A pet plastic degradation enzyme gene, a pet degradation enzyme, a recombinant carrier and application

Abstract

The application discloses a PET plastic degrading enzyme gene, a PET degrading enzyme, a recombinant carrier and application, and particularly relates to the field of biotechnology and environmental microbial technology, and the PET plastic degrading enzyme gene sequence is shown as SEQ ID NO.1, or is obtained after site mutation of the nucleotide sequence shown as SEQ ID NO.1. The nucleotide sequence of the PET degrading enzyme gene after signal peptide is shown as SEQ ID NO.2, or is obtained after site mutation of the nucleotide sequence shown as SEQ ID NO.2. The catalytic function region sequence of the PET degrading enzyme gene is shown as SEQ ID NO.3, or is obtained after mutation of the nucleotide sequence shown as SEQ ID NO.3. The application provides certain ideas and technical references for mining the PET plastic degrading enzyme by using metagenomics, further enriches the types of the PET degrading enzyme, provides valuable degrading enzyme resources for further development of industrialized biodegradation of PET, and provides a solution for realizing green recovery of PET plastic, and eliminating the problems of PET plastic and microplastic pollution in the environment.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and environmental microbiology, specifically to a PET plastic degrading enzyme gene, a PET degrading enzyme, a recombinant vector, and its applications. Background Technology

[0002] Polyethylene terephthalate (PET) is a common polyester polymer material derived from petroleum and polymerized from polyethylene terephthalate (BHET) through ester bonds. PET possesses excellent physical and mechanical properties, making it suitable for various applications in daily life and production, including the food industry, medical devices, transportation, and even aerospace. Most PET materials are used to produce disposable products such as beverage bottles, food packaging bags, medicine bottles, and medical masks and protective clothing used during the COVID-19 pandemic, resulting in a large amount of PET waste. Due to the high cost of recycling PET waste, most of it ends up in landfills, incineration, or dumping into the ocean. This plastic waste, under the influence of ultraviolet radiation and mechanical abrasion from wind, rain, and ocean waves, will form microplastics and even nanoplastics, poisoning the environment and harming human health.

[0003] The demand for and manufacture of PET began after 1940 and quickly surpassed most other synthetic materials, becoming one of the fastest-growing industries globally. In fact, PET has steadily replaced most natural materials, such as wood, metal, ceramics, stone, and leather. However, the disposal of PET waste is not as simple as the materials it replaces. As a petroleum-based macromolecular polymer, PET is known to have very strong chemical stability and mechanical strength, requiring decades or even centuries to fully degrade under natural conditions. Currently, landfill and incineration are still the mainstream disposal methods, often leading to significant ecological and environmental damage. Physical processing methods require high-quality substrate sorting and lack effective methods for handling colored plastics. Furthermore, physical recycling of PET presents safety concerns, as polyester easily decomposes to produce acetaldehyde under high-temperature blowing and stretching, resulting in excessive acetaldehyde content in recycled polyester packaging containers. Excessive acetaldehyde can harm human health, therefore these containers cannot be used for packaging food, pharmaceuticals, and other products, and are mostly relegated to the next lower level of recycling. Chemical recycling of PET generates large amounts of acidic and alkaline wastewater. As a result, the PET recycling industry is not thriving, and a large amount of PET plastic pollution has spread all over the world, making plastic pollution one of the biggest man-made and global challenges facing humanity in the 21st century!

[0004] Microorganisms have long played the role of decomposers in the food chain during tens of thousands of years of evolution. Therefore, researchers naturally pinned their hopes on biodegradation to overcome the challenges and bottlenecks of physicochemical PET recycling. Since the 21st century, the solution to the green recycling of PET waste has been envisioned through biodegradation. To date, approximately thirty enzymes from the carboxylate hydrolase (EC 3.1.1) family have been identified as having the ability to degrade PET. However, their activity levels in degrading PET vary, and only a few enzymes with high activity and development value are readily available. Therefore, finding more novel PET-degrading enzymes is a significant unmet need. Summary of the Invention

[0005] The main problem this invention addresses is to provide a PET plastic degrading enzyme that can degrade PET plastic at lower temperatures, thus solving the problem of the difficulty in green recycling of PET plastic.

[0006] The PET degrading enzyme ZF950 has the nucleotide sequence shown in SEQ ID NO.1. The nucleotide sequence encoding the mature protein ZF950-T35 (signal peptide deactivation) is shown in SEQ ID NO.2. The nucleotide sequence encoding the functional region protein that catalyzes PET degradation is shown in SEQ ID NO.3.

[0007] The discovery of this enzyme leveraged metagenomics, a powerful tool for studying the genetic resources of refractory microorganisms. Metagenomic functional screening techniques were used to mine novel PET plastic-degrading enzyme genes from a metagenomic library of Mount Everest soil. After heterologous expression and protein purification of the screened PET plastic-degrading enzyme genes, its enzyme activity in degrading PET plastic under optimal conditions was measured. This invention provides a framework and technical reference for the discovery of PET-degrading enzymes through metagenomics, further enriches the types of PET-degrading enzymes, and provides valuable enzyme resources for further development of biodegradable PET.

[0008] To achieve the above, the present invention adopts the following technical solution:

[0009] Since the discovery of the first microorganism capable of degrading PET and its secreted PET-degrading enzyme TfH in 2005, a surge of interest in finding PET-degrading enzymes has been sparked, leading to the discovery of approximately thirty PET degradation-related enzymes. Statistical analysis reveals that most of these enzymes belong to the same family—the carboxylester hydrolase (EC 3.1.1) family. Lipases (EC 3.1.1.3), keratinases (EC 3.1.1.74), carboxylesterases (EC 3.1.1.1), PETases (EC 3.1.1.101), and MHETases (EC 3.1.1.102) within this family have all been found to be associated with PET degradation. The carboxylester hydrolase family possesses highly conserved catalytic site sequences, indicating that they share very similar catalytic mechanisms. In various studies, tributyrate has been experimentally demonstrated to be a substrate capable of being hydrolyzed by the vast majority of enzymes in this family, making it a potential substrate for metagenomic functional screening to identify new carboxylester hydrolases.

