Broad-spectrum polyester hydrolase with wide temperature adaptability and application thereof

Abstract

This invention discloses a broad-spectrum polyester hydrolase with wide temperature adaptability and its applications, belonging to the fields of enzyme engineering and biocatalysis. Compared to the wild type, the polyester hydrolase mutant provided by this invention exhibits significantly improved catalytic performance, excellent thermal stability, and strong temperature adaptability, demonstrating good activity across a temperature range of 20-60 °C. It also possesses a broad substrate spectrum, showing good hydrolytic ability not only for PBAT but also for PET and PLLA. This provides a high-performance enzyme catalyst for biodegradable plastics treatment, mixed polyester recycling, and green chemical processes, demonstrating outstanding industrial application prospects. It can be used in fields such as biodegradable plastics, polyester recycling, and green chemical processes.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis, and relates to a broad-spectrum polyester hydrolase with wide temperature adaptability and its application, specifically a heat-resistant broad-spectrum polyester hydrolase that maintains excellent activity over a wide temperature range and its application. Background Technology

[0002] Polyester materials are a class of high-molecular-weight polymers containing ester bonds in their main molecular chain. Typical examples include polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), and polylactic acid (PLA). Due to their excellent mechanical properties, thermal stability, and processability, polyester materials have been widely used in packaging, textiles, agriculture, and disposable products. However, with the continuous growth in polyester product production, the accumulation of its waste in the environment has caused serious resource waste and ecological pollution. Currently, polyester waste recycling mainly relies on two methods: mechanical recycling and chemical recycling. Mechanical recycling uses processes such as crushing, washing, melting, and remolding to transform high-purity plastic scraps or sorted waste into recycled products. However, during multiple processing steps, the polymer chains are easily broken by heat and shear, leading to a decrease in molecular weight and performance, making it difficult to achieve a closed-loop cycle. Chemical recycling depolymerizes polyester into monomers or oligomers through reactions such as transesterification, hydrolysis, or alcoholysis. Theoretically, these monomers can be resynthesized into products with properties comparable to virgin polyester. However, this process typically requires harsh conditions such as high temperature, high pressure, and catalysts, resulting in high energy consumption, high costs, and potential side reactions that affect monomer purity. Therefore, both traditional mechanical and chemical recycling technologies suffer from insufficient environmental friendliness and poor economic feasibility, making it difficult to meet the actual needs of green recycling of polyester materials.

[0003] In recent years, enzymatic recycling technology has gradually demonstrated its unique advantages as an emerging green and sustainable recycling method. This method utilizes specific hydrolytic enzymes (such as PET hydrolase, keratinase, and lipase) to catalyze the ester bond breakage in polyester under mild conditions, efficiently hydrolyzing polyester into high-value monomers or oligomers. The reaction conditions are mild, energy consumption is low, and no toxic byproducts are generated. Research has found that the bacterium *Ideonella sakaiensis*, originating from a landfill in Sakai City, Japan, can secrete PET hydrolase (IsPETase), achieving efficient hydrolysis of PET under natural conditions. [1] FAST-PETase has been obtained through machine learning optimization, and this mutant exhibits good hydrolytic activity under ambient temperature conditions. [2] Furthermore, the directed evolution-modified HotPETase exhibits superior activity and stability under high-temperature conditions. [3]However, one of the biggest technical bottlenecks in current plastic pollution control is the highly mixed nature of plastic waste. Different types of polyester materials (such as PET beverage bottles, PLA lunch boxes, and PBAT mulch films) often exist in a physically mixed state and are difficult to completely separate using traditional methods. Furthermore, PET, PLA (e.g., PLLA), and PBAT have different glass transition temperatures and crystallization characteristics, but currently reported enzymes still suffer from narrow substrate adaptability, insufficient thermal stability, and low degradation efficiency for mixed polyesters. This typically requires multiple different polyesterases to treat the mixed plastic waste, increasing the difficulty and cost of treatment. Currently, research on modifying PET hydrolases mainly focuses on improving thermal stability. While these enzymes can hydrolyze PET at high temperatures (≥60 °C), their catalytic activity is low at medium and low temperatures, making it difficult to efficiently hydrolyze PET and PBAT in natural environments. In contrast, FAST-PETase, with its medium-temperature activity, can effectively hydrolyze PET and PBAT at medium and low temperatures, but due to insufficient thermal stability, it cannot maintain high catalytic activity for extended periods under composting conditions (approximately 55-60 °C). This demonstrates that existing enzyme systems lack an enzyme that exhibits high activity and thermal stability across a wide temperature range for various polyesters. Therefore, there is an urgent need to develop novel polyester hydrolases, employing structural optimization and intelligent design strategies to enhance their thermal stability and broad-spectrum catalytic performance, thereby promoting the green and efficient recycling of polyester plastic waste.

[0004] In view of this, this invention is hereby proposed.

[0005] References

[0006] [1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degradesand assimilates poly(ethylene terephthalate)[J]. Science, 2016, 353: 759.

[0007] [2] Lu H, Diaz DJ, Czarnecki NJ, et al. Machine learning-aidedengineering of hydrolases for PET depolymerization[J]. Nature, 2022, 604(7907): 662–667.

[0008] [3] Bell EL, Smithson R, Kilbride S, et al. Directed evolution of anefficient and thermostable PET depolymerase[J]. Nature Catalysis, 2022, 5(8):673-681. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] To address the shortcomings of existing technologies, this invention develops a high-performance, broad-spectrum polyester hydrolase that better meets the needs of industrial applications. It exhibits high thermal stability and maintains high activity over a wide temperature range (20-60 °C). It can hydrolyze various substrates such as PBAT, PET, and PLLA, and provides its application in the degradation of polyesters.

[0011] Solution for solving the problem

[0012] The objective of this invention is achieved through the following technical solution:

[0013] [1]. A polyester hydrolase mutant comprising a sequence having at least 90% sequence identity with SEQ ID NO:1 and having the following mutations compared to the sequence shown in SEQ ID NO:1: T51A, Q119Y, S214Y and N233K.

[0014] [2]. The polyester hydrolase mutant according to [1] contains a sequence having at least 95%, at least 97%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:3.

