Mutant of a thermophilic pet hydrolase and use thereof

Abstract

The application discloses a mutant of thermophilic PET hydrolytic enzyme and application thereof, and belongs to the technical field of protein engineering and biological catalysis application.The mutant of thermophilic PET hydrolytic enzyme disclosed by the application is H185N / F189I or H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P.The mutant of thermophilic PET hydrolytic enzyme disclosed by the application enhances the PET depolymerization performance of PET hydrolytic enzyme and the durability of long-term use at high temperature.Through coupling a one-step PET pretreatment process, efficient biological depolymerization of real PET waste is realized at an L-grade bioreactor scale.

Description

Technical Field

[0001] This invention relates to the fields of protein engineering and biocatalysis, and more specifically to a mutant of a thermophilic PET hydrolase and its applications. Background Technology

[0002] Polyethylene terephthalate (PET), as the most widely used synthetic polyester, is extensively used in packaging and textiles and is one of the most prevalent plastics in global solid waste. Currently, global PET production exceeds 80 million tons annually, and is projected to exceed 25 billion tons by 2050. The large amount of PET waste dumped into the environment poses a serious threat to global ecosystems and even human health. In recent years, the biodepolymerization of PET has received increasing attention due to its environmental and energy-saving advantages, and is considered a very promising PET recycling strategy. However, the insufficient activity and thermal stability of key biocatalysts, such as PET hydrolases, often limit their efficient depolymerization of PET.

[0003] To address this challenge, researchers employed various protein engineering strategies in the enzyme engineering of PET hydrolases, including rational / semi-rational design, directed evolution, computational de novo design / redesign, ancestor sequence reconstruction, and AI-assisted redesign methods, resulting in several PET hydrolases with significantly enhanced activity and stability. Notably, thermal stability is a crucial performance indicator for PET hydrolases. Since PET depolymerization typically occurs near the glass transition temperature (Tg) of PET (approximately 70 °C), high enzyme activity at high temperatures is essential. Furthermore, PET hydrolases need to maintain high activity over long reaction periods to meet the industrial demand for multi-batch recycling of the enzyme. In previous studies, a mutant ThcCut1-AICCG with improved activity and thermal stability was obtained through rational design based on wild-type keratinase ThcCut1 (Depolymerization of post-consumer PET bottles with engineered cutinase 1 from Thermobifida cellulosilytica, Green Chem., 2022, 24, 5998-6007), but its depolymerization performance on real PET waste was still insufficient.

[0004] In conclusion, in order to obtain highly efficient PET hydrolases suitable for industrial-grade depolymerization applications of real PET waste, realize the economic feasibility of enzyme-catalyzed PET depolymerization and recycling processes, and provide a more effective solution to the increasingly serious plastic pollution problem, it is particularly important to develop PET hydrolases with superior PET depolymerization performance.

[0005] Therefore, providing a mutant of thermophilic PET hydrolase and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a mutant of thermophilic PET hydrolase and its application, specifically providing a PET hydrolase mutant and its application in improving the activity and thermal stability of PET hydrolase and in the efficient depolymerization of real PET waste.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A thermophilic PET hydrolase mutant, namely H185N / F189I (NI) or H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P (NI-M7).

[0009] The amino acid sequence of the PET hydrolase is shown in SEQ ID NO.1;

[0010] The amino acid sequence of H185N / F189I is shown in SEQ ID NO.3;

[0011] The amino acid sequence of H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P is shown in SEQ ID NO.5.

[0012] Furthermore, the relevant biomaterial of the thermophilic PET hydrolase mutant is at least one of the following (1)-(4):

[0013] (1) Nucleic acid molecules encoding mutants of thermophilic PET hydrolase;

[0014] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0015] (3) A recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2);

[0016] (4) Recombinant microorganisms containing the nucleic acid molecules described in (1), recombinant microorganisms containing the expression cassette described in (2), or recombinant microorganisms containing the recombinant vector described in (3).

[0017] Furthermore, the application of the mutant of the thermophilic PET hydrolase or related biomaterials of the mutant of the thermophilic PET hydrolase in improving the activity and thermal stability of PET hydrolase.

