantitatively evaluate environmental pollution, there is a need to develop technologies that specifically stain plastics and detect them using image processing or other methods.

[0007] In view of the above circumstances, this disclosure relates to a protein that can specifically bind to PET; a polynucleotide encoding the protein; a recombinant vector and transformant containing the polynucleotide; a composition containing the protein; and a method for detecting and degrading PET. [Means for solving the problem]

[0008] The means for solving the above problems include the following embodiments. <1> A protein having a mutation at at least one position selected from the group consisting of W17, W51, and W69 in SEQ ID NO: 2, comprising one of the following amino acid sequences (A1) to (A5), and possessing the ability to bind to polyethylene terephthalate. (A1) An amino acid sequence (a1) which has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W17L and W69L mutations added, and which has the W17L and W69L mutations. (A2) An amino acid sequence having 90% or more sequence identity with the amino acid sequence (a2) obtained by adding the W17V, W51F, and W69F mutations to the amino acid sequence of Sequence ID No. 2, and having the aforementioned W17V, W51F, and W69F mutations. (A3) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W17S and W69L mutations added (a3), and having the aforementioned W17S and W69L mutations, (A4) An amino acid sequence (a4) that has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W51L and W69G mutations added, and which has the W51L and W69G mutations. (A5) An amino acid sequence having 90% or more sequence identity with the amino acid sequence (a5) obtained by adding the W51V and W69F mutations to the amino acid sequence of Sequence ID No. 2, and also having the W51V and W69F mutations. <2> The amino acid at the 13th position from the N-terminus of sequence number 2 is H, the amino acid at the 15th position is P, the amino acid at the 22nd position is V, and the amino acid at the 24th position is G. <1> The protein described above. <3> Furthermore, it contains a labeling substance, <1> or <2> The protein described above. <4> The labeling substance is a fluorescent dye or a fluorescent protein. <3> The protein described above. <5> Furthermore, it includes an enzyme domain, <1> ~ <3> The protein described in any one of the items. <6> The enzyme domain is a polyethylene terephthalate degrading enzyme domain. <5> The protein described above. <7> It is a polymer. <1> ~ <6> The protein described in any one of the items. <8> <1> ~ <6> A polynucleotide containing a base sequence that codes for any one of the proteins described in item 1. <9> A recombinant vector comprising the polynucleotide described in <8>. <10> A transformant comprising the recombinant vector described in <9>. <11> A composition comprising the protein described in any one of <1> to <7>. <12> Contacting the protein described in <3> or <4> with polyethylene terephthalate, Detecting the labeling substance, A method for detecting polyethylene terephthalate, comprising: <13> A method for decomposing polyethylene terephthalate, comprising contacting the protein described in <6> with polyethylene terephthalate.

Advantages of the Invention

[0009] According to the present disclosure, a protein that can specifically bind to PET; a polynucleotide encoding the protein; a recombinant vector and a transformant comprising the polynucleotide; a composition comprising the protein; and a method for detecting and decomposing PET are provided.

Brief Description of the Drawings

[0010] [Figure 1] A graph showing the amount of binding of stagRFP-PfChBD2(WT) and stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutants to amorphous PET. [Figure 2] A graph showing the results of fluorescence measurement of each mutant extracted into the soluble fraction in soluble selection. [Figure 3] A graph showing the relationship between the PET concentration and the adsorbed protein concentration in PET adsorption capacity measurement. [Figure 4] A graph showing the results of chitin and cellulose adsorption measurement. [Figure 5] A graph comparing the results of PET, chitin, and cellulose adsorption measurement. [Figure 6]This figure shows the ratio of adsorption amount to PET and adsorption amount to chitin or cellulose for the wild type and each mutant. [Modes for carrying out the invention]

[0011] The embodiments of this disclosure will be described in detail below. However, the embodiments of this disclosure are not limited to the embodiments described below. In the embodiments described below, the components (including elemental steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the embodiments of this disclosure.

[0012] In this disclosure, the numerical range indicated using "~" includes the numbers before and after "~" as the minimum and maximum values, respectively. In numerical ranges described in stages within this disclosure, the upper or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Furthermore, in numerical ranges described within this disclosure, the upper or lower limit of that range may be replaced with the values ​​shown in the examples. In this disclosure, each component may contain multiple types of the corresponding substance. If multiple types of the substance corresponding to each component are present in the composition, the content or amount of each component means the total content or amount of the multiple types of substances present in the composition, unless otherwise specified. In this disclosure, a combination of two or more preferred embodiments is a more preferred embodiment. In this disclosure, "identity" of amino acid sequences refers to the ratio of identical amino acid residues to the total number of amino acid residues when two amino acid sequences to be compared are aligned by inserting gaps as appropriate in one or both sequences, as necessary, to maximize the number of matching amino acid residues. Alignment can be evaluated using the default parameters of BLAST. In this disclosure, "having X% or more sequence identity with the reference amino acid sequence" means that the entire length or a portion of the amino acid sequence being compared has X% or more sequence identity with the entire length of the reference amino acid sequence. In this disclosure, amino acid residues may be indicated by the following single-letter abbreviations in parentheses: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V). In this disclosure, amino acids include natural amino acids, unnatural amino acids, modified amino acids, and their derivatives. In this disclosure, unnatural amino acids refer to amino acids other than the 20 amino acids mentioned above, and include both natural and artificial products. In this disclosure, the amino acid positions of a peptide or protein may be expressed using the single-letter abbreviation of the amino acid and in order of amino acid locus from the N-terminus. For example, "SEQ ID NO: 2 W17" refers to the 17th tryptophan position from the N-terminus of SEQ ID NO: 2. In this disclosure, amino acid mutations in peptides or proteins may be expressed in the following order: the single-letter designation of the amino acid before the mutation, the amino acid locus from the N-terminus, and the single-letter designation of the amino acid after the mutation. For example, "the W17L mutation was applied to the amino acid sequence of SEQ ID NO: 2" means that the 17th tryptophan from the N-terminus of SEQ ID NO: 2 has been replaced with leucine. In this disclosure, "protein containing the amino acid sequence of X" means that all or part of the amino acid sequence of the protein is the amino acid sequence of X. That is, the protein may consist only of the amino acid sequence of X, or it may have an additional sequence in addition to the amino acid sequence of X.

[0013] <Protein> The protein of this disclosure has a mutation at at least one position selected from the group consisting of W17, W51, and W69 in SEQ ID NO: 2, and comprises one of the following amino acid sequences (A1) to (A5), and has the ability to bind to polyethylene terephthalate. (A1) An amino acid sequence having 90% or more sequence identity with (a1) the amino acid sequence of SEQ ID NO: 2 with the W17L and W69L mutations added, and having the aforementioned W17L and W69L mutations. (A2) An amino acid sequence having 90% or more sequence identity with the amino acid sequence (a2) obtained by adding the W17V, W51F, and W69F mutations to the amino acid sequence of Sequence ID No. 2, and having the aforementioned W17V, W51F, and W69F mutations. (A3) An amino acid sequence having 90% or more sequence identity with (a3) ​​the amino acid sequence of SEQ ID NO: 2 with the W17S and W69L mutations added, and having the aforementioned W17S and W69L mutations. (A4) An amino acid sequence having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W51L and W69G mutations added (a4), and having the aforementioned W51L and W69G mutations. (A5) An amino acid sequence having 90% or more sequence identity with the amino acid sequence (a5) obtained by adding the W51V and W69F mutations to the amino acid sequence of Sequence ID No. 2, and also having the W51V and W69F mutations.

[0014] The protein with the amino acid sequence of Sequence ID No. 2 is an amino acid sequence obtained by adding K13H, N15P, E22V, and D24G mutations to the wild-type amino acid sequence (Sequence ID No. 1) of the CBM2 family chitin-binding domain (hereinafter also referred to as "PfChBD2") of a heat-stable chitinase from Pyrococcus furiosus. The amino acid sequences of Sequence ID No. 1 and No. 2 are shown below.

[0015] Sequence ID 1: TTPVPVSGSL EVKVNDWGSG AEYDVTLNLD GQYDWTVKVK LAPGATVGSF WSANKQEGNG YVIFTPVSWN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0016] Sequence ID 2: TTPVPVSGSL EVHVPDWGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF WSANKQEGNG YVIFTPVSWN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0017] The protein disclosed herein contains one of the amino acid sequences (A1) to (A5) having a mutation at at least one position selected from the group consisting of W17, W51, and W69 in SEQ ID NO: 2. Hereinafter, any of the amino acid sequences (a1) to (a5) in (A1) to (A5) below will also be collectively referred to as "amino acid sequence A". (a1) Amino acid sequence obtained by adding W17L and W69L mutations to the amino acid sequence of SEQ ID NO: 2 (a2) Amino acid sequences obtained by adding the W17V, W51F, and W69F mutations to the amino acid sequence of SEQ ID NO: 2 (a3) Amino acid sequence obtained by adding W17S and W69L mutations to the amino acid sequence of SEQ ID NO: 2 (a4) Amino acid sequence obtained by adding W51L and W69G mutations to the amino acid sequence of SEQ ID NO: 2 (a5) Amino acid sequence obtained by adding W51V and W69F mutations to the amino acid sequence of SEQ ID NO: 2

[0018] The inventors found that four mutations in the amino acid sequence of PfChBD2, K13H, N15P, E22V, and D24G, improved PET binding ability compared to the wild type. However, the mutants with these four mutations still retained binding affinity to chitin, indicating room for improvement in PET specificity. The inventors further investigated proteins with PET-specific binding ability and conceived of adding a mutation to at least one of the amino acid sequences of Sequence ID No. 2, selected from the group consisting of W17, W51, and W69. The three tryptophan residues W17, W51, and W69 may be hydrophobic residues that play an important role in the binding of chitinase to chitin. On the other hand, it was found that any of the mutations specified by (a1) to (a5) at these sites reduced affinity to chitin while maintaining high affinity to PET.