[0015] [3]. An isolated polynucleotide, wherein the polynucleotide encodes a polyester hydrolase mutant as described in [1] or [2].

[0016] [4]. An expression vector comprising a polynucleotide as described in [3].

[0017] [5]. A host cell, wherein the host cell comprises a polyester hydrolase mutant as described in [1] or [2], an isolated polynucleotide as described in [3], or a recombinant expression vector as described in [4];

[0018] Optionally, the selected recombinant host cell is a microbial cell;

[0019] Optionally, the recombinant host cell is derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, Pichia, or Corynebacterium.

[0020] Preferably, the recombinant host cell is derived from Escherichia coli or Pichia pastoris.

[0021] [6]. A cell culture comprising recombinant host cells as described in [5].

[0022] [7]. A product for degrading polyester material comprising a polyester hydrolase mutant as described in [1] or [2], a polynucleotide as described in [4], a recombinant expression vector as described in [5], a recombinant host cell as described in [6], and / or a cell culture as described in [7].

[0023] [8]. According to the product described in [7], the polyester material comprises polyester and / or copolyester;

[0024] Optionally, the polyester material includes at least one of polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), and polylactic acid (PLLA).

[0025] [9]. A method for degrading polyester material, comprising contacting the polyester material with a polyester hydrolase mutant as described in [1] or [2], a recombinant host cell as described in [6], or a cell culture as described in [7] under conditions for degrading polyester material;

[0026] Optionally, the polyester material includes polyester and / or copolyester;

[0027] Optionally, the polyester material includes at least one of polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), and polylactic acid (PLLA).

[0028]

[10] . The method according to [9], wherein the conditions include incubation at a temperature of 20 to 60 °C, preferably at a temperature of 30 to 60 °C.

[0029] Invention Beneficial effects:

[0030] This invention utilizes site-directed mutagenesis, combinatorial mutagenesis, and saturation mutagenesis techniques to systematically optimize the PET hydrolase ThermoPETase (TS-PETase), obtaining a high-performance mutant ThermoPETase. N233K / T51A / S214Y / Q119Y .

[0031] In some embodiments, the mutant ThermoPETase N233K / T51A / S214Y / Q119Y The catalytic activity of this enzyme is enhanced, with its hydrolytic activity against PBAT at 30 °C being 8 times that of the wild type, and its activity against PBAT in the low-temperature range of 20-37 °C approaching the level of the known highly efficient enzyme FAST-PETase; the thermostability of this enzyme is significantly improved, T m The activity level is 20.4 °C higher than the wild type, and it maintains high activity at 50-60 °C, making it more suitable for continuous industrial reaction environments. This enzyme has a broad substrate spectrum, exhibiting high degradation capabilities for polyesters such as PET and PLLA. At 60 °C, its PET degradation activity is 20 times higher than TS-PETase, demonstrating excellent thermal stability and providing strong technical support for the efficient biohydrolysis of PET. Its PLLA degradation efficiency is 6 times higher than TS-PETase, and it maintains stable catalytic performance even at temperatures above the PLLA glass transition temperature (55 °C). The broad substrate applicability of this enzyme can meet the biodegradation and recycling needs of various polyesters. Attached Figure Description

[0032] Figure 1 The mutated amino acids and sites (a) during the modification process from TS-PETase to HotPETase and FAST-PETase in Example 2, as well as the PBAT hydrolytic activity and T of TS-PETase, FAST-PETase, HotPETase, and mutants, are shown. m Value (b).

[0033] Figure 2 The PBAT hydrolysis activity and Tb of mutants based on hotspot mutation sites (a) and calculated predicted mutation sites (b) in Example 3 at 30°C. m Values. Enzyme activity and T at 30 °C and 50 °C based on a combination of high activity and high thermostability. m Characterization of values ​​(c) and the single-point mutations that enhance substrate activity based on the combination of highly active three mutants at 30 °C, showing PBAT hydrolysis activity and T m Value (d).

[0034] Figure 3The enzyme activity and T of the saturation mutation at amino acid position 119 in Example 4 m Value (a) and protein melting curve of TS-M3-Q119Y (b).

[0035] Figure 4 The relative enzyme activity and T of the combined mutant in Example 5 at 30 °C for PBAT hydrolysis are shown. m Values ​​(a), activity determination of PBAT hydrolysis at different temperatures by FAST-PETase, BtaPBATase and HotPETase (b), and changes in the hydrolysis of PBAT films catalyzed by BtaPBATase, FAST-PETase and HotPETase over time at 30 °C and 60 °C (c).

[0036] Figure 5 The hydrolytic activity of BtaPBATase for low-crystallinity PET and PLLA in Example 6 is shown.

[0037] Figure 6 The SDS-PAGE (a) of BtaPBATase, Endo H and Endo H enzymes digested in the Escherichia coli and Pichia pastoris expression system in Example 7, and the hydrolytic activity of BtaPBATase, Endo H and Endo H enzymes digested in the Escherichia coli and Pichia pastoris expression system at different temperatures on PBAT (b). Detailed Implementation

[0038] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0039] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0040] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0041] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0042] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0043] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0044] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0045] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0046] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still included, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0047] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, Joseph Frank et al. “Molecular Cloning: A Laboratory Manual.” (2001). (Hereinafter referred to as “Sambrook”). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0048] In this specification, the terms "peptide" or "protein" are polymers of amino acid residues linked by peptide bonds, whether naturally occurring or synthetically produced.

[0049] In this specification, the terms "mutant" or "variant" refer to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide. Substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. Exemplarily, a "mutant" in this invention is a polypeptide with enhanced hydrolytic activity in polyester materials.

[0050] In this invention, a "mutation" can be an addition, deletion, or substitution of amino acids at one or more positions corresponding to the sequence shown in SEQ ID NO:1 or SEQ ID NO:2 that does not affect the activity of the polyester hydrolase. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-critical regions, does not substantially alter its biological activity; for example, appropriately replacing, adding, or deleting certain amino acids results in sequences that do not affect their activity.

[0051] As used herein, the terms “corresponding” and “corresponding” have the meanings commonly understood by those skilled in the art. Specifically, “corresponding” and “corresponding” refer to the positions in one sequence that correspond to a specified position in another sequence after homology or sequence identity alignment.