[0018] Furthermore, the application of the mutant of the thermophilic PET hydrolase or related biomaterials of the mutant of the thermophilic PET hydrolase in the efficient depolymerization of real PET waste.

[0019] Furthermore, the mutant of the thermophilic PET hydrolase or related biomaterials of the mutant of the thermophilic PET hydrolase can achieve efficient depolymerization of real PET waste on a bioreactor scale by coupling a one-step PET pretreatment process.

[0020] Furthermore, the one-step PET pretreatment process involves grinding and pulverizing the PET bottle flakes at high speed using a high-speed rotary grinder.

[0021] As can be seen from the above technical solution, compared with the prior art, this invention discloses a mutant of thermophilic PET hydrolase and its application. Based on the previously developed PET hydrolase, through synergistic optimization of substrate binding key sites and computational-aided design of thermal stability sites, multiple key sites and mutants that enhance the activity and thermal stability of PET hydrolase have been discovered and obtained, thereby enhancing the PET depolymerization performance of PET hydrolase and its durability for long-term use at high temperatures. By coupling a one-step PET pretreatment process, efficient biodepolymerization of real PET waste is achieved on an L-scale bioreactor basis. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 SDS-PAGE protein gel electrophoresis images of PET hydrolase C5 and its mutants NI and NI-M7.

[0024] Figure 2 The HPLC chromatograms are of TPA and MHET, the enzymatic depolymerization products of PET.

[0025] Figure 3 Temperature-dependent curves of the activity of PET hydrolase and mutant depolymerization of Gf-PET membrane.

[0026] Figure 4 The reaction time process for PET hydrolase and mutant depolymerization of real PET waste pc-PET powder.

[0027] Figure 5Thermal inactivation curves of PET hydrolase and mutants under long-term high-temperature reaction conditions at 70 °C to simulate actual industrial applications.

[0028] Figure 6 Using Gf-PET film as substrate inv Kinetic analysis of MM enzyme reaction.

[0029] Figure 7 For PC-PET powder as substrate inv Kinetic analysis of MM enzyme reaction.

[0030] Figure 8 The reaction process of depolymerizing real PET waste pc-PET powder using the PET hydrolase mutant NI on a bioreactor scale.

[0031] Figure 9 The reaction process of depolymerizing real PET waste pc-PET powder by the PET hydrolase mutant NI-M7 on a bioreactor scale. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: Construction and site-directed mutagenesis of PET hydrolase

[0034] Previous laboratory work on the gene source of the PET hydrolase ThcCut1-AICCG (C5) (Patent Application No.: 202210771873.7).

[0035] The amino acid sequence of PET hydrolase C5 is shown in SEQ ID NO.1.

[0036] MANPYERGPNPTDALLEAS S GPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTAT E ASIAWLGERIASHGFVVITI D TIT T LDGPDSRAEQLNAALN H MINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWH LNKNWSSVTVPTLIIGADLDTIAP V AT H AKP F YNSLPSSISKAYLELCGATHIAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVCEYRSTCPF; SEQ ID NO.1。

[0037] The nucleotide sequence of PET hydrolase C5 is shown in SEQ ID NO.2.

[0038] atggcgaatccgtatgaacgcggcccgaatccgaccgatgcgctgttagaagcgagc agc ggtccatttagcgtgagcgaagaaaatgtgagccgcctgagcgcgagcggctttggtggtggtaccatttattatccgcgcgaaaataatacctacggcgcggtggcgattagcccgggttataccgccacc gaa gcgagcattgcgtggctgggtgaacgcattgcgagccatggttttgtggtgattaccatt gat accattacc acc ctggatggaccggatagccgcgcggaacaattaaatgcggcgttaaat cac atgattaaccgcgcgagcagcaccgtgcgcagtagaattgatagcagccgtctggcggttatgggccatagcatgggcggtggtggtaccttacgtttggcgagccaacgcccagatttaaaagcggcgattccgttgaccccatggcat ctg aataaaaattggagcagcgtgaccgtgccgaccctgattattggcgcggatctggataccattgcgccg gtg gcgact cat gcgaaacca ttttataatagcctgccgagcagcattagcaaagcgtatctggaactgtgcggcgcgacccatatagcgccgaatattccgaataaaattatcggcaaatacagcgtggcgtgg SEQ ID NO.2.

