Protein, polynucleotide, recombinant vector, transformant, composition for decomposing polyethylene terephthalate, and method for producing recycled products

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

[Technical Field]

[0001] The present disclosure relates to a protein, a polynucleotide, a recombinant vector, a transformant, a composition for degrading polyethylene terephthalate, and a method for producing a recycled product. [Background technology]

[0002] Addressing environmental pollution caused by plastics and building a sustainable society are becoming increasingly important. Among these, polyethylene terephthalate (PET) accounts for approximately 4% of the plastics produced in Japan and is used in everyday items such as beverage bottles and clothing. Currently, most PET is incinerated or converted into carpets and other products, but recycling is desirable for sustainable use. Currently, two methods of recycling PET are used: physical recycling, in which the plastic is crushed and heat-treated to reshape it, and chemical recycling, in which the plastic is decomposed using chemical catalysts and then repolymerized. However, the former method can cause problems such as deterioration of physical properties and coloration, while the latter requires the use of highly hazardous chemicals and high-temperature processing. PET degradation technology using enzymes, which can reduce the amount of corrosive chemicals and heat required, is a technology that will become important in society in the future.

[0003] As a PET-degrading enzyme, a mutant with improved activity of IsPETase derived from a PET-utilizing bacterium has been reported (see Patent Document 1). In addition, Cut190 (see Patent Document 2), which originates from a cutinase that degrades the waxy layer of cutin, and LCC, another cutinase mutant with improved heat resistance and PET-degrading activity, have also been reported (see Patent Documents 3 and 4).

[0004] On the other hand, the ester bond of PET can also be degraded by esterases other than those mentioned above. For example, the LipIAF5.2 enzyme derived from an environmental genome, which was discovered as a lipase in 2009 (see Non-Patent Document 1), was reported to have PET-degrading activity in 2018 and was named PET2 (see Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 168811 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-119670 [Patent Document 3] International Publication No. 2012 / 099018 [Patent Document 4] Special Publication No. 2019-527060 [Non-patent literature]

[0006] [Non-Patent Document 1] Meilleur, C.; Hupe, JF; Juteau, P.; Shareck, F. Isolation and Characterization of a New Alkali-Thermostable Lipase Cloned from a Metagenomic Library. J. Ind. Microbiol. Biotechnol. 2009, 36, 853-61. [Non-patent document 2] Danso, D.; Schmeisser, C.; Chow, J.; Zimmermann, W.; Wei, R.; Leggewie, C.; Li, X.; Hazen, T.; Streit, WR New Insights into the Function and Global Distribution of Polyethylene Terephthalate (Pet)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes. Appl. Environ. Microbiol. 2018, 84, No. e02773-17. Summary of the Invention [Problem to be solved by the invention]

[0007] Previously known PET-degrading enzymes each have their own challenges when used for recycling. For example, IsPETase has a problem with low heat resistance. Furthermore, Cut190 and LCC are calcium ion dependent, which is a disadvantage for recycling, where it is desirable to minimize impurities. Furthermore, PET2's heat resistance and PET-degrading activity were not as high as those of other enzymes.

[0008] The present disclosure relates to providing a novel protein having excellent 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 comprising the protein, and a method for producing recycled products using the protein. [Means for solving the problem]

[0009] Means for solving the above problems include the following aspects. <1> A protein having polyethylene terephthalate hydrolysis activity, comprising any one 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 relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C and G62C mutations; (B) an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1; (B1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C, G62C, S129P, and T270P mutations; (C) an amino acid sequence having the following mutations relative to the amino acid sequence of SEQ ID NO: 1: R20C, G62C, S129P, T270P, G153A, E83K, and F78R; or (C1) An amino acid sequence having 90% or more sequence identity with an amino acid sequence having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO: 1, and having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R. <2> The optimum temperature for the polyethylene terephthalate hydrolysis activity at pH 7.0 is higher than 60.0°C. <1> The protein described in <3> The melting temperature is 70.0°C or higher. <1> or <2> The protein described in <4> The polyethylene terephthalate hydrolysis activity is calcium ion independent. <1> ~ <3> The protein according to any one of the preceding claims. <5> At pH 7.0, it has a higher polyethylene terephthalate hydrolysis activity than a protein having the amino acid sequence of SEQ ID NO: 1. <1> ~ <4> The protein according to any one of the preceding claims. <6> The two cysteine ​​residues at the R20C and G62C mutations form a disulfide bond. <1> ~ <5> The protein according to any one of the preceding claims. <7> Contains any of the following amino acid sequences: <1> ~ <6> The protein according to any one of the preceding claims. (A) an amino acid sequence having R20C and G62C mutations relative to the amino acid sequence of SEQ ID NO: 1; (B) an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1; or (C) Amino acid sequence having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO: 1. <8> <1> ~ <7> A polynucleotide comprising a base sequence encoding the protein according to any one of claims 1 to 4. <9> <8> A recombinant vector comprising the polynucleotide described in . <10> <9> A transformant comprising the recombinant vector described in 1. <11> <1> ~ <7> A composition for degrading polyethylene terephthalate, comprising the protein according to any one of claims 1 to 4. <12> <1> ~ <7> 10. A method for producing a recycled product, comprising decomposing polyethylene terephthalate with the protein according to any one of claims 1 to 9. [Effects of the Invention]

[0010] According to the present disclosure, there are provided a novel protein having excellent 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 comprising the protein, and a method for producing recycled products using the protein. [Brief explanation of the drawings]

[0011] [Figure 1] This figure shows an overview of PET hydrolysis by PET hydrolase, as well as an overview of PET and its hydrolysis products. PET is a water-insoluble synthetic polymer with crystalline and amorphous regions. PET hydrolase binds to the PET surface and captures molecular chains in the mobile range of the amorphous regions, incorporating them into its active site. PET chains are hydrolyzed into bis(hydroxyethyl) terephthalate (BHET), (hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG). [Figure 2A] The evaluation of wild-type PET2 is shown in Figure 2A. Thermostability was assessed by residual activity. After 1 hour of treatment at temperatures ranging 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 concentration of produced BHET (μM) was quantified and divided by the enzyme concentration (0.1 μM) and reaction time (600 seconds) to calculate activity. [Figure 2B]Figure 2B shows the pH dependence of BHET hydrolysis activity at 60°C for 10 minutes. The concentration of MHET produced (μM) was quantified and divided by the enzyme concentration (0.1 μM) and reaction time (600 seconds) to calculate activity. The concentration of MHET produced in the absence of PET2 was subtracted as background. [Figure 2C] Figure 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). The signal at 230 nm was used for melting temperature (Tm) analysis. [Figure 2D] Figure 2D shows the signal change at 230 nm during the temperature increase. [Figure 3A] The Tm and amorphous PET hydrolysis activity of each PET2 mutant are shown. Figure 3A shows the homology modeling structure of wild-type PET2. The positions of the mutations are indicated by bars. [Figure 3B] The Tm and amorphous PET hydrolysis activity of each PET2 mutant are shown. Figure 3B summarizes single mutations and cysteine ​​pairings for disulfide bond formation. Mutations based on wild-type or mutant IsPETase (◆), proline mutations (●), alanine mutations (▲), and cysteine ​​pairing mutations (▼) are plotted alongside the wild-type (WT) (■). Amorphous PET disks (0.32 cm, 4.0 mg) were incubated with 0.1 μM enzyme in 50 mM sodium phosphate buffer (pH 7.0) at 60 °C for 60 min. The produced TPA, MHET, and BHET were summed. Activity values ​​were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 min). [Figure 3C] The Tm and amorphous PET hydrolysis activity of each PET2 mutant are shown. Figure 3C shows an overview of the mutation integration. The order of mutations is indicated by arrows. Activity assay conditions were the same as those shown in Figure 3B. [Figure 4]X-ray crystal structures of PET2 mutants are shown. (A) X-ray crystal structure of PET2(2M). The B-chain molecule of the asymmetric unit is shown. The catalytic triad (S175, D221, and H253) and the amino acid residues to be mutated are indicated. (B) X-ray crystal structure of PET2(7M). The catalytic triad (S175, D221, and H253) and the mutated amino acid residues are indicated. (C) Surface charges of PET2 wild-type, PET2(7M), and IsPETase at pH 7.0. The PET2 wild-type structure was reconstructed by back-mutation of the PET2(2M) mutant structure. In each structure, the catalytic pocket is indicated by a dashed circle. [Figure 5A] Figure 5 shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. Figure 5A shows a bright-field image of the PET thin film. Scale = 2 μm. [Figure 5B] Figure 5B shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. Figure 5B shows an example of the binding and dissociation of Alexa Fluor 555-labeled PET2(7M) at 50 pM. The binding time estimated from the time course of signal intensity is 0.6 seconds. [Figure 5C] Figure 5C shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. The fluorescence image of a PET thin film stained with 10 nM PET2(7M) was taken from the same field of view as in Figure 5A. Scale = 2 μm. [Figure 5D] Figure 5D shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. The cumulative number of bound molecules changes over time. Individual trajectories for each movie are shown for PET2 wild-type and PET2(7M). The binding rate constant was calculated from the time required for binding of 100 molecules. [Figure 5E] Figure 5D shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. Figure 5E shows the distribution of binding times for PET2 wild-type, fitted with a double exponential decay function. The proportions of slow and fast dissociation events were calculated from the area under the fitted curve. [Figure 5F] Figure 5F shows single-molecule fluorescence imaging of the binding and dissociation of PET2 wild-type and PET2(7M) on a PET thin film. The distribution of binding times for PET2(7M) was fitted with a double exponential decay function. The proportions of slow and fast dissociation events were calculated using the same method as for the wild-type. [Figure 6A] The temperature dependence and time course of activity are shown. Figure 6A shows the temperature dependence of the hydrolytic activity of amorphous PET. The temperature dependence is shown for PET2 wild-type (■), PET2(4M) (●), PET2(5M) (▲), and PET2(7M) (▼). The concentrations of TPA, MHET, and BHET were summed and shown as the sum of the products. Activity was calculated by dividing the sum of the products (μM) by the enzyme concentration (0.1 μM) and the reaction time (60 min). The maximum activity of PET2(7M) at 68°C was 6.8-fold higher than that of the wild-type at 60°C. The plot shows the mean and standard deviation of triplicate measurements. [Figure 6B] The temperature dependence and time course of activity are shown. Figure 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. The plot shows the average and standard deviation of triplicate measurements. [Figure 6C] The temperature dependence and time course of activity are shown. Figure 6C shows the filled plots of TPA, MHET, and BHET produced from amorphous PET films by PET2 wild-type (left) and PET2(7M) (right). The total amount of product is consistent with Figure 6B. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, modes for carrying out 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, components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and they do not limit the embodiments of the present disclosure.