[0052] In some embodiments, the "mutation" of this invention may be selected from "conservative mutations." In this invention, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0053] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0054] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed.

[0055] The methods disclosed herein for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0056] In this specification, the term "polynucleotide" refers to a polymer composed of nucleotides. Polynucleotides can be in the form of individual fragments or as a component of a larger nucleotide sequence structure, derived from a nucleotide sequence isolated at least once in number or concentration, and capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, T, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0057] In this specification, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components removed from at least one other component naturally associated with them.

[0058] In this specification, the term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] In this specification, the term "expression vector" refers to a DNA sequence operatively linked to a suitable control sequence for expressing a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired exogenous polypeptide. Recombinant expression vectors may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence transcribed into mRNA and translated into a protein; and iii) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used.

[0060] In this specification, the term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., by means of the mutant polypeptide of the present invention, the polynucleotide encoding the mutant polypeptide, or a recombinant expression vector.

[0061] In this specification, the term "cell culture" refers to a combination of cells and cell culture medium, wherein the cells are cultured in a cell culture medium outside the organism.

[0062] Polyester hydrolase mutant

[0063] In a first aspect of the invention, a polyester hydrolase mutant is provided, the mutant comprising a sequence having at least 90% sequence identity with SEQ ID NO:1 and having the following mutations compared to the sequence shown in SEQ ID NO:1: T51A, Q119Y, S214Y, and N233K. These mutations significantly improve the thermal stability and medium-to-high temperature catalytic performance of the polyester hydrolase mutant. Furthermore, the polyester hydrolase mutant exhibits good hydrolytic ability for various polyester materials, such as PET, PBAT, and PLLA, and can be used for the degradation of mixtures containing multiple polyester materials.

[0064] In some specific embodiments, the polyester hydrolase is a modification based on the sequence shown in SEQ ID NO:2. The sequence shown in SEQ ID NO:2 is obtained by truncating the first to 28th amino acid residues from the N-terminus of the sequence shown in SEQ ID NO:1. In the art, the first amino acid residue of the sequence shown in SEQ ID NO:2 is numbered as the 29th residue. Therefore, A47, T51, A65, Q119, S125, P181, S207, S214, D220, and N233 in this document actually correspond to the 19th, 23rd, 37th, 91st, 97th, 153rd, 179th, 186th, 192nd, and 195th amino acid residues of the sequence shown in SEQ ID NO:2. Other sites mentioned in this document also conform to the above correspondence.

[0065] In some specific embodiments, the polyester hydrolase comprises a sequence having at least 95%, at least 97%, or at least 99% sequence identity with the sequence shown in SEQ ID NO:3.

[0066] In some exemplary embodiments, the polyester hydrolase can be expressed in different host cells, such as Escherichia coli and Pichia pastoris. When the polyester hydrolase shown in SEQ ID NO:3 is expressed in Pichia pastoris, the enzyme undergoes N-glycosylation modification, which affects the binding of the enzyme to the substrate. Therefore, the mutant in this invention also contains mutations at sites that can remove the influence of N-glycosylation modification, such as mutations at sites N37, N114, N138, N173, N190, N205, N212, N264, N277, and N288. Through these mutations, while maintaining the catalytic efficiency of the polyester hydrolase shown in SEQ ID NO:3, the hydrolytic activity of the polyester hydrolase shown in SEQ ID NO:3 under high temperature conditions can be further enhanced.

[0067] biomaterials

[0068] In one aspect, the present invention provides an isolated polynucleotide encoding a polyester hydrolase mutant as described above.

[0069] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. Polynucleotides encoding mutants of this invention include: a coding sequence encoding only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0070] In some embodiments, the polynucleotide includes a promoter operatively linked to a coding sequence. The coding sequence may be codon-optimized for the cell in which it is expressed.

[0071] In another aspect, the present invention provides a recombinant expression vector that expresses a polyester hydrolase mutant as described above or contains a polynucleotide as described above.

[0072] In another aspect, this disclosure provides a recombinant host cell comprising the modified Dda helicase as described above, the polynucleotide as described above, or the expression vector as described above.

[0073] In some alternative embodiments, the recombinant host cell is derived from microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Enterobacteria*, *Salmonella*, *Streptomyces*, *Pseudomonas*, *Brevibacterium*, *Bacillus*, *Pichia*, or *Corynebacterium*. In some preferred embodiments, the host cell is derived from the genera Escherichia and Pichia, such as Escherichia coli and Pichia pastoris, including Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Trans T1, Escherichia coli BL21(DE3), Pichia pastoris X-33, Pichia pastoris GS115, Pichia pastoris KM71, etc.

[0074] In some preferred embodiments, using Pichia pastoris as the host cell can achieve stable production of the polyester hydrolase mutant.

[0075] Furthermore, this disclosure provides cell cultures comprising the recombinant host cells described above.

[0076] Products and Uses

[0077] In one aspect, the present invention provides a product for degrading polyester materials, comprising the polyester hydrolase mutant as described above, the polynucleotide as described above, the recombinant expression vector as described above, the recombinant host cell as described above, and the cell culture as described above.

[0078] In some implementations, the products include enzyme preparations, composite biomaterials, microbial preparations for the biorecycling of plastic waste, and preparations with efficient polyester material biorecycling functions.

[0079] In another aspect, the present invention provides a method for degrading polyester materials, said polyester materials comprising mixtures of polyesters and / or copolyesters, such as containing at least one of polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), polylactic acid (PLLA), or mixtures thereof.

[0080] The time required to degrade polyester materials can vary depending on the polyester material itself (i.e., the properties and source of the plastic product, its composition, shape, etc.), the specific enzyme and the enzymes used, as well as various process parameters (i.e., temperature, pH, additional reagents, stirring, etc.).

[0081] In some preferred embodiments, the PET, PBAT, and PLLA are in powder form. In some embodiments, each gram of PET, PBAT, or PLLA is subjected to a depolymerization reaction using at least 25U, at least 26U, at least 27U, at least 28U, at least 29U, at least 30U, at least 31U, at least 32U, at least 33U, at least 34U, at least 35U, or at least 36U of the polyester hydrolase mutant described above.