[0019] The proteins of this disclosure include amino acid sequences having 90% or more sequence identity with amino acid sequence A, and preferably include amino acid sequences having 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with amino acid sequence A. The amino acid sequence A, i.e., the sequences of amino acid sequences (a1) to (a5), are shown below.

[0020] Amino acid sequence (a1): Mutations in W17L and W69L (SEQ ID NO: 3) TTPVPVSGSL EVHVPDLGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF WSANKQEGNG YVIFTPVSLN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0021] Amino acid sequence (a2): Mutations in W17V, W51F, and W69F (SEQ ID NO: 4) TTPVPVSGSL EVHVPDVGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF FSANKQEGNG YVIFTPVSFN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0022] Amino acid sequence (a3): Mutations in W17S and W69L (SEQ ID NO: 5) TTPVPVSGSL EVHVPDSGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF WSANKQEGNG YVIFTPVSLN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0023] Amino acid sequence (a4): Mutations in W51L and W69G (SEQ ID NO: 6) TTPVPVSGSL EVHVPDWGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF LSANKQEGNG YVIFTPVSGN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0024] Amino acid sequence (a5): Mutations in W51V and W69F (SEQ ID NO: 7) TTPVPVSGSL EVHVPDWGSG AVYGVTLNLD GQYDWTVKVK LAPGATVGSF VSANKQEGNG YVIFTPVSFN KGPTATFGFI VNGPQGDKVE EITLEINGQV I

[0025] Among the amino acid sequences (a1) to (a5), amino acid sequence (a1) or (a2), or an amino acid sequence having 90% or more sequence identity with these, is preferred. From the viewpoint of having a tendency for high maximum adsorption capacity to PET, amino acid sequence (a1) or an amino acid sequence having 90% or more sequence identity with it is particularly preferred, and from the viewpoint of having high adsorption capacity to PET and a tendency for low dissociation rate, amino acid sequence (a2) or an amino acid sequence having 90% or more sequence identity with it is particularly preferred.

[0026] The protein of this disclosure preferably has H as the amino acid at the 13th position from the N-terminus of SEQ ID NO: 2, P as the amino acid at the 15th position, V as the amino acid at the 22nd position, and G as the amino acid at the 24th position. That is, the protein of this disclosure preferably maintains the K13H, N15P, E22V, and D24G mutations relative to SEQ ID NO: 1. "Corresponding amino acids" means the amino acids at the positions corresponding to each position in SEQ ID NO: 2 when SEQ ID NO: 2 and a comparison amino acid sequence are aligned by inserting gaps as appropriate in one or both of them as necessary to maximize the number of matching amino acid residues.

[0027] The proteins of this disclosure may or may not contain mutations other than those specified in (a1) to (a5) of SEQ ID NO: 2, as long as the above sequence identity is satisfied. For example, the proteins of this disclosure may contain an amino acid sequence in which one or more amino acids are deleted, substituted, or added to amino acid sequence A. In this case, the number of amino acids deleted, substituted, or added is not limited as long as the protein has the ability to bind to PET, and may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1. The positions of the deleted, substituted, or added amino acids may be any of the positions 1 to 12, 14, 16, 18 to 21, 23, 25 to 50, 52 to 68, and 70 to 101 from the N-terminus in SEQ ID NO: 2.

[0028] The proteins of this disclosure may have additional amino acid sequences at the N-terminus or C-terminus of the amino acid sequence corresponding to SEQ ID NO: 2. Examples of additional amino acid sequences include sequences for constructing the proteins of this disclosure by recombinant technology, and other functional sequences. Examples of such additional amino acid sequences include amino acids that serve as the starting point for protein synthesis (e.g., N-terminal methionine residues), N-terminal signal sequences (e.g., signal sequences for protein secretion in E. coli), tag sequences (e.g., histidine tags, GST tags, FLAG tags, etc.), protease recognition sequences (e.g., TEV protease recognition sequences), etc. Furthermore, the proteins of this disclosure may also contain functional substances and / or functional domains, such as labeling substances and / or enzyme domains, as described later.

[0029] The protein of this disclosure may consist of 91 to 1000 amino acids, 96 to 500 amino acids, or 101 to 300 amino acids.

[0030] The proteins of this disclosure have the ability to bind to PET. The conditions under which the proteins of this disclosure have the ability to bind to PET are not particularly limited; they only need to have the ability to bind to PET under any of the conditions. For example, the protein of this disclosure may have the ability to bind to PET under any of the following temperature conditions. The protein of this disclosure may have the ability to bind to PET at, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. Preferably, the protein of this disclosure may have the ability to bind to PET at least at 25°C, more preferably at 25°C to 40°C, even more preferably at 25°C to 50°C, and particularly preferably at a temperature range of 25°C to 60°C. Furthermore, for example, the protein of this disclosure may have the ability to bind to PET under any pH condition. The protein of this disclosure may have the ability to bind to PET at pH 4, 5, 6, 7, 8, 9, or 10. Preferably, the protein of this disclosure may have the ability to bind to PET at least pH 8, more preferably at least pH 6 to 8, even more preferably at least pH 5 to 9, and particularly preferably at least pH 4 to 10.

[0031] In this disclosure, the binding ability to PET is confirmed by the following method: A washed PET film or PET powder is mixed with a protein solution containing the test protein, and a binding reaction is carried out at a predetermined temperature for 1 hour. The supernatant is collected, and the amount of protein in the solution that has been reduced by binding to PET is calculated by measuring the decrease in absorbance at 280 nm derived from the protein. Furthermore, if the protein of this disclosure contains a labeling substance, the amount of protein in the solution may be calculated by detecting the labeling substance and creating a calibration curve. If the PET binding ability obtained by the above method exceeds the margin of error, it is determined that the protein has the ability to bind to PET.

[0032] For example, the amount of protein bound per 1 μM of enzyme and 1 mg of PET (substrate) after a reaction of the protein of this disclosure with a sufficient amount of PET at 25°C, pH 8, for 1 hour (nmol protein / mg substrate / μM enzyme) is preferably 0.3 nmol or more, more preferably 0.5 nmol or more, even more preferably 0.8 nmol or more, and particularly preferably 1.0 nmol or more. Here, "sufficient amount" means that the substrate concentration in the reaction system is sufficient for the protein. The same applies to the following descriptions.

[0033] The reaction of the protein disclosed herein with PET at 25°C, pH 8, and 1 hour results in B maxis preferably 0.070 nmol or more, more preferably 0.075 nmol or more, and even more preferably 0.080 nmol or more. The above B max has no particular upper limit and may be 0.500 nmol or less. Regarding the protein of the present disclosure, the K d obtained by reacting with PET at 25°C, pH 8 for 1 hour is preferably 5.50 mg / mL or less, more preferably 5.00 mg / mL or less, even more preferably 4.50 mg / mL or less, particularly preferably 4.00 mg / mL or less, and extremely preferably 3.50 mg / mL or less. The above K d has no particular lower limit and may be 1.00 mg / mL. Regarding the protein of the present disclosure, the B max / K d obtained by reacting with PET at 25°C, pH 8 for 1 hour is preferably 0.015 nmol·mL / mg or more, more preferably 0.020 nmol·mL / mg or more, even more preferably 0.025 nmol·mL / mg or more, and particularly preferably 0.030 nmol·mL / mg or more. The above B max / K d has no particular upper limit and may be 0.100 nmol·mL / mg. Note that B max represents the maximum adsorption amount, and K d represents the dissociation constant.

[0034] Regarding the protein of the present disclosure, the ratio of the PET adsorption amount to the chitin adsorption amount (PET adsorption amount / chitin adsorption amount) obtained by reacting with a sufficient amount of substrate at 25°C, pH 8 for 1 hour is preferably 20 or more, more preferably 30 or more, and even more preferably 40 or more.

[0035] Regarding the protein of the present disclosure, the ratio of the PET adsorption amount to the cellulose adsorption amount (PET adsorption amount / cellulose adsorption amount) obtained by reacting with a sufficient amount of substrate at 25°C, pH 8 for 1 hour is preferably 4 or more, more preferably 7 or more, and even more preferably 10 or more.

[0036] From the viewpoint of superior protein productivity, the protein of this disclosure is preferably water-soluble.

[0037] In one embodiment, the protein of the disclosure may further contain a labeling substance, which is a substance that serves as a label to make the protein of the disclosure detectable. When the protein of the disclosure contains a labeling substance, the protein of the disclosure bound to a PET becomes easily detectable. Examples of labeling substances include fluorescent dyes, fluorescent proteins, and alkynyl groups (for example, alkynyl groups as tags for Raman spectroscopy). Among these, fluorescent dyes or fluorescent proteins are preferred. Examples of fluorescent dyes include fluorescein, azo, rhodamine, coumarin, pyrene, and cyanine. Examples of fluorescent proteins include red fluorescent protein (RFP), orange fluorescent protein (mOrange), yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), and blue fluorescent protein (BFP). For example, stagRFP is a well-known red fluorescent protein (Nature Communications (2020) 11:1848).

[0038] In one embodiment, the protein of this disclosure may further include an enzyme domain. An "enzyme domain" is a domain having enzymatic activity. In this embodiment, the enzyme domain is a domain that contains any of the amino acid sequences (A1) to (A5) and is linked to a domain that has the ability to bind to PET (hereinafter also referred to as the "PET-binding domain"). In other words, the protein of this disclosure may be a protein in which an enzyme domain and a PET-binding domain are linked.

[0039] The type of enzyme in the enzyme domain is not particularly limited; for example, a PET-degrading enzyme (e.g., PET hydrolase) domain can be used. For example, if the protein of this disclosure is a protein in which a PET-degrading enzyme domain and a PET-binding domain are linked, the PET-binding domain ensures binding to PET, and the PET-degrading enzyme domain enables the degradation of PET, thus enabling efficient degradation of PET.