[0010] Therefore, a carboxylester hydrolase screening plate was designed using tributyrate as a substrate. Through metagenomic functional screening, positive clones with carboxylester hydrolase activity were first screened from the high-quality Everest library constructed in our laboratory. The selected positive clones were then expressed, and after cell lysis, they were incubated with powdered PET plastic at 37°C and pH 8.0 for 4 days, with cell lysis buffer added intermittently. The carboxylester hydrolases with PET-degrading activity degraded PET into products such as TPA, MHET, and BHET; their chemical structures are shown in [reference needed]. Figure 1 These products contain benzene rings and can be detected by ultraviolet absorption. Therefore, the final design involved collecting the reaction solution, extracting it with ethyl acetate, rotary evaporating, resuspending it, and finally detecting the absorption peaks of TPA, MHET, and BHET using high-performance liquid chromatography (HPLC) to screen for positive clones with PET degradation activity. Ultimately, after screening approximately 6 million positive clones, 14 positive clones encoding carboxylester hydrolase activity were obtained. These clones underwent protein expression, cell disruption, whole-cell catalysis, and HPLC detection, ultimately revealing one positive clone encoding carboxylester hydrolase with PET degradation activity, designated ZF950.

[0011] To further identify the gene encoding ZF950, our laboratory constructed a subcloning library and, using the same screening method, obtained a subclone of the PET-degrading enzyme ZF950. Sequencing and bioinformatics analysis yielded a full-length ORF of 1362 bp. This sequence has a GC content of 61%, encodes 453 amino acids, and has a predicted isoelectric point of 6.50. The predicted ORF was uploaded to the NCBI database for BALSTX homology comparison. This PET plastic-degrading enzyme showed the highest identity (57.36%) with IsPETase from Ideonella sakaiensis 201-F6 and its mutants. Furthermore, a phylogenetic tree was constructed using this protein sequence along with other reported enzymes with good PET-degrading effects, revealing that this enzyme belongs to the same family as IsPETase. Homology modeling was used to construct the tertiary structure of this protein, and protein superposition analysis was performed with IsPETase. Multiple sequence comparisons revealed that this protein possesses the same catalytic site and conserved regions as the PETase family, as well as an additional disulfide bond spanning the catalytic site compared to other carboxyl ester hydrolases. Furthermore, experiments showed that this PET-degrading enzyme exhibits the same characteristics as IsPETase, the representative enzyme of the PETase family, namely, the ability to degrade PET plastic at lower temperatures and a substrate preference for PET. However, multiple sequence comparisons also revealed that this enzyme does not belong to any of the three current classifications within the PETase family.

[0012] Ultimately, this invention provides a PET plastic degrading enzyme gene sequence, the full length of which is 1362 bp, and its complete sequence is shown in SEQ ID NO.1. The nucleotide sequence of the PET degrading enzyme after removing the signal peptide is shown in SEQ ID NO.2. The catalytic functional region sequence is shown in SEQ ID NO.3.

[0013] The nucleotide sequence shown in SEQ ID NO.2 is obtained by removing the nucleotide sequence encoding the signal peptide from the nucleotide sequence shown in SEQ ID NO.1. The nucleotide sequence shown in SEQ ID NO.3 is obtained by removing the nucleotides encoding the signal peptide region (1-105), the nucleotides encoding region A (106-495), and the loop portion (496-546) connecting the A and B domains from the nucleotide sequence shown in SEQ ID NO.1, leaving the remaining nucleotide sequence.

[0014] This invention also discloses two pairs of specific primers, ZF950-T35-F / ZF950-T35-R and ZF950-BF / ZF950-BR, designed for amplifying the target gene by constructing a recombinant plasmid together with the expression vector pET28a(+). The primer sequences of ZF950-T35-F / ZF950-T35-R are ZF950-T35-F (SEQ ID NO.4): aattCATATGGCGGTTGTGTTCTCAGAAAA and ZF950-T35-R (SEQ ID NO.5): aattAAGCTTATACGGGCAGGTCGAC, used to amplify the nucleotide sequence shown in SEQ ID NO.2. The primer sequences for ZF950-BF / ZF950-BR are ZF950-BR (SEQ ID NO.6): aattCATATGAACCAGTATCAGCGCG and ZF950-BR (SEQ ID NO.7): aattAAGCTTATACGGGCAGGTCGAC, used to amplify the nucleotide sequence shown in SEQ ID NO.3.

[0015] This invention also discloses a method for inducing expression and purifying the above-mentioned PET plastic degrading enzyme gene, comprising:

[0016] (1) The recombinant plasmids pET-28a(+)-zf950-t35 and pET-28a(+)-zf950-b were induced to express at low temperature by adding the inducer IPTG to liquid LB medium;

[0017] (2) After low-temperature induction of expression of recombinant plasmids pET-28a(+)-zf950-t35 and pET-28a(+)-zf950-b in Escherichia coli BL21(DE3), pure PET plastic degrading enzymes ZF950-T35 and ZF950-B were obtained by nickel column affinity chromatography.

[0018] This invention also discloses a method for determining the enzyme activity of the above-mentioned PET plastic degrading enzyme, comprising the following:

[0019] (1) By measuring the optimal pH and temperature of PET degrading enzyme, the appropriate conditions for co-incubation of PET degrading enzyme and PET were determined.

[0020] (2) The yield of PET degrading enzyme was measured when PET was co-incubated with PET under optimal pH and temperature conditions. The curve of product yield accumulated over time was used to determine the time range in which the enzyme activity of PET degrading enzyme was relatively stable.

[0021] (3) The enzyme activity of PET degrading enzyme was calculated by co-incubating PET plastic with PET plastic under the optimal pH and temperature conditions for a period of time when the enzyme activity measured in (2) was relatively stable. Finally, the enzyme activity of PET degrading enzyme was calculated by measuring the amount of product released. The unit of measurement is the number of milligrams of product released per milligram of enzyme per hour.

[0022] The present invention also discloses an example of PET plastic degrading enzyme and PET sheet degradation, including the following: PET degrading enzyme and PET sheet are co-incubated at pH 8.0 and 35°C for 48 hours, and then the degradation of PET sheet is observed under an optical microscope, with a negative control.

[0023] The present invention also discloses a PET plastic degrading enzyme, the amino acid sequence of which is encoded by the nucleotide sequence described in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 above.

[0024] This invention also discloses a recombinant vector containing the nucleotide sequence shown in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3. The vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus. In this invention, a nucleotide sequence encoding a PET plastic degrading enzyme can be inserted into the vector to form a recombinant vector containing the polynucleotides described in this invention. "Vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.

[0025] The present invention has the following beneficial effects:

[0026] This invention screened a positive clone with PET degrading activity from a metagenomic library of Mount Everest soil using functional screening. Subclones were constructed, and bioinformatics analysis was applied to identify potential PET-degrading enzyme genes within the positive clone. Furthermore, the enzyme activity levels of these PET-degrading enzymes were experimentally determined. Compared to existing technologies, this invention provides insights and technical guidance for metagenomic mining of PET-degrading enzymes, enriches the variety of PET-degrading enzymes, provides valuable enzyme resources for the further development of industrial-scale biodegradable PET, and offers a solution for achieving green recycling of PET plastics and eliminating PET and microplastic pollution in the environment. Attached Figure Description

[0027] Figure 1 The present invention provides the structural formulas for PET and its degradation products;

[0028] Figure 2 This invention provides a functional screening clone of carboxylate hydrolase activity.