[0013] In the present disclosure, the term "process" and similar terms include processes that are independent of other processes, as well as processes that cannot be clearly distinguished from other processes, as long as the purpose of the process is achieved. In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples.

[0014] In the present disclosure, the "identity" of amino acid sequences refers to the percentage of matching amino acid residues in the total number of amino acid residues when two amino acid sequences to be compared are aligned, with appropriate gaps inserted in one or both sequences as needed to maximize the number of matching amino acid residues. Alignment can be performed using well-known alignment tools such as BLAST, FASTA, and CLUSTAL W. For example, alignment can be evaluated using the default parameters of BLAST.

[0015] In the present disclosure, "having X% or more sequence identity" with a reference amino acid sequence means that the entire length or a portion of the amino acid sequence being compared has X% or more sequence identity with the entire length of the reference amino acid sequence.

[0016] In this disclosure, amino acid residues may be indicated by the following single-letter or three-letter codes in parentheses: 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), and valine (V, Val).

[0017] In the present disclosure, amino acid mutations in peptides or proteins may be represented by the single-letter designation of the amino acid before the mutation, the amino acid position number from the N-terminus, and the single-letter designation of the amino acid after the mutation, in that order. For example, "having an R20C mutation in the amino acid sequence of SEQ ID NO: 1" means that the arginine at the 20th position counting from the N-terminus of SEQ ID NO: 1 has been replaced with a cysteine.

[0018] In the present disclosure, a "protein comprising the amino acid sequence of X" means that all or part of the amino acid sequence of the protein is the amino acid sequence of X. That is, the protein may consist of only the amino acid sequence of X, or may have, in addition to the amino acid sequence of X, an additional sequence (e.g., a sequence for protein expression and recovery, such as a signal sequence or tag sequence) at one or both of the N-terminus and C-terminus of the amino acid of X. Such additional sequences, such as signal sequences and tag sequences, are well known to those skilled in the art. The same applies to nucleotide sequences; for example, a "polynucleotide comprising the nucleotide sequence of Y" means that all or part of the nucleotide sequence of the polynucleotide is the nucleotide sequence of Y.

[0019] In this disclosure, the "PET hydrolysis activity" of a protein is defined as the value obtained by subjecting the target protein and PET to specific reaction conditions, analyzing the concentration of degradation products by HPLC, and dividing the sum of the degradation product concentrations by the protein concentration and reaction time. Unless otherwise specified, the PET hydrolysis activity in this disclosure refers to the activity at pH 7.0. Column: Phenomenex Luna C18 (2) Eluent: Gradient from 20 mM sodium phosphate buffer (pH 7.0) to methanol Flow rate: 1ml / min. Column temperature: 25℃ Detection: 210nm Injection volume: 10μL

[0020] Decomposition products of PET due to hydrolysis include bis(hydroxyethyl) terephthalate (BHET), (hydroxyethyl) terephthalate (MHET), terephthalic acid (TPA), and ethylene glycol (EG), as shown in Figure 1. The degradation products of PET by the proteins of the present disclosure may be all of these, some of these, or partial degradation products formed by linking these.

[0021] <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 relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C and G62C mutations; (B) an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1; (B1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C, G62C, S129P, and T270P mutations; (C) an amino acid sequence having the following mutations relative to the amino acid sequence of SEQ ID NO: 1: R20C, G62C, S129P, T270P, G153A, E83K, and F78R; or (C1) An amino acid sequence having 90% or more sequence identity with an amino acid sequence having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO: 1, and having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R.

[0022] Hereinafter, for convenience, the amino acid sequence of (A) (SEQ ID NO: 2) will also be referred to as "amino acid sequence A," the amino acid sequence of (A1) (excluding amino acid sequence A) will also be referred to as "amino acid sequence A1," the amino acid sequence of (B) (SEQ ID NO: 3) will also be referred to as "amino acid sequence B," the amino acid sequence of (B1) (excluding amino acid sequence B) will also be referred to as "amino acid sequence B1," the amino acid sequence of (C) (SEQ ID NO: 4) will also be referred to as "amino acid sequence C," and the amino acid sequence of (C1) (excluding amino acid sequence C) will also be referred to as "amino acid sequence C1."

[0023] SEQ ID NO: 1 shows the amino acid sequence of wild-type PET2. Hereinafter, a protein having the amino acid sequence of SEQ ID NO: 1 is also referred to as wild-type PET2. The inventors have found that this protein, which has at least R20C and G62C mutations compared to wild-type PET2, has excellent heat resistance and PET hydrolysis activity. Amino acid sequence A has at least the mutations R20C and G62C compared to wild-type PET2. The mutations R20C and G62C are located away from the active site of PET2, but it has surprisingly been found that these mutations can improve both heat resistance and activity. Without being bound by theory, it is speculated that the mutations R20C and G62C form disulfide bonds with the cysteine ​​residue pairs, improving the stability of the protein itself. Amino acid sequence B has at least the following mutations compared to the wild-type PET2: R20C, G62C, S129P, and T270P. Without being bound by theory, it is speculated that S129P and T270P introduce cyclic proline residues into the flexible region (unstable site) of the protein, thereby stabilizing the protein structure. Amino acid sequence C contains at least the following mutations compared to wild-type PET2: R20C, G62C, S129P, T270P, G153A, E83K, and F78R. Without being bound by theory, the G153A mutation is speculated to contribute to structural stabilization by substituting alanine for glycine in the α-helix of PET2. The F78R mutation is speculated to enhance PET hydrolysis activity by altering the surface charge. The E83K mutation also improves heat resistance, although the mechanism behind this is unclear.

[0024] The amino acid sequence of SEQ ID NO: 1 (amino acid sequence of wild-type PET2) and amino acid sequences A to C are shown below. The boxed letters indicate the mutation sites.

[0025] [Table 1]

[0026] The protein of the present disclosure comprises any one of amino acid sequences A to C, or has 90% or more sequence identity to any one of amino acid sequences A to C, preferably 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 sequence identity, and has the specific mutation described above. The protein of the present disclosure may also comprise an amino acid sequence that has 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 sequence identity to amino acid sequence A. The protein of the present disclosure is a protein that comprises the amino acid sequence described above and has PET hydrolysis activity. The PET hydrolysis activity at pH 7.0 is preferably higher than that of wild-type PET2. In this case, the PET hydrolysis activity is compared using the method described below.

[0027] For example, the protein of the present disclosure may have an amino acid sequence in which one or more amino acids are deleted, substituted, or added to any of amino acid sequences A to C while maintaining the above amino acid sequence identity, and has the specific mutations described above, so long as the protein has PET hydrolysis activity. In this case, the number of amino acids deleted, substituted, or added is not limited as long as the protein has PET hydrolysis activity, and may be, for example, 1 to 28, 1 to 25, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. The positions of the amino acids to be deleted, substituted, or added may be selected from the ranges corresponding to, for example, positions 1 to 147, positions 149 to 193, positions 195 to 225, and positions 227 to 281 from the N-terminus of SEQ ID NO:1.

[0028] In one aspect, amino acid A1 has a sequence identity of 90% or more with an amino acid sequence having R20C and G62C mutations relative to the amino acid sequence of SEQ ID NO: 1, and 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 a sequence identity of 90% or more with an amino acid sequence having R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1, and may have the R20C, G62C, S129P, and T270P mutations and at least one mutation selected from the group consisting of G153A, E83K, and F78R.

[0029] In one aspect, the protein of the present disclosure is a protein that has PET hydrolysis and preferably includes any one of the amino acid sequences B, B1, C, or C1, and more preferably includes any one of the amino acid sequences C or C1.

[0030] 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, more preferably a protein comprising any one of amino acid sequences B or C, and even more preferably a protein comprising amino acid sequence C.

[0031] The proteins of the present disclosure may have an additional amino acid sequence at the N-terminus or C-terminus, provided that the additional amino acid sequence does not impair PET hydrolysis activity. Examples of additional amino acid sequences include sequences for recombinantly producing the proteins of the present disclosure. Examples of such additional sequences include an amino acid sequence that serves as the initiation point for protein synthesis (e.g., an N-terminal methionine residue), an N-terminal signal sequence (e.g., a signal sequence for protein secretion in E. coli), a tag sequence (e.g., a histidine tag, a GST tag, a FLAG tag), a protease recognition sequence (e.g., a TEV protease recognition sequence), and the like. In one embodiment, the additional amino acid sequence may be the amino acid sequence of SEQ ID NO: 6 added to the N-terminus, or a combination of the amino acid sequence of SEQ ID NO: 7 added to the C-terminus and the amino acid sequence of SEQ ID NO: 8 added to the C-terminus of SEQ ID NO: 7, or a combination thereof. SEQ ID NOs: 6 to 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, and may be, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5 per terminus. For example, the protein of the present disclosure may have the above-mentioned additional amino acid sequence added to the N-terminus or C-terminus of a sequence consisting of any of the amino acid sequences A, A1, B, B1, C, or C1, as long as the PET hydrolysis activity is not impaired. Details of the additional amino acid sequence are as described above.