[0040] Various enzymes are known as PET-degrading enzymes, for example, International Publication No. 2019 / 168811; Japanese Patent Publication No. 2015-119670; International Publication No. 2012 / 099018; Japanese Patent Publication No. 2019-527060; Meilleur, C.; Hupe, JF; Juteau, P.; Shareck, F. Isolation and Characterization of a New Alkali-Thermostable Lipase Cloned from a Metagenomic Library. J. Ind. Microbiol. Biotechnol. 2009, 36, 853-61; Danso, D.; Schmeisser, C.; Chow, J.; Zimmermann, W.; Wei, R.; Leggewie, C.; Li, X.; Hazen, T.; Streit, WR New Insights into the Function and Global Distribution of Polyethylene Terephthalate Examples of enzymes include those described in (Pet)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes. Appl. Environ. Microbiol. 2018, 84, No. e02773-17.

[0041] The optimal pH (the pH at which activity is highest) for the PET hydrolysis activity of PET-degrading enzymes is not particularly limited, but it is practically preferable to be near neutral. For example, the optimal pH is preferably 5.0 to 9.5, more preferably 5.5 to 9.0, and may also be 6.0 to 8.0.

[0042] Since PET has a glass transition temperature of around 70°C, and it is expected that the decomposition efficiency of PET will increase when decomposed at high temperatures, the optimal temperature for the PET hydrolysis activity of PET-degrading enzymes is preferably above 60°C, and more preferably above 65°C, at pH 7. Furthermore, from the viewpoint of performing decomposition with low energy within a range where PET decomposition is promoted, the optimal temperature for the PET hydrolysis activity of PET-degrading enzymes may be 90°C or lower, 85°C or lower, or 80°C or lower. The above optimal temperature is measured as follows: Two amorphous PET discs (thickness 0.25 mm, total surface area 0.32 cm²) 2 To a total mass of 4.0 mg, add 250 μL of 0.1 μM protein in a 50 mM sodium phosphate solution (pH 7.0) and incubate for 60 minutes at each temperature. Analyze the degradation product concentrations by HPLC, and obtain the activity value by dividing the total degradation product concentrations by the protein concentration and reaction time.

[0043] Examples of degradation products of PET by hydrolysis include bis(hydroxyethyl) terephthalate (BHET), mono(hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG). The degradation products of PET by PET-degrading enzymes may be all of these, some of these, or partial degradation products formed by linking these products together.

[0044] The melting temperature (Tm) of the PET-degrading enzyme is preferably 70.0°C or higher, more preferably 72.0°C or higher, and even more preferably 74.0°C or higher. Since it is desirable to perform the degradation at a low energy level within the range where the degradation of PET is promoted, the melting temperature (Tm) of the PET-degrading enzyme may be 100.0°C or lower, 95.0°C or lower, or 90.0°C or lower. In this disclosure, the melting temperature (Tm) of a protein is measured by circular dichroism (CD) spectroscopy using the following procedure: Prepare a 13 μM protein solution in a 10 mM sodium phosphate solution (pH 7.0). Measure the CD spectrum of the protein at 250–200 nm at 20°C. Track the protein signal at 230 nm while heating the protein at a rate of 1°C / min from 20°C. Measure the CD spectrum of the denatured protein at 250–200 nm at 95°C. Estimate the Tm of the protein by curve fitting of the temperature dependence of the signal at 230 nm.

[0045] In one embodiment, the protein of the disclosure is preferably a polymer. By making the protein of the disclosure a polymer, its binding ability to PET can be increased. For example, the protein may be a 2-10-mer, a 3-8-mer, or a 4-6-mer. The polymer may be a homopolymer or a heteropolymer.

[0046] <Polynucleotides> The polynucleotides of this disclosure include a base sequence encoding the protein of this disclosure. The polynucleotide may be any polynucleotide encoding the protein of this disclosure. The nucleic acid sequence of the polynucleotide can be varied within the range of codon degeneracy. When a recombinant vector is introduced into a host to obtain a transformant and express the protein, it is preferable to use codons that are frequently used in the host.

[0047] <Recombinant Vectors> The recombinant vectors of this disclosure comprise the polynucleotides. The recombinant vectors may be any recombinant vectors capable of expressing the proteins of this disclosure. Examples of vectors include plasmid vectors; viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses; and Agrobacterium vectors.

[0048] The vector may contain various nucleic acid sequences, such as regulatory sequences including promoters, enhancers, and polyadenylation signals; restriction enzyme cleavage sites; replication start sites; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins.

[0049] Examples of promoters include the TEF promoter, TRPC promoter, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and GPD promoter.

[0050] Examples of restriction enzyme cleavage sites include AccI, BamHI, EcoRI, HincII, HindIII, I-SceI, KpnI, PstI, SacI, SalI, SmaI, SphI, Sse8387I, TaqI, XbaI, Esp3I, BsaI, SpeI, AscI, and PacI. The vector may also contain a multi-cloning site with multiple restriction enzyme cleavage sites.

[0051] Examples of drug resistance genes include chloramphenicol resistance genes, tetracycline resistance genes, neomycin resistance genes, erythromycin resistance genes, spectinomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, and puromycin resistance genes.

[0052] The vector may be obtained commercially or prepared. The vector can be prepared according to known genetic engineering techniques, using PCR, restriction enzyme cleavage, DNA ligation technology, in vitro transcription and translation technology, etc.

[0053] <Transformant> The transformant of the present disclosure contains the recombinant vector. The host is not particularly limited as long as it can express the protein of the present disclosure, and is selected in combination with the recombinant vector used. It is convenient to use microbial cells as the host, and for example, prokaryotes (e.g., Escherichia coli), eukaryotes (e.g., yeast), etc. may be used. When producing a protein using an expression vector and a host, methods well-known to those skilled in the art can be adopted for the conditions in transformation, expression, and recovery.

[0054] <Composition> The composition of the present disclosure contains the protein of the present disclosure. The protein of the present disclosure may be contained in the composition in an unpurified state (e.g., a state containing a host expressing the protein, or a state containing a crude protein solution secreted from a host expressing the protein), or in a purified state, and is preferably contained in the composition in a purified state.

[0055] In addition to the protein of the present disclosure, the composition of the present disclosure may contain a buffer solution suitable for protein stabilization, etc. As the buffer solution, a buffer solution that maintains the liquid property of the composition near neutrality is preferable, and examples thereof include phosphate buffer, sodium phosphate buffer, Tris-HCl buffer, HEPES, etc. The composition of the present disclosure may contain additives such as stabilizers in addition to the protein of the present disclosure and the buffer solution.

[0056] <Method for detecting PET> In one embodiment, there is provided a method for detecting PET, including contacting the protein of the present disclosure containing a labeling substance with PET and detecting the labeling substance by fluorescence. According to such a method, since the protein of the present disclosure binds to PET, PET can be efficiently detected by detecting the labeling substance. Details of the labeling substance are as described above.

[0057] <Method for decomposing PET> In one embodiment, there is provided a method for decomposing PET, including contacting the protein of the present disclosure containing a PET-degrading enzyme domain with PET. According to such a method, since the protein of the present disclosure binds to PET, PET can be efficiently decomposed. The temperature and reaction time for decomposing PET can be appropriately set within a range suitable for the decomposition of PET. Details of the PET-degrading enzyme domain are as described above. [Examples]

[0058] Next, embodiments of the present disclosure will be specifically described by way of examples, but the embodiments of the present disclosure are not limited to these examples.

[0059] [Reference Example 1] 1. Background and Objectives In nature, some carbohydrate-binding modules (CBMs) in cellulases and chitinases enhance the enzyme's ability to bind to crystalline cellulose and chitin, thereby improving their degradation activity. The CAZy (Carbohydrate-Active EnZymes) database classifies CBM families based on amino acid sequence homology, and currently there are 96 families. Furthermore, CBMs are classified into three types: Type A, which can bind to insoluble carbohydrates such as crystalline cellulose; Type B, which can bind to the interior of various glycans excluding insoluble substrates; and Type C, which can bind to the ends of glycans. In particular, Type A CBMs form a planar interaction surface because the aromatic residues exposed on the surface bind to the flat surfaces of crystalline cellulose and chitin. CBMs may have affinity for PET, and it has been suggested that the exposed aromatic amino acid residues of Type A CBMs play an important role in their interaction with PET. It is thought that the exposed aromatic amino acids of Type A CBMs can interact with the PET surface, just as they can with the flat surfaces of crystalline cellulose and chitin. Therefore, as a strategy to improve the enzyme's ability to bind to PET, an approach is being taken to fuse CBM with the enzyme.

[0060] The CBM2 family chitin-binding domain (PfChBD2) of the heat-stable chitinase derived from Pyrococcus furiosus is a type A CBM. PfChBD2 exhibits high heat resistance, maintaining its structure even at 85°C, which is higher than the glass transition temperature of PET, making it suitable as a template structure for PET-binding domains.

[0061] The inventors attempted to develop a PET-binding protein by artificially evolving PfChBD2 using saturation mutation introduction and phage display as a template. Saturation mutations were introduced into the PfChBD2 gene incorporated into a phage vector to construct a plasmid library. A phage library displaying the PfChBM2 mutant was created from the plasmid library using E. coli. Affinity selection (biopanning) with amorphous PET film fragments was performed on the phage library to create a PET-binding protein. As a result, the PET-binding mutant PfChBD2-K270H-N272P-E279V-D281G was discovered. As will be described later, the K270H, N272P, E279V, and D281G mutations are indicated by amino acid loci relative to the chitinase from which PfChBD2 originates, and correspond to K13H, N15P, E22V, and D24G of PfChBD2, respectively. The amino acid sequence of the PET adsorption domain of wild-type PfChBD2 is shown in Sequence ID No. 1, and the amino acid sequence of the PET adsorption domain of the PfChBD2-K270H-N272P-E279V-D281G mutant is shown in Sequence ID No. 2.