[0029] Figure 3 This invention provides HPLC detection results for whole-cell catalysis.

[0030] Figure 4 This invention provides a subcloning screening method for PET-degrading enzymes.

[0031] Figure 5 This invention provides the ZF950 phylogenetic tree analysis;

[0032] Figure 6 This is a comparison diagram of the superposition of ZF950 and IsPETase (PDB ID: 5XJH) proteins provided by this invention;

[0033] Figure 7 This is a sequence alignment diagram of ZF950 and IsPETase provided by this invention;

[0034] Figure 8 This invention provides a PETase classification method;

[0035] Figure 9 The present invention provides the agarose gel electrophoresis detection of ZF950-T35 and ZF950-B PCR products;

[0036] Figure 10 The recombinant plasmid successfully constructed by colony PCR selection provided by this invention;

[0037] Figure 11 This invention provides the SDS-PAGE detection of ZF950-T35 / ZF950-B protein;

[0038] Figure 12 This invention provides the effect of different temperatures on the activity and stability of PET-degrading enzymes.

[0039] Figure 13 This invention provides the effect of different pH values ​​on the activity and stability of PET-degrading enzymes.

[0040] Figure 14 These are the standard curves of the MHET and BHET standards provided by this invention;

[0041] Figure 15 These are the PET degradation curves and HPLC chromatograms of PET degradation products provided by this invention;

[0042] Figure 16 This is a diagram showing the degradation results of ZF950-B PET sheets provided by the present invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The various terms and phrases used in this invention have their general meanings known to those skilled in the art. The present invention provides a general or specific description of the materials and experimental methods used in the experiments. Although many materials and operating methods used to achieve the objectives of the present invention are known in the art, the present invention will still be described in as much detail as possible herein. Unless otherwise specified, the instruments, reagents, materials, etc., involved in the following embodiments are all conventional instruments, reagents, materials, etc., already existing in the prior art; and unless otherwise specified, they are all conventional experimental methods, detection methods, etc., already existing in the prior art.

[0044] Example 1: Functional screening of PET-degrading enzymes and acquisition of related coding genes

[0045] The Mount Everest soil metagenomic library: Soil samples were taken from the Mount Everest region of Tibet (28.21°N, 86.56°E, altitude 4276 meters). This region has rugged terrain, unique environmental conditions, and is sensitive to climate change, resulting in a rich diversity and abundance of microorganisms in the soil. The Tibetan soil metagenomic library contains approximately 1.04 × 10⁷ library clones, with an average inserted target fragment of 38,000 bp, encompassing a vast amount of microbial genetic resources.

[0046] Screening Principle: PET is a high-molecular-weight polyester compound polymerized from BHET via ester bonds. Carboxylester hydrolases are functional enzymes that accelerate the hydrolysis of ester bonds. Currently discovered enzymes capable of degrading PET all belong to the carboxylester hydrolases family, and tributyrate is commonly used as a substrate for screening carboxylester hydrolases. By spreading library bacteria on a carboxylester hydrolases screening plate containing tributyrate, a clear zone appears around a single clone of library bacteria exhibiting carboxylester hydrolases activity. Positive clones with carboxylester hydrolases activity are thus selected. A schematic diagram of the plate screening is shown below. Figure 2 (The image shows the esterase hydrolysis zone formed by a positive clone with glycerol tribose degradation activity). After incubation with PET, PET-degrading enzymes generate products such as TPA, MHET, and BHET. TPA, MHET, and BHET, containing benzene rings, can be detected by liquid chromatography. Positive clones with carboxylate hydrolase activity obtained through expression screening were incubated with powdered PET for 48 hours after cell lysis. The reaction solution was extracted with ethyl acetate, and the resulting solution was analyzed using high-performance liquid chromatography (HPLC) to screen for carboxylate hydrolases with PET degradation activity.

[0047] Therefore, a strategy can be implemented to first screen carboxyl ester hydrolases from the metagenomic library of Mount Everest soil, and then screen PET-degrading enzymes from the carboxyl ester hydrolases. The experimental method is as follows:

[0048] 1.1 Functional screening yielded positive clones of degradable PET plastic.

[0049] 1.1.1 Screening for positive clones with tricresyl tributylate degradation activity using plate activity

[0050] (1) Preliminary experiment: Before screening, the library bacteria were diluted into multiple concentration gradients, spread on screening plates, and the dilution of about 2000 single colonies was recorded as the dilution factor for the formal experiment.

[0051] (2) Preparation of screening plates: 1% glyceryl tartrate, 0.1% Tween 80, 1.5% gum arabic, and 2% agar powder were added to ordinary LB medium to prepare a screening medium for carboxylate hydrolases. The prepared medium was sterilized at 120℃ and 103.4kPa for 20 minutes, then cooled to 50-60℃. Chl was added to a final concentration of 12.5μg / mL and Amp was added to 100μg / mL. After shaking well, the medium was immediately used to prepare screening plates (Φ15cm).

[0052] (3) Screening of positive clones: The library bacterial culture diluted to an appropriate multiple is evenly spread on a plate (each plate contains approximately 2000 Cosmids single clones). The plates are incubated at 37°C for 3-5 days. The presence of clear zones formed by esterification around the single clones is visually observed to select those with glyceryl tartrate degradation activity. The selected single clones are then horizontally transferred to a new plate for verification. The presence of clear zones formed by esterification is observed, and Cosmids plasmids are extracted and digested with enzymes to eliminate false positives.

[0053] (4) Preservation of positive clones: For clones that are confirmed to be positive, they are cultured overnight in tubes and then stored in glycerol with a final concentration of 20% and frozen at -80°C.

[0054] 1.2.2 Screening for PET degradation activity

[0055] (1) Protein expression: Seed culture of the above positive clones was obtained by in vitro culture at 37℃, 225 rpm / min for 12-16 h. The seed culture was inoculated at 1% into 100 ml of LB liquid medium containing Chl at a final concentration of 12.5 μg / mL and Amp at a final concentration of 100 μg / mL. The culture was carried out at 37℃ and 225 rpm / min until OD600≈0.6. Then IPTG at a final concentration of 0.5 mM was added, and the protein was expressed at 18℃ and 180 rpm / min.