[0032] The two cysteine ​​residues at the R20C and G62C mutation sites preferably form a disulfide bond. The presence or absence of disulfide bond formation can be confirmed by X-ray crystal structure analysis.

[0033] The optimal 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 preferably near neutral in practice. For example, the optimal 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.

[0034] Since PET has a glass transition temperature around 70°C and PET degradation at high temperatures is expected to increase degradation efficiency, the optimal temperature for PET hydrolysis activity of the protein of the present disclosure (the temperature at which activity is highest) is preferably higher than the optimal temperature for PET hydrolysis activity of wild-type PET2. From this perspective, the optimal temperature for PET hydrolysis activity of the protein of the present disclosure at pH 7.0 is preferably above 60.0°C, and more preferably 65.0°C or higher. Furthermore, from the perspective of performing decomposition with low energy within a range that promotes PET degradation, the optimal temperature for PET hydrolysis activity of the protein of the present disclosure may be 90.0°C or lower, 85.0°C or lower, or 80.0°C or lower. The optimum temperature is measured as follows: Two amorphous PET disks (0.25 mm thick, total surface area 0.32 cm) are placed on the substrate. 2 To a total mass of 4.0 mg, add 250 μL of 0.1 μM protein in 50 mM sodium phosphate solution (pH 7.0) and incubate at each temperature for 60 minutes. Analyze the concentration of degradation products by HPLC, and obtain the activity value by dividing the total concentration of degradation products by the protein concentration and reaction time.

[0035] From a similar perspective, the melting temperature (Tm) of the protein of the present disclosure is preferably higher than the Tm of wild-type PET2. 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 decomposition with low energy within a range that promotes PET decomposition, the melting temperature (Tm) of the protein of the present disclosure may be 100.0°C or lower, 95.0°C or lower, or 90.0°C or lower. In this disclosure, the melting temperature (Tm) of a protein is measured by circular dichroism (CD) spectroscopy using the following procedure: Prepare a 13 μM protein solution in 10 mM sodium phosphate solution (pH 7.0). Measure the CD spectrum of the protein from 250 to 200 nm at 20°C. Monitor the protein signal at 230 nm while heating the protein from 20°C at a rate of 1°C / min. Measure the CD spectrum of the denatured protein from 250 to 200 nm at 95°C. Estimate the protein Tm by curve fitting the temperature dependence of the signal at 230 nm.

[0036] From the viewpoint of convenience when using the protein of the present disclosure for recycling purposes, it is preferable that the PET hydrolysis activity of the protein is calcium ion independent. In this disclosure, the "calcium ion-independent" PET hydrolysis activity of a protein is confirmed by the following procedure: 250 μL of a 0.1 μM protein solution in bis-Tris-HCl (pH 7.0) containing 0, 10, 100, and 300 mM CaCl2 is applied to two amorphous PET films (0.25 mm thick, 0.32 cm2 total surface area). 2 The mixture was incubated with 4.0 mg of calcium hydroxide (total mass) at 60°C for 60 minutes. After incubation, 200 μL of the 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 then mixed with 68.6 μL of 1 M sodium phosphate buffer (pH 7.0) to precipitate calcium phosphate. The precipitate was removed by treating at 15,000 g for 3 minutes using a 0.22 μm spin column filter. The concentration of degradation products was analyzed by HPLC, and the total concentration of degradation products was divided by the protein concentration and reaction time to obtain the activity value. PET hydrolysis activity was determined to be calcium-independent if it did not change by 50% or more compared to 0 mM at CaCl2 concentrations of 10, 100, or 300 mM.

[0037] The PET hydrolysis activity of the protein of the present disclosure at pH 7.0 is preferably higher than the hydrolysis activity of wild-type PET2 at pH 7.0; for example, it is preferably 2.0 times or more higher than the hydrolysis activity of wild-type PET2 at pH 7.0, more preferably 3.0 times or more higher, even more preferably 4.0 times or more higher, and particularly preferably 5.0 times or more higher. The PET hydrolysis activity of the wild-type and mutant PET2 proteins was compared by comparing their activity at the optimum temperature for each protein. The reaction conditions were as follows: Two amorphous PET disks (0.25 mm thick, 0.32 cm2 in total surface area) were used. 2 To a total mass of 4.0 mg, add 250 μL of 0.1 μM protein in 50 mM sodium phosphate solution (pH 7.0) and incubate at the optimal temperature for 60 minutes. Analyze the degradation product concentration by HPLC, and obtain the activity value by dividing the total degradation product concentration by the protein concentration and reaction time.

[0038] The proteins of the present disclosure can be produced using 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 then introduced into a host to obtain a transformant. The resulting transformant is cultured to produce the target protein. The resulting protein can be recovered by conventional methods to obtain the protein of the present disclosure.

[0039] The use of the protein of the present disclosure is not particularly limited, and it can be used, for example, to decompose and recycle PET. Furthermore, the degradation products of the protein of the present disclosure can be further reacted with a decomposing enzyme such as MHETase, and used to produce terephthalic acid or ethylene glycol.

[0040] <Polynucleotides, recombinant vectors, and transformants> The polynucleotides of the present disclosure include nucleotide sequences that encode the proteins of the present disclosure. The recombinant vector of the present disclosure comprises the polynucleotide. The transformant of the present disclosure contains the recombinant vector.

[0041] The polynucleotide may be any polynucleotide encoding a protein of the present disclosure. The nucleic acid sequence of the polynucleotide may be varied within the range of codon degeneracy. When a recombinant vector is introduced into a host to obtain a transformant and express a protein, it is preferable to use codons that are frequently used in the host. In one embodiment, when an expression system using Escherichia coli as a host is used, the TGC codon may be used for the cysteine ​​residues R20C and G62C, the CCG codon for the proline residues S129P and T270P, the GCG codon for the alanine residue G153A, the AAA codon for the lysine residue E83K, and the CGC codon for the arginine residue F78R.

[0042] The recombinant vector may be any recombinant vector capable of expressing the protein of the present disclosure. Preferred recombinant vectors include plasmids, phages, etc. The expression vector has a promoter necessary for expression and may also have other components such as a terminator, a selection marker (such as a 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 in accordance with the recombinant vector to be used. Microbial cells are conveniently used as the host, and for example, prokaryotes (e.g., Escherichia coli) or eukaryotes (e.g., yeast) may be used. When producing a protein using an expression vector and a host, conditions for transformation, expression, and recovery can be selected from methods well known to those skilled in the art.

[0043] <Composition for decomposing polyethylene terephthalate> The PET-degrading composition of the present disclosure includes the protein of the present disclosure. The protein of the present disclosure may be contained in the composition in an unpurified state (e.g., a state containing a host expressing the protein, or a state containing a crude protein solution secreted from a host expressing the protein) or in a purified state, and is preferably contained in the composition in a purified state.

[0044] In addition to the protein of the present disclosure, the composition for decomposing PET may contain a buffer suitable for stabilizing the protein. The buffer is preferably one that maintains the solution at approximately neutral pH, such as phosphate buffer, sodium phosphate buffer, Tris-HCl buffer, and HEPES.

[0045] The PET decomposition composition may contain salts such as calcium salts, but considering recycling applications, it is preferable that the salt content be as low as possible. In the PET decomposition composition, the salt concentration is preferably 50 mM or less, more preferably 30 mM or less, and even more preferably 20 mM or less.

[0046] The composition for decomposing PET may contain additives such as stabilizers in addition to the protein and buffer solution of the present disclosure.

[0047] <Manufacturing methods for recycled products> The method for producing recycled products of the present disclosure includes decomposing PET using the protein of the present disclosure. PET decomposition can be achieved by contacting PET with the protein of the present disclosure. For example, PET decomposition can be performed using the aforementioned PET decomposition composition. As the PET, for example, waste PET can be used. The decomposition reaction is preferably carried out under conditions close to the aforementioned optimum pH and optimum temperature. For example, the decomposition reaction is preferably carried out at a pH of 5.0 to 9.5, more preferably at a pH of 5.5 to 9.0, and may be carried out at a pH of 6.0 to 8.0. The decomposition reaction is preferably carried out in a buffer appropriate for maintaining the above pH. Examples of the buffer include those described above. The decomposition reaction is also preferably carried out at a temperature above 60.0°C, more preferably 65.0°C or higher. The upper limit of the temperature for the decomposition reaction 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 degradation is not particularly limited as long as sufficient degradation is possible for the production of recycled products. Since the use of the protein of the present disclosure enables efficient PET hydrolysis, a reduction in reaction time is expected. For example, the reaction time may be within 3 days, 2 days, 24 hours, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour. From the perspective of sufficient degradation, the reaction time may be 10 minutes or more, or 30 minutes or more. It is preferable that the reaction product is decomposed to a state where it can be sufficiently repolymerized (for example, until 70% by mass or more, 80% by mass or more, or 90% by mass or more of the PET substrate is decomposed).

[0048] In one embodiment, after PET decomposition, the decomposition products are recovered. The recovery method is not particularly limited and can be performed by a known method. After recovery, the decomposition products may be purified by a known method such as separation, extraction, adsorption, concentration, or filtration. The decomposition products can be recycled by reusing them to synthesize polymers through repolymerization, and can be reused to produce PET or other polymers. [Example]

[0049] Next, embodiments of the present disclosure will be described in detail using examples, but the embodiments of the present disclosure are not limited to these examples.

