JP2023058381A - Protein, polynucleotide, recombinant vector, transformant, composition for decomposing polyethylene terephthalate, and manufacturing method for recycled products - Google Patents

Abstract

To provide a novel protein with excellent heat resistance and PET hydrolysis activity, to provide a polynucleotide comprising a nucleic acid sequence encoding the protein, to provide a recombinant vector comprising the polynucleotide, to provide a transformant comprising the recombinant vector, to provide a composition for decomposing polyethylene terephthalate comprising the protein, and to provide a method for producing a recycled product using the protein.SOLUTION: The protein of the present invention has PET hydrolytic activity and has at least R20C and G62C mutations relative to PET2 wild type.SELECTED DRAWING: Figure 6A

Description

(1) Akihiko Nakamura, Naoya Kobayashi, Nobuyasu Koga, and Ryota Iino, Positive Charge Introduction on the Surface of Thermostabilized PET Hydraulic PET Hydrochloride Binding and Degradation, ACS Catal. 2021, 11, 8550-8564 (Released on June 29, 2021) (2) The 3rd ExCELLS Symposium Online Poster (Released on December 21, 2020) (3) Date July 1, 2021 (4) Press release dated July 1, 2021 (Shizuoka University website) (5) July 2021 Press release dated 1st (website of Institute for Molecular Chemistry, National Institutes of Natural Sciences)

The present disclosure relates to proteins, polynucleotides, recombinant vectors, transformants, compositions for degrading polyethylene terephthalate, and methods for producing recycled products.

The solution of environmental pollution caused by plastics and the construction of a sustainable society are being emphasized. Among them, polyethylene terephthalate (PET) accounts for about 4% of domestically produced plastics, and is used in everyday items such as drink bottles and clothing. At present, most of PET is incinerated or converted into carpets, etc., but it is desirable to reuse it for sustainable use. PET recycling currently in practical use employs two methods: physical recycling in which PET is pulverized and then heat-treated and remolded, and chemical recycling in which PET is decomposed with a chemical catalyst and then repolymerized. However, the former poses problems of deterioration in physical properties and coloration, while the latter requires highly dangerous chemicals and high-temperature treatment. PET decomposition technology using enzymes that can reduce the amount of highly corrosive chemicals used and the amount of heat required will be an important technology in the future society.

As a PET-degrading enzyme, a mutant with improved activity of IsPETase derived from PET-utilizing bacteria has been reported (see Patent Document 1). In addition, Cut190 (see Patent Document 2), which originates from a cutinase that degrades cutin, which is a wax layer, and another cutinase, LCC, as a mutant with improved heat resistance and PET decomposition activity have been reported (see Patent Document 2). See Patent Documents 3 and 4).

On the other hand, ester bonds of PET can be decomposed by esterases other than those described above. For example, the environmental genome-derived LipIAF5.2 enzyme (see Non-Patent Document 1), which was discovered as a lipase in 2009, was reported to have PET degradation activity in 2018 and was named PET2 (Non-Patent Document 2).

WO2019/168811 JP 2015-119670 A WO2012/099018 Japanese Patent Publication No. 2019-527060

Meilleur, C.; Hupe, J. F.; Juteau, P.; Shareck, F. Isolation and Characterization of a New Alkali-Thermostable Lipase Cloned from a Metagenomic Library. J. Ind. Microbiol. Danso, D.; Schmeisser, C.; Chow, J.; Zimmermann, W.; Wei, R.; Distribution of Polyethylene Terephthalate (Pet)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes. Appl. Environ. Microbiol. 2018, 84, No. e02773-17.

Conventionally known PET degrading enzymes have their respective problems when used for recycling. For example, IsPETase has a problem of low heat resistance. Also, Cut190 and LCC are dependent on calcium ions, which is disadvantageous for use in recycling where it is desired to reduce impurities as much as possible. Furthermore, it cannot be said that PET2 has high heat resistance and PET decomposition activity as compared with other enzymes.

The present disclosure provides a novel protein excellent in heat resistance and PET hydrolysis activity, a polynucleotide comprising a nucleic acid sequence encoding the protein, a recombinant vector comprising the polynucleotide, a transformant comprising the recombinant vector, the protein and a method for producing a recycled product using the protein.

Means for solving the above problems include the following aspects.
<1> A protein having polyethylene terephthalate hydrolysis activity and comprising any of the following amino acid sequences.
(A) an amino acid sequence having mutations R20C and G62C relative to the amino acid sequence of SEQ ID NO: 1;
(A1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having R20C and G62C mutations with respect to the amino acid sequence of SEQ ID NO: 1, and having said R20C and G62C mutations;
(B) an amino acid sequence having mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO: 1;
(B1) has a sequence identity of 90% or more with the amino acid sequence having R20C, G62C, S129P, and T270P mutations with respect to the amino acid sequence of SEQ ID NO: 1, and the R20C, G62C, S129P, and T270P mutations an amino acid sequence having
(C) an amino acid sequence having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO:1; or (C1) R20C, G62C, relative to the amino acid sequence of SEQ ID NO:1; An amino acid sequence having 90% or more sequence identity with an amino acid sequence having mutations S129P, T270P, G153A, E83K, and F78R, and having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R .
<2> The protein according to <1>, wherein the optimum temperature for the polyethylene terephthalate hydrolysis activity at pH 7.0 is above 60.0°C.
<3> The protein according to <1> or <2>, which has a melting temperature of 70.0°C or higher.
<4> The protein according to any one of <1> to <3>, wherein the polyethylene terephthalate hydrolysis activity is independent of calcium ions.
<5> The protein according to any one of <1> to <4>, which has higher polyethylene terephthalate hydrolysis activity than the protein having the amino acid sequence of SEQ ID NO: 1 at pH 7.0.
<6> The protein according to any one of <1> to <5>, wherein the two cysteine residues at the R20C and G62C mutations form a disulfide bond.
<7> The protein according to any one of <1> to <6>, comprising any one of the following amino acid sequences.
(A) an amino acid sequence having mutations R20C and G62C relative to the amino acid sequence of SEQ ID NO: 1;
(B) an amino acid sequence having the mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO:1; or (C) R20C, G62C, S129P, T270P, G153A, Amino acid sequence with E83K and F78R mutations.
<8> A polynucleotide comprising a base sequence encoding the protein according to any one of <1> to <7>.
<9> A recombinant vector comprising the polynucleotide according to <8>.
<10> A transformant containing the recombinant vector of <9>.
<11> A composition for decomposing polyethylene terephthalate, comprising the protein according to any one of <1> to <7>.
<12> A method for producing a recycled product, comprising decomposing polyethylene terephthalate with the protein according to any one of <1> to <7>.

According to the present disclosure, a novel protein excellent in heat resistance and PET hydrolysis activity, a polynucleotide comprising a nucleic acid sequence encoding the protein, a recombinant vector comprising the polynucleotide, a transformant comprising the recombinant vector, A composition for decomposing polyethylene terephthalate containing the protein and a method for producing a recycled product using the protein are provided.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline|summary of PET hydrolysis by a PET hydrolase, and the outline|summary of PET and its hydrolyzate. PET is a water-insoluble synthetic polymer with crystalline and amorphous regions. The PET hydrolase binds to the PET surface and captures the molecular chains in the motion range of the amorphous region and incorporates them into the active site. PET chains are hydrolyzed to bis(hydroxyethyl)terephthalate (BHET), (hydroxyethyl)terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG). Evaluation of PET2 wild type is shown. FIG. 2A shows thermal stability determined from residual activity. After treatment for 1 hour at each temperature from 40 to 90°C at pH 7.0, 1 mM BHET was hydrolyzed with 0.1 µM enzyme at 60°C for 10 minutes at pH 7.0. The MHET concentration (μM) produced was quantified and activity was calculated by dividing by the enzyme concentration (0.1 μM) and reaction time (600 seconds). Evaluation of PET2 wild type is shown. FIG. 2B shows the pH dependence of BHET hydrolysis activity at 60° C. for 10 minutes. The MHET concentration (μM) produced was quantified and activity was calculated by dividing by the enzyme concentration (0.1 μM) and reaction time (600 seconds). MHET concentrations produced in the absence of PET2 were subtracted as background. Evaluation of PET2 wild type is shown. FIG. 2C shows the CD spectra of PET2 wild type in 10 mM sodium phosphate solution (pH 7.0) at 20° C. (solid line) and 95° C. (dashed line). Signals at 230 nm were used for melting temperature (Tm) analysis. Evaluation of PET2 wild type is shown. FIG. 2D shows the signal change at 230 nm during heating. The Tm and amorphous PET hydrolytic activity of each variant of PET2 are shown. FIG. 3A shows the homology modeling structure of PET2 wild type. Mutation positions are indicated by bars. The Tm and amorphous PET hydrolytic activity of each variant of PET2 are shown. FIG. 3B presents a schematic of single mutations and cysteine pairs for disulfide bond formation. IsPETase wild-type or mutant-based mutations (♦), proline mutations (●), alanine mutations (▴), cysteine versus mutations (▼) are plotted with wild type (WT) (▪). Amorphous PET discs (0.32 cm ² , 4.0 mg) were incubated with 0.1 μM enzyme in 50 mM sodium phosphate buffer (pH 7.0) at 60° C. for 60 minutes. TPA, MHET and BHET produced were totaled. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 min). The Tm and amorphous PET hydrolytic activity of each variant of PET2 are shown. FIG. 3C presents an overview of mutation integration. The order of mutations is indicated by arrows. The activity analysis conditions are the same as those shown in FIG. 3B. Figure 2 shows the X-ray crystal structure of PET2 mutants. (A) X-ray crystal structure of PET2(2M). A B-chain molecule of the asymmetric unit is shown. The catalytic triad (S175, D221, and H253) and amino acid residues undergoing mutation are indicated. (B) X-ray crystal structure of PET2(7M). The catalytic triad (S175, D221, and H253) and mutated amino acid residues are indicated. (C) Surface charge of PET2 wild-type, PET2(7M) and IsPETase at pH 7.0. The PET2 wild-type structure was reconstructed by backmutation of the PET2(2M) mutant structure. In each structure, catalyst pockets are indicated by dashed circles. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5A shows a bright field image of the PET thin film. Scale = 2 μm. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5B shows an example of binding and dissociation of Alexa Fluor 555 labeled PET2 (7M) at 50 pM. The binding time estimated from the change in signal intensity over time is 0.6 seconds. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5C shows a fluorescence image of a PET thin film stained with 10 nM PET2 (7M). The same field of view as in FIG. 5A was observed. Scale = 2 μm. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5D shows the cumulative number of bound molecules over time. Individual trajectories for each movie of PET2 wild-type and PET2(7M) are shown. A binding rate constant was calculated from the time required for binding of 100 molecules. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5E shows the distribution of PET2 wild-type binding times fitted with a double exponential decay function. The ratio of slow and fast dissociation events was calculated from the area of the fitting curve. Single-molecule fluorescence imaging of binding and dissociation of PET2 wild-type and PET2(7M) on PET thin films. FIG. 5F shows the distribution of binding times of PET2(7M) fitted with a double exponential decay function. The ratio of slow and fast dissociation events was calculated by the same method as for the wild type. Temperature dependence and time course of activity are shown. FIG. 6A shows the temperature dependence of amorphous PET hydrolysis activity. The temperature dependence of PET2 wild-type (▪), PET2(4M) (●), PET2(5M) (▴), PET2(7M) (▴) is shown. Concentrations of TPA, MHET and BHET are summed and presented as total product. Activity was calculated by dividing total product (μM) by enzyme concentration (0.1 μM) and reaction time (60 min). The maximal activity of PET2 (7M) at 68°C was 6.8 times that of the wild type at 60°C. Plots show the mean and standard deviation of triplicate determinations. Temperature dependence and time course of activity are shown. FIG. 6B shows the time course of amorphous PET hydrolysis activity. PET2 wild type and PET2 (7M) were reacted at 60°C and 68°C, respectively. Plots show the mean and standard deviation of triplicate determinations. Temperature dependence and time course of activity are shown. FIG. 6C represents filled plots of TPA, MHET, and BHET generated from amorphous PET films with PET2 wild-type (left) and PET2(7M) (right). The total amount of product is consistent with Figure 6B.