[0082] In some specific implementations, the enzyme activity unit (U) is defined as the amount of enzyme that hydrolyzes to produce 1 μmol of 4-nitrophenol per minute using p-butyl nitrobenzene (pNPB) as a substrate under normal temperature, pH 8.0, and 50 mM phosphate buffer.

[0083] In some implementations, the hydrolysis method is carried out under conditions including ambient temperature, for example, 20–60 °C. These temperatures are conducive to the contact between the polyester hydrolase mutant and the polyester material in the mixture and to the hydrolysis process.

[0084] In some specific implementations, incubation is carried out at a temperature of 20–60 °C, especially at 30–60 °C, and more preferably at 30–50 °C.

[0085] In some specific embodiments, the enzyme reacts with the polyester material at a pH of 6.5 to 9.0, preferably 7.5 to 9.0. The pH of the system can be adjusted using a phosphate buffer, for example, using a 0.05-0.5 M phosphate buffer with a pH of 7.5 to 9.0, more preferably a 0.05 M phosphate buffer with a pH of 8.0.

[0086] In some embodiments, the polyester material may be pretreated before contact with the polyester hydrolase mutant to physically alter its structure, thereby increasing the contact surface between the polyester material and the polyester hydrolase mutant.

[0087] Example

[0088] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0089] The following description, in conjunction with the accompanying drawings and examples, further explains the specific implementation process of the present invention. However, it is worth noting that the implementation and protection of the present invention are not limited to the following embodiments. It should also be noted that the enzyme protein used in the implementation of the present invention has PBAT hydrolytic activity and was developed and prepared independently by the inventor's research institution; the other chemicals and equipment are all commercially available conventional products.

[0090] Example 1: Construction of recombinant plasmid of polyester hydrolase mutant

[0091] This embodiment takes the existing PET hydrolase TS-PETase as a starting point, and through cloning, expression and purification, analyzes its structure, performs computational design and site-directed mutagenesis to improve the enzyme's catalytic activity over a wide temperature range and the hydrolytic activity of other polyesters, thereby enhancing its industrial application value.

[0092] (1) Chemical transformation of plasmids

[0093] The target protein plasmid from BGI Genomics was centrifuged for 2 min (12000 rpm), then dissolved thoroughly in 40 µL of sterile water. The dissolved plasmid was stored at -20 ℃ for later use. An ice bath was prepared, and E. coli BL21(DE3) competent cells were removed from the -80 ℃ freezer and immediately placed in an ice bath for slow thawing. 2 µL of the dissolved plasmid was added to the competent cells, gently mixed with a pipette tip, and then incubated on ice for 30 min. Subsequently, the competent cells containing the plasmid were heat-shocked in a preheated water bath at 42 ℃ for 45 s, then removed and incubated on ice again for 2 min. Then, 800 µL of liquid LB medium was added, gently mixed with a pipette, and the mixture was incubated on a shaker at 37 ℃ (200 r / min) for 1 h for recovery and culture.

[0094] (2) Plate culture

[0095] During the resuscitation of competent cells, 100 mL of solid LB medium is dissolved beforehand. When the temperature drops to a suitable level for handling (the bottom of the flask is not hot to the touch), the medium is transferred to a laminar flow hood. Under aseptic conditions, 100 µL of antibiotic solution is added and mixed well. Then, the medium is poured into disposable sterile agar plates. Approximately 5 plates can be prepared from 100 mL of medium. The plates are left to solidify in the laminar flow hood with the fan on (they can be sterilized under ultraviolet light if appropriate).

[0096] After resuscitation, the bacterial culture was centrifuged for 2 minutes (8000 rpm), 700 µL of supernatant was discarded, and 50 µL of the resuspended bacterial cells were added to the surface of an LB agar plate. The bacterial culture was then evenly dispersed using a spreader sterilized with an alcohol lamp and cooled to room temperature. The plate was inverted and incubated at 37 °C for 12–16 h. Since the plasmid carries an antibiotic resistance gene, the appearance of a single colony on the plate indicates successful transformation of the plasmid into E. coli BL21(DE3) competent cells, after which subsequent experimental procedures can be performed.

[0097] (3) Expand cultivation

[0098] Pick a single colony from the plate and inoculate it into 10 mL of liquid LB medium, adding 10 µL of antibiotic solution and gently mixing. Place the tube in a 37 °C shaking incubator and incubate overnight at 200 rpm. Then, transfer 10 mL of the culture (approximately 1% inoculum) to 1 L of liquid LB medium and add 1 mL of antibiotic. Place the shake flask in a 37 °C shaking incubator and continue incubating at 200 rpm for 4 h. Transfer 500 µL of the bacterial culture from the shake flask to a centrifuge tube and measure its absorbance using a UV spectrophotometer, with deionized water as a blank control. The OD value of the bacterial culture should be between 0.6 and 0.8, indicating that the bacteria are in the exponential growth phase and the optimal time for induction of expression.

[0099] (4) Induced expression

[0100] Under aseptic conditions in a laminar flow hood, add 1 mL of IPTG inducer (100 mg / mL, final concentration 0.5 mmol / L) to the flask. After the operation, place the flask in an 18 ℃ shaking incubator and incubate at 180 r / min for 18 h to promote proper enzyme folding.

[0101] (5) Cell disruption

[0102] After the bacterial strain was expressed, the cells were centrifuged at 4000 rpm (3400×g) for 40 min to remove the culture medium and retain the cells. Next, 10 mM imidazole was added, and the centrifuged cells were collected and resuspended. The cells were then homogenized using an autoclave at 4 °C. After cell lysis, the cells were centrifuged at 12000 rpm (5400×g) for 40 min to remove cell debris, and the supernatant was collected.

[0103] (6) Enzyme purification

[0104] The supernatant was transferred to a 3 mL Ni-NTA agarose column equilibrated with 10 mM imidazole and rotated at 4 °C for 1 h to ensure complete binding of the target protein to the Ni-NTA agarose gel column. The column was washed continuously with 50 mM and 100 mM imidazole. The wash solution was checked with Coomassie Brilliant Blue G-250 solution; the absence of blue discoloration (with imidazole solution and Coomassie Brilliant Blue G-250 solution serving as a blank control) indicated complete elution of contaminating proteins. The target protein was then collected by elution with 300 mM imidazole.