[0050] In the following examples, because a signal sequence (27 amino acids) is used to express and secrete the PET2 mutants in E. coli, the amino acid position numbers from the N-terminus are indicated as the position numbers from the N-terminus including the signal sequence. For example, in the above-mentioned protein of the present disclosure, the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R with respect to SEQ ID NO: 1 correspond to the mutations R47C, G89C, S156P, T297P, G180A, E110K, and F105R with respect to the wild-type PET2, respectively, in the following examples. The amino acid sequence of PET2(7M) used in the examples (SEQ ID NO:5) (including an N-terminal signal sequence and a C-terminal protease recognition site and histidine tag) is shown below, as well as the signal sequence (SEQ ID NO:6; positions 1 to 27 of SEQ ID NO:5), protease recognition sequence (SEQ ID NO:7; positions 309 to 315 of SEQ ID NO:5), and histidine tag (SEQ ID NO:8; positions 316 to 321 of SEQ ID NO:5) contained therein. The boxed letters in SEQ ID NO:5 represent the mutation site, and the shaded letters represent the signal sequence and tag sequence containing the protease recognition site and histidine tag from the N-terminus. PET2(2M) used in the examples has only the F105R and E110K mutations shown in SEQ ID NO:5. PET2(4M) used in the examples has only the F105R, E110K, S156P, and T297P mutations shown in SEQ ID NO:5. PET2(5M) used in the examples has only the following mutations among those shown in SEQ ID NO:5: F105R, E110K, S156P, T297P, and G180A.

[0051] [Table 2]

[0052] The nucleotide sequence of PET2(7M) (SEQ ID NO: 9) (including the termination codon) corresponding to SEQ ID NO: 5 is shown below.

[0053] [Table 3]

[0054] 1. Introduction Plastics, which are made up of synthetic polymers, are relatively inexpensive and easy to process, and are widely used in modern society. (1) However, from the viewpoint of sustainability, environmental pollution caused by these materials is a serious problem. In particular, polyethylene terephthalate (PET) is mass-produced and widely used in beverage bottles, clothing, packaging materials, etc. (2) PET can form a physically and chemically stable crystalline structure. (3) PET is classified as an engineering plastic. Due to this stability, PET is thought to remain in the environment for a long period of time. (4) .

[0055] PET is highly resistant to biodegradation, but its molecular chain is composed of carboxylic acid esters of terephthalic acid (TPA) and ethylene glycol (EG). Therefore, esterases should have the ability to hydrolyze PET. The first enzyme reported as an efficient PET hydrolase was cutinase (TfH) from Thermobifida fusca. (5) Cutinase (EC3.1.1.74) is generally registered as a hydrolase of plant wax layers (cutin), but Muller et al. have 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 a PET hydrolase (IsPETase), was discovered. (6) Since then, much research has been conducted on IsPETase, including determining its structure and investigating its reaction mechanism. (7-10) IsPETase is classified as a PET hydrolase (EC3.1.1.101), but its low thermostability makes it difficult to use industrially. Therefore, various mutants with improved thermostability have been reported. (9、11)From this perspective, some cutinases have higher thermostability than IsPETase and are suitable templates for constructing thermostable PET hydrolases. The most promising PET hydrolases include leaf-branch compost cutinase (LCC). (12) Furthermore, the cutinase from Thermobifida fusca (TfCut2) has high thermostability and activity. (13) A cutinase (Cut190) from Saccharomonospora viridis has also been engineered to have improved thermostability and activity. (14、15) Furthermore, lipase (EC 3.1.1.3) is also a type of carboxylic acid ester hydrolase, and PET (16) , poly(tetramethylene succinate) (17) , poly(ω-hydroxyalkanoic acid) (18) Recently, lipases have been cloned from metagenomic libraries of gelatin-degrading reactors. (19) was rediscovered as PET2, a PET hydrolase (20) PET2 is a thermostable enzyme discovered from a gene library obtained from an artificial high-temperature environment, and is one of the candidates for a thermostable PET hydrolase.

[0056] PET is a water-insoluble synthetic polymer, and its enzymatic degradation is a heterogeneous reaction that occurs at the solid-liquid interface (see Figure 1). Furthermore, since PET has amorphous regions that are easily degraded and crystalline regions that are difficult to degrade, PET hydrolase is expected to preferentially hydrolyze the amorphous regions of PET, especially the mobile regions. (12、13、21) Among natural polymers, cellulose, the main component of plant cell walls, and chitin, a component of crustacean and fungal cell walls, are also water-insoluble crystalline polysaccharides. (22、23)For efficient degradation of these water-insoluble polymers, binding of enzymes to solid surfaces is important. Therefore, cellulases and chitinases, which have high decomposition activity for crystalline cellulose and crystalline chitin, have domain structures that allow them to bind with high affinity to the crystalline polymer surface. (24) Consistent with this finding, IsPETase has more basic amino acid residues on the enzyme surface compared to other carboxylesterases, and it has been reported that these positively charged residues contribute to PET degradation activity. (25) .

[0057] Binding affinities to solid substrates are typically quantitatively compared using dissociation constants (or binding constants) obtained by biochemical adsorption measurements. However, because this constant is the ratio of the dissociation rate constant to the association rate constant, it is difficult to clearly identify which rate constant is responsible for the change in dissociation constant based solely on biochemical measurements. Therefore, in the analysis of cellulases and chitinases, the association and dissociation rate constants have been directly measured using single-molecule fluorescence imaging. (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 bacterial and fungal cellulases.

[0058] 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. Next, we introduced up to seven mutations into the PET2 wild-type by screening single mutations and combining single mutations. As a result, we successfully identified PET2 mutants with significantly improved thermal stability and PET hydrolysis activity. X-ray crystallography was used to determine the structures of the mutant enzymes, and the structural changes resulting from the mutations that contribute to thermal stability and adsorption properties were verified. To elucidate the mechanism behind the enhanced activity, we used single-molecule fluorescence imaging to compare the adsorption properties of the best-performing mutant with those of the wild-type on PET thin films. Finally, we evaluated the temperature dependence of the degradation activity of the mutant enzymes and tested their long-term thermal stability through 24-hour degradation experiments.

[0059] 2. Materials and Methods 2.1 Homology modeling of the wild-type PET2 structure for selection of mutation sites SWISS-MODEL Server (29) Using the crystal structure of IsPETase (PDB ID: 6EQE), a homology modeling structure of wild-type PET2 was constructed. The crystal structure of IsPETase and the modeling structure of wild-type PET2 were then aligned using Pymol, and mutation positions in PET2 corresponding to the unique basic residues and effective mutation positions of IsPETase were selected.

[0060] 2.2 Plasmid construction and enzyme purification Alkaline thermostable lipase lipIAF5-2 (UniProt: C3RYL0) (19) , the gene for the enzyme recently renamed PET2 because it has PET hydrolysis activity. (20)A gene was synthesized in which the nucleotide sequence α was fused to a TEV protease recognition sequence (ENLYFQG) and a histidine-6 ​​tag at the C-terminus. This gene was ligated into the pET27b plasmid at the NdeI-NotI site. The lipIAF5-2 signal sequence replaced the pelB leader sequence of pET27b. OverExpress E. coli C41(DE3) (Lucigen) was transformed with this plasmid and plated on an LB plate containing 50 μg / mL kanamycin. Colonies were collected in 20 mL of super broth medium (2.5 w / v% tryptone, 1.5 w / v% yeast extract, 0.5 w / v% sodium chloride) and inoculated into 700 mL of super broth medium containing 50 μg / mL kanamycin. The flask was shaken at 140 rpm at 37°C for 3 hours and then cooled on ice for 30 minutes. 700 μL of 1 M IPTG was added to the flask and incubated at 16° C. and 140 rpm for 16 hours. Cells were harvested by centrifugation at 3000 g for 20 minutes.

[0061] The harvested 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 away with 50 mL of the same buffer containing 50 mM imidazole. The purified enzyme was eluted with buffer containing 100 mM imidazole. The enzyme was concentrated to 500 μL at 8,000 g using a Vivaspin 20 (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. 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 using a Vivaspin 20 (molecular weight cutoff = 10K) and loaded onto a Superdex 75 Increase (Cytiva) column equilibrated with 10 mM sodium phosphate buffer (pH 7.0). The purity of each fraction was confirmed by SDS-PAGE, and three fractions containing high concentrations of pure enzyme were collected in glass vials. The enzyme concentration was calculated from the difference in absorbance at 280 nm and 340 nm. The molar extinction coefficient of the wild-type enzyme was ε 280 =52,420M -1 cm -1 It was.

[0062] The PET2 mutant genes were constructed by PCR. Using a primer pair containing the mutation, the product was ligated using NEBuilder HiFi Assembly (New England Biolabs) according to the manufacturer's instructions. The ligated plasmid was transformed into Tuner (DE3) competent cells (Novagen). The cells were incubated at 37°C for 1 hour and then 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 and 140 rpm for 16 hours. The plasmid was purified from the cells, and the sequence of the coding region was confirmed. The mutant enzymes were purified using the same procedure as the wild-type. The molar extinction coefficients of the mutants containing R47C-G89C or L265C-A295C were ε 280 =52,540M -1 cm -1 and the molar extinction coefficient of the mutant containing Y262C-L298C is ε 280 =51,280M -1 cm -1 and the molar extinction coefficients of the mutants containing W174H or Q134Y are ε 280 =46,730M -1 cm -1 or ε 280 =53,600M -1 cm -1 It was.

[0063] The enzyme for crystallization was purified using a slightly different procedure. After affinity purification using the histidine-6 ​​tag, the enzyme solution was eluted from NAP10 with 10 mM sodium phosphate buffer (pH 7.0) and loaded onto a 5 mL Toyopearl DEAE-650 column equilibrated with 10 mM sodium phosphate buffer (pH 7.0). The unbound enzyme was eluted with the same buffer and concentrated at 8000 g using a Vivaspin 20 column (molecular weight cutoff = 10K). 500 μL of the enzyme solution was loaded onto a Superdex 75 Increase column (Cytiva) equilibrated with 10 mM Tris-HCl buffer (pH 7.5). The eluted enzyme was concentrated at 8000 g using a Vivaspin 20 column (molecular weight cutoff = 10K).