Hereinafter, modes for implementing embodiments of the present disclosure will be described in detail. However, the embodiments of the present disclosure are not limited to the following embodiments. In the following embodiments, the constituent elements (including element steps and the like) are not essential unless otherwise specified. The same applies to numerical values and their ranges, which do not limit the embodiments of the present disclosure.

In the present disclosure, the term "process" and equivalent terms include a process that is independent of other processes, and even if the process cannot be clearly distinguished from other processes, if the purpose of the process is achieved, This step is also included.
In the present disclosure, the numerical range indicated using "-" includes the numerical values before and after "-" as the minimum and maximum values, respectively.
In the numerical ranges described step by step in the present disclosure, the upper limit or lower limit of one numerical range may be replaced with the upper or lower limit of another numerical range described step by step. . Moreover, in the numerical ranges described in the present disclosure, the upper or lower limits of the numerical ranges may be replaced with the values shown in the examples.

In the present disclosure, the "identity" of the amino acid sequences means that the two amino acid sequences to be compared are aligned with appropriate gaps inserted in one or both of them so that the number of matching amino acid residues is maximized. is the ratio of the number of matching amino acid residues to the total number of amino acid residues. Alignments can be performed using well-known alignment tools such as BLAST, FASTA, CLUSTAL W, and the like. For example, alignments can be evaluated with the default parameters of BLAST.

In the present disclosure, "having X% or more sequence identity" with a reference amino acid sequence means that the full length or part of the amino acid sequence to be compared is X% or more of the full length of the reference amino acid sequence. It means having identity.

In the present disclosure, amino acid residues may be designated by their single-letter or three-letter abbreviations in parentheses below.
Alanine (A, Ala), Arginine (R, Arg), Asparagine (N, Asn), Aspartic acid (D, Asp), Cysteine (C, Cys), Glutamine (Q, Gln), Glutamic acid (E, Glu), Glycine (G, Gly), Histidine (H, His), Isoleucine (I, Ile), Leucine (L, Leu), Lysine (K, Lys), Methionine (M, Met), Phenylalanine (F, Phe), Proline (P, Pro), Serine (S, Ser), Threonine (T, Thr), Tryptophan (W, Trp), Tyrosine (Y, Tyr), Valine (V, Val).

In the present disclosure, amino acid mutations in peptides or proteins may be indicated in the order of the one-letter code for the amino acid before mutation, the amino acid position number from the N-terminus, and the one-letter code for the amino acid after mutation. For example, "having an R20C mutation in the amino acid sequence of SEQ ID NO: 1" means that the 20th arginine counted from the N-terminus of SEQ ID NO: 1 is substituted with cysteine.

In the present disclosure, "a protein comprising an 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, and in addition to the amino acid sequence of X, additional sequences at one or both of the N-terminus and C-terminus of the amino acid of X (for example, sequences, such as signal sequences, tag sequences, etc.). Additional sequences such as signal sequences and tag sequences are well known to those skilled in the art. The same applies to base sequences. For example, "a polynucleotide containing a Y base sequence" means that all or part of the base sequence of the polynucleotide is a Y base sequence.

In the present disclosure, the "PET hydrolysis activity" of a protein is obtained by placing the protein to be measured and PET under predetermined reaction conditions, analyzing the concentration of degradation products by HPLC, and determining the total concentration of degradation products as the protein concentration and reaction A value obtained by dividing by time. PET hydrolytic activity in this disclosure represents activity at pH 7.0 unless otherwise specified.
Column: Phenomenex Luna C18 (2)
Eluent: gradient flow rate from 20 mM sodium phosphate buffer (pH 7.0) to methanol: 1 ml/min.
Column temperature: 25°C
Detection: 210 nm
Injection volume: 10 μL

Hydrolytic degradation products of PET include bis(hydroxyethyl) terephthalate (BHET), (hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG), as shown in FIG. be done. The degradation products of PET by the protein of the present disclosure may be all of these, some of them, or partial degradation products in which these are linked.

<Protein>
The protein of the present disclosure is a protein having polyethylene terephthalate hydrolysis activity and comprising any of the following amino acid sequences.
(A) an amino acid sequence having R20C and G62C mutations relative to the amino acid sequence of SEQ ID NO: 1;
(A1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having R20C and G62C mutations with respect to the amino acid sequence of SEQ ID NO: 1, and having said R20C and G62C mutations;
(B) an amino acid sequence having mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO: 1;
(B1) has a sequence identity of 90% or more with the amino acid sequence having R20C, G62C, S129P, and T270P mutations with respect to the amino acid sequence of SEQ ID NO: 1, and the R20C, G62C, S129P, and T270P mutations an amino acid sequence having
(C) an amino acid sequence having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO:1; or (C1) R20C, G62C, relative to the amino acid sequence of SEQ ID NO:1; An amino acid sequence having 90% or more sequence identity with an amino acid sequence having mutations S129P, T270P, G153A, E83K, and F78R, and having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R .

Hereinafter, for convenience, the amino acid sequence of (A) (SEQ ID NO: 2) is also referred to as "amino acid sequence A", the amino acid sequence of (A1) (excluding amino acid sequence A) is also referred to as "amino acid sequence A1", and the above ( The amino acid sequence of B) (SEQ ID NO: 3) is also referred to as "amino acid sequence B", the amino acid sequence of (B1) (excluding amino acid sequence B) is also referred to as "amino acid sequence B1", and the amino acid sequence of (C) ( SEQ ID NO: 4) is also referred to as "amino acid sequence C", and the amino acid sequence of (C1) above (excluding amino acid sequence C) is also referred to as "amino acid sequence C1".

SEQ ID NO: 1 shows the amino acid sequence of PET2 wild type. Hereinafter, the protein having the amino acid sequence of SEQ ID NO: 1 is also referred to as PET2 wild type. The inventors found that the above protein, which has at least R20C and G62C mutations relative to PET2 wild type, has excellent thermostability and PET hydrolytic activity.
Amino acid sequence A has at least the R20C and G62C mutations relative to the PET2 wild type. Although the R20C and G62C mutations are located away from the active site of PET2, it was surprisingly found that these mutations can improve both heat resistance and activity. Without being bound by theory, it is speculated that the R20C and G62C mutations form a disulfide bond with the cysteine residue pair and improve the stability of the protein itself.
Amino acid sequence B has at least the R20C, G62C, S129P, and T270P mutations relative to the PET2 wild type. Although not bound by theory, it is speculated that S129P and T270P stabilize the protein structure by introducing a cyclic proline residue into the flexible region (unstable site) of the protein.
Amino acid sequence C has at least the R20C, G62C, S129P, T270P, G153A, E83K and F78R mutations relative to the PET2 wild type. Without being bound by theory, it is speculated that the G153A mutation contributes to structural stabilization by substituting alanine for glycine in the α-helix of PET2. The F78R mutation is speculated to enhance PET hydrolytic activity by altering the surface charge. Also, the E83K mutation improves heat resistance, but the mechanism is not clear.

The amino acid sequence of SEQ ID NO: 1 (PET2 wild-type amino acid sequence) and amino acid sequences A to C are shown below. Enclosed letters represent mutation sites.

A protein of the present disclosure comprises the sequence of amino acid sequences A-C or has 90% or more sequence identity with any one of amino acid sequences A-C, preferably 91% or more, 92% or more, Include amino acid sequences having 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater sequence identity and having the specific mutations described above. The protein of the present disclosure is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of amino acid sequence A It may also contain amino acid sequences with % or more sequence identity. The protein of the present disclosure is a protein containing the above amino acid sequence and having PET hydrolyzing activity. The PET hydrolyzing activity is preferably higher than that of PET2 wild type at pH 7.0. The comparison of PET hydrolysis activity in this case is performed by the method described later.

For example, the protein of the present disclosure maintains the above amino acid sequence identity and has one or more amino acids deleted from any of amino acid sequences A to C, as long as it has PET hydrolysis activity. It may contain substituted or added amino acid sequences and have the specific mutations described above. In this case, the number of amino acids to be deleted, substituted, or added is not limited as long as the protein has PET hydrolysis activity. 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2. The amino acid positions to be deleted, substituted, or added are selected from the range corresponding to, for example, 1 to 147, 149 to 193, 195 to 225, and 227 to 281 from the N-terminus of SEQ ID NO: 1. good too.

In one embodiment, amino acid A1 has 90% or more sequence identity with an amino acid sequence having R20C and G62C mutations with respect to the amino acid sequence of SEQ ID NO: 1, and has said R20C and G62C mutations, and It may further have at least one mutation selected from the group consisting of S129P, T270P, G153A, E83K, and F78R.
In one aspect, amino acid B1 has 90% or more sequence identity with an amino acid sequence having mutations R20C, G62C, S129P, and T270P with respect to the amino acid sequence of SEQ ID NO: 1, and said R20C, G62C, S129P , and T270P mutations, and at least one mutation selected from the group consisting of G153A, E83K, and F78R.

In one aspect, the protein of the present disclosure is a protein that has PET hydrolysis and preferably comprises the amino acid sequence of any of the amino acid sequences B, B1, C, or C1, and any of the amino acid sequences C or C1 More preferably, it is a protein comprising the amino acid sequence of

In one aspect, the protein of the present disclosure has PET hydrolysis activity and is preferably a protein comprising any one of amino acid sequences A to C, and any amino acid sequence of amino acid sequence B or C. A protein containing the amino acid sequence C is more preferable.