[0062] 2. Gene generation of stagRFP-PfChBD2 To confirm the binding ability to amorphous PET of the three most abundant generations of PfChBD2-K270H-N272P-E279V-D281G mutants obtained by biopanning, an expression system was constructed using E. coli as the host. The pET27b vector, containing a kanamycin resistance gene and a His tag, was used as the protein expression vector. Using PCR and ligation reactions, the WT and mutant genes of PfChBD2 were inserted into plasmid DNA containing the fluorescent protein stagRFP and a TEV protease recognition sequence in pET27b. stagRFP is a red fluorescent protein with approximately twice the fluorescence intensity of tagRFP-T and a maximum absorption wavelength of 555 nm. The ligated plasmids were transformed into E. coli Tuner (DE3) strain. Plasmids were extracted from the cultured E. coli, and sequencing analysis confirmed the acquisition of the target gene.

[0063] (1) PCR for generating the stagRFP-PfChBD2 gene <Method> Primers were designed to insert the PfChBD2 gene into pET27b plasmid DNA containing the stagRFP gene and TEV protease recognition sequence (Table 1). The pET27b plasmid DNA containing the stagRFP gene and TEV protease recognition sequence, along with the WT and K270H-N272P-E279V-D281G mutant DNA of PfChBD2, were used as template DNA. PCR solutions were prepared using the template DNA and primers according to Table 2. PCR reactions were performed in a thermal cycler at 98°C for 10 seconds and 68°C for 40 seconds for 30 cycles. Loading buffer was added to the PCR reaction samples, and the samples were applied to 2% and 1% agarose gels, respectively, and electrophoresis was performed at 100V for 25 minutes. The gel was stained with MIDORI Green Xtra and the band size was confirmed. All bands were cut out from the gel and placed in microcentrifuge tubes. Gel extraction was performed using the Wizard® SV Gel and PCR Clean-Up System. The DNA concentration of the purified DNA sample was measured using a spectrophotometer.

[0064] [Table 1]

[0065] [Table 2]

[0066] <Result> The expected DNA sizes were 6085 bp for the pET27b plasmid DNA containing the stagRFP gene and the TEV protease recognition sequence, and 328 bp for PfChBD2. Bands were observed around 6 kbp and 300 bp on the gel, indicating amplification of the target gene. Gel extraction yielded 25 μL each of DNA samples with DNA concentrations of 44.7 ng / μL from the stagRFP gene, 161 ng / μL from the WT of PfChBD2, and 50 ng / μL from PfChBD2-K270H-N272P-E279V-D281G.

[0067] (2) Ligation and transformation of the stagRFP gene and the PfChBD2 gene <Method> 1 μL each of the stagRFP gene and PfChBD2 DNA samples obtained in (1) were added to 2 μL of NEBuilder HiFi DNA Assembly Master Mix, for a total of 4 μL. This mixture was incubated in a thermal cycler at 50°C for 1 hour for ligation. 0.8 μL of the ligation reaction mixture was mixed with 50 μL of electrocompetent cell Tuner(DE3) strain, and the mixture was placed in an electroporation cuvette. The Tuner(DE3) strain is a lacZY deletion mutant that can induce IPTG (isopropyl β-D-thiogalactopyranoside) concentration-dependent protein expression. Electroporation was performed, and 200 μL of SOC medium was added to suspend the cells. The suspension was collected in a microtube and incubated at 37°C at 1,000 rpm for 1 hour. The suspension was inoculated onto LB agar medium supplemented with 50 μg / mL kanamycin and incubated at 37°C for 1 day. One colony was selected from the agar plates and inoculated into LB liquid medium supplemented with 50 μg / mL of kanamycin and into agar plates, respectively. The cultures were incubated at 37°C and 300 rpm for 1 day. The tubes containing the bacterial cultures were collected and centrifuged at 6,000 g for 3 minutes. The supernatant was removed, and plasmid extraction was performed from the precipitated E. coli using the QIA prep Spin Miniprep Kit. The DNA concentration of the purified plasmids was measured using a spectrophotometer, and the plasmid DNA samples were sequenced.

[0068] <Result> Single colonies were formed on LB agar medium supplemented with 50 μg / mL kanamycin. 50 μL each of plasmid DNA samples were obtained: one with PfChBD2(WT) inserted at a DNA concentration of 30 ng / μL, and another with PfChBD2-K270H-N272P-E279V-D281G inserted at a DNA concentration of 260 ng / μL. Sequence analysis confirmed that the PfChBD2 gene was accurately inserted. The obtained plasmid DNAs are referred to as stagRFP-PfChBD2-TEV-His and stagRFP-PfChBD2-K270H-N272P-E279V-D281G-TEV-His.

[0069] 3. Expression and purification of stagRFP-PfChBD2 To perform binding experiments to amorphous PET using the wild-type PfChBD2 (WT) and the K270H-N272P-E279V-D281G mutant, we prepared WT PfChBD2 and the K270H-N272P-E279V-D281G mutant with the fluorescent protein stagRFP fused to its N-terminus. E. coli Tuner (DE3) strains incorporating stagRFP-PfChBD2-TEV-His and stagRFP-PfChBD2-K270H-N272P-E279V-D281G-TEV-His were cultured in liquid medium, and the target protein was produced by inducing protein expression using IPTG. The produced protein was then purified using affinity chromatography and gel filtration chromatography.

[0070] (1) Expression of stagRFP-PfChBD2 and stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutants <Method> Colonies of stagRFP-PfChBD2-TEV-His and stagRFP-PfChBD2-K270H-N272P-E279V-D281G-TEV-His, grown on agar plates, were inoculated into 10 mL of LB medium supplemented with 50 μg / mL kanamycin, respectively, and incubated at 37°C and 300 rpm for 1 day. The cultured bacterial broths were then inoculated into 700 mL of Super Broth medium supplemented with 50 μg / mL kanamycin, respectively, and incubated at 37°C and 130 rpm for 3 hours. After incubation, the media were cooled in ice water for 30 minutes. 350 μL of 1 M IPTG was added to each of the 700 mL media, and incubated at 20°C and 130 rpm for 1 day. Each medium was centrifuged at 3,000 g for 10 minutes, and the precipitate was collected. The precipitate was stored frozen at -80°C.

[0071] <Result> 6.1 g of precipitate of stagRFP-PfChBD2-TEV-His and 3.0 g of precipitate of stagRFP-PfChBD2-K270H-N272P-E279V-D281G-TEV-His were obtained. Both recovered precipitates were red in color.

[0072] (2) Purification by affinity chromatography <Method> 10 mL of 50 mM sodium phosphate and 100 mM sodium chloride buffer (Buffer 1) was added to 10 g of the precipitate obtained in (1), and the precipitate was broken up using an ultrasonic lithograph for 20 minutes. The lithified solution was centrifuged at 20,000 g for 15 minutes, and the supernatant was collected. The supernatant was loaded onto a 3 mL Ni-NTA column, and the flow-through (FT) was collected. 130 mL of buffer was passed through the column, and 3-6 mL of the flow-through was collected. Subsequently, 30 mL of 50 mM imidazole, 50 mM sodium phosphate, and 100 mM sodium chloride buffer were passed through the column, and the same procedure was performed. After that, 30 mL of 100 mM imidazole, 50 mM sodium phosphate, and 100 mM sodium chloride buffer were passed through the column, and 3 mL of each was collected in a test tube. The absorbance of each was measured, and the band size of those showing absorbance was confirmed by SDS-PAGE. Fractions showing a band were collected in VIVASPIN20 (30,000MWCO) and concentrated to 700 μL. The concentrated protein solution was replaced with Buffer 1 using illustra NAP-10 Columns to obtain 1.4 mL of protein solution. Then, 80 μL of 3 mg / mL TEV protease was added and incubated at 16°C for 1 day. The protein solution was passed through a 0.5 mL Ni-NTA column. The sample that flowed out was passed back into the column and collected. Buffer 1 was passed through in 500 μL increments, and the protein solution that flowed out was collected from each increment. This procedure was repeated until a total of 3 mL of Buffer 1 was passed through. The absorbance of each fraction was measured, and those showing absorbance at a wavelength of 280 nm were collected in VIVASPIN20 (30,000MWCO) and concentrated by centrifugation until the protein solution was 500 μL or less.

[0073] <Result> The absorbance of each fraction obtained by affinity chromatography was measured. The expected band size was 40 kDa. From the SDS-PAGE results, a band was observed between 35 kDa and 45 kDa, indicating that the target protein was present in fractions No. 2 to 10. After loading the TEV protease-treated protein solution onto a Ni-NTA column, each fraction was collected using buffer 1 and its absorbance was measured. 0.5 mL of concentrated protein solution was obtained from fraction No. 17 of the WT protein and fraction No. 14 of the K270H-N272P-E279V-D281G mutant.

[0074] (3) Purification by gel filtration chromatography <Method> Gel filtration chromatography was performed using a Superdex 75 Increase 10 / 300GL column. The protein solutions obtained in (2), concentrated to less than 500 μL, were loaded onto the column. Purification was performed using 50 mM sodium phosphate buffer (pH 8) at a flow rate of 0.5 ml / min and 0.5 mL per fraction. After purification, the band size of the absorbable fractions was confirmed by SDS-PAGE. The fractions in which bands were confirmed were collected in VIVASPIN20 (30,000 MWCO) and concentrated to less than 1 mL. The absorbance at 280 nm relative to 340 nm of the concentrated protein solution was measured using a spectrophotometer. The measured absorbance and molar extinction coefficient (54,860 M) were used. -1 cm -1 The protein concentration was determined from the following:

[0075] <Result> The expected band size is 40 kDa. SDS-PAGE confirmed the presence of a band between 35 kDa and 45 kDa, indicating that the target protein had been purified. Fractions No. 5-19 were recovered from stagRFP-PfChBD2, and fractions No. 3-16 were recovered from stagRFP-PfChBD2-K270H-N272P-E279V-D281G, and were concentrated. Approximately 800 μL each of stagRFP-PfChBD2 with a protein concentration of 40.9 μM and stagRFP-PfChBD2-K270H-N272P-E279V-D281G with a protein concentration of 25.9 μM were obtained.