[0056] (2) Cell collection and resuspension: After protein expression, the bacterial culture was divided into 50ml centrifuge tubes and centrifuged at 8000-10000rpm / min, 4℃ for 15min. The supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in distilled water and centrifuged 2-3 times to wash the LB culture medium as thoroughly as possible. The collected bacterial cells were resuspended in 5ml PBS and 10μL of the bacterial culture was diluted 20-40 times (the dilution factor can be adjusted according to the actual situation). The OD value of the diluted bacterial culture was measured using a spectrophotometer. 600 If the value at the specified location is 0.1-0.9, the concentration of the resuspended cells can be adjusted according to the dilution factor.

[0057] (3) Cell disruption: Using a Φ3mm probe, sonicate for 20 minutes at 50% maximum ultrasonic power, 2 seconds of sonication, 3 seconds of interval, and in an ice bath.

[0058] (4) Whole-cell catalysis: The broken bacterial solution was directly used as the bioreactor for PET degradation, powdered PET plastic was added, and the mixture was incubated at 37°C and 180 rpm / min for 4 days.

[0059] (5) HPLC detection of products: After adding 2M concentrated hydrochloric acid to the reaction solution until the pH value is 2.0, add 1-2 volumes of ethyl acetate, shake, and let stand. Pour the upper ethyl acetate extract into a round-bottom flask and evaporate it on a rotary evaporator until there is no liquid in the flask. Add 1 mL of the prepared heavy solution to the round-bottom flask to dissolve the concentrate. Collect the heavy solution and centrifuge it at 12000 rpm / min for 15 min. Take the supernatant and filter it through a 22 μm filter. Measure the UV absorption of each numbered product by HPLC. The HPLC detection results of whole-cell catalysis are shown in the figure. Figure 3 (The positive clones obtained from the first step of screening that degrade tricresyl ester were all expressed for protein. After cell disruption, they were incubated with powdered PET plastic at 35°C and pH 8.0 for 48 hours. Finally, the degradation products were detected by liquid chromatography and compared with product standards.)

[0060] 1.2 Construction of PET degrading enzyme subclones

[0061] Plasmids from positive clones with PET plastic degradation activity obtained in section 1.1 were extracted and partially digested with Sau3AI. Finally, 2-3 kbp DNA fragments were recovered using a gel. The partial digestion results are shown below. Figure 4 (A) (Figure (A) shows the positive clone with PET degradation activity after Cosmid plasmid extraction, partial digestion with Sau3AI, and detection by agarose gel electrophoresis). The digested DNA fragments were compared with the digested pWEB-TNC using T4 DNA Ligase. TMVector ligation was used to construct subclonal libraries. Using the same screening method as described in 1.1, positive subclones with carboxylester hydrolase activity were selected, such as... Figure 4 As shown in (B) (Figure (B) is a diagram of the screening of subclone libraries). Plasmids for positive subclones were extracted and verified by enzyme digestion. The results were detected by agarose gel electrophoresis. The results are shown in [Figure B]. Figure 4 (C) (Figure (C) is a diagram showing the plasmid extracted from the screened positive subclones and verified by enzyme digestion with BamHI). Figure 4 (C) Lane M is the DL5000 DNA Marker, lane 1 is the circular pWEB-TNC vector, lane 2 is the undigested subclone plasmid, and lane 3 is the product verified by enzyme digestion. Lane 3 shows a DNA band of approximately 5800 bp (vector), a band of approximately 1700 bp, and a band of approximately 1000 bp. The approximately 1700 bp and 1000 bp bands are due to the presence of a BamHI restriction site in the insert. Therefore, the size of the insert is as expected and will be sent to Qingke Biotechnology for sequencing. Detailed procedures are as follows:

[0062] (1) Preparation of dephosphorylated vector: Extract the pWEB-TNC self-ligated vector and digest it with BamHI for 1 hour. Take 2 μL of the digested plasmid solution and 2 μL of the undigested plasmid solution as controls, and check the completeness of digestion with an agarose gel containing EB. If the digestion is complete, heat at 65℃ for 15 minutes to inactivate it. Finally, dephosphorylate the plasmid DNA according to the system in Table 2-1.

[0063] Table 1 Dephosphorylation system

[0064]

[0065] The above mixture was placed in a 1.5 mL centrifuge tube and incubated at 37 °C for 30 min, followed by heating at 65 °C for 30 min to inactivate Alkaline Phosphatase. 2.5 μL of 3 M NaCl (final concentration 150 mM) was added. Approximately 2.5 times the volume of pre-cooled ethanol was added, and the mixture was incubated at -20 °C for 30–60 min, followed by centrifugation at 10,000 rpm / min. The precipitate was washed with 200 μL of 70% cold ethanol and then dried. Finally, the precipitate was dissolved in TE buffer (<20 μL).

[0066] (2) Partial digestion and recovery of Cosmids: Sau3A I was diluted at a ratio of 1:500 and then used to perform incomplete digestion of Cosmids DNA containing the carboxyl ester hydrolase gene. The digestion system is shown in Table 2-2.

[0067] Table 2 Partial enzyme digestion of positive clones

[0068]

[0069] Mix the above mixture in a micro-EP tube and incubate at 37 °C for 40 min. Immediately place the tube on ice and add 0.5 M EDTA (pH 8.0) at a volume ratio of 1:10 to terminate the enzyme digestion reaction. Prepare an agarose gel using a 1.5 mm comb, and add the mixture to DNA loading buffer for DNA gel electrophoresis. Observe the diffuse bands using a gel imaging system, and cut a DNA band of 2000-3000 nm from the agarose gel and weigh it in a 2.0 mL centrifuge tube (removing as much excess as possible). Finally, purify and recover the plasmid according to the gel extraction kit instructions.

[0070] (3) Preparation of competent cells: First, the frozen E. coli EPI100-T1 cells were prepared. R Streak inoculation onto LB agar plates and incubate at 37°C for 16-20 hours to revitalize the bacterial strain. Select relatively large and round single colonies and inoculate them into 12 mL test tubes containing 5 mL LB, incubate at 37°C with shaking at 225 rpm / min for 8-12 hours. Inoculate 1% of the culture into 250 mL shake flasks containing 100 mL LB, and incubate at 37°C with shaking at 225 rpm / min until OD (outlet count) is reached. 600 ≈0.4, immediately remove the shake flask and place it on ice, then centrifuge at 4 ℃ and 4500 rpm / min for 5 min. Discard the supernatant and resuspend the bacterial cells in 5 mL of pre-chilled 0.1 M CaCl2. Finally, divide the cells into 100 μL portions in 1.5 mL centrifuge tubes and store at -80 ℃.

[0071] (4) Subcloning library construction and functional screening:

[0072] The dephosphorylated vector fragments prepared in (1) and (2) and some enzyme-digested gene fragments were mixed in a sterile, clean microcentrifuge tube, and the components were added according to the system in Table 3.