[0105] (7) Ultrafiltration Concentration

[0106] Ultrafiltration tubes (10 kD cutoff) were soaked in 0.2 M NaOH for 3 h before use, then rinsed with deionized water. The eluted target protein was added to the ultrafiltration tubes, balanced on a balance, and centrifuged at 6000 rpm for 15 min at a time until the total volume of collected target protein was reduced to approximately 1 ml. The liquid was then transferred to 1.5 ml centrifuge tubes. Subsequently, the protein was desalted using a HiTrap desalting column with 50 mM phosphate buffer (pH = 8.0). Finally, the desalted protein was stored on ice.

[0107] (8) Protein concentration determination

[0108] Protein concentrations were determined using a BCA assay kit. Bovine serum albumin (BSA) standards were diluted to concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL using diluent. The protein samples to be tested were also diluted to these concentration ranges. 20 μL of the standards and the test samples were added to each well of a 96-well plate. 200 μL of BCA working solution was added to each well using a pipette, and the mixture was incubated at 37 °C for 30 min. The absorbance was measured at 562 nm using a microplate reader. A standard curve was then plotted with the standard concentration on the x-axis and the absorbance on the y-axis. The protein concentration of the test samples was calculated from the standard curve. Each test was performed in triplicate.

[0109] Example 2: Determination of the activity and thermal stability of a single-point mutant of polyester hydrolase

[0110] HotPETase evolved from TS-PETase through four rounds of directed evolution, introducing a total of 18 mutation sites. Its melting temperature (T) m The temperature was increased from 56.6 °C to 82.5 °C, enabling efficient depolymerization of crystalline PET at 70 °C. FAST-PETase, on the other hand, was obtained by introducing R224Q and N233K mutations into TS-PETase. Figure 1 a), its T m The value was 63.4 °C, and the hydrolytic activity of PBAT at 30 °C was significantly higher than that of TS-PETase. To verify the effect of different mutation sites on the thermostability and low-temperature catalytic activity of polyester hydrolases, TS-PETase was selected as the basic enzyme (SEQ ID NO:2), and single-point mutations were performed on amino acids at positions 224 and 233 to obtain mutant TS-PETase. R224Q and TS-PETase N233K .

[0111] Enzyme thermal stability determination: The melting temperature (T0) of the enzyme was determined using differential scanning calorimetry (DSF). m A protein sample solution at a concentration of 0.2 mg / ml was filled into a NanoDSF standard capillary tube (NanoTemper Technologies) and detected on a Prometheus NT.48 device controlled by PR.ThermControl software (version 2.1.2). Detection conditions were as follows: excitation power preset to F330 and F350 with fluorescence readings above 2000 RFU, and sample heated at a rate of 0.5 °C / min between 40 °C and 80 °C.

[0112] Enzyme activity assay: Using PBAT as a substrate, the reaction was carried out at 30 °C in 50 mM PB buffer (pH 8.0). The concentration of hydrolysis products was then measured using a UV-Vis spectrophotometer at 240 nm. The products were identified as soluble aromatic hydrolysis products, all possessing the same molar extinction coefficient (17,000 M). -1 cm -1 ), and expressed as aromatic product equiv.

[0113] The results showed that ( Figure 1 b) TS-PETase N233K T m The value was 63.9 °C, comparable to FAST-PETase, and its PBAT hydrolysis activity at 30 °C was 10 times higher than TS-PETase, even exceeding that of FAST-PETase. TS-PETase... R224Q T m The value was 56.0 °C, lower than TS-PETase. N233K The mutant exhibits significantly reduced PBAT hydrolytic activity at 30 °C, retaining only 40% of TS-PETase, which is far lower than TS-PETase. N233K To further evaluate the effect of the N233K mutation site on low- and medium-temperature activity, the 233K mutation was introduced into HotPETase, resulting in HotPETase. C233K Its PBAT hydrolytic activity at 30 °C was measured. HotPETase's activity at 30 °C was similar to TS-PETase, but when its cysteine ​​(C) at position 233 was mutated to lysine (K), HotPETase... C233K The PBAT hydrolysis activity of this enzyme is approximately 3.3 times higher than that of HotPETase, but due to the mutation at this site disrupting the disulfide bond, T... mThe temperature decreased by 5.5 °C. These results further demonstrate that the mutation at position 233 to lysine (K) contributes to increased PBAT hydrolysis activity at 30 °C.

[0114] Example 3: Determination of the activity and thermal stability of polyester hydrolase combinatorial mutants

[0115] Based on the experimental results of Example 2, TS-PETase N233K It exhibits good PBAT hydrolysis activity at 30 °C, therefore TS-PETase is used. N233K Starting with (TS-N233K), further two-point combination mutations were performed, targeting hotspot sites P181V, S207R, S214Y, Q119K, S213E, R90T, R224L, S58A, S61V, N241C, M154G, K252M, and T270Q. Figure 2 a) and two-point mutation experiments were conducted to verify the predicted sites A47R, T51A, A65G, V68I, S125R, V134L, S141P, Q182L, D220N, and N246F. Figure 2 b). Thermal stability test results show that, compared to TS-N233K, S214Y (ΔT) is significantly improved. m = +10.4 °C), P181V (ΔT) m =+1.3 °C), S213E (ΔT) m =+1.3 °C), S207R (ΔT) m =+0.8 °C), S61V (ΔT) m =+1.0 °C), M154G (ΔT) m =+0.9 °C), R224L (ΔT) m =+0.7 °C), S58A (ΔT) m =+0.7 °C) and R90T (ΔT) m =+0.4 °C), D220N (ΔT) m Both the S125R (+1.2 °C) and S125R (+0.4 °C) mutations increased T mActivity tests showed that, compared to TS-N233K, the PBAT hydrolysis activities of each mutant at 30 °C exhibited significant differences in their effects on enzyme catalytic performance. Q119K showed the most significant promoting effect on catalytic activity, increasing it by 46%; A65R, T51A, and A47R increased enzyme activity by 24%, 19%, and 15%, respectively; in addition, S207R and N241C also showed some degree of activity enhancement, increasing by 13% and 11%, respectively. The S214Y mutation was most significant in improving thermal stability, while the Q119K mutation showed the best performance in improving activity at room temperature.