[0064] 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. The enzyme was purified using the same procedure as for activity measurements. The purified enzyme was reduced with 1 mM DTT for 1 h at 25 °C, 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 h at 25 °C. The enzyme to Alexa Fluor 555 ratio was 1:3 (molar ratio). The mixture was concentrated at 8000 g using a VIVAspin 20, followed by removal of unreacted dye using a 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 molar extinction coefficient of Alexa Fluor 555 is ε 556 =158,000M -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. After correction, the labeling efficiencies of PET2 wild-type-C309 and PET2(7M)-C309 were 109% and 122%, respectively. As a control experiment, labeling reactions were also performed with PET2 wild-type and PET2(7M) lacking C309. The labeling efficiencies of PET2 wild-type and PET2(7M) were 3.4% and 7.1%, respectively, confirming that C309 reacted primarily with Alexa Fluor 555 maleimide.

[0065] 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 h and then cooled on ice. The enzyme was then diluted to 200 nM with 200 mM sodium phosphate solution (pH 7.0). 25 μL of the enzyme solution was mixed with an equal volume of 2 mM BHET in ultrapure water and incubated at 60°C for 10 min. 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 separated using a gradient from 20 mM sodium phosphate buffer (pH 7.0) to methanol. 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 s).

[0066] The BHET hydrolysis activity of PET2 wild-type was also measured in various buffer solutions with different pH values ​​(sodium citrate buffer for pH 3.0, sodium acetate buffer for pH 4.0 and pH 5.0, sodium phosphate buffer for pH 6.0 and pH 7.0, Tris-HCl buffer for pH 8.0, glycine-NaOH buffer for pH 9.0, and 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 min. Spontaneous degradation of BHET in the absence of enzyme was subtracted as background. The procedures for product separation and detection were the same as those described above. Activity values ​​were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (600 s).

[0067] 2.4 PET film decomposition activity Amorphous PET film (thickness = 0.25 mm, code ES301445) and biaxially oriented (crystalline) PET film (thickness = 0.25 mm, code ES301450) were purchased from Goodfellow. The PET film was punched into circular disks (diameter 3 mm, surface area 0.16 cm). 2 The PET disks were then washed by immersion in 20% ethanol (2.0 mg / disk, 2.0 mg / amorphous disk, 2.3 mg / crystalline disk). The washed disks were then dried at room temperature and stored in glass vials.

[0068] Two PET discs (0.32 cm 2 A 4.0 mg amorphous disk was 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 the degradation. The reaction mixture was incubated at 60°C for 60 min. The supernatant was collected and analyzed for product concentration by HPLC using the same procedure as for the analysis of BHET hydrolysis. Activity values ​​were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and the reaction time (60 min).

[0069] The optimum reaction temperature was evaluated in the range of 60°C to 80°C using the same procedure as above. Activity values ​​were calculated by dividing the sum of the product concentrations (μM) by the enzyme concentration (0.1 μM) and the reaction time (60 min). Activity measurements were also performed at a moderate temperature (30°C) for 960 min (16 h) using the same procedure. Activity values ​​were calculated by dividing the sum of the product concentrations (μM) by the enzyme concentration (0.1 μM) and the reaction time (960 min). For the long-term degradation assay, two PET disks (0.32 cm) were used. 2 , 4.0 mg of amorphous disks, or 4.6 mg of crystalline disks) were incubated with 250 μL of 0.1 μM PET2 wild-type in 50 mM sodium phosphate solution (pH 7.0) at 60°C, and with 0.1 μM PET2 (7M) in 50 mM sodium phosphate solution (pH 7.0) at 68°C for 1, 2, 4, 8, 16, and 24 hours.

[0070] 2.5 Thermal stability measurement by CD spectroscopy A quartz cell was filled with 400 μL of 13 μM enzyme in 10 mM sodium phosphate solution (pH 7.0), and the CD spectrum of the enzyme was measured at 20 °C from 250 to 200 nm using a J-1500KS equipped with a Peltier thermostatic cell holder (JASCO). The signal at 230 nm was tracked 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 was measured at 95 °C from 250 to 200 nm. The melting temperature (Tm) of the enzyme was estimated by curve fitting the temperature dependence of the signal at 230 nm using denatured protein analysis software (JASCO).

[0071] 2.6 X-ray crystal structure analysis One microliter of purified PET2 (2M) (F105R-E110K mutant) (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. One microliter 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. Immediately before diffraction measurement, both crystals were immersed in a reservoir containing 30% ethylene glycol by volume. SWISS-MODEL server (29) The phases were determined by molecular replacement with phaser using the homology modeling structure of PET2 wild type constructed with the Phenix refine program. (30) The model structure was refined using Coot (31) This was improved by PDB2PQR. (32) Calculate the protonation state of the enzyme at pH 7.0 using APBS (33) The surface charge was calculated using and plotted using Pymol.

[0072] 2.7 Single-molecule fluorescence imaging of binding and dissociation Observations were 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 from the PET film. After centrifugation at 20,000 g for 5 min, the supernatant was removed and the precipitate was suspended in 5 mL of acetone. After centrifugation at 20,000 g for 5 min, the supernatant was removed and the precipitate was completely dried at 70°C for 1 h and then dissolved in 1 mL of hexafluoro-2-propanol. The PET solution was diluted to 5 mg / mL with hexafluoro-2-propanol. 50 μL of the diluted PET solution was spin-coated at 3000 rpm for 10 s onto a cover glass that had been incubated overnight in 10 M KOH and washed, followed by thorough rinsing with ultrapure water. The PET-spin-coated cover glass was 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 in 50 mM sodium phosphate solution (pH 7.0) and dropped onto the PET thin film on the cover glass. The thin film was then covered with a topcoat wall to retain the enzyme solution. The power density was 0.25 μW / μm. 2 The dye on the sample surface was excited at 532 nm using a laser, 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 binding of 100 enzyme molecules was measured. The binding rate constant was then estimated by dividing 100 (molecules) by the required time, and then 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. Therefore, the unit of the binding rate constant is s -1 μm -2 M -1 The average area of ​​the PET thin film within the field of view was 163 ± 31 μm for PET2 wild type and PET2(7M), respectively. 2 and 169±19 μm 2The dissociation rate constant was obtained by fitting the distribution of the association times of the individual molecules with a sum of double exponential decay functions. The ratio of slow to fast dissociation events was estimated from the area under the fitted curve.

[0073] To confirm the photobleaching time of Alexa Fluor 555, 50 μL of 100 pM PET2-Alexa Fluor 555 was directly spin-coated onto a cover glass at 3000 rpm for 10 seconds. Under the same conditions as for single-molecule imaging of binding and dissociation, 20 μL of 50 mM sodium phosphate solution (pH 7.0) was added dropwise, and the enzyme molecules tightly bound to the cover glass were observed. The photobleaching time distribution for each spot was fitted with a single exponential decay function, and the time constant was estimated to be 38.1 seconds.

[0074] 2.8 Degradation assay of PET thin films used for single molecule imaging PET thin films were prepared using the same method as for single-molecule imaging. A 50 μL drop of 0.1 μM PET2 wild-type or PET2 (7M) in 50 mM sodium phosphate solution (pH 7.0) was added to the film and incubated at room temperature (approximately 26 °C) for 60 min. To prevent evaporation of the droplets, the PET thin-film-coated coverslips were covered with a Petri dish. After incubation, the droplets were collected and the product concentrations were analyzed 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).

[0075] 2.9 Measurement of Alexa Fluor 555-labeled enzyme activity Two hundred fifty microliters of 0.1 μM PET2 wild-type or PET2 (7M), either labeled with Alexa Fluor 555 or unlabeled, in 50 mM sodium phosphate solution (pH 7.0) was applied to two amorphous PET film discs (0.32 cm). 2The mixture was incubated with 4.0 mg of ATP at 60°C for 60 minutes. After incubation, the supernatant was collected and analyzed for product concentration by HPLC. Activity was calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and the reaction time (60 minutes).

[0076] 2.10 Ca2+-dependent PET degradation activity by wild-type PET2 2+ dependence Two hundred fifty microliters of 0.1 μM PET2 wild-type in bis-Tris-HCl (pH 7.0) containing 0, 10, 100, and 300 mM CaCl2 was applied to two amorphous PET film discs (0.32 cm). 2 The mixture was combined with 4.0 mg of ATP and incubated at 60°C for 60 minutes. After incubation, 200 μL of the 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 then mixed with 68.6 μL of 1 M sodium phosphate buffer (pH 7.0) to precipitate calcium phosphate. The precipitate was removed by treatment at 15,000 g for 3 minutes using a 0.22 μm spin column filter, and 10 μL of the flow-through was injected into HPLC. Activity values ​​were calculated by dividing the total product concentration (μM) by the enzyme concentration (0.1 μM) and reaction time (60 minutes).

[0077] 2.11 Evaluation of crystallinity of PET film by differential scanning calorimetry (DSC) and X-ray diffraction (XRD) Amorphous PET film and biaxially oriented crystalline PET film were punched out to 5 mm diameter and washed using the same procedure as for the decomposition activity measurement. One disk was placed in an aluminum dish and heat flow was measured using a differential scanning calorimeter (DSC) (Model DSC-60, Shimadzu Corporation). The disk was heated from 30 °C to 290 °C at 10 °C / min. To measure the regenerated PET from 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 allowed to dry at room temperature for 2 hours. This procedure was repeated twice. Heat flow was measured for both PET films using the same procedure. Thermal crystallization and thermal melting were estimated from the peak areas, and the endotherm (ΔH) was calculated from the difference between the heat of crystallization and the heat of fusion. The heat of fusion (ΔH0) of the perfectly crystalline PET was 140.1 J / g. The crystallinity of both PET films was calculated from ΔH and ΔH0. (34) The estimated crystallinity values ​​for the amorphous PET, crystalline PET, and recycled PET used in this study were 0.02%, 59.3%, and 5.89%, respectively.