[0073] Table 3. Connection system between target fragment and vector

[0074]

[0075] The mixed connection system was placed in a refrigerator at 4 °C and reacted overnight for 16 h, and then inactivated by heating at 65 °C.

[0076] Transformation: Add 10 μL of the ligation product to a 1.5 mL centrifuge tube containing 100 μL of competent cells, gently pipette to mix, and incubate on ice for 30 min. After incubation on ice, place the centrifuge tube in a 42℃ water bath for 90 s for heat shock, then immediately remove the tube and incubate on ice for 2 min. Finally, add 900 μL of sterile LB liquid medium and incubate at 37℃, 210 rpm / min for 60 min. Spread the transformed product evenly on a carboxylester hydrolase selection plate containing 12.5 μg / mL Chl and 100 μg / mL Amp, place in a clean bench to dry the surface liquid, and incubate upside down at 37℃ for 3-5 days. Observe visually for the presence of a clear zone.

[0077] Example 2 Bioinformatics Analysis of PET Degrading Enzymes

[0078] The sequencing results were assembled and analyzed using SnapGene 3.2.1 software, yielding a full-length 1362 bp ORF. This sequence has a GC content of 61%, encodes 452 amino acids, predicts a protein isoelectric point of 6.50, and contains a BamH I restriction site, verifying its... Figure 4 (C) Results. BLASTx homology comparison revealed that this hydrolase had the highest identity (57.36%) with IsPETase from Ideonella sakaiensis 201-F6 and its mutants. Yoshida et al. isolated Ideonella sakaiensis 201-F6 from a landfill and reported that its secreted extracellular enzyme IsPETase could degrade PET (Yoshida, et al., 2016). IsPETase is currently the most extensively studied strain in the field of PET biodegradation and is also the only reported enzyme capable of degrading PET at relatively low temperatures. Therefore, BLASTx homology comparison further indicates that this hydrolase is an undiscovered PET-degrading enzyme that may have the same function as IsPETase. Our laboratory named it ZF950, and its encoding gene was named zf950.

[0079] ZF950 was compared with currently published PET-degrading enzymes with good performance using MAGA-X software in ClustalW, and a phylogenetic tree was constructed for both. The results showed that ZF950 is most closely related to IsPETase, and it is preliminarily determined that it may belong to the PETase family. Figure 5 ( Figure 5 This is a phylogenetic tree constructed by zf950 and other carboxylate hydrolase genes with good activity in degrading PET plastics.

[0080] SignalP-5.0 analysis revealed a 35-amino acid signal peptide at the N-terminus of the protein. Homology modeling using Alphafold2 generated the tertiary structure of ZF950. The tertiary structure of ZF950 was then subjected to protein superposition analysis with the highly homologous IsPETase (PDB ID: 5XJH), and the sequences were compared. The results of the protein superposition analysis are shown below. Figure 6 (ZF950 shares 53.7% identity with IsPETase, making it the only enzyme gene discovered in this invention to be matched by BLASTx. In the figure, the white area represents the A region of ZF950, the red area represents the α-helix region of ZF950, the yellow area represents the β-sheet region of the B region of ZF950, and the light cyan area represents 5XJH. The catalytic triplet of ZF950 is represented by orange, and the catalytic triplet of 5XJH is represented by light yellow; the DS1 of ZF950 is represented by a pink stick model, and the DS1 of IsPETase is represented by a light purple stick model.) See sequence comparison [link to diagram]. Figure 7 (In the diagram, residues forming disulfide bonds are marked with green numbers, catalytic residues are marked with blue dots, residues related to substrate binding are marked with green dots, and oxyanion holes are marked with orange dots.) The tertiary structure of ZF950 reveals that the protein first folds into a region consisting of 10 β-sheets near the N-terminal signal peptide, then connects to a region consisting of 8 α-helices and 8 β-sheets via a longer loop. The protein superposition shows that ZF950 has a catalytic site consistent with 5XJH and a disulfide bond spanning the catalytic site, which is relatively unique compared to other types of carboxylester hydrolases.

[0081] Finally, based on the classification method of Seongjoon Joo et al., ZF950 was found to belong to the PETase family, but not to any of the three current classifications within this family. Enzymes in the PETase family are distinguished from other carboxylate hydrolases by the presence or absence of an extra disulfide bond spanning the catalytic site, and by their greater substrate preference for PET at room temperature compared to other carboxylate hydrolases with PET-degrading activity. These characteristics allow them to degrade PET at room temperature. Within this family, the amino acid composition of the IsPETase three-segment binding pocket is also referenced; the classification method is described in [link to classification]. Figure 8This classification method, proposed by Joo et al., reveals that ZF950 leans towards Type IIa in its catalytic site, Subsite I, and Subsite II regions, but with slight differences. Finally, in its Extended Loop region, ZF950 exhibits a novel amino acid composition, suggesting that it represents a completely new type of PETase enzyme.

[0082] Example 3 Construction and heterologous expression of recombinant plasmids

[0083] A carboxylate hydrolase gene with PET-degrading activity was screened from the Mount Everest Library. The gene expressing this PET-degrading enzyme was captured using a subcloning library and sequenced at Beijing Qingke Biotechnology Co., Ltd. Bioinformatics analysis revealed that the protein expressed by this gene has two regions, A and B, connected by a long loop. The required active pocket for PET degradation is located in region B, away from region A. Literature review indicated that PET degradation first requires the enzyme to fix the hydrophobic active pocket to the PET fiber surface through hydrophobic interactions. Studies have shown that improving the accessibility of the substrate PET to the enzyme's active pocket can significantly enhance the enzyme's PET degradation ability. Based on the protein conformation modeled using ZF950 homology, it was found that when region B performs PET degradation activity, region A may oscillate in the buffer solution, causing region B to detach from the PET surface. Therefore, it is inferred that the presence of region A may affect the degradation level of the PET-degrading enzyme. To confirm this hypothesis, recombinant plasmids pET28a(+)-zf950-t35 (with the ZF950 signal peptide removed) and pET28a(+)-zf950-b (with the B region retained but the first 182 amino acids removed) were constructed. These recombinant plasmids were then transfected into competent E. coli BL21 (DE3) cells for heterologous expression. Detailed experiments are as follows:

[0084] 3.1 Construction of recombinant plasmids for PET plastic degrading enzymes

[0085] (1) Primer design

[0086] Primers were designed using SnapGene 3.2.1, removing the stop codon of the target gene to add a 6 × His tag to the C-terminus of the expression product. The final primer design results are shown in Table 4.

[0087] Table 4 Primers for amplifying genes zf950-t35 and zf950-b

[0088]

[0089] Note: Protective bases of primers are indicated by lowercase letters, and restriction enzyme sites are indicated by bold font.