[0116] To obtain a PBAT hydrolase that combines high thermal stability with low-temperature catalytic performance, a three-point combination mutation was used for superposition mutation, specifically, increasing the T value starting at TS-N233K-Q119K. m This improves thermal stability, but T m The activity of mutation sites with increased values ​​decreased, therefore the standard was set at T. m Mutation sites that improve activity but reduce it by no more than 30% were used for combined mutation experiments for validation, specifically S207R, S58A, D220N, P181V, S125R, and M154G. Another strategy involved adding mutation sites that improve low-temperature activity to the thermally stable TS-N233K-S214Y, with the standard being an activity increase of more than 10% but a reduction in T... m The value decreased by no more than 1 ℃, so A47R, T51A, A65R, Q119K, and S207R were selected for experimental verification. The results showed ( Figure 2 c) The combined mutants starting with TS-N233K-Q119K showed lower hydrolytic activity at 30 °C than the starting enzyme, and limited improvement in thermostability; while in the combined mutants based on TS-N233K-S214Y, the T51A mutation significantly improved low-temperature activity, with its PBAT hydrolytic activity at 30 °C increasing by approximately 61% compared to the basal enzyme, and T... m No decrease was observed in the activity at 50°C. Comparison of different mutation combinations revealed that the superposition of the Q119K mutation in the S214Y background led to a decrease in activity of approximately 52%, indicating a possible conformational antagonism between the two.

[0117] To further screen for the best-performing combined mutants, four low-temperature activity-related mutation sites—A47R, A65R, Q119K, and S207R—were introduced, starting with TS-N233K-S214Y-T51A. Experimental results ( Figure 2 d) indicates that the T of this four-point combination mutant mThe values ​​did not increase further, and the enzyme activities all decreased. Through systematic screening and combination optimization, the three-point mutant TS-N233K-S214Y-T51A(M3) using TS-PETase as a template was determined to have the best performance. It exhibited excellent PBAT hydrolytic activity under both low temperature (30 °C) and high temperature (50 °C) conditions, with a melting temperature T m It reached 73.7 °C, and its overall performance was significantly better than other mutants.

[0118] Example 4: Saturation mutation of polyester hydrolase for detecting PBAT hydrolytic activity

[0119] Based on the experimental results of Example 3, the Q119K mutation significantly improved the PBAT hydrolytic activity of the TS-N233K mutant at 30 °C. However, when this mutation coexisted with S214Y, an antagonistic effect of decreased activity occurred. To mitigate this adverse effect and further improve the enzyme's low-temperature catalytic ability, a saturation mutation was performed on the 119th amino acid in this example. Using the mutant TS-M3 (N233K, S214Y, T51A) as a template, site-directed mutagenesis was used to replace the 119th amino acid with the remaining 19 amino acid residues, constructing a saturation mutant library. All mutants were fermented for expression, purified, and their PBAT hydrolytic activity was measured at 30 °C.

[0120] Experimental results ( Figure 3 a) indicates that when the 119th amino acid is mutated from glutamine (Q) to tyrosine (Y), the low-temperature activity of the mutant is comparable to that of TS-M3, without a significant decrease. Simultaneously, its melting temperature (T0) remains relatively constant. m The temperature increased from 73.7 °C to 77.1 °C. Figure 3 (b) indicates that this mutation helps improve low-temperature catalytic performance while maintaining high thermal stability. In summary, the Q119Y mutation effectively enhances low-temperature catalytic performance while maintaining high thermal stability, making it an effective mutation strategy for optimizing the balance between polyester hydrolase activity and stability.

[0121] Example 5: Determination of the hydrolytic performance of the polyester hydrolase mutant BtaPBATase on PBAT

[0122] To obtain a polyester hydrolase possessing both high thermal stability and broad-spectrum temperature activity, this embodiment employs stepwise combination mutagenesis based on TS-PETase. First, the N233K mutation, which enhances enzyme activity, is introduced into TS-PETase to obtain mutant M1. Second, the S214Y mutation, which improves thermal stability, is introduced into M1 to obtain M2, whose T... mThe thermal stability value increased significantly, but the catalytic activity decreased, demonstrating a typical trade-off between thermal stability and activity. To balance these two aspects, the activity-enhancing mutant T51A was further introduced into M2 to form M3, maintaining thermal stability while improving activity. Finally, the mutant site Q119Y obtained through saturated mutation screening was superimposed on M3 to obtain the mutant M4 with the best overall performance. Given that the obtained optimal mutant exhibits a relatively wide temperature range during PBAT hydrolysis, this invention names it BtaPBATase (broad-temperature-active PBAThydrolase) to highlight its significant wide-temperature-range catalytic characteristics.

[0123] Evolutionary analysis results as follows Figure 4 As shown in Figure a, the relative enzyme activities of M1, M2, M3, and M4 at 30 °C were 10.0, 4.9, 7.9, and 8.0 times that of the wild type, respectively, corresponding to T... m The values ​​increased by 7.4, 17.2, 17.1, and 20.4 °C, respectively. The resulting M4 exhibited high thermal stability and high catalytic activity. Further testing of the PBAT hydrolysis activities of FAST-PETase, HotPETase, and BtaPBATase within the 20-70 °C range showed that ( Figure 4 b) BtaPBATase maintains high catalytic activity within the temperature range of 20-60 °C, exhibiting broad temperature adaptability. In the low-temperature range (20-37 °C), the activity of BtaPBATase is slightly lower than that of FAST-PETase. In the medium-temperature range (30-37 °C), the activity of BtaPBATase is basically consistent with that of FAST-PETase, indicating that it still possesses good catalytic efficiency under mild reaction conditions. As the reaction temperature increases to 50-60 °C, the relative activity of BtaPBATase is significantly higher than that of FAST-PETase and HotPETase, comprehensively demonstrating its excellent thermal stability and medium-to-high temperature catalytic ability, providing potential application prospects for the industrial degradation of PBAT.