[0039] The gene was cloned into the NdeI and XhoI restriction sites of the pET-22b(+) vector, which carries a C-terminal hexahistine tag, and then transformed into E. coli Trans10 or BL21(DE3) competent cells (TransGenBiotech, China) for gene cloning and heterologous expression, respectively.

[0040] Site-directed mutagenesis experiments were performed according to the method described in the Fast Mutagenesis System Site-Directed Mutagenesis Kit (TransGen Biotech, Beijing, China). Variants were constructed by PCR amplification of full-length plasmids using the 2×TransStart FastPfu FlyPCR SuperMix polymerase premix (TransGen Biotech, Beijing, China) as recommended in the method. The sequences encoding the mutant amino acids and the homologous sequences for DNA assembly were introduced using PCR primers (BGI, Beijing, China).

[0041] The PCR reaction system was as follows: the total reaction volume was 50 μL, with 25 μL of 2×TransStart FastPfu FlyPCR SuperMix, 20 μL of ddH2O, 2 μL each of forward and reverse primers, and 1 μL of template added. The reaction conditions were: 98 ℃ pre-denaturation for 1 min; 98 ℃ denaturation for 10 s, 60 ℃ annealing for 5 s, and 72 ℃ extension for 65 s, for a total of 35 cycles; followed by a final extension at 72 ℃ for 1 min. Finally, the mixture was stored at 4 ℃.

[0042] The PCR products were treated with DpnI (New England Biolabs, USA) to digest the original DNA template. 1 µL of DpnI and 5 µL of Cut Smart Buffer were added to the amplification product, and the mixture was incubated at 37°C for 2 h. The DpnI-treated amplified fragments were then purified using the FastPureGel DNA Extraction Mini Kit (Vazyme, Nanjing, China).

[0043] The purified product was subjected to homologous recombination using 2×Basic Assembly Mix (TransGen Biotech, Beijing, China). The ligation system consisted of 5 μL of the target mutant plasmid fragment and 5 μL of 2×Gibson Assemble Mix, and ligation was performed at 50 °C for 15 min.

[0044] Finally, the ligation product was introduced into *E. coli* Trans10 competent cells (TransGen Biotech, Beijing, China). Transformation was performed using the *E. coli* chemical transformation method, with the following steps:

[0045] (1) Take Trans 10 competent cells and place them on ice to thaw. Add all 10 μL of the above ligation product and incubate on ice for 30 min.

[0046] (2) Heat shock at 42 ℃ for 30 s, and immediately ice bath for 2 min.

[0047] (3) Add 500 μL of antibiotic-free LB medium that has been pre-ice bathed, and then revive in a shaker at 37 ℃ and 180 rpm for 1 h.

[0048] (4) After the recovery is complete, take 100 μL and spread it on an LB plate containing Amp resistance.

[0049] (5) Place the plate in a 37 ℃ incubator and incubate overnight for about 12 to 16 hours.

[0050] After initial screening using ampicillin, the introduced mutations were validated by Sanger sequencing (BGI, Beijing, China). All genes that were validated by sequencing were used to extract plasmid DNA using the EasyPure Plasmid MiniPrep Kit (TransGen Biotech, Beijing, China) and introduced into E. coli BL21(DE3) competent cells (TransGen Biotech, China) for subsequent expression.

[0051] Two mutants based on PET hydrolase C5 were ultimately obtained. The PET hydrolase mutant NI (H185N / F189I) was created by mutating His at position 185 to Asn and Phe at position 189 to Ile, based on the template enzyme PET hydrolase C5. Its amino acid sequence is shown in SEQ ID NO.3.

[0052] MANPYERGPNPTDALLEASSGPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTATEASIAWLGERIASHGFVVITIDTITTLDGPDSRAEQLNAALNHMINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWHLNKNWSSVTVPTLIIGADLDTIAPVAT N AKP I YNSLPSSISKAYLELCGATHIAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVCEYRSTCPF; SEQ ID NO.3.

[0053] Its nucleotide sequence is shown in SEQ ID NO.4.