[0078] X-ray diffraction (XRD) of the PET film was performed using a RINT-Ultima III (Rigaku Corporation). The PET film was fixed to the stage, and X-ray diffraction (40 mA, 40 kV) from a copper target was measured at 25°C with diffraction angles of 3° to 90°. Amorphous PET showed no peak, while crystalline PET showed a strong peak corresponding to the

[0100] crystal plane.

[0079] 3.Results 3.1 Thermal stability and pH optimum of wild-type PET2 PET2 wild-type was incubated with BHET, a moderately water-soluble PET building block, and the resulting MHET was quantified by HPLC (TPA was not detected under these test conditions). The thermal stability of PET2 wild-type was assessed by examining the residual activity after 1 hour at temperatures between 40 and 90°C at pH 7.0. Up to 65°C, the activity was 1.3 s. -1The decomposition activity of β-glucan was maintained. On the other hand, almost no activity was detected at temperatures above 70°C (Figure 2A). Decomposition activity was also measured at 60°C and pH 3.0 to 10.0, and the highest activity was observed at pH 6.0 and 60°C (Figure 2B). Therefore, 60°C and pH 7.0 were used as the standard reaction conditions.

[0080] Next, the CD spectra of wild-type PET2 at pH 7.0 were compared at 20°C and 95°C (Figure 2C). The CD spectrum of wild-type PET2 at 20°C showed a mixture of α-helix and β-sheet, with a downward peak at approximately 230 nm. The CD spectrum at 95°C appeared to be random coil in the short wavelength range, but signals derived from secondary structures remained. Because the difference in signals at 222 nm and 215 nm, corresponding to α-helix and β-sheet, respectively, was not significant between 20°C and 95°C, the Tm of wild-type PET2 was calculated based on the change in the signal at 230 nm due to tryptophan residues. (35) As a result, the Tm of wild-type PET2 was estimated to be 69.0°C (Fig. 2D), which was consistent with the degradation activity after heat treatment (Fig. 2A).

[0081] The thermal stability and PET degradation activity of cutinases LCC, TfCut2, and Cut190 depend on Ca 2+ It has been reported that the effect is enhanced in the presence of (36-38) , PET2 wild-type PET degradation activity Ca 2+ In the wild-type PET2, the production concentration of the product was measured at 10 mM and 100 mM Ca. 2+ In the presence of Ca 2+ Even with 300 mM CaCl2, the PET degradation activity was only 1.1 times higher than in the absence of CaCl2. 2+ Based on these results, the PET degradation activity of PET2 mutants was measured in the absence of CaCl in the following experiments.

[0082] 3.2 Single mutation screening To improve the thermal stability and activity of PET2, single mutations were introduced based on the homology model structure (Fig. 3A), and the effects of the mutations were examined. The Tm and amorphous PET degradation activity of the wild-type and mutant enzymes were measured, and the values ​​were plotted (Fig. 3B). First, the basic amino acid residues characteristic of IsPETase were identified. (11) , and single mutations reported to enhance activity in IsPETase (39) The F105R and E110K mutations increased Tm by more than 1°C compared to the wild type, and enhanced PET degradation activity by 1.5-fold and 1.4-fold, respectively. The L298R mutation, which is located on the same surface as F105R and E110K but is not present in IsPETase, reduced Tm and activity. (39) The Q183R and Q134Y mutations increased the activity. However, the Q183R mutation decreased the Tm, and the Q134Y mutation significantly prolonged the elution time during size-exclusion chromatography (SEC) and significantly reduced the yield after purification. The S202Q and S155D mutations increased the Tm but decreased the activity.

[0083] Based on the homology modeling structure, we searched for amino acid residues compatible with the dihedral angle of proline and introduced proline mutations (Fig. 3B, ●). The Tm values ​​of the S156P and T297P mutations were 1–2°C higher than those of the wild-type, whereas the Tm values ​​of the D53P and A192P mutations were lower than those of the wild-type. The activity of these mutants was not significantly different from that of the wild-type, except for A192P, which showed less than half the activity of the wild-type.

[0084] 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 contains the corresponding glycine residues (Gly177-Gly180). We mutated these Gly177-Gly180 to alanine one by one (Figure 3B, ▲). The G180A mutation was one of the most effective mutations, significantly improving Tm (+2.6°C) and activity (1.5-fold). Meanwhile, the other three mutations reduced Tm or activity compared to wild-type.

[0085] Furthermore, we tested four pairs of cysteine ​​mutations near the N- or C-terminus of PET2 (Figure 3B, ▼). The Tm of the R47C-G89C mutation pair was 72.0°C, 3°C higher than the wild-type Tm. The C-terminal cysteine ​​mutation pairs (Y262C-L298C and L265C-A295C) also showed Tms 1–2°C higher than the wild-type Tm, but the activity of these mutation pairs was approximately half that of the wild-type. We also attempted the T64C-T86C mutation, but the purification yield was very low, and we were unable to obtain sufficient sample for characterization.

[0086] 3.3 Combining Mutations Single mutations that showed higher Tm or activity than the wild type were integrated (Figure 3C). The PET2 F105R-E110K mutant (PET2(2M)) showed a Tm of 70.7 °C and produced amorphous PET in 0.71 min. -1 These values ​​are higher than those of each single mutant. The PET2 F105R-E110K-S156P-T297P mutant (PET2(4M)) showed a Tm 1.6°C higher than those of the two mutants mentioned above, but the activity was 0.13 min -1 The improvement was only 0.71 min -1 From 0.84 min -1The further introduction of G180A (PET2(5M)) significantly improved the activity (1.4-fold compared with PET2(4M)), but only slightly increased the Tm. Furthermore, the 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 Tm 6.7°C higher at 60°C and threefold higher activity than the wild-type. Among these, the disulfide bond resulting from the R47C-G89C mutation significantly increased the Tm and activity, despite its location away from the active site. Therefore, it was considered to be one of the most effective mutations.

[0087] 3.4 X-ray crystal structures of PET2 mutants The X-ray crystal structures of PET2(2M) and PET2(7M) were solved at 1.3 Å and 1.8 Å resolution, respectively. Crystals of wild-type PET2 were not available. Therefore, the wild-type structure of PET2 was reconstructed by back-mutating PET2(2M) in Pymol. The root-mean-square deviation (RMSD) of the overall structure between PET2(7M) and PET2(2M) was 0.241 Å, indicating that these structures were highly similar. The electron density maps of the PET2 mutants were sufficient to recognize the side chains of the mutated residues (Figures 4A and 4B). The introduction of two proline mutations (S156P and T297P) did not alter the loop structure, suggesting that these positions are suitable for the dihedral angles of proline, as expected. Mutation of Gly180 to alanine did not alter the α-helical structure containing Ala180, but the adjacent α-helix appeared to be more stable (Figures 4A and 4B, right). The wild-type PET2 has six cysteine ​​residues in its sequence, and two native disulfide bonds (Cys218-Cys255 and Cys289-Cys306) were observed in the PET2(2M) structure. Another pair of cysteine ​​residues (Cys43 and Cys39) was present in the N-terminal disordered region (Ala28 to Asn44) and was not observed. In the PET2(7M) structure, the pair of cysteine ​​residues introduced at the N-terminus (R47C-G89C) clearly formed a disulfide bond (Figure 4B). These mutations caused a slight shift in the two-loop structure (Figure 4A and Figure 4B, left), and the electron density for Tyr45 was not observed. The N-terminal residues (Ala28 to Asn44) could not be modeled in either PET2(2M) or PET2(7M) due to unclear electron density maps, suggesting that the N-terminal residues are disordered in PET2.

[0088] Next, the wild-type PET2 structure was reconstructed by back-mutation of the PET2(2M) structure, and the surface charge at pH 7.0 was compared with that of PET2(7M) and IsPETase (Fig. 4C). In the wild-type PET2, neutral to negative charges were observed around Phe105 and Glu110 (Fig. 4C, left). On the other hand, in PET2(7M) (Fig. 4C, center), the positively charged region was larger than that of the wild-type PET2, and was comparable to that of IsPETase (Fig. 4C, right).

[0089] PET2 was originally discovered as a lipase (19) The structure of the catalytic triad of PET2 (2M) was compared with that of lipases. The lipase from Thermomyces lanuginosa has a lid domain that covers the catalytic triad. (47) The catalytic triad of PET2(2M) was solvent-exposed, similar to 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, not a lipase.

[0090] To explain the calcium-independence of PET2's PET degradation activity, Cut190 (15) The calcium binding sites of LCC, IsPETase, and PET2 (2M) were compared. Binding site 1 is essentially composed of the carbonyl group of the main chain, and binds Ca. 2+ can bind to LCC and PET2. In binding site 2, the aspartic acid (Asp250) and two glutamic acids (Glu220 and Glu296) of Cut190 are changed to threonine, glutamine, and serine in PET2, respectively. 2+ The affinity of PET2 to Cut190 is lower than that of Cut190. Furthermore, binding site 3 of Cut190 is completely occupied by a loop in PET2. Therefore, from a structural point of view, the calcium-independence of the PET degradation activity of PET2 is reasonable.

[0091] 3.5 Characterization of PET2 binding and dissociation by single-molecule fluorescence imaging Using single-molecule fluorescence imaging, we compared 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 efficiencies of PET2 wild-type and PET2 wild-type-C309-Alexa Fluor 555, as well as PET2(7M) and PET2(7M)-C309-Alexa Fluor 555, were similar, suggesting that dye labeling does not affect activity. Furthermore, the labeling efficiencies of PET2 wild-type and PET2(7M), which lack Cys309, were only 3.4% and 7.1%, respectively, suggesting that the dye primarily reacts with Cys309 under these labeling conditions. The original PET film was highly autofluorescent, making single-molecule imaging of the dye-labeled PET2 enzyme difficult. Although the reason for this autofluorescence is unclear, the autofluorescence was successfully reduced by dissolving the PET in hexafluoro-2-propanol and precipitating it with acetone.