The protein of the present disclosure may have an additional amino acid sequence at the N-terminus or C-terminus as long as it does not impair the PET hydrolysis activity. Additional amino acid sequences include additional sequences for recombinant production of proteins of the present disclosure. Examples of such additional sequences include amino acids that serve as starting points for protein synthesis (N-terminal methionine residue, etc.), N-terminal signal sequences (eg, signal sequences for protein secretion in E. coli), tag sequences (eg, histidine tags). , GST tag, FLAG tag, etc.), protease recognition sequences (eg, TEV protease recognition sequences), and the like. In one aspect, the additional amino acid sequence may be the amino acid sequence of SEQ ID NO: 6 added to the N-terminus, and the amino acid sequence of SEQ ID NO: 7 added to the C-terminus and the C-terminus of SEQ ID NO: 7. It may be a combination with the added amino acid sequence of SEQ ID NO: 8, or a combination thereof. SEQ ID NOs:6-8 are described in the Examples section.
The number of amino acids in the additional amino acid sequence is not limited as long as the protein has PET hydrolysis activity. It may be ˜30, 1-25, 1-20, 1-15, 1-10, or 1-5.
For example, the protein of the present disclosure can be added to the N-terminus or C-terminus of a sequence consisting of any one of the amino acid sequences A, A1, B, B1, C, or C1 to the extent that the PET hydrolysis activity is not impaired. A specific amino acid sequence may be added. Additional amino acid sequence details are provided above.

The two cysteine residues at the R20C and G62C mutations preferably form a disulfide bond. The presence or absence of disulfide bond formation can be confirmed by X-ray crystal structure analysis.

The optimum pH for the PET hydrolysis activity of the protein of the present disclosure (the pH at which the activity is highest) is not particularly limited, and is practically preferably near neutral. For example, the optimum pH is preferably pH 5.0 to 9.5, more preferably pH 5.5 to 9.0, and may be pH 6.0 to 8.0.

PET has a glass transition temperature around 70 ° C., and it is expected that decomposition efficiency will increase when PET is decomposed at a high temperature. The elevated temperature) is preferably above the optimum temperature for PET hydrolysis activity of PET2 wild type. From this point of view, the optimum temperature for the PET hydrolysis activity of the protein of the present disclosure is preferably above 60.0°C, more preferably 65.0°C or higher at pH 7.0. In addition, from the viewpoint of decomposing PET with low energy, the optimum temperature for the PET hydrolysis activity of the protein of the present disclosure may be 90.0 ° C. or less, and may be 85.0 ° C. ° C. or lower, or 80.0 ° C. or lower.
The optimum temperature is measured as follows. Two amorphous PET discs (thickness 0.25 mm, total surface area 0.32 cm ² , total mass 4.0 mg) were loaded with 250 μL of 0.1 μM protein in 50 mM sodium phosphate solution (pH 7.0). and incubate for 60 minutes at each temperature. Analyze the degradation product concentration by HPLC and divide the total degradation product concentration by the protein concentration and reaction time to obtain the activity value.

From a similar point of view, the melting temperature (Tm) of the protein of the present disclosure is preferably higher than that of PET2 wild type. The Tm of the protein of the present disclosure 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 decomposition at low energy within the range where the decomposition of PET is promoted, the melting temperature (Tm) of the protein of the present disclosure may be 100.0° C. or less, and may be 95.0° C. or less. It may be 90.0° C. or lower.
In the present disclosure, the melting temperature (Tm) of proteins is measured by circular dichroism (CD) spectrum according to the following procedure. A 13 μM protein solution in 10 mM sodium phosphate solution (pH 7.0) is prepared. CD spectra of proteins are measured at 250-200 nm at 20°C. The protein signal is followed at 230 nm as the protein is heated from 20° C. at a rate of 1° C./min. CD spectra of denatured proteins are measured at 250-200 nm at 95°C. Protein Tm is estimated by curve fitting the temperature dependence of the signal at 230 nm.

From the viewpoint of convenience when using the protein of the present disclosure for recycling applications, it is preferred that the PET hydrolysis activity of the protein be calcium ion independent.
In the present disclosure, the fact that the PET hydrolysis activity of protein is "independent of calcium ions" is confirmed by the following procedure. Two pieces of amorphous PET _film (0.25 mm thickness, surface area total 0.32 cm ² , total mass 4.0 mg) at 60° C. for 60 minutes. After incubation, 200 μL of supernatant is collected and mixed with 60 μL of ultrapure water or CaCl ₂ solution to adjust the CaCl ₂ concentration to 231 mM. The mixture is further mixed with 68.6 μL of 1 M sodium phosphate buffer (pH 7.0) to precipitate calcium phosphate. The precipitate is removed using a 0.22 μm spin column filter at 15,000 g for 3 minutes. Analyze the degradation product concentration by HPLC and divide the total degradation product concentration by the protein concentration and reaction time to obtain the activity value. When the PET hydrolysis activity does not change by 50% or more when the PET hydrolysis activity is 10, 100, or 300 mM compared to when the _CaCl concentration is 0 mM, the PET hydrolysis activity is Judged to be calcium independent.

The PET hydrolysis activity of the protein of the present disclosure at pH 7.0 is preferably higher than the hydrolysis activity of PET2 wild type at pH 7.0, for example, 2.0 times or more than the hydrolysis activity of PET2 wild type at pH 7.0. It is preferably high, more preferably 3.0 times or more, more preferably 4.0 times or more, and particularly preferably 5.0 times or more.
A comparison of the PET hydrolytic activity of the PET2 wild type and mutants is made by comparing the activity of each protein at the optimum temperature. The reaction conditions in this case are as follows. Two amorphous PET discs (thickness 0.25 mm, total surface area 0.32 cm ² , total mass 4.0 mg) were loaded with 250 μL of 0.1 μM protein in 50 mM sodium phosphate solution (pH 7.0). and incubate at the optimum temperature for 60 minutes. Analyze the degradation product concentration by HPLC and divide the total degradation product concentration by the protein concentration and reaction time to obtain the activity value.

The protein of the present disclosure can be produced by known genetic engineering techniques using polynucleotides. For example, a polynucleotide encoding the amino acid sequence of the target protein is prepared, incorporated into an expression vector to obtain a recombinant vector, and introduced into a host to obtain a transformant. The target protein is produced by culturing the resulting transformant. The protein of the present disclosure can be obtained by collecting the obtained protein by a conventional method.

Applications of the protein of the present disclosure are not particularly limited, and can be used, for example, to degrade and recycle PET. In addition, the decomposition product of the protein of the present disclosure can be used for the production of terephthalic acid and ethylene glycol by reacting it with a degrading enzyme such as MHETase.

<Polynucleotide, Recombinant Vector, Transformant>
Polynucleotides of this disclosure include base sequences that encode proteins of this disclosure.
A recombinant vector of the present disclosure comprises the polynucleotide.
A transformant of the present disclosure contains the recombinant vector.

A polynucleotide may be any polynucleotide that encodes a protein of the present disclosure. The nucleic acid sequence of a polynucleotide can vary within codon degeneracy. When introducing a recombinant vector into a host to obtain a transformant and expressing a protein, it is preferable to adopt codons that are frequently used in the host. In one aspect, when using an expression system using E. coli as a host, a TGC codon for the cysteine residues of R20C and G62C, a CCG codon for the proline residues of S129P and T270P, a GCG codon for the alanine residue of G153A, and a lysine residue of E83K An AAA codon may be used as the base, and a CGC codon may be used as the arginine residue of F78R.

A recombinant vector may be any recombinant vector capable of expressing a protein of the present disclosure. Preferred recombinant vectors include plasmids, phages and the like. The expression vector has a promoter required for expression and may have other components such as a terminator and a selectable marker (drug resistance gene, etc.).
The host is not particularly limited as long as it can express the protein of the present disclosure, and is selected together with the recombinant vector to be used. It is convenient to use microbial cells as hosts, and for example, prokaryotes (eg, E. coli), eukaryotes (eg, yeast), and the like may be used.
Methods well known to those skilled in the art can be adopted for the conditions for transformation, expression, and recovery when producing a protein using an expression vector and host.

<Composition for decomposition of polyethylene terephthalate>
A PET degrading composition of the present disclosure comprises a protein of the present disclosure. The proteins of the present disclosure may be included in the composition in an unpurified state (e.g., containing a protein-expressing host, or containing a crude protein fluid secreted from a protein-expressing host), or in a purified state. may be contained in the composition, preferably in a purified state.

The composition for PET decomposition may contain, in addition to the protein of the present disclosure, buffers and the like suitable for protein stabilization. As the buffer, a buffer that maintains a solution near neutrality is preferred, and examples thereof include phosphate buffer, sodium phosphate buffer, Tris-HCl buffer, HEPES and the like.

The PET decomposition composition may contain salts such as calcium salts, but the salt content is preferably as low as possible in consideration of recycling applications. In the composition for decomposing PET, the salt concentration is preferably 50 mM or less, more preferably 30 mM or less, and even more preferably 20 mM or less.

The composition for PET decomposition may contain additives such as stabilizers in addition to the protein and buffer solution of the present disclosure.

<Manufacturing method for recycled products>
The method of manufacturing a recycled product of the present disclosure includes degrading PET with the protein of the present disclosure. Degradation of PET can be performed by contacting PET with the protein of the present disclosure. For example, PET may be decomposed using the aforementioned PET decomposing composition.
Waste PET, for example, can be used as PET.
The decomposition reaction is preferably carried out under conditions close to the optimum pH and optimum temperature described above. For example, the decomposition reaction is preferably carried out in the range of pH 5.0-9.5, more preferably in the range of pH 5.5-9.0, and may be carried out in the range of pH 6.0-8.0. The decomposition reaction is preferably carried out in a suitable buffer to maintain the above pH. Buffers include the buffers described above. Also, the decomposition reaction is preferably carried out at a temperature above 60.0°C, more preferably above 65.0°C. The upper limit of the decomposition reaction temperature is preferably less than 100.0°C, more preferably less than 95.0°C, and even more preferably less than 90.0°C.
The reaction time for decomposition is not particularly limited as long as sufficient decomposition for production of recycled products is possible. Efficient PET hydrolysis is possible using the protein of the present disclosure, and shortening of the reaction time is expected. For example, the reaction time may be within 3 days, within 2 days, within 24 hours, within 18 hours, within 12 hours, within 6 hours, within 3 hours, or 2 hours. It may be within 1 hour. From the viewpoint of sufficient decomposition, the reaction time may be 10 minutes or longer, or 30 minutes or longer. The reaction product is preferably decomposed to a sufficiently repolymerizable state (for example, until 70% by mass or more, 80% by mass or more, or 90% by mass or more of the substrate PET is decomposed).

In one embodiment, after degradation of PET, degradation products are recovered. The recovery method is not particularly limited, and can be performed by a known method. After recovery, it may be purified by known methods such as separation, extraction, adsorption, concentration, and filtration.
Recycled products can be produced by reusing the degradation products to synthesize polymers by repolymerization. The degradation products may be reused for the production of PET and may be reused for the production of other polymers.

EXAMPLES Next, the embodiments of the present disclosure will be specifically described with reference to examples, but the embodiments of the present disclosure are not limited to these examples.