[0054] atggcgaatccgtatgaacgcggcccgaatccgaccgatgcgctgttagaagcgagcagcggtccatttagcgtgagcgaagaaaatgtgagccgcctgagcgcgagcggctttggtggtggtaccatttattatccgcgcgaaaataatacctacggcgcggtggcgattagcccgggttataccgccaccgaagcgagcattgcgtggctgggtgaacgcattgcgagccatggttttgtggtgattaccattgataccattaccaccctggatggaccggatagccgcgcggaacaattaaatgcggcgttaaatcacatgattaaccgcgcgagcagcaccgtgcgcagtagaattgatagcagccgtctggcggttatgggccatagcatgggcggtggtggtaccttacgtttggcgagccaacgcccagatttaaaagcggcgattccgttgaccccatggcatctgaataaaaattggagcagcgtgaccgtgccgaccctgattattggcgcggatctggataccattgcgccggtggcgact aac gcgaaacca att tataatagcctgccgagcagcattagcaaagcgtatctggaactgtgcggcgcgacccatatagcgccgaatattccgaataaaattatcggcaaatacagcgtggcgtggctgaaacgctttgtggataatgatacccgctatacccagtttctgtgcccgggcccacgtgatggcttatttggtgaagtttgcgaatatcgcagcacctgcccgttt;SEQ ID NO.4。

[0055] The PET hydrolase mutant NI-M7 (H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P) is based on the template enzyme PET hydrolase C5, with the following mutations: His at position 185 is mutated to Asn, Phe at position 189 is mutated to Ile, Ser at position 20 is mutated to Arg, Glu at position 65 is mutated to Gln, Asp at position 86 is mutated to Asn, Thr at position 90 is mutated to Pro, His at position 107 is mutated to Tyr, Leu at position 158 is mutated to Tyr, and Val at position 182 is mutated to Pro. Its amino acid sequence is shown in SEQ ID NO. 5.

[0056] MANPYERGPNPTDALLEAS R GPFSVSEENVSRLSASGFGGGTIYYPRENNTYGAVAISPGYTAT Q ASIAWLGERIASHGFVVITI N TIT P LDGPDSRAEQLNAALN Y MINRASSTVRSRIDSSRLAVMGHSMGGGGTLRLASQRPDLKAAIPLTPWH Y NKNWSSVTVPTLIIGADLDTIAP P AT N AKP I YNSLPSSISKAYLELCGATHIAPNIPNKIIGKYSVAWLKRFVDNDTRYTQFLCPGPRDGLFGEVCEYRSTCPF; SEQ ID NO.5.

[0057] Its nucleotide sequence is shown in SEQ ID NO.6.

[0058] atggcgaatccgtatgaacgcggcccgaatccgaccgatgcgctgttagaagcgagc cgc ggtccatttagcgtgagcgaagaaaatgtgagccgcctgagcgcgagcggctttggtggtggtaccatttattatccgcgcgaaaataatacctacggcgcggtggcgattagcccgggttataccgccacc cag gcgagcattgcgtggctgggtgaacgcattgcgagccatggttttgtggtgattaccatt aataccattacc ccg ctggatggaccggatagccgcgcggaacaattaaatgcggcgttaaat tat atgattaaccgcgcgagcagcaccgtgcgcagtagaattgatagcagccgtctggcggttatgggccatagcatgggcggtggtggtaccttacgtttggcgagccaacgcccagatttaaaagcggcgattccgttgaccccatggcat tat aataaaaattggagcagcgtgaccgtgccgaccctgattattggcgcggatctggataccattgcgccg ccg gcgact aac gcgaaacca att tataatagcctgccgagcagcattagcaaagcgtatctggaactgtgcggcgcgacccatatagcgccgaatattccgaataaaattatcggcaaatacagcgtggcgtgg SEQ ID NO.6.

[0059] The point mutations used to construct all the above sites are shown in Table 1. Among them, primers SEQ ID NO.7-SEQ ID NO.10 were used to generate the PET hydrolase mutant NI based on PET hydrolase C5, and the remaining primers were used to generate the mutant NI-M7 based on the mutant NI.