[0092] For single-molecule imaging of dye-labeled PET2 enzyme, acetone-precipitated PET was redissolved in hexafluoro-2-propanol, spin-coated onto glass coverslips, and completely dried to form thin PET films with some residual cleavage. At room temperature, PET2(7M) produced 2.6-fold higher product concentrations than PET2 wild-type. The appearance of the thin PET films was significantly different from that of the original amorphous PET films (Figure 5A). The difference in activity between PET2 wild-type and PET2(7M) toward the thin PET films was similar to that observed with amorphous PET films at 60 °C. Therefore, we believe that thin PET films on glass coverslips can be used as a model substrate for single-molecule imaging of PET2 binding and dissociation. At low concentrations (50 pM) of dye-labeled PET2 wild-type and PET2(7M), fluorescent signals from individual molecules were clearly observed (Figure 5B). After single-molecule observation, the PET thin film was stained with a high concentration (10 nM) of dye-labeled enzyme (Figure 5C). This image was then overlaid with a single-molecule video of PET2 to identify the location of the PET thin film. The dye-labeled enzyme bound only to the PET thin film, not to the cover glass. Next, the binding rate constants and binding times (dissociation rate constants) of PET2 wild-type and PET2(7M) were analyzed and compared. To calculate the binding rate constant, the cumulative number of bound molecules was counted as a function of time in each video, and the time required for binding of 100 enzyme molecules was measured (Figure 5D). 100 (molecules) was then divided by the required time (seconds), and the resulting value was further calculated as the area (μm) of the PET thin film within the field of view. 2 The binding rate constant was estimated by dividing by the enzyme concentration in solution (M). The binding rate constant for PET2 wild type was (2.8 ± 1.6) × 10 8 s -1 μm -2 M -1 The binding rate constant of PET2(7M) is (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 that of the wild type.

[0093] On the other hand, there was no significant difference in the dissociation rate constant between PET2 wild-type and PET2(7M). The distribution of binding times for PET2 wild-type was better fitted to the sum of biexponential decay functions than to a single exponential decay function, suggesting the existence of at least two PET binding states (Figure 5E, Table 1). The fast dissociation rate constant was 4.3 s. -1 and the rate constant for slow dissociation is 0.71 s -1 From the area of ​​the fitted equation, the proportions of fast and slow dissociation were calculated to be 69.3% and 30.7%, respectively. The distribution of binding times of PET2(7M) also fitted well to a double exponential decay function, with the rate constants (proportions) of fast and slow dissociation being 5.0 s -1 (85.2%), 0.54s -1 (14.8%) (Figure 5F, Table 1). For both PET2 wild-type and PET2(7M), these dissociation rate constants were significantly higher than the rate constant for photobleaching of Alexa Fluor 555 (0.026 s -1 ) was much larger than that of the fast and slow components. Therefore, photobleaching of Alexa Fluor 555 is not the cause of the fast and slow components.

[0094] [Table 1]

[0095] Table 1 Note: a: Cys309 was additionally introduced at the C-terminus for Alexa Fluor 555 labeling. b: The binding rate constant was calculated from the time required for binding of 100 enzyme molecules and further normalized by the area covered by PET in the field of view and the enzyme concentration in solution. c: Dissociation rate constants were calculated by fitting the distribution of association times of individual molecules with the sum of two exponential decay functions. d: The ratio of fast to slow dissociation events was calculated from the area of ​​the fitting equation. e: Values ​​are the mean ± SD from five independent videos.

[0096] 3.6 Temperature dependence of activity and long-term stability The temperature dependence of amorphous PET degradation activity in wild-type and mutants was measured, and the optimum temperature at which the highest activity was observed was determined. -1 The highest activity was observed for PET2(7M), and activity decreased at higher temperatures (Fig. 6A). On the other hand, the optimum temperature shifted to above 60°C with increasing number of mutations. PET2(7M) showed a growth rate of 2.7 min at 68°C. -1 The activity of PET2(7M) was twice as high as that at 60°C (Figure 6A). PET2(7M) retained detectable activity even at 80°C, whereas the wild-type and the other two mutants (4M and 5M) were inactivated. This result indicates that the R47C-G89C mutation in PET2(7M) significantly contributes to the thermostability of the final mutant. Furthermore, a comparison of the activity of PET2(5M) and PET2(7M) revealed that the R47C-G89C mutation also improved activity at each temperature. To confirm the difference in activity at intermediate temperatures, we also measured the hydrolysis of amorphous PET disks by PET2 wild-type and PET2(7M) at 30°C. While the hydrolysis activity of PET2 wild-type and PET2(7M) was very low at 30°C, the activity of PET2(7M) was three times higher than that of the wild-type. Therefore, the difference was similar to that observed at 60°C.

[0097] Next, we compared the PET degradation by PET2 wild-type and PET2(7M) at their respective optimum temperatures of 60°C and 68°C for incubation times up to 24 h. For amorphous PET degradation, the amount of product produced by PET2(7M) consistently remained more than three times higher than that produced by PET2 wild-type (Figure 6B). Importantly, PET2(7M) maintained a nearly constant degradation rate from 1 to 24 h, suggesting long-term thermal stability at its optimum temperature of 68°C. We also investigated the degradation of crystalline PET. PET2(7M) consistently maintained twice the activity of PET2 wild-type. Again, PET2(7M) maintained a nearly constant degradation rate from 1 to 24 h. However, 0.1 μM PET2 wild-type and PET2(7M) only produced 1.7 μM and 4.5 μM of product, respectively, from crystalline PET after 24 h of incubation. 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) .

[0098] 4. Discussion The upper reaction temperature limit (65°C) of wild-type PET2 estimated from the residual activity (Figure 2A) was in good agreement with the Tm (69.0°C) estimated from the change in CD signal at 230 nm (Figure 2D). Furthermore, previous studies have shown that the lipase activity of wild-type PET2 is 60°C and that activity decreases at higher temperatures. (The original name of this lipase was LipIAF5.2, but for convenience, we use the name PET2 here.) (19)These results suggest that the tryptophan residue signal at 230 nm correlates very well with the catalytic activity of wild-type PET2. We found that the signal at 230 nm of the PET2 W174H mutant was much weaker than that of the wild-type. Therefore, the characteristic signal at 230 nm of PET2 may be due to Trp174, which is located next to the Ser175 residue in the catalytic triad, which is consistent with the correlation described above. On the other hand, the optimum pH for BHET hydrolysis by PET2 was approximately 7 in this study (Figure 2B). Previous studies have shown that the optimum pH for p-nitrophenyl myristate hydrolysis was 10.5. Because BHET spontaneously decomposes to MHET at high pH even in the absence of PET2, this difference is likely due to differences in substrate stability.

[0099] Interestingly, the lipase activity of PET2 was inhibited by only 30% by 1 mM ethylenediaminetetraacetic acid (EDTA) or 1% Tween 80, but was completely abolished by 1 mM ethylene glycol tetraacetic acid (EGTA) or 1% Triton X-100. (19) EGTA and EDTA differ in the structure of the linking group that connects the two pairs of acetate groups. The ethylene glycol structure of EGTA may bind near the active site of PET2 and prevent substrate binding. Furthermore, among the surfactants tested, only Triton X-100 has an aromatic ring in its structure. These results suggest that PET2 has a binding affinity for ethylene glycol and aromatic rings. In this study, Ca was found to be involved in the PET degradation activity of wild-type PET2. 2+ No concentration dependence was observed. In the PET2(2M) structure, the potential Ca(2+) domain corresponds to site 1 of Cut190. 2+ This result is reasonable considering that only the binding site exists, and therefore the ion chelating ability of EGTA and EDTA is unrelated to the change in lipase activity of PET2.

[0100] The results for single mutations in PET2 suggest that mutations that increased the thermostability and activity of IsPETase were not very effective for PET2. In particular, the W174H mutation increased the Tm by 5°C compared to the wild type, but reduced the activity by 4-fold (Figure 3B). In contrast, mutations in the surface charge of PET2 (F105R and E110K), which mimic the surface charge modification of IsPETase, enhanced activity. Interestingly, these mutations also increased the Tm of PET2. The F105R mutation replaces a hydrophobic residue on the enzyme surface with a positively charged residue. Similar mutations have been reported to increase the thermostability of acetylcholinesterase. (49) On the other hand, the reason why the E110K mutation thermostabilizes PET2 is unclear. G180A is one of the most effective mutations, improving both the Tm and activity of PET2. The α-helical structures adjacent to Gly180 and Ala180 are slightly different between PET2(2M) and PET2(7M) (Figure 4A and 4B, right). In PET2(7M), the main chains of Arg138 and Arg139 are slightly shifted to form an ideal α-helical structure. This may contribute to the improved thermal stability and activity. On the other hand, the G177A and G179A mutations decreased the Tm and activity. This is presumably due to the side chains of these alanine residues being too close to the main-chain carbonyl oxygen atoms of Pro100 and Trp199, as confirmed by the crystal structure of PET2(2M) (Figure 4A). The R47C-G89C mutation formed a disulfide bond and increased Tm even more than Y262C-L298C and L265C-A295C (Figure 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. Therefore, disulfide bond formation in the N-terminal region is thought to provide more effective thermal stabilization than disulfide bond formation in the C-terminal region. In the case of LCC and Cut190, Ca 2+ Disulfide bond formation at binding site 2 improved stability (12、15)Because PET2 and IsPETase already have a disulfide bond in this vicinity, we did not attempt to introduce a new disulfide bond at this site in this study.