[0090] The positive subclone plasmids obtained through screening and sequencing verification were used as PCR amplification templates. Two upstream and downstream primers, ZF950-BF and ZF950-BR, listed in Table 4, were used to amplify the target gene encoding the protein ZF950-B, which was named zf950-b. Two upstream and downstream primers, ZF950-T35-F and ZF950-T35-R, were used to amplify the target gene encoding the protein ZF950-T35, which was named zf950-t35. The PCR amplification systems (50 μL) for both are shown in Table 5.

[0091] Table 5. ZF950-T35 / ZF950-B PCR Amplification System

[0092]

[0093] The PCR amplification procedures for the target genes zf950-b and zf950-t35 are shown in Table 6.

[0094] Table 6. PCR reaction conditions for ZF950-T35 / ZF950-B

[0095]

[0096] After the PCR amplification reaction, the PCR amplification products were subjected to agarose gel electrophoresis to check whether the size of the amplified target gene fragment met expectations, and the PCR amplification products were purified. The agarose gel electrophoresis results are shown below. Figure 9 (Lane M is a 5000bp DNA marker, lane 1 is the zf950-t35 PCR product, and lane 2 is the zf950-b PCR product).

[0097] (2) Preparation of vector and target gene fragment

[0098] Take frozen E. coli containing pET28a(+) plasmid, streak it on LB solid medium containing a final concentration of 50 μg / mL Kana, and incubate it in a constant temperature incubator at 37 ℃ for 12-16 hours. After that, pick a single colony and inoculate it into 5 mL of LB liquid medium containing a working concentration of 50 μg / mL Kana. Incubate it in a shaker at 37 ℃ and 225 rpm / min for 12-16 hours. Extract the plasmid according to the instructions of the plasmid miniprep kit, and check the plasmid size and concentration by gel electrophoresis before use.

[0099] Take the purified PCR product (1) and the prepared vector plasmid for double enzyme digestion to prepare ligation reaction product. The enzyme digestion system is shown in Table 7-9 below.

[0100] Table 7 pET-28a(+) vector digestion system

[0101]

[0102] Table 8. Enzyme digestion system for zf950-b PCR products

[0103]

[0104] Table 9 Enzyme digestion system for zf950-t35 PCR products

[0105]

[0106] Following the NEB restriction endonuclease instructions, digestion was performed at 37 °C for 60 min, followed by inactivation at 65 °C for 20 min. The digested products were examined by electrophoresis, and correctly sized bands were extracted and purified using a plasmid recovery kit.

[0107] (3) Preparation of E. coli BL21 (DE3) competent cells by CaCl2 method

[0108] Following the preparation process of the chemically competent cells in 1.2 (3), E. coli BL21 (DE3) was prepared and then dispensed into 100 μL tubes.

[0109] (4) Ligation and transformation of vector and target gene

[0110] Add the purified vector and plasmid of the insert fragment from (2) to the ligation system according to Tables 10 and 11, and gently mix with a pipette tip. Incubate at 4 ℃ for 18-24 hours, then remove and inactivate at 65 ℃ for 20 min. Following the culture transfer steps in this paper, transfer the ligation product to E. coli BL21 (DE3) competent cells and spread evenly on the surface of LB solid medium containing a final concentration of 50 μg / mL Kana. Finally, incubate at 37 ℃ for 12-16 hours. Select single clones for colony PCR. Refer to Table 6 of this example for primers and PCR conditions, but reduce the number of cycles. Perform agarose gel electrophoresis on the colony PCR products. Select single clones with the correct band size and corresponding number, culture in test tubes, and send to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results of the agarose gel electrophoresis of colony PCR are shown in [link to a table]. Figure 10 (The middle image shows a 5000bp DNA marker; the DNA marker to the left is the PCR product of zf950-t35, and the DNA marker to the right is the PCR product of zf950-b).

[0111] Table 10 Ligation system of pET-28a(+) vector and zf950-t35 gene fragment

[0112]

[0113] Table 11 Ligation system of pET-28a(+) vector and zf950-b gene fragment

[0114]

[0115] 3.2 Induced expression of recombinant plasmids

[0116] Recombinant strains with correct sequencing results and verified lack of mutations were used for induction of expression. *E. coli* BL21 / pET28a(+)-zf950-b and *E. coli* BL21 / pET28a(+)-zf950-t35 were incubated in tubes at 37 ℃ and 225 rpm / min for 12-16 hours. The bacterial culture was then added at 1% to a shake flask containing 400 mL of LB medium (Kana resistant), and cultured at 37 ℃ and 225 rpm / min with shaking until OD reached. 600 ≈0.6. Remove the shake flask and place it in an ice bath. After cooling, add IPTG to a final concentration of 0.5 mM and induce expression for 20-24 hours at 25 ℃ and 180 rpm / min.

[0117] 3.3 Affinity chromatography purification and protein electrophoresis verification of recombinase

[0118] (1) Cell disruption:

[0119] Take the bacterial culture after induction (3.2), centrifuge at 10000 rpm / min, 4 ℃ for 15 min, remove the supernatant and collect the bacterial cells. Resuspend the bacterial cells in sterile distilled water and centrifuge twice under the same conditions, washing off the LB medium. Resuspend the bacterial cells in PBS and centrifuge once more under the same conditions, collecting the bacterial cells. Finally, add 10 mL of PBS to resuspend the bacterial cells, take a small amount of the bacterial culture, dilute it, and determine the final dilution factor using a spectrophotometer.

[0120] The bacterial culture, adjusted to a suitable bacterial concentration, was dispensed into 12 mL sterilized centrifuge tubes, and proteins were released using an ultrasonic cell disruptor. The disruption conditions were: total working time 20 min, 3 mm ultrasonic probe, 2 s ultrasonic cycles with 3 s intervals, and maximum ultrasonic power 50%. Finally, the disrupted bacterial cells were centrifuged at 4 ℃ and 12000 rpm / min for 30 min to separate the supernatant from the precipitate.

[0121] (2) Affinity chromatography purification:

[0122] ① Open the caps at both ends of the pre-packed column to allow the preservation solution to flow out.

[0123] ② Wash the chromatography column with 5-10 column volumes of ddH2O;

[0124] ③ Rinse the chromatography column with 5-10 column volumes of PBS to equilibrate the nickel column;

[0125] ④ Add the crude enzyme solution to the chromatography column. When adding the sample, pay attention to controlling the flow rate of the enzyme solution in the chromatography column to ensure that His and Ni in the enzyme solution are in harmony. 2+ Fully integrate;

[0126] ⑤ Pass the column through 5-10 column volumes of washing buffer to wash away impurities from the crude enzyme;

[0127] ⑥ Pass the column through different concentrations of imidazole solution (10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM) and collect each component;

[0128] ⑦ After elution, pass the column with 5-10 column volumes of ddH2O, then pass it with 5-10 column volumes of 20% ethanol, and finally add 20% ethanol to the pre-packed column and seal it for storage.