In the following examples, a signal sequence (27 amino acids) for expression and secretion of PET2 mutants in E. coli is used, so the amino acid position number from the N-terminus is the position number from the N-terminus containing the signal sequence. is represented by For example, the R20C, G62C, S129P, T270P, G153A, E83K, and F78R mutations relative to SEQ ID NO: 1 in the proteins of the present disclosure described above are, in the examples below, R47C, G89C, S156P, respectively, relative to PET2 wild type. Corresponds to the T297P, G180A, E110K, and F105R mutations. The amino acid sequence (SEQ ID NO: 5) of PET2 (7M) used in the Examples (including the N-terminal signal sequence and the C-terminal protease recognition site and histidine tag), and the signal sequence contained therein (SEQ ID NO: 6 1 to 27 of SEQ ID NO: 5), a protease recognition sequence (SEQ ID NO: 7; 309 to 315 of SEQ ID NO: 5) and a histidine tag (SEQ ID NO: 8: 316 to 321 of SEQ ID NO: 5) are shown below. Enclosed letters in SEQ ID NO: 5 represent the mutation sites, and shaded letters represent the signal sequence, protease recognition site and tag sequence including histidine tag from the N-terminal side. PET2(2M) used in the examples has only F105R and E110K among the mutations shown in SEQ ID NO:5. PET2(4M) used in the examples has only F105R, E110K, S156P and T297P among the mutations shown in SEQ ID NO:5. PET2(5M) used in the examples has only F105R, E110K, S156P, T297P, and G180A among the mutations displayed in SEQ ID NO:5.

The base sequence (SEQ ID NO: 9) of PET2 (7M) corresponding to SEQ ID NO: 5 (including a termination codon) is shown below.

1. Introduction Plastics, which are composed of synthetic polymers, are relatively cheap and easy to process, and are widely used in modern society ⁽¹⁾ . However, from the viewpoint of sustainability, environmental pollution by these has become a serious problem. In particular, polyethylene terephthalate (PET) is produced in large quantities and is widely used for beverage bottles, clothing, packaging, etc. ⁽²⁾ . PET can form a physically and chemically stable crystalline structure ⁽³⁾ and is classified as an engineering plastic. This stability is believed to allow PET to persist in the environment for long periods ⁽⁴⁾ .

Although PET is highly resistant to biodegradation, its molecular chain is composed of carboxylic acid esters of terephthalic acid (TPA) and ethylene glycol (EG). Therefore, an esterase should have the ability to hydrolytically degrade PET. The first reported efficient PET hydrolase was cutinase (TfH) from Thermobifida fusca ⁽⁵⁾ . Although cutinase (EC 3.1.1.74) is generally registered as a hydrolase of plant waxy layers (cutin), Muller et al. also found that TfH has PET hydrolysis activity. . In 2016, Ideonella sakaiensis 201-F6, a bacterium that uses PET as a carbon source and produces PET hydrolase (IsPETase) was discovered ⁽⁶⁾ . Since then, many studies on IsPETase have been actively carried out, such as determination of the structure of IsPETase and reaction mechanism ^(7-10) . IsPETase is classified as a PET hydrolase (EC 3.1.1.101), but its low thermostability makes industrial use difficult, and various mutants with improved thermostability have been reported. ^{(9, 11)} . From this point of view, it can be said that some cutinases are more thermostable than IsPETase and are suitable templates for constructing thermostable PET hydrolases. The most promising PET hydrolase is leaf-branch compost cutinase (LCC) ⁽¹²⁾ . Thermobifida fusca-derived cutinase (TfCut2) has high thermostability and activity ⁽¹³⁾ , and Saccharomonospora viridis-derived cutinase (Cut190) has also been produced with improved thermostability and activity ^{(14, 15)} . In addition, lipases (EC 3.1.1.3) are also part of the carboxylic acid ester hydrolases, PET ⁽¹⁶⁾ , poly(tetramethylene succinate) ⁽¹⁷⁾ , poly(ω-hydroxyalkanoic acid) ^{( 18)} have hydrolytic activity on polyesters. Recently, an enzyme cloned as a lipase from a gelatinolysis reactor metagenomic library ⁽¹⁹⁾ was rediscovered as PET hydrolase, PET2 ⁽²⁰⁾ . PET2 is a thermostable enzyme discovered from a gene library obtained from an artificial high-temperature environment, and is one of candidates for thermostable PET hydrolase.

PET is a water-insoluble synthetic polymer, and its enzymatic degradation is a heterogeneous reaction occurring at the solid-liquid interface (see FIG. 1). Furthermore, since PET has an amorphous region that is easily degraded and a crystalline region that is not easily degraded, PET hydrolases are expected to preferentially hydrolyze the amorphous region of PET, especially the mobile region ^{( 12, 13, 21)} . Among natural polymers, cellulose, a major constituent of plant cell walls, and chitin, a component of crustacean and fungal cell walls, are also water-insoluble crystalline polysaccharides ^{(22, 23)} . Enzyme binding to the solid surface is important for efficient degradation of these water-insoluble polymers. Therefore, cellulases and chitinases, which have high degradative activity on crystalline cellulose and crystalline chitin, have domain structures for binding with high affinity to the surface of crystalline polymers ⁽²⁴⁾ . Consistent with this finding, IsPETase has more basic amino acid residues on the enzyme surface compared to other carboxyesterases, and these positively charged residues were reported to contribute to PET degradation activity. ⁽²⁵⁾ .

Binding affinities to solid substrates are typically compared quantitatively using dissociation constants (or binding constants) obtained by biochemical adsorption measurements. However, since this constant is the ratio of the dissociation rate constant to the association rate constant, it is difficult to unambiguously identify which rate constant is responsible for the change in dissociation constant based on biochemical measurements alone. is. Therefore, cellulase and chitinase assays have used single-molecule fluorescence imaging to directly measure the association and dissociation rate constants ^(26-28) . Single-molecule analysis allowed us to identify amino acid residues important for binding and differences in the contribution of each domain to cellulose binding between cellulases from bacteria and fungi. rice field.

In this study, we first investigated the thermal stability and pH optimum of the PET2 wild-type (WT) reaction using bis(2-hydroxyethyl)terephthalate (BHET) as a water-soluble model substrate. Up to 7 mutations were then introduced into PET2 wild type based on single mutation screening and single mutation combination. As a result, we succeeded in identifying a PET2 mutant with greatly improved thermostability and PET hydrolysis activity. The structure of the mutated enzyme was determined by X-ray crystallography to verify structural changes due to mutations that contribute to thermostability and adsorption properties. To elucidate the mechanism by which the degradation activity is enhanced, single-molecule fluorescence imaging was used to compare the adsorption properties of the best-performing mutant and wild-type on PET thin films. Finally, we evaluated the temperature dependence of the degradation activity of the mutant enzymes and tested long-term thermal stability by degradation experiments up to 24 hours.

2. Materials and Methods 2.1 Homology Modeling of PET2 Wild-type Structure for Selection of Mutation Positions Using the SWISS-MODEL server ⁽²⁹⁾ , the crystal structure of IsPETase (PDB ID: 6EQE) was used as a template to model the homology of PET2 wild-type. A modeling structure was constructed. The crystal structure of IsPETase and the modeling structure of PET2 wild type were then aligned in Pymol, and mutation positions of PET2 corresponding to the unique basic residues of IsPETase and the positions of valid mutations were selected.

2.2 Plasmid Construction and Enzyme Purification The gene ⁽²⁰⁾ of the enzyme registered as alkaline thermostable lipase lipIAF5-2 (UniProt: C3RYL0) ⁽¹⁹⁾ and recently renamed PET2 due to its PET hydrolyzing activity was transformed into C A gene was synthesized fused at the end with a TEV protease recognition sequence (ENLYFQG) and a histidine-6 tag sequence. This gene was ligated into the pET27b plasmid at the NdeI-NotI sites. The signal sequence of lipIAF5-2 was used instead of the pelB leader sequence of pET27b. Over ExpressE. E. coli C41(DE3) (Lucigen) was transformed with this plasmid and plated on LB plates containing 50 μg/mL kanamycin. Colonies were collected in 20 mL super broth medium (2.5 w/v % tryptone, 1.5 w/v % yeast extract, 0.5 w/v % sodium chloride) and transferred to 700 mL super broth medium containing 50 µg/mL kanamycin. inoculated. The flask was shaken at 140 rpm and 37° C. for 3 hours, followed by ice cooling for 30 minutes. 700 μL of 1 M IPTG was added to the flask and incubated at 16° C., 140 rpm for 16 hours. Cells were harvested by centrifugation at 3000 g for 20 minutes.

Collected cells were suspended in 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride at a concentration of 1 g/mL. Cells were disrupted by sonication and the suspension was centrifuged at 20,000 g for 20 minutes. The supernatant was loaded onto 5 mL of Ni-NTA agarose (Qiagen) equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride. The unbound fraction was washed with 50 mL of the same buffer and contaminants were washed with 50 mL of the same buffer containing 50 mM imidazole. Purified enzyme was eluted with a buffer containing 100 mM imidazole. The enzyme was concentrated to 500 μL at 8000 g with Vivaspin20 (molecular weight cutoff=10K; Sartorius) and loaded onto a NAP10 column (Cytiva) equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride. bottom. 100 μL of 5 mg/mL TEV protease was added per mL of 400 μM enzyme and incubated at 16° C. for 16 hours. The enzyme/TEV protease mixture was loaded onto 1 mL of Ni-NTA agarose equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 100 mM sodium chloride and unbound enzyme was eluted with the same buffer. The eluted enzyme was concentrated to 500 μL at 8000 g with Vivaspin 20 (molecular weight cutoff=10 K) and loaded onto Superdex 75 Increase (Cytiva) equilibrated with 10 mM sodium phosphate buffer (pH 7.0). The purity of each fraction was checked by SDS-PAGE and 3 fractions containing high concentrations of pure enzyme were collected in glass vials. Enzyme concentration was calculated from the difference in absorbance at 280 nm and 340 nm. The molecular extinction coefficient of wild-type was ε ₂₈₀ =52,420 M ⁻¹ cm ⁻¹ .

Genes for PET2 mutants were constructed by PCR. A primer pair containing the mutation was used and the product was ligated by the NEBuilder HiFi Assembly (New England Biolabs) according to the manufacturer's instructions. Ligated plasmids were transformed into Tuner (DE3) competent cells (Novagen) and the cells were incubated at 37° C. for 1 hour before being plated on LB agar plates containing 50 μg/mL kanamycin. A single colony was inoculated into 10 mL of LB medium and incubated at 37° C., 140 rpm for 16 hours. Plasmids were purified from cells and the sequence of the coding region was confirmed. The mutant enzyme was purified using the same procedure as the wild type. The molecular extinction coefficient for variants containing R47C-G89C or L265C-A295C is ε ₂₈₀ =52,540 M ⁻¹ cm ⁻¹ and for variants containing Y262C-L298C is ε ₂₈₀ =51,280 M ⁻¹ . cm ⁻¹ and the molecular extinction coefficients of variants containing W174H or Q134Y were ε ₂₈₀ =46,730 M ⁻¹ cm ⁻¹ or ε ₂₈₀ =53,600 M ⁻¹ cm ⁻¹ , respectively.