[0060] Table 1 Summary of Mutant Primers

[0061]

[0062] Example 2 Protein Expression and Purification

[0063] Single colonies of *E. coli* strain BL21(DE3) with the introduced enzyme and its mutant gene were inoculated into 4 ml of LB medium containing 0.1 mg / mL ampicillin and cultured overnight at 37°C and 180 rpm. The overnight culture was then inoculated into 250 ml shake flasks containing 50 mL of LB medium and cultured at 37°C and 180 rpm until the OD600 reached 0.8. Protein expression was induced by adding 0.2 mM isopropyl-β-D-thiogalactoside (IPTG), and the temperature was lowered to 16°C and cultured for 20 hours.

[0064] The induced cell cultures were centrifuged at 3000×g and 4°C for 20 min to separate cells and supernatant. The collected cells were resuspended in lysis buffer (100 mM K₂HPO₄-KH₂PO₄, pH 8.0, 300 mM NaCl) and lysed in an ice-water bath using an ultrasonic cell disruptor (SCIENTZ, JY92-IIN, China). The resulting cell lysates were centrifuged at 3000×g and 4°C for 20 min to obtain a supernatant containing soluble proteins.

[0065] The supernatant was purified by affinity chromatography using a Ni-NTA agarose gel. Unbound proteins were washed away with washing buffer (100 mM K2HPO4-KH2PO4, pH 8.0, 300 mM NaCl, 50 mM imidazole), and the target protein was eluted with elution buffer (100 mM K2HPO4-KH2PO4, pH 8.0, 300 mM NaCl, 250 mM imidazole). The buffer was replaced with storage buffer (100 mM K2HPO4-KH2PO4, pH 8.0, 300 mM NaCl) using an Amicon Ultra-15 centrifuge and filter system (10 kDa, Millipore, USA). The purified protein was verified by SDS-PAGE gel electrophoresis (e.g., ...). Figure 1 (As shown). Protein concentration was determined at 280 nm using a NanoPhotometer-N50 (IMPLEN, Germany). The purified enzyme was stored at 4°C.

[0066] Example 3 Preparation of PET substrate

[0067] (1) Amorphous PET film (Gf-PET film): purchased from Goodfellow (product number: ES30-FM-000145, thickness: 0.25 mm, UK), and made into uniform discs (⌀5 mm, about 6 mg) using a punch for later use.

[0068] (2) Post-consumer authentic PET waste powder (pc-PET powder): Post-consumer authentic PET comes from used commercial drinking water bottles (Yibao, China). Only the bottle body is used, the neck and bottom are removed, and the bottles are washed with water and ethanol and dried at room temperature. The bottle body is then cut into PET sheets of approximately 1 cm × 1 cm. The PET sheets are pulverized into particles with a particle size of less than 0.5 mm using a high-speed rotary grinder (AM500S, Ant Instruments, China) equipped with an annular sieve ring (0.5 mm aperture).

[0069] The preparation method of the aforementioned pc-PET powder is the one-step pretreatment process described in this paper, which involves directly pulverizing real post-consumer PET waste into PET powder through high-speed grinding, without the need for other complex pretreatment steps. The pc-PET powder was used as a substrate for activity verification of PET hydrolase mutants and for depolymerization performance verification in an L-scale reactor.

[0070] Example 4: Activity-temperature correlation study of PET hydrolase mutant

[0071] The activity of the PET hydrolase mutant was verified by reaction using Gf-PET membrane and pc-PET powder as substrates, respectively. The reaction system is as follows:

[0072] (1) Add one Gf-PET membrane (6 mg) and the final concentration of 1 μM pure enzyme to 1 ml of 100 mM phosphate buffer (pH 8.0), shake the reaction at 70 °C or other appropriate temperature, take samples at 12 h or 24 h intervals, and terminate the reaction by adding termination buffer (160 mM phosphate buffer, pH 2.5, containing 20% ​​v / v DMSO).

[0073] (2) Alternatively, add 6 mg of pc-PET particles and 1 μM of pure enzyme to 1 ml of 100 mM phosphate buffer (pH 8.0), shake the reaction at 70 °C, take samples at 12 h or 24 h intervals, and terminate the reaction by adding a stop buffer.