[0076] 4. Measurement of binding amount to PET To confirm the PET binding ability of PfChBD2(WT) and PfChBD2-K270H-N272P-E279V-D281G, purified proteins were incubated with amorphous PET. After centrifugation, the amount of protein in the supernatant was measured from the absorbance at the maximum absorption wavelength of 555 nm of the fluorescent protein stagRFP fused to the PfChBD2 contained in the supernatant. The amount of protein bound per 1 mg of substrate was also determined by measuring the absorbance of the substrate alone and the protein alone. Protein quantification using the red fluorescent protein stagRFP was performed because the amorphous PET substrate showed absorption at 280 nm, which presented problems with measurement accuracy.

[0077] (1) Creation of a calibration curve for stagRFP-PfChBD2 <Method> Purified stagRFP-PfChBD2 was mixed with 50 mM sodium phosphate buffer (pH 8) to final concentrations of 0.5 μM, 1 μM, and 1.5 μM. Absorbance was measured, and a calibration curve was created from the results. In subsequent experiments, protein concentrations were measured using this calibration curve.

[0078] (2) Measurement of binding ability of stagRFP-PfChBD2 and stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutant <Method> Solutions were prepared using purified stagRFP-PfChBD2 and stagRFP-PfChBD2-K270H-N272P-E279V-D281G and substrates according to Table 3. Amorphous PET was used as the substrate. Similarly, solutions containing only the substrate and only the protein were also prepared. Three samples of each solution were prepared. The prepared solutions were incubated at 25°C for 1 hour. The substrate was precipitated by centrifugation, and the absorbance of the supernatant was measured.

[0079] [Table 3]

[0080] <Result> Based on the absorbance measurements, the concentrations of stagRFP-PfChBD2 and stagRFP-PfChBD2-K270H-N272P-E279V-D281G in each solution were determined using the calibration curve obtained in (1). The amount of protein bound to the substrate (nmol) was determined by taking the difference in protein concentration between the solution containing both the substrate and protein and the solution containing only the substrate. The binding amount of stagRFP-PfChBD2(WT) to amorphous PET was 0.3 (±0.64) nmol / mg, while that of the stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutant was 3.87 (±0.86) nmol / mg (Figure 1).

[0081] <Consideration> Binding experiments revealed that stagRFP-PfChBD2(WT) hardly bound to amorphous PET. However, stagRFP-PfChBD2-K270H-N272P-E279V-D281G, which uses three generations of PfChBD2 mutants obtained by biopanning, acquired the ability to bind to amorphous PET.

[0082] The table below shows the amino acid sequence (SEQ ID NO: 12) and gene sequence (SEQ ID NO: 13) of the stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutant. In the table below, the N-terminus (amino acids 1-244, bases 1-732) represents the red fluorescent protein stagRFP. The shaded area (amino acids 245-247, bases 733-741) represents the linker sequence. The bold and underlined C-terminus (amino acids 248-348, bases 742-1044) represents the PfChBD2 mutant. Boxed text indicates mutation points.

[0083] [Table 4]

[0084] The table below shows the mutation sites in the stagRFP-PfChBD2-K270H-N272P-E279V-D281G mutant. In this reference example, the mutant amino acid numbers (270, 272, 279, and 281) in the K270H-N272P-E279V-D281G mutant are shown in terms of amino acid loci relative to the chitinase from which PfChBD2 originates. PfChBD2 is the C-terminal domain of the chitinase, and the number of amino acids at the N-terminus differs between stagRFP-PfChBD2-K270H-N272P-E279V-D281G and the chitinase. Therefore, the amino acid numbers 270, 272, 279, and 281 mentioned above correspond to the 260th, 262nd, 269th, and 271st amino acids from the N-terminus in stagRFP-PfChBD2-K270H-N272P-E279V-D281G, respectively.

[0085] [Table 5]

[0086] In the above amino acid sequence, each mutation in the K270H-N272P-E279V-D281G mutant (i.e., the mutations at positions 260, 262, 269, and 271 from the N-terminus of Sequence ID No. 12) corresponds to the K13H, N15P, E22V, and D24G mutations in PfChBD2, respectively.

[0087] [Example 1] 1. Purpose We developed a PET-binding protein using the PET-binding mutant PfChBD2-K270H-N272P-E279V-D281G (hereinafter also referred to as "4M") of the chitin-binding domain (PfChBD2) of thermostable chitinase from Pyrococcus furiosus as a template protein. PfChBD2-4M is a mutant that has improved PET adsorption capacity compared to the wild type and decreased affinity for the original substrate, chitin. However, the remaining adsorption capacity to chitin was a challenge. Therefore, we aimed to create a protein that reduces chitin adsorption capacity and adsorbs to PET, and performed mutation introduction and screening.

[0088] 2. Construction of the PfChBD2-4M-W274X-W308X-W326X plasmid library Here, we used saturated mutagenesis to randomly introduce mutations into the codons W274, W308, and W326 of the PfChBD2 gene, creating a saturated mutant library of PfChBD2-4M-W274X-W308X-W326X. In saturated mutagenesis, primers are designed to introduce random mutations into the codons corresponding to the amino acid loci to be mutated, and then PCR is performed. This PCR allows for the random generation of codons corresponding to the target amino acid loci. In primer design, we used codon NNK to introduce random mutations into the codons. N represents any base from A, T, G, and C, and K represents any base from G and T, and these bases are synthesized randomly. By using codon NNK, the variability in the ratio of encoded amino acids is suppressed, and all 20 types of amino acids can be covered. In addition, the probability of stop codon generation can be suppressed to 3.125%.

[0089] fADL-1e was used as a plasmid for phage expression that displays PfChBD2-4M-W274X-W308X-W326X on its surface. fADL-1e is a phage vector, and when fADL-1e, with the target gene inserted upstream of the g3 protein gene, is transformed into E. coli (SS320 strain), it is possible to express M13 phages that display the target protein on the g3 protein surface. fADL-1e incorporates a kanamycin resistance gene as a selection marker. In addition, a trypsin cleavage site was inserted between the target gene and the g3 protein gene to recover phages adsorbed to PET. All phage handling procedures in this study were performed using a safety cabinet.

[0090] (1) Saturation mutation introduction into W274 of PfChBD-4M_fADL-1e <Method> PfChBD2-4M_fADL-1e as template DNA (1 ng μL) -11 μL of 10 μM primer mix was mixed with 0.75 μL of 10 μM primer mix, and ultrapure water was added to make a total volume of 12.5 μL. 12.5 μL of 2× KOD One PCR Master mix was added to the mixture, and the PCR reaction was performed on a thermal cycler under the conditions of 98°C for 10 seconds and 68°C for 40 seconds for 30 cycles. The primers used are shown in Table 6. Loading buffer was added to the PCR reaction sample, applied to a 1% agarose gel, and electrophoresis was performed at 100 V for 25 minutes. The gel was stained with MIDORI Green Xtra to confirm the band size, and all bands were cut out from the gel and placed in microcentrifuge tubes. Gel extraction was performed using the Wizard SV Gel and PCR Clean-Up System. The DNA concentration of the purified DNA sample was measured.

[0091] <Result> The expected DNA size is 8318 bp. A band was observed around 8 kbp on the gel, indicating that the target DNA was amplified. After gel extraction, 25 μL of DNA sample with a DNA concentration of 36.1 ng / μL was obtained. This obtained DNA sample will be designated as PfChBD2-4M-W274X.

[0092] [Table 6]

[0093] (2) Ligation and transformation of PfChBD-4M-W274X_fADL-1e <Method> Equal volumes of NEBuilder HiFi DNA Assembly Master Mix were added to the DNA sample obtained in 2(1), and the mixture was incubated in a thermal cycler at 50°C for 1 hour for ligation. 0.8 μL of the ligation reaction solution was mixed with 50 μL of electrocompetent cells (DH10B strain), and the mixture was placed in an electroporation cuvette. Electroporation was performed, and 200 μL of SOC medium was added to suspend the cells. The suspension was collected in a microtube and incubated at 37°C at 1000 rpm for 1 hour. The suspension was inoculated onto LB agar medium supplemented with 50 μg / mL kanamycin and incubated at 37°C for 1 day. These steps were repeated to obtain three agar plates. After counting the colonies that grew on the agar plates, all colonies were collected using a platinum loop into 10 mL of liquid LB medium supplemented with 50 μg / mL kanamycin, and incubated overnight at 37°C at 1000 rpm. 3 mL was collected from each of the three liquid mediums. After centrifugation at 6000g for 3 minutes, the supernatant was removed, and plasmid extraction was performed from the precipitated E. coli using the Wizard Plus SV Minipreps DNA Purification System. The DNA concentration of the purified plasmid was measured.

[0094] <Result> The total number of colonies that grew on three LB agar plates supplemented with 50 μg / mL kanamycin was 3,945. Plasmid extraction yielded a plasmid library of 50 μL with a DNA concentration of 56.8 ng / μL. This plasmid library was designated PfChBD2-4M-W274X_fADL-1e. The estimated coverage of the plasmid library PfChBD2-4M-W274X_fADL-1e, which was created by a single mutation, was 100%.

[0095] (3) Saturation mutation introduction of PfChBD-4M-W274X_fADL-1e to W308 <Method> Using PfChBD2-4M-W274X_fADL-1e as the template DNA, PCR was performed as in 2(1), followed by agarose gel electrophoresis and gel extraction. The DNA concentration of the purified DNA sample was measured. The primers used are shown in Table 6.

[0096] <Result> The expected DNA size is 8318 bp. A band was observed around 8 kbp on the gel, indicating that the target DNA was amplified. After gel extraction, 25 μL of DNA sample with a DNA concentration of 42.9 ng / μL was obtained. This obtained DNA sample will be designated as PfChBD2-4M-W274X-W308X.