Enzyme for crystallization was purified by a slightly different procedure. After affinity purification with a histidine-6 tag, the enzyme solution was eluted from NAP10 with 10 mM sodium phosphate buffer (pH 7.0) and 5 mL of Toyopearl equilibrated with 10 mM sodium phosphate buffer (pH 7.0). Loaded on DEAE-650. Unbound enzyme was eluted with the same buffer and concentrated at 8000 g with Vivaspin 20 (molecular weight cutoff = 10K). 500 μL of enzyme solution was loaded onto Superdex 75 Increase (Cytiva) equilibrated with 10 mM Tris-HCl buffer (pH 7.5). The eluted enzyme was concentrated at 8000 g with Vivaspin 20 (molecular weight cutoff = 10K).

For single-molecule fluorescence imaging, a cysteine residue (Cys309) was added to the C-terminus of PET2 wild-type and PET2(7M) (R47C-G89C-F105R-E110K-S156P-G180A-T297P) for labeling with Alexa Fluor 555 maleimide. added. This enzyme was purified by the same procedure as for activity measurement. The purified enzyme was reduced with 1 mM DTT at 25° C. for 1 hour and the reaction mixture was loaded onto a NAP5 column equilibrated with 10 mM sodium phosphate solution (pH 7.0). Alexa Fluor 555 maleimide dissolved in DMSO was mixed with the enzyme for 1 hour at 25°C. The mixing ratio of the enzyme and Alexa Fluor 555 was 1:3 (molar ratio). Concentration by VIVAspin20 at 8000 g followed by removal of unreacted dye by NAP10 column equilibrated with 10 mM sodium phosphate solution (pH 7.0). After measuring the absorption spectrum in the range of 650 nm to 250 nm, the labeled enzyme was stored at 4°C. The molecular extinction coefficient of Alexa Fluor 555 was ε ₅₅₆ =158,000 M ⁻¹ cm ⁻¹ . From the absorption spectrum, the absorbance of Alexa Fluor 555 at 280 nm was calculated to be 5.5% of the absorbance at 556 nm. The corrected labeling rates of PET2 wild-type-C309 and PET2(7M)-C309 were 109% and 122%, respectively. As a control experiment, labeling reactions against PET2 wild-type and PET2(7M) without C309 were performed together, and the labeling rates of PET2 wild-type and PET2(7M) were 3.4% and 7.1%, respectively. , mainly C309 was confirmed to react with Alexa Fluor 555 maleimide.

2.3 Determination of Thermostability and pH Optimum Using Model Substrates 1 μM PET2 wild type dissolved in 100 mM sodium phosphate solution (pH 7.0) was incubated at various temperatures (40-90° C.) for 1 hour. After that, it was ice-cooled. The enzyme was subsequently diluted to 200 nM with a 200 mM sodium phosphate solution (pH 7.0). 25 μL of enzyme solution was mixed with an equal volume of 2 mM BHET in ultrapure water and incubated at 60° C. for 10 minutes. 10 μL of the reaction mixture was injected onto a LUNA C18 column (Phenomenex) and terephthalic acid (TPA), mono-2-hydroxyethyl terephthalate (MHET), and BHET were added from 20 mM sodium phosphate buffer (pH 7.0) to methanol. separated with a gradient of up to Each compound was detected by absorbance at 210 nm. TPA and BHET were purchased from Tokyo Chemical Industry Co., Ltd., and MHET was purchased from Activa Scientific. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (600 seconds).

The BHET hydrolytic activity of PET2 wild type was also measured in different pH buffers (sodium citrate buffer for pH 3.0, sodium acetate buffer for pH 4.0 and pH 5.0, pH 6.0 and sodium phosphate buffer for pH 7.0, Tris-HCl buffer for pH 8.0, glycine-NaOH buffer for pH 9.0, boric acid-NaOH buffer for pH 10.0) . 25 μL of 200 nM enzyme in 200 mM buffer was mixed with an equal volume of 2 mM BHET in ultrapure water and incubated at 60° C. for 10 minutes. Spontaneous degradation of BHET in the absence of enzyme was subtracted as background. The product separation and detection procedures were the same as above. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (600 seconds).

2.4 PET Film Degradation Activity Amorphous PET films (thickness = 0.25 mm, code ES301445) and biaxially oriented (crystalline) PET films (thickness = 0.25 mm, code ES301450) were purchased from Goodfellow. . This PET film was punched to produce circular discs (3 mm diameter, surface area 0.16 cm ² /disc, 2.0 mg/amorphous disc, 2.3 mg/crystalline disc). The PET disc was then immersed in 20% by volume ethanol, washed, and incubated at 25° C., 20 rpm for 1 hour. The washed discs were dried at room temperature and stored in glass vials.

Two PET discs (0.32 cm ² , 4.0 mg amorphous discs) were inserted into a glass vial and 250 μL of 0.1 μM enzyme dissolved in 50 mM sodium phosphate solution (pH 7.0) was added. to initiate decomposition. The reaction mixture was incubated at 60°C for 60 minutes. Supernatants were collected and analyzed for product concentration by HPLC using the same procedure for analysis of BHET hydrolysis. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 minutes).

The optimum reaction temperature was evaluated in the range of 60°C to 80°C by the same procedure as above. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 minutes). In addition, the activity was measured for 960 minutes (16 hours) at moderate temperature (30° C.) in the same manner. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (960 min). For long-term degradation assays, two PET discs (0.32 cm ² , 4.0 mg amorphous disc or 4.6 mg crystalline disc) were added to 0.1 μM PET2 in 50 mM sodium phosphate solution (pH 7.0). 1, 2, 4, 8, 16 with 250 μL wild type at 60° C. and with 0.1 μM PET2 (7 M) in 50 mM sodium phosphate solution (pH 7.0) at 68° C. , and incubated for 24 hours.

2.5 Thermal stability measurement by CD spectrum J-1500KS equipped with 400 μL of 13 μM enzyme in 10 mM sodium phosphate solution (pH 7.0) in a quartz cell and equipped with a Peltier thermostatic cell holder (JASCO Corporation (JASCO)). was used to measure the CD spectrum of the enzyme at 250-200 nm at 20°C. The signal at 230 nm was followed while the enzyme was heated from 20°C to 95°C at a rate of 1°C/min. The CD spectrum of the denatured enzyme at 250-200 nm was measured at 95°C. The melting temperature (Tm) of the enzyme was estimated by curve fitting the temperature dependence of the signal at 230 nm using denaturing protein analysis software (JASCO).

2.6 X-ray crystallography 1 μL of purified PET2 (2M) (F105R-E110K variant) (40 mg/mL) was mixed with 1 μL of 1 M ammonium sulfate in 100 mM Tris-HCl (pH 8.0) and incubated at 20°C. bottom. 1 μL of purified PET2 (7M) (80 mg/mL) was mixed with 1 μL of 10 w/v % PEG3350 in 100 mM Tris-HCl (pH 7.5) and incubated at 20°C. Both crystals were immersed in a reservoir containing 30% by volume ethylene glycol immediately prior to diffraction measurements. Using the homology modeling structure of PET2 wild type built on the SWISS-MODEL server ⁽²⁹⁾ , phases were determined by molecular replacement with phaser. The model structure was refined using the Phenix refine program ⁽³⁰⁾ and improved with Coot ⁽³¹⁾ . PDB2PQR ⁽³²⁾ was used to calculate the protonation state of the enzyme at pH 7.0 and APBS ⁽³³⁾ to calculate the surface charge and plotted in Pymol.

2.7 Single Molecule Fluorescence Imaging of Binding and Dissociation Observation was performed at 25° C. using the fabricated total internal reflection fluorescence microscope. 50 mg of finely chopped amorphous PET film was dissolved in 1 mL of hexafluoro-2-propanol and precipitated with 20 mL of acetone to remove autofluorescent contaminants on the PET film. After centrifugation at 20,000 g for 5 minutes, the supernatant was removed and the precipitate was suspended in 5 mL of acetone. After centrifugation at 20,000 g for 5 minutes, the supernatant was removed and the precipitate was completely dried at 70° C. for 1 hour and dissolved in 1 mL of hexafluoro-2-propanol. The PET solution was diluted to 5 mg/mL with hexafluoro-2-propanol, and 50 μL of the diluted PET solution was spin-coated at 3000 rpm for 10 seconds onto coverslips that had been incubated overnight in 10 M KOH and washed, After that, it was thoroughly washed with ultrapure water. The PET spin-coated coverslips were dried overnight at room temperature. The PET thin film formed on the cover glass was observed as a bright field image. The enzyme labeled with Alexa Fluor 555 was diluted to 50 pM with a 50 mM sodium phosphate solution (pH 7.0), dropped onto the PET thin film on the cover glass, and then covered with a topcoat wall to retain the enzyme solution. A laser with a power density of 0.25 μW/μm ² excited the dye on the sample surface at 532 nm and fluorescence images were recorded at 5 frames/second for 1000 seconds. After single-molecule observation, the PET thin film was stained with a 10 nM dye-labeled enzyme solution, and the area covered by PET was calculated. For binding rate constant analysis, the cumulative number of bound molecules was counted as a function of time in each movie, and the time required for the binding of 100 enzyme molecules was determined. The binding rate constant was then estimated by dividing 100 (molecules) by the required time and further dividing the resulting value by the area of the PET thin film within the field of view and the enzyme concentration in the solution. The binding rate constant thus has units of s ⁻¹ μm ⁻² M ⁻¹ . The average area of PET thin films in the field was 163±31 μm ² and 169±19 μm ² in PET2 wild-type and PET2(7M), respectively. Dissociation rate constants were obtained by fitting the distribution of binding times of individual molecules with a sum of double exponential decay functions. The ratio of slow to fast dissociation events was estimated from the area of the fitted curve.

To check the photobleaching time of Alexa Fluor 555, 50 μL of 100 pM PET2-Alexa Fluor 555 was directly spin-coated onto the coverslip at 3000 rpm for 10 seconds. After dropping 20 μL of 50 mM sodium phosphate solution (pH 7.0) under the same conditions as for single-molecule imaging of binding and dissociation, enzyme molecules firmly bound to the cover glass were observed. The distribution of photobleaching times for each spot was fitted with a single exponential decay function and the time constant was estimated to be 38.1 seconds.

2.8 Degradation Assay for PET Thin Films Used for Single Molecule Imaging PET thin films were prepared in the same manner as for single molecule imaging. 50 μL of 0.1 μM PET2 wild type or PET2 (7 M) in 50 mM sodium phosphate solution (pH 7.0) was dropped onto the film and incubated for 60 minutes at room temperature (approximately 26° C.). A cover glass coated with a PET thin film was covered with a Petri dish to prevent evaporation of the droplets. After incubation, the droplets were collected and analyzed by HPLC for product concentration. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 min).

2.9 Activity Measurement of Alexa Fluor 555-Labeled Enzyme It was incubated with a disk of amorphous PET film (0.32 cm ² , 4.0 mg) at 60° C. for 60 minutes. After incubation, supernatants were collected and analyzed for product concentration by HPLC. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 min).