[0074] Analysis of PET degradation products was performed using a Thermo Fisher high-performance liquid chromatograph (UHPLC) (Thermo Fisher, UltiMate 3000, USA), which included an autosampler and a UV detector set to 260 nm. A Kinetex XB-C18 HPLC column (100 Å, 5 μm, 50 × 2.1 mm) was used with gradient elution. Mobile phase A was ultrapure water containing 0.1% formic acid (HPLC grade), and mobile phase B was acetonitrile (HPLC grade), with a fixed flow rate of 1.1 mL / min. −1The injection volume was 4 μl. After injection, the mobile phase was set to 13% buffer B and held for 52 s to separate TPA and MHET. Buffer B was then increased to 95% and held for 33 s to separate larger reaction products and contaminants. The column was then rebalanced to 13% buffer B until the total run time was 3 min. In this study, the main PET degradation products were TPA and MHET. Product peaks were confirmed by comparison with commercially available TPA (TCI, Shanghai, China) and MHET (Peter Pharmaceuticals, Shanghai, China) chemical standards. The concentrations of TPA and MHET were calculated using a standard curve. All reaction solutions were filtered through a 0.22 μm aqueous filter membrane to prepare samples. The peak characteristics of the two products are shown below. Figure 2 As shown, the retention time of TPA is 0.340 min and the retention time of MHET is 0.543 min.

[0075] Using Gf-PET film as a substrate, the total amount of depolymerization products released at different reaction temperatures was determined. The results are as follows: Figure 3 As shown, the PET hydrolase mutants NI and NI-M7 exhibit varying degrees of increased activity compared to the original PET hydrolase C5 at different temperatures. NI reaches its highest activity level at 72℃, after which its activity decreases with increasing reaction temperature, reaching near-complete inactivation at 78℃. NI-M7 shows slightly lower activity than NI in the 65-72℃ range, but its optimal temperature is increased to 75℃, and it retains relatively high activity at 78℃ and 80℃.

[0076] Example 5: Reaction time analysis of PET hydrolase mutant depolymerizing real PET waste (pc-PET powder)

[0077] The depolymerization process of PET hydrolase on PC-PET powder was studied in a ml-scale reaction system. 33 mg of substrate (1.1 wt%) was added to 3 ml of 100 mM phosphate buffer (pH 8.0), and the enzyme dosage was 99 μg (3 mg enzyme). -1 PET was subjected to a shaking reaction at 70°C, and samples were taken at specified time points to determine the amount of product generated and released. The percentage of PET depolymerization was assessed by analyzing the total amount of product released in the supernatant.

[0078] The results are as follows Figure 4 As shown, the depolymerization rates and final depolymerization rates of the PET hydrolase mutants NI and NI-M7 were superior to those of the original PET hydrolase C5. NI exhibited the fastest initial depolymerization rate, reaching over 90% depolymerization rate after 24 h of reaction. While NI-M7 had lower initial activity, it maintained a relatively constant depolymerization rate, reaching 90% depolymerization rate after 12 h and a maximum of 95% depolymerization rate at 24 h.

[0079] Example 6: Study on the heat resistance and inactivation properties of PET hydrolase

[0080] The heat inactivation experiment first involves incubating PET hydrolase and mutants at a certain temperature for a period of time, and then measuring their residual activity at 70°C to characterize their heat inactivation resistance and durability under industrial application conditions.

[0081] The specific experimental conditions were as follows: A final concentration of 1 μM pure enzyme was placed in 1 ml of 100 mM phosphate buffer (pH 8.0) and incubated at 70°C with shaking for 250 h. Samples were taken at regular intervals during this period, and a Gf-PET membrane was added. The reaction was carried out at 70°C with shaking for 12 h to determine residual activity. Activity was characterized by the total release of TPA and MHET products. The initial activity without heat inactivation was set as 100%, which was used as a reference standard to compare the residual activity after heat inactivation.

[0082] The results are as follows Figure 5 As shown, the heat resistance of the PET hydrolase mutant NI decreased. The PET hydrolase mutant NI-M7 exhibited excellent heat resistance, significantly improved compared to PET hydrolase C5. Its activity did not decrease after 150 h of heat incubation, and it retained over 90% of its activity after 250 h.

[0083] Example 7 Reaction Kinetic Analysis

[0084] based on inv The MM kinetic equation was used to perform a kinetic analysis of the PET depolymerization reaction. Unlike the traditional Michaelis equation kinetics, the reaction proceeded under substrate saturation conditions.