[0097] (4) Ligation and transformation of PfChBD-4M-W274X-W308X_fADL-1e <Method> Using the DNA samples obtained in 2(3), ligation and transformation were performed in the same manner as in 2(2). These steps were repeated to produce 10 agar plates. After counting the colonies that grew on the agar plates, all colonies were collected using a platinum loop into 10 mL of liquid LB medium supplemented with 50 μg / mL kanamycin, and incubated overnight at 37°C and 1000 rpm. 1 mL was collected from each of the 10 liquid medium plates. Plasmid extraction and DNA concentration measurement were then performed in the same manner as in 2(2).

[0098] <Result> A total of 4,104 colonies were observed on 10 sheets of LB agar medium supplemented with 50 μg / mL kanamycin. Plasmid extraction yielded a plasmid library of 50 μL with a DNA concentration of 91.9 ng / μL. This plasmid library was designated PfChBD2-4M-W274X-W308X_fADL-1e. The estimated coverage of the plasmid library PfChBD2-4M-W274X-W308X_fADL-1e, which was created by two mutations, was 98%.

[0099] (5) Saturation mutation introduction into W326 of PfChBD-4M-W274X-W308X_fADL-1e <Method> Using PfChBD2-4M-W274X-W308X_fADL-1e as the template DNA, PCR was performed as in 2(1), followed by agarose gel electrophoresis and gel extraction. The DNA concentration of the purified DNA sample was measured. The primers used are shown in Table 6.

[0100] <Result> The expected DNA size is 8318 bp. A band was observed around 8 kbp on the gel, indicating that the target DNA was amplified. After gel extraction, 25 μL of DNA sample with a DNA concentration of 31 ng / μL was obtained. This obtained DNA sample will be designated as PfChBD2-4M-W274X-W308X-W326X.

[0101] (6) Ligation and transformation of PfChBD-4M-W274X-W308X-W326X_fADL-1e <Method> Using the DNA samples obtained in 2(5), ligation and transformation were performed in the same manner as in 2(2). These procedures were repeated to produce 20 agar plates. After counting the colonies that grew on the agar plates, all colonies were collected using a platinum loop into 10 mL of liquid LB medium supplemented with 50 μg / mL kanamycin, and incubated overnight at 37°C and 1000 rpm. 1 mL was collected from each of the 20 liquid medium plates. Plasmid extraction and DNA concentration measurement were then performed in the same manner as in 2(2).

[0102] <Result> The total number of colonies that grew on 20 sheets of LB agar medium supplemented with 50 μg / mL kanamycin was 24,366. Plasmid extraction yielded a plasmid library of 50 μL with a DNA concentration of 56.7 ng / μL. This plasmid library was designated PfChBD2-4M-W274X-W308X-W326X_fADL-1e. The estimated coverage of the plasmid library PfChBD2-4M-W274X-W308X-W326X_fADL-1e, which was created by three mutations, was 52%.

[0103] 3. Affinity selection using phage display method To select PfChBD2 mutants that bind to PET using phage display, a phage display library was constructed by expressing and purifying phages that displayed the PfChBD2 mutant on their surface. Biopanning was performed using the obtained phage display library and PET film. In biopanning, the cycle of adsorption, washing, recovery, and growth on amorphous PET film pieces was repeated, varying the washing intensity and temperature, to select phages that adsorbed to PET. The SS320 strain was used as the host for phage expression. The SS320 strain possesses the F factor and is a suitable E. coli strain for constructing a phage display library.

[0104] (1) Expression and purification of the phage display library (0th generation) <Method> 0.5 μL of the PfChBD2-4M-W274X-W308X-W326X_fADL-1e plasmid library was mixed with 50 μL of electrocompetent cells (SS320 strain), and the mixture was placed in an electroporation cuvette. Electroporation was performed, and 200 μL of SOC medium was added to suspend the mixture. The suspension was collected in a microtube and incubated at 37°C and 1000 rpm for 1 hour. The suspension was inoculated into 2×YT liquid medium supplemented with 50 μg / mL of kanamycin and incubated at 37°C and 300 rpm for 1 day.

[0105] 10 mL of bacterial culture was centrifuged at 13,000 g for 5 minutes to precipitate E. coli. The supernatant was transferred to a new tube, and 5×PEG / NaCl was added to the supernatant in a 4:1 ratio. The mixture was mixed by inversion, and the tube was incubated on ice for 1 hour. The phage was precipitated by centrifuging at 13,000 g for 10 minutes. The supernatant was removed, and after centrifugation, the supernatant was completely removed. 120 μL of Tris Buffer Saline: 50 mM Tris-HCl pH 7.5 was added to the precipitated phage, resuspended by vortexing, and incubated on ice for 1 hour. The phage solution was collected by vortexing again and centrifugation at 13,000 g for 1 minute.

[0106] A blank was taken using 1×TBS buffer, and the absorbance at 269 nm and 320 nm was measured. The number of virions in the phage solution was calculated using the following formula, with the length of the phage vector + insert (8303 bp) as the number of bases per virion.

[0107]

number

[0108] <Result> Table 7 shows the absorbance of the phage solution after PEG precipitation. The virion number of the phage can be calculated from the difference in absorbance between 269 nm and 320 nm to 1930 × 10⁶. 10 It was found that 120 μL of phage solution with a virions / mL concentration was obtained. The phage obtained here was designated PfChBD2-4M-W274X-W308X-W326X-gene0.

[0109] [Table 7]

[0110] (2) Preparation of bacterial cells (SS320 strain) <Method> SS320 was incubated in 2×YT liquid medium at 37°C and 250 rpm for 1 day. A mixture of culture medium and fresh 2×YT liquid medium was prepared in a 1:10 ratio and incubated at 37°C and 250 rpm for 1 hour. Absorbance at 600 nm was measured and adjusted to an absorbance of 0.5–1.0. Phages were infected within 1 hour at room temperature.

[0111] (3) Biopanning <Method> In a 1.5 mL tube, the PfChBD2-4M-W274X-W308X-W326X-gene0 phage obtained in 1 M sodium phosphate buffer pH 8, 3(1), and ultrapure water were added to achieve the composition shown in Table 8. As the amorphous PET film, a 0.25 mm thick Amorphous PET film from Goodfellow was cut into 5.5 mm diameter discs, washed in 20% ethanol at 20 rpm for 1 hour, rinsed with 20% ethanol, and dried.

[0112] [Table 8]

[0113] In the binding step, the solution containing the prepared amorphous PET film was incubated at 25°C at 1,000 rpm for 1 hour to bind the phage to the amorphous PET film. The supernatant was then transferred to a new tube and stored. In the washing step, 500 μL of 50 mM sodium phosphate buffer was added for washing, and the solution was incubated at 25°C at 1,000 rpm for 5 minutes. The washing solution was transferred to a new tube and stored. One round consisted of 5 washings. In the elution step, 500 μL of 0.25% trypsin solution was added to eluate the phage bound to the PET film, and the solution was incubated at 37°C at 1,000 rpm for 20 minutes. The eluate containing the phage was stored in a new tube. Trypsin treatment was also performed on the supernatant and washing solution by adding trypsin to a final concentration of 0.25%.

[0114] In the infection and amplification step, equal volumes of the SS320 culture medium obtained in 3(2) were added to the supernatant, washing solution, and phage eluate, and the phages were infected with Escherichia coli (SS320 strain) at 37°C and 1000 rpm for 30 minutes. After centrifugation at 13000 g for 5 minutes, the supernatant was removed so that the final volume was 200 μL. After suspending the precipitated Escherichia coli, it was inoculated onto 2×YT agar medium supplemented with 50 μg / mL kanamycin and incubated at 37°C for 1 day. Colonies that grew on the agar medium inoculated with the phage eluate and the infected Escherichia coli were all collected using a platinum loop and placed in 2×YT liquid medium supplemented with 50 μg / mL kanamycin and incubated at 37°C and 300 rpm for 1 day. The bacterial culture medium was centrifuged at 13,000 g for 2 minutes to precipitate the Escherichia coli. The phages were purified by PEG precipitation using the supernatant as in 3(1). Plasmid extraction was also performed using the precipitate. The phage display libraries and plasmid libraries obtained from these methods were saved as a new generation.

[0115] The four steps up to this point constituted one round, and a total of three rounds were performed. In the second round, the same procedure was repeated using the new generation obtained in the previous round, with the number of washes increased to 10. In the next third round, the same procedure as in the second round was performed, but the incubation temperature conditions in the binding and washing steps were changed from 25°C to 60°C.

[0116] <Result> The absorbance of phages obtained by PEG precipitation in rounds 1, 2, and 3 was measured. The number of virions in the phage solution obtained in each round was determined by calculating the number of bases per virion based on the length of the phage vector + insert (8303 bp) from the difference in absorbance at 269 nm and 320 nm (Table 7). It was confirmed that phages were obtained in each round. In addition, plasmid DNA was obtained from plasmid extraction performed in parallel.

[0117] When examining the colony formation in rounds 2 and 3 of biopanning, more colonies were observed from the phage solution in the elution step than from the washing solution in the 10th washing step. This suggests that biopanning selects phages that adsorb to PET. In round 3, 425 colonies were formed when E. coli infected with the eluted phages were inoculated onto 2×YT agar medium supplemented with 50 μg / mL kanamycin. After collecting the resulting colonies and culturing them, the obtained phages and plasmid DNA were divided into three generations and named PfChBD2-4M-W274X-W308X-W326X-gene3.

[0118] 4. Soluble Selection Proteins selected by affinity screening using phage display may contain mutants that are difficult to purify as soluble proteins. Soluble selection aimed to select highly soluble proteins.

[0119] The gene portion of the PfChBD2 mutant was amplified by PCR from the PfChBD2-4M-W274X-W308X-W326X-gene3 plasmid DNA obtained by affinity selection using phage display. Using PCR and ligation reactions, the gene of the PfChBD2 mutant was inserted into plasmid DNA in which the fluorescent protein stagRFP and a TEV recognition sequence were inserted into pET27b. StagRFP is a red fluorescent protein with a maximum absorption wavelength of 555 nm and a maximum fluorescence wavelength of 579 nm. The ligated plasmid was transformed into E. coli Tuner (DE3) strain. After protein expression, protein extraction was performed, and the solubility of the protein was evaluated by fluorescence measurement using the fluorescence of stagRFP in the extract.