[0129] (3) SDS-PAGE gel detection of recombinase:

[0130] Based on the predicted molecular weights of ZF950-T35 and ZF950-B proteins, a 12% polyacrylamide separation gel and a 5% stacking gel were selected to detect the purification results. The protein gel was stained with protein staining solution for 6-12 hours (the destaining solution can be reused; the staining time should be adjusted according to the number of uses to ensure the protein gel color and dye are almost identical). After overnight destaining with protein destaining solution, the results were observed and preserved using a gel imaging system. The results are shown in [Figure number missing]. Figure 11 (M: Protein Ladder; 1: pET28a(+) empty vector control; 2: Recombinase supernatant; 3: Recombinase precipitate; 4: Purified protein. The left image is the SDS-PAGE image of ZF950-T35, and the right image is the SDS-PAGE image of ZF950-B).

[0131] Example 4: Effects of temperature and pH on the activity and stability of PET plastic degrading enzymes

[0132] A method was developed to determine the optimal temperature and pH for PET-degrading enzymes. Using p-nitrophenylbutyrate as a substrate, the enzyme was co-incubated with p-nitrophenylbutyrate at different temperatures or pH values ​​within the same reaction time. Finally, the optimal values ​​were measured using a spectrophotometer. 405 The amount of p-nitrophenol produced was measured to determine the optimal temperature and pH of the enzyme.

[0133] The reaction mixture contained 200 μL of 0.5 mM p-nitrophenylbutyrate, 750 μL of 0.05 M pH buffer, and 50 μL of protein at a concentration of 0.1 μmol / mL, for a total reaction volume of 1 mL.

[0134] In the experiments, after preheating at the corresponding reaction temperature for 1 min, the purified enzyme was added to initiate the reaction. Then, after incubation at the corresponding temperature or pH for 10 min, 100 μL of 2 M HCl was added to terminate the reaction. Finally, the absorbance was measured at 405 nm using a microplate reader. A blank control was performed for each measurement under different conditions to correct for errors caused by non-enzymatic hydrolysis.

[0135] (1) Effects of different temperatures on the activity and stability of PET degrading enzymes

[0136] Under pH 8.0 control conditions, referring to the above reaction system, the hydrolytic activity of ZF950-T35 and ZF950-B on p-nitrophenol butyrate was measured at temperature gradients of 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, and 60 ℃. The temperature range was then narrowed down to gradually determine the optimal temperature for enzyme activity determination. Using the highest enzyme activity as 100%, relative enzyme activity versus temperature was calculated and plotted as a curve. Three parallel and control experiments were performed for each condition.

[0137] Under substrate-free conditions at pH 8.0, a protein concentration of 0.1 μmol / mL, and at 30 °C, 40 °C, 50 °C, and 60 °C for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively, 200 μL of 0.5 mM p-nitrophenylbutyrate was added, and the mixture was incubated at 40 °C for 10 min. The reaction was then terminated by adding 100 μL of 2 M HCl. Enzyme activity was calculated using a spectrophotometer, with the highest enzyme activity defined as 100%. Relative enzyme activities under other conditions were calculated. Three parallel experiments and a control were performed for each condition.

[0138] (2) Effects of different pH values ​​on the activity and stability of PET degrading enzymes

[0139] Under controlled temperature of 40 ℃, referring to the above reaction system, the hydrolytic activities of ZF950-T35 and ZF950-B on p-nitrophenylbutyrate were measured at pH gradients of 3, 4, 5, 6, 7, 8, 9, and 10. The pH range was then narrowed down to gradually determine the optimal pH for enzyme activity. A curve was plotted using the highest enzyme activity as 100% and relative enzyme activity against pH. Three parallel and control experiments were conducted for each condition.

[0140] 50 μL of 0.1 μmol / mL protein was added to different buffer solutions at pH 6, 7, 8, and 9 and incubated at 40 °C for 40 min. Then, 200 μL of 0.5 mM p-nitrophenylbutyrate was added, and the reaction was allowed to proceed for 10 min. The reaction was terminated by adding 100 μL of 2M HCl. Enzyme activity was calculated using a spectrophotometer. The highest enzyme activity was taken as 100%, and the relative enzyme activity under other conditions was calculated. Three parallel experiments and a control were performed for each condition.

[0141] The effects of temperature on the enzyme activities of ZF950-T35 and ZF950-B are shown in the following figures. Figure 12 (A) and (B), the optimal temperature for both ZF950-T35 and ZF950-B is around 35 ℃. ZF950-B rapidly loses its enzyme activity at 50 ℃, but ZF950-T35 still retains nearly 40% relative enzyme activity at 60 ℃. The effect of temperature on the stability of ZF950-T35 and ZF950-B is shown in [reference needed]. Figure 12 Both (C) and (D) showed relatively stable enzyme activity after incubation at 30 ℃ and 40 ℃ for 60 min. Furthermore, ZF950-T35 exhibited slightly better thermal stability than ZF950-B. This result may be due to the presence of an additional disulfide bond in ZF950-T35 compared to ZF950-B. However, it is evident that the temperature range exceeding 50% for both ZF950-T35 and ZF950-B is quite narrow.

[0142] The effects of pH on the enzyme activities of ZF950-T35 and ZF950-B are shown in the figures below. Figure 13 (A) and (B), ZF950-T35 reached its maximum activity at pH 7.0, while ZF950-B reached its maximum activity at pH 9.0. Both almost completely lost their ability to degrade p-nitrophenylbutyrate at lower acidities (pH ≤ 5) and pH values ​​above 10. The effect of pH on the stability of ZF950-T35 and ZF950-B is shown in [reference needed]. Figure 13 (C) and (D) Although ZF950-B has the highest enzyme activity at pH 9.0, it is not stable. Choosing pH 8.0 as the reaction condition is the best choice. Under this condition, the enzyme activity of ZF950-B is only slightly lower than that at pH 9.0, but it is more stable.

[0143] Example 5: Determination of enzyme activity of PET plastic degrading enzymes

[0144] 5.1 Plotting the molar concentration standard curves of MHET and BHET standards

[0145] Current research on the degradation mechanism of IsPETase has revealed that PET biodegradation by IsPETase mainly produces MHET and BHET, as well as small amounts of terephthalic acid and ethylene glycol. Therefore, a standard curve of peak area versus molar concentration for MHET and BHET standards can be plotted by HPLC to calculate the amount of products generated by co-incubation of PET with the isPETase.