[0085] The reaction using Gf-PET membrane as a substrate was carried out in 0.5 ml of 100 mM phosphate buffer (pH 8.0), with one Gf-PET membrane (6 mg, 12 g L) added. -1 Add 0-1.2 μM enzyme and the reaction mixture, and incubate with shaking at 70°C for 1 h. The reaction is then terminated by adding 0.5 mL of stop buffer.

[0086] The reaction using pc-PET powder as a substrate was carried out in 1 ml of 100 mM phosphate buffer (pH 8.0), and the substrate loading was maintained at 12 g / L by adding 12 mg of pc-PET particles. -1 The reaction mixture was incubated with 0-2.5 μM enzyme at 70°C with shaking for 1 h, and the reaction was terminated by adding 1 mL of stop buffer. Data were fitted to Origin.

[0087] in invKm represents the enzyme concentration at which the enzyme-catalyzed reaction rate reaches half of the maximum reaction rate. inv V max This represents the maximum rate that an enzyme-catalyzed reaction can achieve under substrate saturation conditions, kcat( inv Vmax / S0 * () indicates the catalytic rate when all attackable sites on the PET surface are covered by the enzyme.

[0088] The calculation results of each dynamic parameter are shown in Table 2.

[0089] Table 2 Calculation results of enzyme kinetic parameters according to the anti-Michaelis equation

[0090]

[0091] Using Gf-PET film as substrate inv MM dynamics curves as follows Figure 6 As shown, NI and NI-M7 inv The Km values ​​were 0.346 and 0.199 μM, respectively, while C5 had a Km value of 0.352 μM. This indicates that NI-M7 exhibits the strongest substrate affinity, significantly enhanced compared to both C5 and NI. For the catalytic rate, the kcat values ​​for NI and NI-M7 were 48.29 and 40.78 nmol g, respectively. -1 s -1 C5, however, has a concentration of only 19.28 nmol g. -1 s -1 Both variants exhibited significantly higher catalytic rates than C5, increasing by 150% and 112%, respectively. The enhanced PET depolymerization activity of the mutant NI compared to C5 was primarily due to the increased catalytic rate. The enhanced depolymerization activity after introducing a thermally stable mutant site was mainly attributed to improved substrate affinity. Variant NI-M7 demonstrated the highest catalytic efficiency (kcat / inv Km), which is 273% and 47% higher than C5 and NI, respectively.

[0092] PC-PET granules as substrate inv MM dynamics curves as follows Figure 7 As shown, the kinetics of depolymerization of PC-PET particles are similar to those of Gf-PET membranes, but the catalytic rate is significantly enhanced due to the increased substrate specific surface area. The kcat values ​​of NI and NI-M7 are 463.57 and 301.19 nmol g, respectively. -1 s -1 C5, however, has a concentration of only 200.66 nmol g. -1 s -1The catalytic rates of both C5 and C6 were significantly higher than those of C5, increasing by 131% and 50%, respectively. Furthermore, due to the increased substrate specific surface area, the number of attackable sites on the surface increased, leading to a significant increase in the amount of enzyme that could bind, resulting in… inv Km increases. (NI and NI-M7) inv The Km values ​​were 0.995 and 0.614 μM, respectively, while C5 was 0.990 μM. NI-M7 exhibited the strongest substrate affinity, significantly enhanced compared to both C5 and NI. The increased activity of the mutant NI compared to C5 when depolymerizing pc-PET particles was also due to the increased catalytic rate. The enhanced depolymerization activity after introducing a thermally stable mutant site was attributed to improved substrate affinity. The variant NI-M7 also showed the highest catalytic efficiency (kcat / ) for pc-PET. inv Km) is 142% higher than C5 and slightly higher than NI, thus exhibiting the best depolymerization performance.

[0093] Example 8 Performance of PET hydrolase mutant under L-scale bioreactor conditions

[0094] To evaluate the application potential of mutants NI and NI-M7 in large-scale reactions, biodepolymerization of post-consumer PET bottles was validated in an L-stage bioreactor.