[0124] In this embodiment, enzyme activity units (U) are defined as the amount of enzyme that hydrolyzes 1 μmol of 4-nitrophenol per minute using pNPB as a substrate in 50 mM phosphate buffer at room temperature, pH 8.0. Specific activity (U / mg) represents the number of enzyme activity units per milligram of protein. Product concentration was determined by UV spectrophotometry at room temperature (23±2°C) by monitoring the change in absorbance of 4-nitrophenol at 405 nm, with a molar extinction coefficient of 18,000 M. -1 ·cm -1In the experiment, PBAT films were cut into 2×0.5 cm samples and placed in a 5 mL reaction system containing 4 mL of 50 mM phosphate buffer (pH 8.0). The total enzyme activity was 0.72 U, and hydrolysis analysis was performed at 30 °C and 60 °C, respectively.

[0125] To further analyze the actual hydrolytic activity of BtaPBATase, FAST-PETase, and HotPETAse on PBAT films, PBAT films prepared by hot pressing of PBAT particles were selected as substrates. The hydrolytic activity of BtaPBATase under near-real-world application conditions was evaluated. Experimental results are as follows: Figure 4 As shown in Figure c, at 60 °C, the hydrolysis of the PBAT film was significantly accelerated after the addition of BtaPBATase. After 6 h of reaction, the film began to disintegrate and form obvious fragments; by 8 h, the fragments were further refined and gradually transformed into granules. When the reaction proceeded to 12 h, the reaction system was completely clear, indicating that the PBAT film had been completely hydrolyzed. At 30 °C, due to the lower reaction temperature, BtaPBATase had little effect on the PBAT film in the early stages of the reaction, but after 6 h, the film was still observed to gradually disintegrate and form fragments. With the extension of reaction time, the fragments further hydrolyzed into granular substances after approximately 68 h, and finally achieved complete hydrolysis at approximately 76 h. In contrast, FAST-PETase, due to its lower thermal stability, rapidly deactivated at 60 °C, and the PBAT film remained largely intact, with no obvious hydrolysis observed. Under reaction conditions of 30 °C, the PBAT film began to break down and form large fragments after 6 h of reaction. These fragments remained predominantly present until 68 h, and only a small number of particles appeared in the reaction system after the reaction time was extended to 76 h. The overall hydrolysis process was significantly slower than that of BtaPBATase. Although HotPETase exhibits higher thermal stability, its catalytic hydrolysis activity for PBAT is lower than that of BtaPBATase. Under reaction conditions of 30 °C, the PBAT film of HotPETase only showed significant breakage and formed large fragments after 56 h, and particle formation was not observed until 76 h of continued reaction. Under reaction conditions of 60 °C, the hydrolysis effect of HotPETase was not significant in the early stages, and the PBAT film gradually hydrolyzed into particles only after 44 h of reaction.

[0126] The above results indicate that, under different temperature conditions, BtaPBATase exhibits a faster hydrolysis rate and a higher degree of hydrolysis for PBAT films compared to FAST-PETase and HotPETase.

[0127] Example 6: Determination of the hydrolytic performance of the polyester hydrolase mutant BtaPBATase on PLLA and PET

[0128] To evaluate the hydrolytic performance of the polyester hydrolase mutant BtaPBATase obtained in this invention on different polyester substrates, hydrolysis reactions were carried out using PLLA and PET as substrates, respectively.

[0129] Hydrolysis experiments were conducted using low-crystallinity PET powder (Goodfellow) as a substrate under the optimal reaction conditions of BtaPBATase. The reaction system consisted of 1 mL of phosphate buffer at pH 8.0, the temperature was set at 60°C, the PET powder concentration was 5 mg / mL, and 0.18 U of total BtaPBATase enzyme activity was added. Five reaction time gradients (6 h, 12 h, 24 h, 36 h, and 48 h) were set to investigate the accumulation pattern of hydrolysis products. After the reaction was completed, it was terminated by heating to 100 °C, and an equal volume of 0.1% trifluoroacetic acid aqueous solution was added. The mixture was then filtered through a 0.2 μm polyethersulfone needle filter to remove residual PET particles. Product analysis was performed using an HPLC system equipped with an Eclipse Plus C18 column. Mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and mobile phase B was methanol. The elution gradient was: 30% to 50% phase B for 10 min, followed by a 50% phase B hold for 5 min, for a total analysis time of 15 min. The flow rate was 1.0 mL / min, the detection wavelength was 240 nm, and the column temperature was 40 ℃. The results showed that ( Figure 5 a) The accumulation of hydrolysis products by BtaPBATase in low-crystallinity PET exhibits a distinct phased pattern. In the initial 0-6 h of the reaction, hydrolysis products accumulate rapidly; as the reaction time increases, the accumulation rate gradually decreases, reaching a peak at 48 h, demonstrating stable and controllable hydrolysis behavior. Under the same reaction conditions and time, the hydrolysis efficiency of BtaPBATase is consistently higher than that of the TS-PETase control group. At each time point, the concentration of hydrolysis products in the BtaPBATase reaction system is significantly higher than that of TS-PETase, indicating that BtaPBATase has a more efficient hydrolysis capability for low-crystallinity PET.

[0130] PLLA powder was used as the substrate for PLLA hydrolysis experiments under the optimal reaction conditions of BtaPBATase. 5 mg of PLLA powder was added to an EP tube containing 1 mL of phosphate buffer (pH 8.0) and sonicated for 5 min to ensure thorough substrate dispersion. BtaPBATase was then added to bring the total enzyme activity to 0.18 U, with a blank control (no enzyme added). The reaction was carried out at 55 °C and 800 rpm in a thermostatic mixer with six reaction time gradients: 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h. After the reaction, unreacted substrate was removed by filtration through a 0.22 μm polyethersulfone membrane. The supernatant was analyzed by high-performance liquid chromatography (HPLC) using a Carbomix H-NP5 organic acid column from SciFen Technologies, with 5 mM sulfuric acid as the mobile phase, a column temperature of 50 °C, and a flow rate of 0.6 mL / min. A standard curve was established using lactic acid standards, and the concentration of lactic acid produced by the enzymatic reaction was calculated based on the peak area of ​​the product. The results showed that ( Figure 5 (b) During the 72-hour continuous reaction period, the lactic acid yields of the self-hydrolysis group, the TS-PETase group, and the BtaPBATase group all showed a gradual increasing trend with the extension of reaction time. Notably, TS-PETase exhibited poor thermal stability; its catalytic activity was significantly inhibited at 55 °C, resulting in no significant difference in lactic acid product accumulation compared to the self-hydrolysis group. In stark contrast, BtaPBATase demonstrated excellent catalytic hydrolysis activity for PLLA, significantly superior to TS-PETase. After 72 hours of reaction, the lactic acid yield of the BtaPBATase group reached 0.5 mM, five times that of the self-hydrolysis group and the TS-PETase group, indicating that BtaPBATase has a highly efficient hydrolysis capacity for PLLA.