[0101] Single-molecule fluorescence imaging revealed that the association rate constant of PET2(7M) was 2.7-fold higher than that of wild-type PET2 (Figure 5D, Table 1). Meanwhile, both enzymes had fast and slow components in their dissociation rate constants, and the values ​​and ratios of these components were similar for both enzymes (Figure 5E, Figure 5F, Table 1). We initially predicted that the enhanced activity of the mutants was due to strong cation-π interactions between lysine or arginine residues on the surface of the mutants 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 the surface charge mutation. Based on the ratio of the fast and slow dissociation components, we then calculated the binding rate constants for these components (Table 1). As a result, for wild-type PET2, the values ​​for the fast and slow components were 1.9 × 10 8 s -1 μm -2 M -1 and 0.86 x 10 8 s -1 μm -2 M -1 In addition, for PET2 (7M), the values ​​for the fast and slow components were 6.4 × 10 8 s -1 μm -2 M -1 , 1.1×10 8 s -1 μm -2 M -1 We further calculated the dissociation constants of the fast and slow components based on the ratio of their association and dissociation rate constants (Table 1). For PET2 wild-type, the values ​​were 2.3 × 10 for the fast and slow components, respectively. -8 μm 2 M, 0.83 x 10 -8 μm 2 M. For PET2 (7M), the values ​​were 0.78 × 10 for the fast and slow components, respectively. -8 μm 2M, 0.49 x 10 -8 μm 2 M. Thus, the binding rate constant of the fast component of PET2(7M) was 3.4 times higher than that of PET2 wild-type, and the dissociation constant was 2.9 times lower than that of PET2 wild-type. Interestingly, the activity of PET2(7M) toward amorphous PET films at 60 °C was 3.2 times higher than that of PET2 wild-type (Figure 6A), and the activity toward thin PET films at room temperature was 2.6 times higher. The difference in activity between PET2 wild-type and PET2(7M) was comparable to the difference in the binding rate constant and dissociation constant of the fast component. Thus, the mutant possesses more positive charges than PET2 wild-type at pH 7.0 (Figure 4C), and the PET surface possesses negative charges. (51) One plausible explanation is that PET2(7M) is electrostatically attracted to the PET surface, increasing the local enzyme concentration near the PET surface and facilitating fast binding. It has been reported that Rhizopus oryzae lipase efficiently binds to aliphatic polymers (polybutylene succinate, polybutylene adipate, and polylactic acid) but not to PET. (52) These results suggest that binding to PET requires different interactions. To further increase the binding rate constant of PET2, it may be necessary to modify the surface shape of the enzyme to fit the PET surface. For example, the cellulose binding domain (CBM) has a finely tuned planar surface that fits the flat surface of crystalline cellulose. (53) Interestingly, it has been previously reported that CBMs strongly interact with PET surfaces. (54) For example, T. fusca cutinase fused with the CBM-W68Y mutant of Cellulomonas fimi showed 1.5-fold higher product concentration after 24 h of incubation. (55) Furthermore, Thermomyces cellullosylitica cutinase fused with the binding domain of Alcaligenes faecalis polyhydroxybutyrate depolymerase showed a three-fold higher product concentration than that without the binding domain. (56)Another approach to improve binding affinity to PET surfaces has been reported, using a fusion protein of hydrophobin and cutinase. (57) Although the mechanism of the fast and slow components is still unclear, the association rate constant and dissociation constant of the slow component were not significantly different between PET2 wild-type and PET2(7M), suggesting that the slow component does not contribute to PET degradation (Table 1).

[0102] In this study, PET2(7M) showed long-term thermal stability and was able to decompose amorphous PET films (1.3 cm) with 0.1 μM enzyme. 2 129 μM of product was produced from a PET2 mutant (16 mg / mL, 16 mg / mL) at 68°C for 24 hours (Figure 6B). Here, we compare this PET2 mutant with other previously reported PET hydrolases. A rationally engineered 0.5 μM IsPETase mutant (S121E-D186H-R280A) showed a DSC profile of 2.0 cm, similar to that of the amorphous PET we used. 2 / mL of amorphous PET produced approximately 70 μM of product after incubation at 40°C for 1 day. (58) Therefore, PET2(7M) produced a larger amount of product than the IsPETase mutant. Similarly, the PE-H(Y250S) mutant, a novel enzyme belonging to type IIa PET hydrolases, (59) produced much less product from amorphous PET than PET2 (7M). On the other hand, 2 μM of the Cut190 mutant (Q138A-D250C-E296C-Q123H-N202H) produced 17.5 mM of product from the same amorphous PET used in our study after 3 days of incubation at 70°C. (15) This amount is much larger than ours, and the activity of PET2 (7M) would need to be doubled to achieve the same production. Also, 1.5 nmol of TfCut2 expressed in Bacillus subtilis was cultured at 70°C for 24 hours (rate 23.5 min). -1 ) after 120 hours, 9.9 mg of amorphous PET chips were decomposed, and after 120 hours, the chips were almost completely decomposed. (13)To further improve the activity of PET2(7M), the following strategies may be effective. During the degradation of amorphous PET films by PET2 wild-type and PET2(7M), a relatively large amount of MHET remained in solution as a product (Figure 6C). The fusion enzyme of IsPETase and MHETase showed a synergistic effect on PET degradation. (60) To completely degrade MHET to TPA, it would be effective to create a fusion enzyme with MHETase. Regarding the substrate, crushing PET is an effective method to increase the accessible surface area and promote degradation. In fact, the dissociation constant of TfCut2 for PET nanoparticles (<100 nm) is low, 0.043 mg / mL, as determined by isothermal titration calorimetry. -1 It was decided that (61) Furthermore, the dissociation constant of PET nanoparticles immobilized in agarose gel (average diameter = 164 nm) was 0.031 mg / mL by turbidimetry. -1 was (62) Increasing the accessible surface of the substrate is a promising means to improve enzyme binding. Furthermore, Tournier et al. used native LCC to produce 93.2 mg of TPA equivalent product per mg of enzyme from a 2 mg / mL suspension of amorphous PET powder (smaller than a 500 μm mesh) in 1 hour (approximately 270 min). -1 equivalent to (12) They also successfully created a thermostable LCC mutant (F243I-D238C-S283C-Y127G) that hydrolyzed 90% of 20 kg of waste PET powder in less than 10 hours. (12) .

[0103] From the perspective of enzymes, in order to further improve the activity per enzyme, so-called "processive catalysis," in which the enzyme repeats the catalytic cycle without dissociating from the polymer substrate, is desirable. For example, in the case of polycaprolactone, almost complete degradation has been achieved in one day by utilizing processive degradation by an enzyme embedded in the resin. (63)For the processive degradation of PET, which has strong hydrophobic interactions between polymer chains, it is desirable for PET hydrolases to have thermal stability above the glass transition temperature of PET. Furthermore, it is desirable for PET hydrolases to have the ability to move unidirectionally along a single polymer chain without dissociation, similar to processive cellulases and chitinases. (64) .

[0104] In conclusion, we successfully improved the PET hydrolysis activity of the PET2 enzyme by thermostabilizing and surface charge modification. X-ray crystal structure analysis revealed that the proline mutations maintained the original loop structure, while the paired cysteine ​​residue mutations formed a disulfide bond at the N-terminus of PET2. Although some of the mutations that increased the activity of IsPETase had no effect on PET2, single-molecule imaging analysis revealed that surface charge modification performed using IsPETase as a reference increased the binding rate constant of PET2, contributing to the improved activity. Among these, the introduction of two cysteine ​​residues, R47C and G89C, significantly increased the Tm and improved the activity of PET2, making it one of the most useful mutations. A combination of seven mutations increased the Tm of PET2 by 6.7°C and the optimum reaction temperature by 8°C. PET2(7M) could serve as a template for the construction of even more efficient PET hydrolases.

[0105] Accession code The PDB IDs of PET2(2M)(F105R-E110K) and PET2(7M)(R47CG89C-F105R-E110K-S156P-G180A-T297P) are 7EC8 and 7ECB, respectively.

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Claims

1. A protein having polyethylene terephthalate hydrolysis activity, comprising any one of the following amino acid sequences: (A) an amino acid sequence having only 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 only R20C and G62C mutations relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C and G62C mutations; (B) an amino acid sequence having only the mutations R20C, G62C, S129P, and T270P relative to the amino acid sequence of SEQ ID NO: 1; (B1) an amino acid sequence having 90% or more sequence identity with an amino acid sequence having only the R20C, G62C, S129P, and T270P mutations relative to the amino acid sequence of SEQ ID NO: 1, and having the R20C, G62C, S129P, and T270P mutations; (C) an amino acid sequence having only the following mutations relative to the amino acid sequence of SEQ ID NO: 1: R20C, G62C, S129P, T270P, G153A, E83K, and F78R; or (C1) An amino acid sequence having 90% or more sequence identity with an amino acid sequence having only the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO: 1, and having the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R.

2. The protein according to claim 1, wherein the optimum temperature for the polyethylene terephthalate hydrolysis activity at pH 7.0 is greater than 60.0°C.

3. The protein according to claim 1 or 2, having a melting temperature of 70.0°C or higher.

4. The protein according to any one of claims 1 to 3, wherein the 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 at pH 7.0 than a protein having the amino acid sequence of SEQ ID NO:

1.

6. The protein according to any one of claims 1 to 5, wherein the two cysteine ​​residues at the R20C and G62C mutation sites form a disulfide bond.

7. The protein according to any one of claims 1 to 6, comprising any one of the following amino acid sequences: (A) an amino acid sequence having only R20C and G62C mutations relative to the amino acid sequence of SEQ ID NO: 1; (B) an amino acid sequence having only the following mutations relative to the amino acid sequence of SEQ ID NO: 1: R20C, G62C, S129P, and T270P; or (C) An amino acid sequence having only the mutations R20C, G62C, S129P, T270P, G153A, E83K, and F78R relative to the amino acid sequence of SEQ ID NO:

1.

8. A polynucleotide comprising a base sequence encoding the protein according to any one of claims 1 to 7.

9. A recombinant vector comprising the polynucleotide of claim 8.

10. A transformant comprising the recombinant vector according to claim 9.

11. A composition for decomposing polyethylene terephthalate, comprising the protein according to any one of claims 1 to 7.

12. A method for producing a recycled product, comprising decomposing polyethylene terephthalate with the protein according to any one of claims 1 to 7.

Images