_2.10 Ca ²⁺ dependence of PET degrading activity by PET2 wild type A disk of amorphous PET film (0.32 cm ² , 4.0 mg) was combined and incubated at 60° C. for 60 minutes. After incubation, 200 μL of supernatant was collected and mixed with 60 μL of ultrapure water or _CaCl2 solution to adjust the _CaCl2 concentration to 231 mM. The mixture was further mixed with 68.6 μL of 1 M sodium phosphate buffer (pH 7.0) to precipitate calcium phosphate. Precipitates were removed using a 0.22 μm spin column filter at 15,000 g for 3 minutes and 10 μL of flow-through was injected into the HPLC. Activity values were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 min).

2.11 Crystallinity evaluation of PET films by differential scanning calorimetry (DSC) and X-ray diffraction (XRD) Amorphous PET films and biaxially oriented crystalline PET films were punched to a diameter of 5 mm, and the same procedure as for decomposition activity measurement was performed. washed with One disk was placed in an aluminum dish and the heat flow was measured with a differential scanning calorimeter (DSC) (model DSC-60, Shimadzu Corporation). The disc was heated from 30°C to 290°C at 10°C/min. For measurement of regenerated PET from the hexafluoro-2-propanol solution, 50 μL of the 50 mg/μL PET solution used in single-molecule imaging was dropped onto an aluminum dish and dried at room temperature for 2 hours. The heat flow was measured with the same procedure for both PET films. Thermal crystallization and thermal melting were estimated from the peak area, and the endothermic amount (ΔH) was calculated from the difference between the heat of crystallization and the heat of fusion. The heat of fusion (ΔH ₀ ) of fully crystalline PET was 140.1 J/g, and the degree of crystallinity of both PET films was calculated from ΔH and ΔH ₀ ⁽³⁴⁾ . The crystallinity estimates for amorphous PET, crystalline PET, and recycled PET used in this study were 0.02%, 59.3%, and 5.89%, respectively.

X-ray diffraction (XRD) of the PET film was performed using RINT-Ultima III (Rigaku Corporation). The PET film was fixed on a stage, and X-ray diffraction from a copper target (40 mA, 40 kV) was measured at diffraction angles of 3° to 90° at 25°C. Amorphous PET showed no peak and crystalline PET showed a strong peak corresponding to the [100] crystal plane.

3. Results 3.1 PET2 Wild-type Thermostability and pH Optimum
PET2 wild-type was incubated with BHET, a moderately water-soluble PET building block, and the MHET produced was quantified by HPLC (TPA was not detected under the conditions tested). The thermostability of PET2 wild-type was evaluated by examining the residual activity after treatment for 1 hour at each temperature between 40 and 90° C. at pH 7.0. A decomposition activity of 1.3 s ^-1 was maintained up to 65°C. At temperatures above 70° C., on the other hand, almost no activity was detected (FIG. 2A). Degradation activity was also measured at pH 3.0 to pH 10.0 at 60° C., and the highest activity was observed at pH 6.0 and 60° C. (FIG. 2B). Therefore, the standard reaction conditions were 60° C. and pH 7.0.

Next, the CD spectra of PET2 wild type at pH 7.0 were compared at 20°C and 95°C (Fig. 2C). The CD spectrum of PET2 wild-type at 20° C. showed a mixture of α-helices and β-sheets, and a downward peak was observed at about 230 nm. The CD spectrum at 95° C. appeared random coil-like in the short wavelength range, but also retained signals from secondary structures. Since the signal difference at 222 nm, 215 nm corresponding to α-helix and β-sheet, respectively, was not very large between 20 °C and 95 °C, the Tm of PET2 wild-type was based on the signal change at 230 nm due to tryptophan residues. calculated ⁽³⁵⁾ . As a result, the Tm of PET2 wild type was estimated to be 69.0° C. (FIG. 2D), which was consistent with the degradation activity after heat treatment (FIG. 2A).

Since the thermostability and PET degrading activity ^of cutinase LCC, TfCut2, and Cut190 have been reported to be enhanced in the presence of Ca ^(36-38) , we explored the Ca ²⁺ dependence of the PET degrading activity of PET2 wild-type. tested. For PET2 wild-type, the concentration of product produced was only 1.1-fold higher in the presence of 10 mM and 100 mM Ca ²⁺ than in the absence of Ca ²⁺ . Even at 300 mM _CaCl2 , the PET degrading activity was comparable to that in the absence of Ca2 ⁺ . From these results, the following experiments measure the PET degrading activity of PET2 mutants in the absence of _CaCl2 .

3.2 Screening of Single Mutations To improve the thermostability and activity of PET2, single mutations were introduced based on the homology model structure (Fig. 3A) and the effects of the mutations were investigated. The Tm and amorphous PET degradation activity of the wild-type and mutant enzymes were measured and plotted (Fig. 3B). First, a basic amino acid residue ⁽¹¹⁾ characteristic of IsPETase and a single mutation ⁽³⁹⁾ reported to enhance the activity of IsPETase were introduced (Fig. 3B, ♦). As a result, the F105R and E110K mutations increased the Tm by more than 1° C. and enhanced the PET degradation activity by 1.5 and 1.4 times, respectively, compared to the wild type. The L298R mutation, which is coplanar with F105R and E110K but not present in IsPETase, decreased Tm and activity. Of the mutations based on modified IsPETase mutations ⁽³⁹⁾ , Q183R and Q134Y enhanced activity. However, the Q183R mutation lowered the Tm and the Q134Y mutation significantly prolonged elution time during size exclusion chromatography (SEC) and greatly reduced yield after purification. Also, the S202Q and S155D mutations increased the Tm but decreased the activity.

Based on the homology modeling structure, amino acid residues matching the proline dihedral angle were searched and proline mutations were introduced (FIG. 3B, ●). The Tm values for the S156P and T297P mutations were 1-2°C higher than the wild type, while the Tm values for the D53P and A192P mutations were lower than the wild type. The activities of these mutants were not significantly different from the wild-type except for A192P, which showed less than half the activity of the wild-type.

In many PET degrading enzymes, four consecutive glycine residues form an α-helix near the serine residue (Ser175 in PET2) of the catalytic triad, and PET2 also forms the corresponding glycine residues (Gly177 to Gly180). have. This Gly177 to Gly180 was mutated to alanine one by one (FIG. 3B, ▲). The G180A mutation was one of the most effective mutations, greatly improving Tm (+2.6°C) and activity (1.5-fold). On the other hand, the other three mutations reduced Tm or activity below wild type.

In addition, four pairs of cysteine mutations near the N-terminus or C-terminus of PET2 were tested (Fig. 3B, ▼). The Tm of the R47C-G89C mutant pair was 72.0°C, 3°C higher than that of the wild type. The C-terminal cysteine mutation pairs (Y262C-L298C and L265C-A295C) also exhibited Tms 1-2°C higher than that of the wild type, but the activity of these mutation pairs was reduced to about half that of the wild type. The T64C-T86C mutation was also attempted, but the purification yield was very low and sufficient sample was not obtained for characterization.

3.3 Combination of Mutations Single mutations that showed a higher Tm or activity than the wild type were integrated (Fig. 3C). The PET2 F105R-E110K mutant (PET2(2M)) exhibited a Tm of 70.7° C. and hydrolyzed amorphous PET at 0.71 min ⁻¹ . These values are higher than those of the respective single mutations. The PET2 F105R-E110K-S156P-T297P mutant (PET2(4M)) showed a Tm 1.6° C. higher than that of the above two mutants, but the activity was only improved by 0.13 min ⁻¹ (0. 71 min ⁻¹ to 0.84 min ⁻¹ ). Further introduction of G180A (PET2(5M)) significantly improved the activity (1.4-fold that of PET2(4M)), but only slightly increased the Tm. Furthermore, an additional disulfide bond increased the Tm by 3.1°C and slightly increased the activity. The resulting R47C-G89C-F105R-E110K-S156P-G180A-T297P mutant (PET2(7M)) exhibited a 6.7°C higher Tm and a 3-fold higher activity than the wild type at 60°C. Among them, the disulfide bond based on the R47C-G89C mutation greatly increased the Tm, and the disulfide bond also improved the activity despite being located away from the active site, making it the most effective. considered to be one of the major mutations.

3.4 X-Ray Crystal Structures of PET2 Mutants The X-ray crystal structures of PET2(2M) and PET2(7M) were resolved at 1.3 Å and 1.8 Å resolution, respectively. No crystals of PET2 wild type were obtained. For this reason, the PET2 wild-type structure was reconstructed by backmutation of PET2(2M) with Pymol. The root mean square deviation (RMSD) of the overall structure between PET2(7M) and PET2(2M) was 0.241 Å, indicating that the structures were very similar. Electron density maps of PET2 mutants were sufficient to recognize the side chains of the mutated residues (Figures 4A and 4B). Since the introduction of two proline mutations (S156P and T297P) did not alter the loop structure, these positions were considered suitable for proline dihedral angles, as expected. Mutation of Gly180 to alanine did not alter the α-helical structure itself involving this Ala180, but the adjacent α-helix appeared to be more stable (FIGS. 4A and 4B, right). PET2 wild-type has six cysteine residues in the sequence and two natural disulfide bonds (Cys218-Cys255 and Cys289-Cys306) were observed in the PET2(2M) structure. Another cysteine residue pair (Cys43 and Cys39) was present in the N-terminal disordered region (Ala28 to Asn44) and could not be observed. In the PET2(7M) structure, the cysteine residue pair (R47C-G89C) introduced at the N-terminus clearly formed a disulfide bond (Fig. 4B). These mutations caused a slight shift in the two-loop structure (FIGS. 4A and 4B, left) and no electron density of Tyr45 could be observed. The N-terminal residues (Ala28 to Asn44) could not be modeled due to unclear electron density maps in both PET2(2M) and PET2(7M), suggesting that the N-terminal residues are disordered in PET2. rice field.

The PET2 wild-type structure was then reconstructed by backmutation of the PET2(2M) structure and the surface charge at pH 7.0 was compared to that of PET2(7M) and IsPETase (Fig. 4C). Neutral to negative charges were observed around Phe105 and Glu110 in PET2 wild type (Fig. 4C, left). On the other hand, PET2(7M) (Fig. 4C, center) had a larger positive charge area than PET2 wild type and was comparable to that of IsPETase (Fig. 4C, right).

Since PET2 was originally discovered as a lipase ⁽¹⁹⁾ , the structure around the catalytic triad of PET2(2M) was also compared with lipases. The Thermomyces lanuginose lipase has a lid domain covering the catalytic triad ⁽⁴⁷⁾ , whereas the catalytic triad of PET2(2M) was solvent exposed like the Bacillus subtilis LipA. The overall RMSD of PET2 (2M) relative to IsPETase (PDB ID: 6ANE) was 0.552 Å and the overall RMSD relative to LipA (PDB ID: 1I6W) was 15.4 Å. Therefore, from a structural point of view, PET2 is a PET hydrolase rather than a lipase.