[0120] (1) Creation of the stagRFP-PfChBD2-4M-W274X-W308X-W326X library <Method> Primers were designed to insert the PfChBD2 mutant gene into the pET27b plasmid DNA containing the stagRFP gene and TEV recognition sequence (Table 9). The pET27b plasmid DNA containing the stagRFP gene and TEV recognition sequence and the PfChBD2-4M-W274X-W308X-W326X-gene3 plasmid DNA were used as template DNA. Using the template DNA and primers, PCR solutions were prepared according to Table 10. PCR reactions were performed in a thermal cycler under conditions of 98°C for 10 seconds and 68°C for 40 seconds for 30 cycles. Loading buffer was added to the PCR reaction sample and applied to 2% and 1% agarose gels, respectively, and electrophoresis was performed at 100V for 25 minutes. The gels were stained with MIDORI Green Xtra and the band size was confirmed. All bands were cut from the gel and placed in microtubes. Gel extraction was performed using the Wizard SV Gel and PCR Clean-Up System. The DNA concentration of the purified DNA sample was measured using an absorbance meter.

[0121] 1 μL each of the two obtained DNA samples was added to 2 μL of NEBuilder HiFi DNA Assembly Master Mix (total 4 μL), and the mixture was incubated in a thermal cycler at 50°C for 1 hour for ligation. Using the ligation reaction mixture as template DNA, PCR solutions were prepared according to Table 10, and the PCR reaction was performed. The primers used are shown in Table 9. Loading Buffer was added to the PCR reaction mixture samples, applied to a 1% agarose gel, and electrophoresis was performed at 100V for 25 minutes. The gel was stained with MIDORI Green Xtra, and the band size was confirmed. All bands were excised from the gel, and gel extraction was performed. The DNA concentration of the purified DNA samples was measured using a spectrophotometer. An equal volume of NEBuilder HiFi DNA Assembly Master Mix was added to the DNA samples obtained here, and the mixture was incubated in a thermal cycler at 50°C for 1 hour for ligation.

[0122] [Table 9]

[0123] [Table 10]

[0124] <Result> The expected DNA sizes were 6,085 bp for the stagRFP gene and the pET27b plasmid DNA with the TEV recognition sequence inserted, and 328 bp for the PfChBD2 mutant. Bands were observed on the gel at approximately 6 kbp and 300 bp, indicating amplification of the target gene. Gel extraction yielded 25 μL each of DNA samples with DNA concentrations of 40.3 ng / μL from the stagRFP gene and 91.4 ng / μL from the PfChBD2 mutant gene.

[0125] After the ligation reaction, a PCR reaction was performed again, and the expected DNA size of the PCR product was 6398 bp. Since bands were observed around 6 kbp on the gel, it was determined that the target gene had been amplified. Gel extraction yielded 25 μL of a 42 ng / μL DNA sample. The obtained ligation reaction mixture was then used as the stagRFP-PfChBD2-4M-W274X-W308X-W326X plasmid library.

[0126] (2) Soluble selection (1 mL scale) <Method> 0.8 μL of the stagRFP-PfChBD2-4M-W274X-W308X-W326X plasmid library was mixed with 50 μL of electrocompetent cell Tuner(DE3) strain, and the mixture was placed in an electroporation cuvette. The Tuner(DE3) strain is a lacZY deletion mutant that can induce IPTG concentration-dependent protein expression. Electroporation was performed, and the mixture was suspended in 200 μL of SOC medium. The suspension was collected in a microtube and incubated at 37°C at 1000 rpm for 1 hour. The suspension was inoculated onto LB agar medium supplemented with 50 μg / mL kanamycin and incubated at 37°C for 1 day. Colonies that formed on the agar medium were selected one by one and inoculated onto 1 mL of Super Broth liquid medium supplemented with 50 μg / mL kanamycin, which was dispensed onto a 96-well plate. After culturing at 37°C and 1000 rpm for 3.5 hours, 10 μL of 100 mM IPTG was added, and the mixture was incubated overnight at 37°C and 1000 rpm. In addition, the cells were inoculated into LB agar medium containing 50 μg / mL kanamycin at the same time as inoculating into liquid medium, and incubated at 37°C for 1 day.

[0127] A 96-well plate was centrifuged at 3100 g for 10 minutes, and all of the supernatant of the cell culture medium was removed. 200 μL of Bug Buster Protein Extraction Reagent was added to the precipitate, and the plate was incubated at 1000 rpm for 20 minutes. The 96-well plate was centrifuged at 4°C and 3100 g for 25 minutes, and 100 μL of the supernatant was collected in a black 96-well microplate, taking care not to touch the precipitate. Fluorescence measurements of the protein extract were performed using a microplate reader (Infinite 200 PRO M Nano+). The fluorescence measurement conditions are shown in Table 11. Mutants showing fluorescence intensity of 3000 or higher were selected. This soluble selection was performed on 456 colonies. The selected mutants were inoculated from the cells grown on LB agar medium into LB liquid medium supplemented with 10 mL of kanamycin 50 μg / mL. After incubation overnight at 37°C and 1000 rpm, plasmid extraction was performed using the same procedure as in 2(2). To confirm the mutation, purified plasmid DNA samples were sequenced. Eurofins Genomics' DNA sequencing service was used for the sequencing analysis.

[0128] [Table 11]

[0129] <Result> By culturing the cells on a 96-well plate and inducing protein expression, most of the bacterial cells turned red, confirming that protein expression was occurring. Fluorescence measurements revealed 11 mutants with fluorescence intensity above 3000. Table 12 shows the results of sequence analysis of these mutants. Hereafter, PfChBD2-4M-W274X1-W308X2-W326X3 will be denoted as X1X2X3 (e.g., PfChBD2-4M-W274L-W308W-W326L is LWL). Eleven mutants were identified in the nucleotide sequence, and ten mutants in the amino acid sequence.

[0130] [Table 12]

[0131] (3) Soluble selection (10 mL scale) <Method> Following the same procedure as in 2(2), the plasmid DNA of the mutant obtained in 4(2) was transformed and inoculated onto LB agar medium supplemented with 50 μg / mL kanamycin, and incubated at 37°C for 1 day. One colony was selected and inoculated into 5 mL of Super Broth liquid medium supplemented with 50 μg / mL kanamycin, and incubated at 37°C and 1000 rpm for 1 day. 9 mL of fresh liquid medium and 1 mL of bacterial culture medium were mixed and incubated at 37°C and 1000 rpm for 1.5 hours, then cooled in ice water for 30 minutes. 10 μL of 1 M IPTG was added and incubated at 16°C and 1000 rpm for 1 day.

[0132] The bacterial culture was centrifuged at 6000 g for 3 minutes, and the supernatant was completely removed. 700 μL of 50 mM sodium phosphate and 100 mM sodium chloride buffer were added to the precipitate, and the suspension was resuspended. The suspension was transferred to a 1.5 mL tube and disrupted using an ultrasonic disruptor for 10 minutes. The lysates were centrifuged at 20000 g for 20 minutes, and 100 μL of the supernatant was collected in a black 96-well microplate, taking care not to touch the precipitate. After collection, the fluorescence of the protein extract was measured as in 4(2). In addition, the template protein PfChBD2-4M was used as a positive control, and the difficult-to-purify mutant PfChBD2-K270T-N272P-E279L-D281R(TPLR) was used as a negative control.

[0133] <Result> After culturing, protein expression was performed, and all bacterial cells turned red, confirming that protein expression was occurring. The results of fluorescence measurement are shown in Figure 2. The LWL, WVF, and WLG mutants showed higher fluorescence intensity than PfChBD2-4M. The SWL and VFF mutants showed values ​​close to those of PfChBD2-4M. Based on these findings, the LWL, WVF, WLG, SWL, and VFF mutants were identified as highly soluble mutants.

[0134] (4) Protein expression and purification (700 mL scale) Protein expression and purification were performed on a large scale of 700 mL for the stagRFP-PfChBD2, 4M, LWL, VFF, SWL, WVF, and WLG variants.

[0135] (i) Expression of stagRFP-PfChBD2 and mutants <Method> Following the same procedure as in 2(2), plasmid DNA was transformed and inoculated onto LB agar medium supplemented with 50 μg / mL kanamycin, and incubated at 37°C for 1 day. Colonies formed on the agar medium were collected in 10 mL of LB medium and mixed with 700 mL of Super Broth medium supplemented with 25 μg / mL kanamycin. The mixture was incubated at 37°C at 130 rpm for 4 hours. After incubation, the medium was cooled in ice water for 30 minutes. 700 μL of 1 M IPTG was added to 700 mL of medium and incubated at 16°C at 130 rpm for 20 hours. The culture medium was centrifuged at 3,000 g for 20 minutes, and the precipitate was collected. The precipitate was stored frozen at -80°C.

[0136] <Result> 8.5g of precipitate was recovered from stagRFP-PfChBD2-TEV-His, 8.6g from stagRFP-PfChBD2-K270H-N272P-E279V-D281G-TEV-His(4M), 9.1g from stagRFP-PfChBD2-4M-LWL-TEV-His, 7g from stagRFP-PfChBD2-4M-VFF-TEV-His, 9.5g from stagRFP-PfChBD2-4M-SWL-TEV-His, 7.3g from stagRFP-PfChBD2-4M-WVF-TEV-His, and 9g from stagRFP-PfChBD2-4M-WLG-TEV-His. All precipitates were red in color.