[0131] Example 7: Validation of the activity of BtaPBATase expressed in the Pichia pastoris system

[0132] To improve the expression level of the target protein, this embodiment used the eukaryotic expression system *Pichia pastoris* GS115 to heterologously express BtaPBATase. During the expression process, it was found that the catalytic efficiency of BtaPBATase expressed in *Pichia pastoris* was reduced. After studying and analyzing the expression product, it was speculated that this was due to glycosylation. Therefore, NetNGlyc 1.0 software was used to predict potential N-glycosylation sites of BtaPBATase. The results showed that there are 10 potential N-glycosylation sites in the protein sequence, located at residues N37, N114, N138, N173, N190, N205, N212, N264, N277, and N288. N-glycosylation modification at these sites may help improve the structural stability of the protein, thereby enhancing its hydrolytic activity under high-temperature conditions; however, it may also affect the substrate binding process, thus reducing catalytic efficiency.

[0133] To verify the effect of glycosylation modification on the catalytic performance of the target protein, this example used endoglycosidase H (Endo H) to deglycosylate recombinant BtaPBATase expressed in Pichia pastoris. The analytical results are as follows: Figure 6 As shown. SDS-PAGE analysis results ( Figure 6 a) indicates that the apparent molecular weight of the recombinant protein was significantly reduced after Endo H treatment, and was basically consistent with the molecular weight of the protein obtained from the *E. coli* expression system, suggesting that the recombinant protein expressed by *Pichia pastoris* contains N-linked glycosyl groups that can be cleaved by Endo H. It should be noted that the molecular weight of the deglycosylated protein was still slightly higher than that of the *E. coli*-derived protein, and this difference is speculated to be related to the presence of O-glycosylation or other post-translational modifications in the protein.

[0134] Based on this, the enzyme activity of proteins obtained from different expression systems and proteins after deglycosylation treatment was measured. The reaction system was 1 mL of 50 mM pH 8.0 phosphate buffer (PB buffer), with PBAT powder added to achieve a concentration of 5 mg / mL, resulting in a total enzyme activity of 0.05 U. The reaction was carried out at 800 rpm for 12 h in a constant temperature mixer. The results are as follows: Figure 6As shown in b, compared with the non-glycosylated protein expressed in *E. coli*, the catalytic activity of the glycosylated protein in the *Pichia pastoris* expression system was reduced under conventional reaction conditions, indicating that glycosylation modification may affect substrate binding or catalytic conformation to some extent. However, at 60 °C, the glycosylated protein still maintained a high relative enzyme activity, showing better thermostability. The enzyme activity level of the protein after Endo H deglycosylation was basically consistent with that of the non-glycosylated protein obtained from the *E. coli* expression system, indicating that N-glycosylation modification is one of the important factors leading to the difference in protein catalytic performance under different expression systems. Therefore, by modifying potential N-glycosylation sites to structurally similar amino acid residues or amino acid residues that are not easily glycosylated (such as glutamine or alanine), the BtaPBATase expressed in *Pichia pastoris* can improve the thermostability of the target protein without significantly reducing enzyme catalytic activity.

[0135] The sequences involved in this invention:

[0136] SEQ ID NO:1

[0137] MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPI YGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS

[0138] SEQ ID NO:2

[0139] TNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGW SMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS

[0140] SEQ ID NO:3

[0141] TNPYARGPNPTAASLEASAGPFAVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDYPESRSSQQMAALRQVASLNGTSSSPIYGKVDTARMGVMGW SMGGGGSLISAANNPSLKAAAPQAPWHSSTNFSSVTVPTLIFACENDSIAPVNSYALPIYDSMSRNAKQFLEIKGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTAVSDFRTANCS

[0142] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0143] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A polyester hydrolase mutant comprising a sequence having at least 90% sequence identity with SEQ ID NO:1 and having the following mutations compared to the sequence shown in SEQ ID NO:1: T51A, Q119Y, S214Y and N233K.

2. The polyester hydrolase mutant according to claim 1, comprising a sequence having at least 95%, at least 97%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:

3.

3. An isolated polynucleotide, wherein, The polynucleotide encodes the polyester hydrolase mutant as described in claim 1 or 2.

4. An expression carrier, wherein, The expression vector comprises the polynucleotide as described in claim 3.

5. A host cell, wherein, The host cell comprises the polyester hydrolase mutant as described in claim 1 or 2, the isolated polynucleotide as described in claim 3, or the recombinant expression vector as described in claim 4; Optionally, the selected recombinant host cell is a microbial cell; Optionally, the recombinant host cell is derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, Pichia, or Corynebacterium. Preferably, the recombinant host cell is derived from Escherichia coli or Pichia pastoris.

6. A cell culture comprising the recombinant host cell as described in claim 5.

7. A product for degrading polyester materials, comprising the polyester hydrolase mutant as described in claim 1 or 2, the polynucleotide as described in claim 4, the recombinant expression vector as described in claim 5, the recombinant host cell as described in claim 6, and / or the cell culture as described in claim 7.

8. The product according to claim 7, wherein the polyester material comprises polyester and / or copolyester; Optionally, the polyester material includes at least one of polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), and polylactic acid (PLLA).

9. A method for degrading polyester material, comprising contacting the polyester hydrolase mutant as described in claim 1 or 2, the recombinant host cell as described in claim 6, or the cell culture as described in claim 7 with the polyester material under conditions for degrading polyester material; Optionally, the polyester material includes polyester and / or copolyester; Optionally, the polyester material includes at least one of polyethylene terephthalate (PET), polybutylene adipate / terephthalate (PBAT), and polylactic acid (PLLA).

10. The method according to claim 9, wherein, The conditions include incubation at a temperature of 20~60 ℃, preferably at a temperature of 30~60 ℃.

Images