To illustrate the calcium independence of PET2's PET degrading activity, positions corresponding to the calcium binding site of Cut190 ⁽¹⁵⁾ were compared between LCC, IsPETase and PET2 (2M). Binding site 1 is basically built up by the main chain carbonyl groups and Ca ²⁺ can bind to LCC and PET2. At binding site 2, aspartic acid (Asp250) and two glutamic acids (Glu220 and Glu296) in Cut190 are changed to threonine, glutamine and serine in PET2, respectively. Therefore, the affinity for Ca ²⁺ is lower for PET2 than for Cut190. Also, binding site 3 of Cut190 is completely occupied by a loop in PET2. Therefore, from a structural point of view, the calcium independence of PET2's PET degrading activity is reasonable.

3.5 Characterization of PET2 association and dissociation by single-molecule fluorescence imaging Single-molecule fluorescence imaging was used to compare the association and dissociation rate constants of PET2 wild-type and PET2(7M). A free cysteine residue (Cys309) was inserted between the C-terminus of the enzyme and the TEV protease recognition site and labeled with Alexa Fluor 555 maleimide. The labeling rates of PET2 wild-type-C309 and PET2(7M)-C309 were 109% and 122%, respectively. The PET degradation activities of PET2 wild-type and PET2 wild-type-C309-Alexa Fluor 555, or PET2(7M) and PET2(7M)-C309-Alexa Fluor 555 are similar to each other, and the dye label does not affect the activity. It was considered. Moreover, the labeling rates of PET2 wild-type and PET2(7M) without Cys309 were only 3.4% and 7.1%, respectively, suggesting that the dye mainly reacts with Cys309 under these labeling conditions. The pristine PET film was found to be highly autofluorescent, making single-molecule imaging of the dye-labeled PET2 enzyme difficult. The reason for the autofluorescence is not clear, but the autofluorescence could be reduced by dissolving PET in hexafluoro-2-propanol and precipitating it with acetone.

For single-molecule imaging of the dye-labeled PET2 enzyme, acetone-precipitated PET was redissolved in hexafluoro-2-propanol, spin-coated onto coverslips, and dried completely, leaving some unformed moieties. A thick PET thin film was formed. From PET thin films on coverslips, at room temperature, PET2 (7 M) produced a 2.6-fold higher concentration of product than PET2 wild-type. At this time, the appearance of the PET thin film was significantly different from the original amorphous PET film (Fig. 5A). The difference in activity between PET2 wild-type and PET2(7M) on PET thin films was similar when tested with amorphous PET films at 60°C. Therefore, we thought that PET thin films on coverslips could be used as model substrates for single-molecule imaging of PET2 binding and dissociation. At low concentrations (50 pM) of dye-labeled PET2 wild-type and PET2 (7 M), fluorescence signals from individual molecules were clearly observed (Fig. 5B). After single-molecule observation, the PET thin film was stained with a high concentration (10 nM) of a dye-labeled enzyme (Fig. 5C), and a single-molecule movie of PET2 was superimposed on this image to localize the PET thin film. The dye-labeled enzyme binds only to the PET thin film and not to the coverslip. Next, the binding rate constant and binding time (dissociation rate constant) of PET2 wild type and PET2 (7M) were analyzed and compared. To calculate the binding rate constant, we counted the cumulative number of bound molecules as a function of time in each movie and determined the time required for binding of 100 molecules of enzyme (Fig. 5D). Next, 100 (molecules) is divided by the required time (seconds), and the obtained value is further divided by the area of the PET thin film in the field of view (μm ² ) and the enzyme concentration (M) in the solution to obtain the binding rate constant. was estimated. As a result, the binding rate constant of PET2 wild-type was (2.8±1.6)×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ and that of PET2 (7M) was (7.5±3 .0)×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ (Table 1). Therefore, the binding rate constant of PET2(7M) was 2.7 times higher than wild type.

On the other hand, the dissociation rate constants were not significantly different between PET2 wild type and PET2(7M). The distribution of PET2 wild-type binding times fitted the sum of the double exponential decay functions better than the single exponential decay function, suggesting that there are at least two PET binding states (Fig. 5E, Table 1). The fast dissociation rate constant was 4.3 s ⁻¹ and the slow dissociation rate constant was 0.71 s ⁻¹ . From the area of the fitting formula, the percentages of fast dissociation and slow dissociation were calculated to be 69.3% and 30.7%, respectively. The distribution of binding times of PET2(7M) also fits well with a double exponential decay function, with rate constants (fractions) for fast and slow dissociation of 5.0 ^s (85.2%) and 0.54 s, respectively. ^-1 (14.8%) (Fig. 5F, Table 1). For both PET2 wild-type and PET2(7M), these dissociation rate constants were much greater than the rate constant for photobleaching of Alexa Fluor 555 (0.026 s ⁻¹ ). Therefore, Alexa Fluor 555 photobleaching is not the cause of the fast and slow components.

Table 1 Note:
a: Cys309 was additionally introduced at the C-terminus for Alexa Fluor 555 labeling.
b: Binding rate constants were calculated from the time required for binding of 100 molecules of enzyme and were further normalized by area covered by PET in the field and concentration of enzyme in solution.
c: Dissociation rate constants were calculated by fitting the distribution of binding times of individual molecules with the sum of two exponential decay functions.
d: The ratio of fast and slow dissociation events was calculated from the area of the fitting equation.
e: Values are mean±SD from 5 independent movies.

3.6 Temperature Dependence of Activity and Long-Term Stability The temperature dependence of the amorphous PET degradation activity in wild-type and mutants was measured, and the optimum temperature at which the highest activity was observed was determined. A maximum activity of 0.4 min ⁻¹ was observed for PET2 wild type at 60° C., and the activity decreased at higher temperatures (FIG. 6A). On the other hand, as the number of mutations increased, the optimum temperature shifted to higher than 60°C. PET2(7M) exhibited a maximum activity of 2.7 min ⁻¹ at 68° C., which was two-fold higher than the activity at 60° C. (FIG. 6A). PET2 (7M) also retained detectable activity at 80° C., whereas the wild-type and two other mutants (4M and 5M) were inactivated. This result indicates that the R47C-G89C mutation of PET2(7M) contributes significantly to the thermostability of the final mutant. Moreover, a comparison of the activities of PET2(5M) and PET2(7M) revealed that the R47C-G89C mutation also improved the activity at each temperature. In addition, the hydrolytic activity of amorphous PET discs by PET2 wild-type and PET2(7M) was also measured at 30°C to confirm the difference in activity at moderate temperatures. The hydrolytic activity of PET2 wild-type and PET2(7M) was very low at 30° C., but the activity value of PET2(7M) was three times that of wild-type. Therefore, the difference was similar to that observed at 60°C.

PET degradation by PET2 wild-type and PET2(7M) was then compared at their respective temperature optima of 60° C. and 68° C. with incubation times up to 24 hours. For amorphous PET degradation, the amount of product produced by PET2(7M) remained consistently >3-fold that produced by PET2 wild-type (Fig. 6B). Furthermore, importantly, PET2(7M) maintained a nearly constant degradation rate from 1 to 24 hours, suggesting long-term thermal stability at the optimum temperature of 68 °C. there is Furthermore, the decomposition of crystalline PET was also investigated. PET2(7M) always maintained twice the activity of PET2 wild type. Again, PET2(7M) maintained a nearly constant rate of degradation between 1 and 24 hours. However, PET2 wild-type at 0.1 μM and PET2 (7 M) yielded only 1.7 μM and 4.5 μM of product, respectively, from crystalline PET after 24 hours of reaction. These results suggest that the PET2 enzyme preferentially hydrolyzes the mobile portion of the amorphous region of PET, consistent with previous studies of other PET hydrolases ^{(4, 12 , 13, 48)} .

4. Discussion The maximum reaction temperature (65° C.) of PET2 wild type estimated from residual activity (FIG. 2A) was in good agreement with Tm (69.0° C.) estimated from the change in CD signal at 230 nm (FIG. 2D). . Furthermore, previous studies have shown that the lipase activity of PET2 wild-type is at 60°C and that activity decreases at higher temperatures (the original name for the lipase is LipIAF5.2, but here for convenience PET2 ⁽¹⁹⁾ . These results suggest that the signal of tryptophan residues at 230 nm correlates very well with the catalytic activity of PET2 wild type. We found that the signal at 230 nm of the PET2 W174H mutant was much weaker than the wild type. Therefore, the characteristic signal at 230 nm of PET2 could be attributed to Trp174 located next to the Ser175 residue of the catalytic triad, which is consistent with the correlation described above. On the other hand, the optimum pH for BHET hydrolysis by PET2 was around 7 in this study (Fig. 2B). In previous studies, the optimum pH was 10.5 for p-nitrophenyl myristate hydrolysis. Since BHET spontaneously degrades to MHET even in the absence of PET2 at high pH, this difference is presumed to be due to differences in substrate stability.

Interestingly, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% Tween 80 inhibited the lipase activity of PET2 by only 30%, whereas 1 mM ethylene glycol tetraacetic acid (EGTA) or 1% Triton X-100 completely inhibited PET2 lipase activity. lose ⁽¹⁹⁾ . EGTA and EDTA differ in the structure of the connecting group that connects two pairs of acetic acid groups. The ethylene glycol structure of EGTA may bind near the active site of PET2 and interfere with substrate binding. Also, among the surfactants tested, only Triton X-100 has an aromatic ring in its structure. These results suggest that PET2 has binding affinity for ethylene glycol and aromatic rings. In the present study, no Ca ²⁺ concentration dependence was observed in PET degrading activity in PET2 wild type. This result is reasonable given that there is only a potential Ca ²⁺ -binding site corresponding to site 1 of Cut190 in the PET2(2M) structure. Therefore, the ion chelating ability of EGTA and EDTA is independent of changes in the lipase activity of PET2.

Results for single mutations in PET2 suggest that mutations that increase IsPETase thermostability and activity are less effective against PET2. In particular, the W174H mutant had a 5°C higher Tm than the wild type, but a four-fold decrease in activity (Fig. 3B). In contrast, mutations in the surface charge of PET2 (F105R and E110K) that mimic that of IsPETase enhanced activity. Interestingly, these mutations also increased the Tm of PET2. The F105R mutation replaces hydrophobic residues on the surface of the enzyme with positively charged residues. A similar mutation has been reported to increase the thermostability of acetylcholinesterase ⁽⁴⁹⁾ . On the other hand, it is unclear why the E110K mutation thermostabilizes PET2. G180A is one of the most effective mutations that improved both Tm and activity of PET2. The α-helical structures flanking Gly180 and Ala180 are subtly different between PET2(2M) and PET2(7M) (FIGS. 4A and 4B, right). In PET2(7M), the main chains of Arg138 and Arg139 are slightly shifted to form an ideal α-helix. This may contribute to the improved thermal stability and activity. On the other hand, the G177A and G179A mutations decreased Tm and activity. This is speculated to be due to the fact that the side chains of these alanine residues are too close to the main chain carbonyl oxygen atoms of Pro100 and Trp199, as confirmed by the crystal structure of PET2(2M) (Fig. 4A). The R47C-G89C mutation formed a disulfide bond and increased Tm even more than Y262C-L298C and L265C-A295C (Fig. 3B). In the crystal structure, the electron density of the peptide chain was unclear and the N-terminal residues (Ala28-Asn44) were not modeled. These results suggest that the N-terminal region of PET2 is more flexible than the C-terminal region. Formation of disulfide bonds at the N-terminal region is therefore believed to provide more effective thermal stabilization than disulfide bonds at the C-terminal region. For LCC and Cut190, disulfide bond formation at Ca ²⁺ -binding site 2 enhanced stability ^{(12, 15)} . Since PET2 and IsPETase already have a disulfide bond in this vicinity, we did not attempt to introduce a new disulfide bond at the same site in this study.