[0137] (ii) Purification by affinity chromatography <Method> To 1 g of the precipitate obtained in 4(4)(i), 10 mL of 50 mM sodium phosphate and 100 mM sodium chloride buffer (Buffer 1) were added, and the precipitate was disrupted using an ultrasonic disruptor for 20 minutes. The disrupted solution was centrifuged at 20,000 g for 15 minutes, and the supernatant was collected. The supernatant was loaded onto a 3 mL Ni-NTA column, and the flow-through (FT) was collected. 30 mL of Buffer 1 was passed through the column, and 3 mL was collected in test tubes. Subsequently, 30 mL of 50 mM imidazole, 50 mM sodium phosphate, and 100 mM sodium chloride buffer was passed through the column, and 3 mL was collected in test tubes. Then, 30 mL of 100 mM imidazole, 50 mM sodium phosphate, and 100 mM sodium chloride buffer was passed through the column, and 3 mL was collected in test tubes. The absorbance of each was measured, and the band size of those showing absorbance was confirmed by SDS-PAGE. Fractions showing the target band were collected in VIVASPIN20 (30,000MWCO) and concentrated to 700 μL. The concentrated protein solution was replaced with Buffer 1 using illustra NAP-10 Columns to obtain 1.4 mL of protein solution. Then, 80 μL of 3 mg / mL TEV protease was added and incubated at 16°C for 1 day. The protein solution was passed through a 0.5 mL Ni-NTA column. The effluent sample was passed back into the column and collected. Buffer 1 was added in 500 μL increments, and the effluent protein solution was collected from each increment. This procedure was repeated until a total of 3 mL of Buffer 1 was added. The absorbance of each fraction was measured, and those showing absorbance at a wavelength of 280 nm were collected in VIVASPIN20 (30,000MWCO) and concentrated by centrifugation until the protein solution was 500 μL or less.

[0138] <Result> The absorbance of each fraction obtained by elution with 100 mM imidazole, 50 mM sodium phosphate, and 100 mM sodium chloride buffer using affinity chromatography was measured. The expected band size is 39 kDa. From the SDS-PAGE results, a band was observed between 35 kDa and 45 kDa, indicating that the target protein was present in fractions No. 1-10 for all proteins.

[0139] After loading TEV prosthesis-treated protein solutions onto a Ni-NTA column, the absorbance of each fraction recovered using buffer 1 was measured. 0.5 mL of concentrated protein solution was obtained for each protein.

[0140] (iii) Purification by gel filtration chromatography <Method> Gel filtration chromatography was performed using Superdex® 75 Increase 10 / 300GL as the column. The protein solutions obtained in 4(4)(ii), concentrated to less than 500 μL, were loaded onto the column. Purification was performed using 10 mM sodium phosphate buffer pH 8 at a flow rate of 0.5 ml / min, fractionating 0.5 mL per fraction. After purification, the band size of the fractions showing absorbance was confirmed by SDS-PAGE. The fractions in which bands were confirmed were collected in VIVASPIN 20 (30.000 MWCO) and concentrated to less than 1 mL. The absorbance at 280 nm relative to 340 nm of the concentrated protein solution was measured using an absorbance meter. The protein concentration was determined from the measured absorbance and molar extinction coefficient.

[0141] <Result> The expected band size was 39 kDa. SDS-PAGE confirmed a band between 35 kDa and 45 kDa, indicating that the target protein had been purified. Each fraction containing the target protein was recovered and concentrated to obtain the respective protein solutions (Table 13). All mutants obtained by soluble selection were purifiable as soluble proteins.

[0142] [Table 13]

[0143] 5.Adsorption measurement To confirm the adsorption capacity of mutants obtained by affinity and solubility selection using phage display to PET, the proteins purified in 4(4) were incubated with PET. After centrifugation, the amount of protein in the supernatant was measured from the absorbance at the maximum absorption wavelength (555 nm) of the fluorescent protein stagRFP fused to PfChBD2 in the supernatant. The amount of substrate-bound protein was determined by measuring the absorbance of the substrate alone and the protein alone.

[0144] (1) Preparation of a calibration curve for stagRFP <Method> Protein solutions were prepared by diluting stagRFP-PfChBD2 and the mutant, purified in 4(4), with 50 mM sodium phosphate buffer at pH 8 to 3-4 levels of final concentration between 0 and 3 μM. The absorbance of the prepared protein solutions at 555 nm relative to 650 nm and at 280 nm relative to 340 nm were measured, and a calibration curve was created from the results.

[0145] <Result> A calibration curve was created from the absorbance measurements. In subsequent experiments, this calibration curve was used to measure protein concentrations.

[0146] (2) Measurement of PET adsorption capacity <Method> Using stagRFP-PfChBD2 and mutants purified in 4(4), protein solutions were prepared according to Table 14. 200 μL of enzyme solution was mixed into a 1.5 mL tube containing a weighed Goodfellow semi-crystalline PET. After tapping, the mixture was incubated at 25°C and 1500 rpm for 1 hour. The mixture was then centrifuged at 15000 g for 5 minutes, and the supernatant was collected. The absorbance of the supernatant at 555 nm (referenced to 650 nm) and at 280 nm (referenced to 340 nm) was measured, and the protein concentration of the supernatant was calculated from the calibration curve prepared in 5(1). The same procedure was performed with the buffer and protein solution alone to calculate the amount of protein adsorbed onto the PET. After calculating the amount of adsorbed protein, fitting was performed using the following formula.

[0147]

number

[0148] [Table 14]

[0149] <Result> The results of PET adsorption measurements are shown in Figure 3 and Table 15. All mutants obtained through screening were found to exhibit adsorption capacity to semi-crystalline PET. For the template protein 4M, the four mutants LWL, VFF, SWL, and WLG showed the maximum adsorption amount (B max [nmol]) had improved. Also, LWL, VFF, and WLG showed improvements in their dissociation constants (K d A decrease in [mg / mL] was confirmed. max / K d Comparing the [nmol·mL / mg] values, it was found that LWL and VFF significantly improved their adsorption capacity.

[0150] [Table 15]

[0151] (3) Measurement of adsorption capacity for chitin and cellulose <Method> Using stagRFP-PfChBD2 and mutants purified in 4(4), solutions were prepared according to Table 16. Crystalline chitin and crystalline cellulose were used as substrates. Similarly, solutions containing only the substrate and only the protein were also prepared. The prepared solutions were incubated at 25°C and 1000 rpm for 1 hour. The substrate was precipitated by centrifugation, and the supernatant was collected. As in 5(1), the absorbance of the supernatant was measured, and the amount of protein adsorbed to the substrate was calculated.

[0152] [Table 16]

[0153] <Result> The results of chitin and cellulose adsorption measurements are shown in Figure 4. All mutants obtained through screening showed a significant decrease in adsorption to chitin and cellulose. Furthermore, when comparing the adsorption amount to PET with the adsorption amount to chitin and cellulose, it was found that all mutants significantly improved their specificity to PET (Figures 5 and 6). The PET adsorption amounts in Figures 5 and 6 are the results when the PET concentration was set to 0.2 mg / ml. In Figure 6, the vertical axis (Ratio) shows the value obtained by dividing the PET adsorption amount by the chitin adsorption amount or the cellulose adsorption amount. A ratio of 1 indicates that it is equal to the PET adsorption amount, and a larger ratio indicates higher PET specificity. From these results, it was found that all mutants adsorbed more specifically to PET than to the template protein. The mutations in the constructed mutants W274, W308, and W326 correspond to the mutations in PfChBD2 W17, W51, and W69, respectively.

[0154] 6. Summary Six PfChBD2 mutants were obtained through affinity selection and solubility selection. Adsorption experiments showed that these mutants had almost lost their ability to adsorb to chitin, the original substrate of PfChBD2, but retained amorphous PET binding ability comparable to that of the template PfChBD2-4M. These results indicate that the six mutants have acquired higher substrate specificity to amorphous PET than the template. Furthermore, while the three exposed tryptophan residues of PfChBD2 are important for interaction with chitin, it was suggested that all three amino acid residues do not need to be aromatic amino acids for binding to amorphous PET.

Claims

1. A protein having a mutation at at least one position selected from the group consisting of W17, W51, and W69 in SEQ ID NO: 2, comprising any of the following amino acid sequences (A1) to (A5), and having the ability to bind to polyethylene terephthalate. (A1) An amino acid sequence (a1) which has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W17L and W69L mutations added, and which has the W17L and W69L mutations. (A2) An amino acid sequence (a2) having 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the mutations W17V, W51F, and W69F added, and having the aforementioned W17V, W51F, and W69F mutations, (A3) An amino acid sequence (a3) ​​that has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W17S and W69L mutations added, and which has the W17S and W69L mutations. (A4) An amino acid sequence (a4) which has 90% or more sequence identity with the amino acid sequence of Sequence ID No. 2 with the W51L and W69G mutations added, and which has the W51L and W69G mutations, (A5) An amino acid sequence having 90% or more sequence identity with the amino acid sequence (a5) obtained by adding the W51V and W69F mutations to the amino acid sequence of Sequence ID No. 2, and having the W51V and W69F mutations.

2. The protein according to claim 1, wherein the amino acid at the position corresponding to the 13th position from the N-terminus of sequence number 2 is H, the amino acid at the position corresponding to the 15th position is P, the amino acid at the position corresponding to the 22nd position is V, and the amino acid at the position corresponding to the 24th position is G.

3. The protein according to claim 1, further comprising a labeling substance.

4. The protein according to claim 3, wherein the labeling substance is a fluorescent dye or a fluorescent protein.

5. The protein according to claim 1, further comprising an enzyme domain.

6. The protein according to claim 5, wherein the enzyme domain is a polyethylene terephthalate-degrading enzyme domain.

7. The protein according to claim 1, which is a polymer.

8. A polynucleotide comprising a base sequence encoding a protein according to any one of claims 1 to 6.

9. A recombinant vector comprising the polynucleotide described in claim 8.

10. A transformant comprising the recombinant vector described in claim 9.

11. A composition comprising the protein according to any one of claims 1 to 7.

12. Contacting the protein described in claim 3 or 4 with polyethylene terephthalate, To detect the labeling substance, A method for detecting polyethylene terephthalate, including [specifically, polyethylene terephthalate].

13. A method for decomposing polyethylene terephthalate, comprising contacting the polyethylene terephthalate with the protein described in claim 6.

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