[0095] The depolymerization of pc-PET powder was carried out in a 3 L bioreactor (BXBIO, Shanghai, China) equipped with an automatic temperature and pH control system. 200 ml of purified protein solution (protein concentration 1.5 mg / ml, dissolved in the aforementioned storage buffer) was mixed with 100 g of pc-PET in 700 ml of phosphate buffer (pH 8.0, 100 mM), and the PET depolymerization reaction was carried out under conditions of 10% PET (wt%) and 0.3% enzyme substrate loading (wt%).

[0096] The reaction temperature was maintained at 70°C through the coordinated regulation of the base and heating mantle, and a constant stirring speed of 200 rpm was maintained using a straight-bladed disc turbine. A 20% NaOH (w / w) (5 M) solution was added to the reactor through a pH monitoring system and an automatic feeding system to stabilize the pH value at pH 8.0, and the consumption of NaOH was recorded at regular intervals.

[0097] The depolymerization kinetics of PET were monitored in real time based on the consumption of NaOH. If the product was completely converted to monomers TPA and EG, and TPA contains two carboxyl groups, 1 mol of NaOH was used to titrate 0.5 mol of TPA. The depolymerization conversion rate of PET relative to TPA could be calculated based on the amount of NaOH consumed. Furthermore, samples were collected at different time points and analyzed by HPLC. The depolymerization conversion rate of PET was further adjusted by considering the TPA / MHET ratio (1 mol of NaOH was consumed to titrate 1 mol of the monobasic acid MHET). The final depolymerization conversion rate of PET was determined by the amount of NaOH consumed and the dry weight of the residual PET. To determine the mass of the residual PET, all reaction mixtures, including solid particles, were filtered through qualitative filter paper. The residual solids were washed twice with 5M NaOH solution and deionized water, and then dried overnight at 65°C.

[0098] The reaction processes of PET hydrolase mutants NI and NI-M7 depolymerizing real PET waste under reactor conditions are as follows: Figure 8 and Figure 9 As shown in the figure. The results show that under bioreactor conditions, NI achieves a depolymerization conversion rate of 52.8% in just 2.5 hours, 85.4% in 12 hours, and a final depolymerization conversion rate of 92.3% after 24 hours. In contrast, NI-M7 has a slightly lower reaction rate in the initial stage of depolymerization, requiring 4 hours to reach a depolymerization conversion rate of over 50%, and a depolymerization rate of 75.5% after 12 hours. Although the rate decreases somewhat in the subsequent 12 hours of reaction, NI-M7 still maintains a high reaction rate, and ultimately, the PET depolymerization conversion rate of NI-M7 also reaches 90% after 24 hours of reaction.

[0099] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A mutant of a thermophilic PET hydrolase, characterized in that, For H185N / F189I or H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P; The amino acid sequence of the thermophilic PET hydrolase is shown in SEQ ID NO.1; The amino acid sequence of H185N / F189I is shown in SEQ ID NO.3; The amino acid sequence of H185N / F189I / S20R / E65Q / D86N / T90P / H107Y / L158Y / V182P is shown in SEQ ID NO.

5.

2. The biomaterial related to the mutant of thermophilic PET hydrolase as described in claim 1, characterized in that, It is at least one of the following (1)-(4): (1) Nucleic acid molecules encoding mutants of thermophilic PET hydrolase; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1) or a recombinant vector containing the expression cassette described in (2); (4) Recombinant microorganisms containing the nucleic acid molecules described in (1), recombinant microorganisms containing the expression cassette described in (2), or recombinant microorganisms containing the recombinant vector described in (3).

3. The application of the mutant of the thermophilic PET hydrolase according to claim 1 or the related biomaterials of the mutant of the thermophilic PET hydrolase according to claim 2 in improving the activity and thermal stability of PET hydrolase.

4. The application of the mutant of the thermophilic PET hydrolase of claim 1 or the related biomaterial of the mutant of the thermophilic PET hydrolase of claim 2 in the depolymerization of PET waste.

5. The application according to claim 4, characterized in that, Depolymerization of PET waste was achieved on a bioreactor scale by coupling a one-step PET pretreatment process.

6. The application according to claim 5, characterized in that, The one-step PET pretreatment process involves grinding and pulverizing PET bottle flakes at high speed using a high-speed rotary grinder.

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