[0146] Stock solutions of 5 mM MHET and BHET were prepared separately. 10.45 mg of MHET was dissolved in 10 mL of 16 mM phosphate buffer (pH 2.5) containing 20% ​​DMSO, and 12.71 mg of BHET was dissolved in 10 mL of the same buffer. Two dilution gradients were prepared: 0.01 mM / mL, 0.03 mM / mL, 0.05 mM / mL, 0.07 mM / mL, 0.09 mM / mL and 0.5 mM / mL, 1 mM / mL, 1.5 mM / mL, 2 mM / mL, 2.5 mM / mL, with each concentration performed in triplicate. Finally, the peak areas of the two standards at different concentrations were determined by HPLC, and a standard curve was plotted. The results are shown below. Figure 14 (Figure (A) is the standard curve of MHET peak area versus peak area in the concentration range of 0.01–0.09 mM / mL; Figure (B) is the standard curve of MHET peak area versus peak area in the concentration range of 0.5–2.5 mM / mL; Figure (C) is the standard curve of BHET peak area versus peak area in the concentration range of 0.01–0.09 mM / mL; Figure (D) is the standard curve of BHET peak area versus peak area in the concentration range of 0.01–0.09 mM / mL). R calculated based on the four standard curves... 2 The above standard curves all have good confidence levels, and their values ​​can be obtained by substituting the peak areas measured by HPLC.

[0147] 5.2 Plotting PET degradation curves

[0148] Purified enzyme solutions from ZF950-T35 and ZF950-B were diluted to 0.1 μmol and dispensed into at least nine tubes, in triplicate. 50 mg of powdered PET was added as the reaction substrate and incubated at pH 8.0 and 35 °C. Samples were taken at 4, 8, 16, 28, 38, 48, 58, 68, and 72 h. For sampling, 2 M HCl was added to one tube until the pH reached 2.0, and ethyl acetate was added to extract the solute. The ethyl acetate extract was rotary evaporated until no liquid remained in the flask. After dissolving the concentrate in a heavy solution, the mixture was centrifuged at 12000 rpm for 15 min, the supernatant was collected, and filtered through a 0.22 μm filter. Finally, the product yield was determined by HPLC. Results are shown in [Figure number missing]. Figure 15 (A) It can be seen that the degradation curve of ZF950-B shows that the enzyme maintains good enzyme activity within 38 h, the curve begins to flatten from 38 h to 58 h, and there is almost no product accumulation after 58 h.

[0149] 5.3 Quantitative determination of PET-degrading enzyme activity

[0150] Based on the PET degradation curve results plotted in section 5.2 of this example, ZF950-B and the substrate were incubated with powdered PET at 35 ℃ and pH 8.0 for 38 h, and enzyme activity was calculated in triplicate. ZF950-T35 was calculated under the same conditions as ZF950-B. The calculated average peak area of ​​the MHET product of ZF950-B was 17,369,205, which, when substituted into the linear fitting equation of the standard curve, yielded a molar concentration of 6.0 mM / mL. The average peak area of ​​the BHET product was 10,197,651, which, when substituted into the linear fitting equation of the standard curve, yielded a molar concentration of 5.3 mM / mL. Similarly, the calculated average peak area of ​​the MHET product of ZF950-T35 was 1,722,884, with a molar concentration of 0.045 mM / mL; the average peak area of ​​the BHET product was 1,096,029, with a molar concentration of 0.031 mM / mL. By calculating the yields of MHET and BHET, and converting this to the amount of product produced per milligram of enzyme per hour to measure enzyme activity, the enzyme activity of ZF950-B was found to be 2.2 mg / h / mg. enzyme The enzyme activity of ZF950-T35 is 0.38 mg / h / mg. enzyme The liquid phase spectra of both are visible. Figure 15 (B) (The right side shows the liquid chromatogram of PET degrading enzyme and powdered PET after 38 hours of co-incubation).

[0151] Example 6: Testing of ZF950-B Degradation of PET Film

[0152] PET film was cut into 2 × 2 cm square slices, rinsed with sterile water, then soaked overnight in 75% alcohol, and finally placed in sterilized glass petri dishes and dried in a laminar flow hood. Purified enzyme solutions from ZF950-T35 and ZF950-B were placed together with the PET slices in 12 mL shake tubes and incubated for 4 days under the same conditions. At the end of the second day, enzyme solution was added, along with inactivated pure enzyme solution as a control. After incubation, the film surface was rinsed with 10% SDS solution, followed by repeated rinsing with sterile water. After cleaning, the film surface was observed under a microscope.

[0153] The surface state of the thin film was observed under an optical microscope, and the results are shown below. Figure 16 . Figure 16 (A) is a negative control obtained by co-incubating ZF950-B protein (inactivated at high temperature) with PET (Figure (A) is the negative control). Under the microscope, the cut edges are very smooth and clear, while Figure 16 (B) The edges of the PET film have become jagged and some PET fragments are in a detached state (Figure (B) is an observation of the edge of the PET film under an optical microscope after ZF950-B and PET film were co-incubated for 48 hours). Figure 16 (C) is an image of the inside of the PET film under an optical microscope (Figure (C) is an image of the inside of the PET film after ZF950-B and PET film were co-incubated for 48 hours under an optical microscope). It can be seen that a large number of corrosion pits also appear on the inner surface of the PET film.

[0154] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A gene for a PET plastic degradation enzyme, characterized in that: The gene sequence of the PET plastic degrading enzyme is shown in SEQ ID NO.

1.

2. A gene for a PET plastic degradation enzyme, characterized in that: The nucleotide sequence of the PET degrading enzyme gene after removing the signal peptide is shown in SEQ ID NO.

2.

3. A gene for a PET plastic degradation enzyme, characterized in that: The catalytic functional region sequence of the PET degradase gene is shown in SEQ ID NO.

3.

4. A primer for amplifying the PET plastic degrading enzyme gene according to claim 2, characterized in that: Includes primers with SEQ ID NO.4 and SEQ ID NO.

5.

5. A primer for amplifying the PET plastic degrading enzyme gene according to claim 3, characterized in that: Includes primers with SEQ ID NO.6 and SEQ ID NO.

7.

6. A PET-degrading enzyme, characterized in that: Its amino acid sequence is encoded by the nucleotide sequence described in any one of claims 1-3.

7. A recombinant vector, characterized in that: It contains the nucleotide sequence described in any one of claims 1-3.

8. The recombinant vector according to claim 7, characterized in that: The recombinant vector can be expressed as an Escherichia coli, Pichia pastoris, or Bacillus subtilis.

9. The use of the PET plastic degrading enzyme as described in claim 6 in the degradation of PET plastics and other esterified compounds and compounds polymerized by ester bonds.

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