Single-molecule fluorescence imaging revealed that the binding rate constant of PET2(7M) was 2.7-fold that of PET2 wild-type (Fig. 5D, Table 1). On the other hand, both enzymes had a fast component and a slow component in the dissociation rate constant, and these values and proportions were similar for both enzymes (Fig. 5E, Fig. 5F, Table 1). We initially predicted that the enhanced activity in the mutants was obtained by strong cation-π interactions between the mutant surface lysine or arginine residues and the aromatic ring of PET ⁽⁵⁰⁾ . However, the results of this study suggest that the PET-bound state of PET2(7M) is not stabilized by surface charge mutation. Based on the proportions of the fast-releasing and slow-releasing components, we then calculated the binding rate constants of these components (Table 1). As a result, for PET2 wild type, the values for the fast and slow components were 1.9×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ and 0.86×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ , respectively. there were. For PET2 (7M), the values for the fast and slow components are 6.4×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ and 1.1×10 ⁸ s ⁻¹ μm ⁻² M ⁻¹ , respectively. there were. We also calculated these dissociation constants based on the ratio of the association and dissociation rate constants of the fast and slow components (Table 1). For PET2 wild type, the values were 2.3×10 ⁻⁸ μm ² M and 0.83×10 ⁻⁸ μm ² M for the fast and slow components, respectively. For PET2 (7M), the values were 0.78×10 ⁻⁸ μm ² M and 0.49×10 ⁻⁸ μm ² M for the fast and slow components, respectively. Thus, the association rate constant for the fast component of PET2(7M) is 3.4-fold higher than PET2 wild-type and the dissociation constant is 2.9-fold lower than PET2 wild-type. Interestingly, the activity of PET2 (7M) on amorphous PET films at 60 °C was 3.2-fold higher than that of PET2 wild-type (Fig. 6A), and on PET thin films at room temperature was 2.6-fold higher. . The difference in activity between PET2 wild-type and PET2(7M) was comparable to the difference in the association and dissociation constants of the fast component. Thus, the mutant has more positive charges than the PET2 wild type at pH 7.0 (Fig. 4C), and the PET surface has a negative charge ⁽⁵¹⁾ , so one plausible explanation is that PET2 (7M) A possible explanation is that electrostatic attraction increases the local enzyme concentration near the PET surface, facilitating fast binding. It has been reported that Rhizopus oryzae lipase binds efficiently to aliphatic polymers (polybutylene succinate, polybutylene adipate, polylactic acid) but not to PET ⁽⁵²⁾ . The above results suggest that binding to PET requires different interactions. To further increase the PET2 binding rate constant, it may be necessary to modify the surface geometry of the enzyme to match the PET surface. For example, cellulose binding domains (CBMs) have finely tuned planes to match those of crystalline cellulose ⁽⁵³⁾ . Interestingly, it was previously reported that CBM and PET surfaces interact strongly ⁽⁵⁴⁾ . For example, T . fusca cutinase showed a 1.5-fold higher product concentration after 24 hours of incubation ⁽⁵⁵⁾ . Also, Thermomyces cellulosylitica cutinase fused with the binding domain of Alcaligenes faecalis polyhydroxybutyrate depolymerase showed a 3-fold higher product concentration than without the binding domain ⁽⁵⁶⁾ . Fusion proteins of hydrophobin and cutinase have been reported as another approach to improve binding affinity to PET surfaces ⁽⁵⁷⁾ . Although the mechanism of the fast and slow components is still unclear, both the association and dissociation constants of the slow component were not significantly different between PET2 wild-type and PET2(7M), suggesting that the slow component contributes to PET degradation. It is thought that it does not contribute (Table 1).

In the present study, PET2 (7 M) exhibited long-term thermal stability, with 0.1 μM of the enzyme activating 129 μM from amorphous PET film (1.3 cm ² /mL, 16 mg/mL) at 68° C. for 24 hours of incubation. of product was produced (Fig. 6B). Here, this PET2 mutant is compared with other previously reported PET hydrolases. A rationally engineered 0.5 μM IsPETase variant (S121E-D186H-R280A) showed a ^similar DSC profile to the amorphous PET we used. Incubation at 40° C. for 1 day produced approximately 70 μM of product ⁽⁵⁸⁾ . Therefore, PET2(7M) produced more product than this IsPETase mutant. Similarly, the novel enzyme PE-H (Y250S) mutant ⁽⁵⁹⁾ belonging to the type IIa PET hydrolases produced much less product on amorphous PET compared to PET2 (7M). . On the other hand, 2 μM of the Cut190 variants (Q138A-D250C-E296C-Q123H-N202H) yielded 17.5 mM of product from the same amorphous PET used in our study, upon incubation at 70° C. for 3 days. generated ⁽¹⁵⁾ . This amount is much higher than ours, requiring a 2-fold enhancement of the activity of PET2(7M) to reach the same output. In addition, 1.5 nmol of TfCut2 expressed in Bacillus subtilis decomposed 9.9 mg of amorphous PET chip after 24 hours at 70°C (rate: 23.5 min ^-1 ), and after 120 hours the chip was almost completely decomposed. decomposed ⁽¹³⁾ . To further improve the activity of PET2(7M), the following strategies may be effective. During degradation of amorphous PET films by PET2 wild-type and PET2(7M), a relatively large amount of MHET remained in solution as a product (Fig. 6C). Since IsPETase and MHETase fusion enzymes have shown a synergistic effect on PET degradation ⁽⁶⁰⁾ , the production of fusion enzymes with MHETase would be effective in completely degrading MHET to TPA. With respect to substrates, grinding of PET is an effective way to increase the accessible surface area and promote degradation. Indeed, the dissociation constant of TfCut2 for PET nanoparticles (<100 nm) is low, determined by isothermal titration calorimetry to be 0.043 mg/mL ^{−1 (61)} . Furthermore, the nephelometric dissociation constant for PET nanoparticles immobilized in agarose gel (mean diameter=164 nm) was 0.031 mg/mL ^{−1 (62)} . Increasing the accessible surface of the substrate is a promising means to improve enzyme binding. Furthermore, Tournier et al. produced 93.2 mg TPA-equivalent product per mg enzyme from 2 mg/mL amorphous PET powder suspension (smaller than 500 μm mesh) using native LCC (approximately 270 min ⁻¹ equivalent to) ⁽¹²⁾ . They also successfully generated a thermostable LCC variant (F243I-D238C-S283C-Y127G) that hydrolyzed 90% of 20 kg of waste PET powder in less than 10 hours ⁽¹²⁾ .

From the viewpoint of enzymes, so-called "processive catalysis", in which the enzyme repeats the catalytic cycle without dissociating from the polymer substrate, is desirable in order to further improve the activity per enzyme. For example, in the case of polycaprolactone, almost complete degradation has been achieved in one day using processive degradation by enzymes embedded in resins ⁽⁶³⁾ . Due to the processive degradation of PET, which has strong hydrophobic interactions between polymer chains, it is desirable for PET hydrolases to acquire thermal stability above the glass transition temperature of PET. In addition, PET hydrolases, like processive cellulases and chitinases, should also acquire the ability to move unidirectionally along single polymer chains without dissociation ⁽⁶⁴⁾ .

In conclusion, we succeeded in improving PET hydrolysis activity by thermal stabilization and surface charge modification of PET2, which is a PET hydrolase. X-ray crystallography revealed that the proline mutation maintained the original loop structure and the cysteine residue mutation pair formed a disulfide bond at the N-terminus of PET2. Although some of the mutations corresponding to mutations that increase the activity of IsPETase had no effect on PET2, single-molecule imaging analysis showed that the surface charge modification, performed with IsPETase as a reference, increased the binding rate constant of PET2, It became clear that it contributed to the improvement of activity. Among them, the introduction of two cysteine residues, R47C and G89C, was found to be one of the most useful mutations because it significantly increased Tm and also improved the activity of PET2. The combination of seven mutations increased the Tm of PET2 by 6.7°C and the optimum reaction temperature by 8°C. PET2(7M) can serve as one of the templates for the construction of more efficient PET hydrolases.

The PDB IDs for accession codes PET2(2M) (F105R-E110K) and PET2(7M) (R47CG89C-F105R-E110K-S156P-G180A-T297P) are 7EC8 and 7ECB, respectively.

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Claims (12)

A protein comprising any of the following amino acid sequences, which has polyethylene terephthalate hydrolysis activity.
(A) an amino acid sequence having mutations R20C and G62C relative to the amino acid sequence of SEQ ID NO: 1;
(A1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having R20C and G62C mutations with respect to the amino acid sequence of SEQ ID NO: 1, and having said R20C and G62C mutations;
(B) an amino acid sequence having mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO: 1;
(B1) has a sequence identity of 90% or more with the amino acid sequence having R20C, G62C, S129P, and T270P mutations with respect to the amino acid sequence of SEQ ID NO: 1, and the R20C, G62C, S129P, and T270P mutations an amino acid sequence having
(C) an amino acid sequence having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO:1; or (C1) R20C, G62C, relative to the amino acid sequence of SEQ ID NO:1; An amino acid sequence having 90% or more sequence identity with an amino acid sequence having mutations S129P, T270P, G153A, E83K, and F78R, and having mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R . 2. The protein according to claim 1, wherein the optimum temperature at pH 7.0 for the polyethylene terephthalate hydrolysis activity is above 60.0<0>C. 3. The protein according to claim 1 or 2, which has a melting temperature of 70.0°C or higher. The protein according to any one of claims 1 to 3, wherein said polyethylene terephthalate hydrolysis activity is calcium ion independent. 5. The protein according to any one of claims 1 to 4, which has a higher polyethylene terephthalate hydrolysis activity than the protein having the amino acid sequence of SEQ ID NO: 1 at pH 7.0. The protein of any one of claims 1-5, wherein the two cysteine residues at the R20C and G62C mutations form a disulfide bond. 7. The protein according to any one of claims 1 to 6, comprising any of the following amino acid sequences:
(A) an amino acid sequence having mutations R20C and G62C relative to the amino acid sequence of SEQ ID NO: 1;
(B) an amino acid sequence having the mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO:1; or (C) R20C, G62C, S129P, T270P, G153A, Amino acid sequence with E83K and F78R mutations. A polynucleotide comprising a nucleotide sequence encoding the protein according to any one of claims 1 to 7. A recombinant vector comprising the polynucleotide of claim 8 . A transformant containing the recombinant vector according to claim 9 . A composition for decomposing polyethylene terephthalate, comprising the protein according to any one of claims 1 to 7. A method for producing a recycled product, comprising decomposing polyethylene terephthalate with the protein according to any one of claims 1 to 7.

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