WO2025007436A1 - 一种深海来源的PET水解酶dsPETase05及其应用 - Google Patents

一种深海来源的PET水解酶dsPETase05及其应用 Download PDF

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WO2025007436A1
WO2025007436A1 PCT/CN2023/122672 CN2023122672W WO2025007436A1 WO 2025007436 A1 WO2025007436 A1 WO 2025007436A1 CN 2023122672 W CN2023122672 W CN 2023122672W WO 2025007436 A1 WO2025007436 A1 WO 2025007436A1
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dspetase05
pet
vector
hydrolase
reaction
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French (fr)
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李盛英
刘琨
孟亮
李登辉
张成松
陆瑞
姬倩悦
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Bgi Research Qingdao
Shandong University
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Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

  • the invention belongs to the technical field of biochemistry and enzyme engineering, and particularly relates to a PET hydrolase derived from deep sea and application thereof.
  • PET is a type of polyester plastic made from terephthalic acid and ethylene glycol. It is widely used in many fields such as the textile industry and food and beverage packaging. Traditional PET processing methods such as mechanical crushing, thermoplastic reprocessing, and chemical decomposition are expensive and will cause secondary pollution to the environment. In recent years, bioenzymatic degradation based on PET hydrolases has shown great application potential.
  • PET6 from the halophilic bacterium Vibrio gazogenes has been shown to tolerate 0-2.5 M NaCl, but its activity is much lower than that of IsPETase (Weigert et al., 2022). Therefore, there is currently a gap in the search for enzymes that can exhibit high PET hydrolysis activity under high-salt conditions.
  • the purpose of the present invention is to solve the problem that the existing enzymes with high PET hydrolysis activity cannot tolerate high-salt environments, and to provide a PET hydrolase dsPETase05 mined and identified from the metagenome of a hot spring at 1,199 meters in the deep sea.
  • the enzyme can exhibit high hydrolysis activity for PET in a high-salt or high-temperature environment.
  • the technical solution adopted by the present invention to solve its technical problem is: a deep-sea derived PET hydrolase dsPETase05, whose amino acid sequence is shown in SEQ ID No.1.
  • the present invention also provides a gene encoding the PET hydrolase dsPETase05, whose nucleotide sequence is shown in SEQ ID No.2.
  • the present invention also provides a carrier of the PET hydrolase dsPETase05, wherein comprising the gene.
  • the vector is a eukaryotic vector or a prokaryotic vector.
  • the vector is a plasmid vector or a viral vector.
  • the present invention also provides a host cell comprising the vector.
  • the host cell is a bacterium.
  • the present invention further provides the use of the PET hydrolase dsPETase05, the vector, and the host cell in degrading PET under high salt or high temperature conditions.
  • the present invention further provides a PET degrading agent, which contains at least one of the PET hydrolase dsPETase05, the surface carrier, and the host cell.
  • the present invention has the following beneficial effects: the PET hydrolase dsPETase05 provided by the present invention can show efficient PET hydrolysis ability under various high salt concentrations or high temperature conditions to generate monohydroxyethyl terephthalic acid (MHET), terephthalic acid (TPA) and ethylene glycol (EG), thereby achieving enzymatic degradation of PET.
  • the dsPETase05 of the present invention is halophilic and can show higher activity and stability under high salt conditions; at the same time, the halophilic property of the enzyme makes it better suitable for high-salt industrial wastewater and sewage treatment, so the present invention has important significance in the fields of environmental protection and catalysis.
  • Figure 1 shows the catalytic hydrolysis of PET by dsPETase05 under different NaCl conditions
  • FIG2 is a plasmid map of pDSP05 expressing dsPETase05;
  • FIG3 is an SDS-PAGE analysis of purified dsPETase05
  • FIG4 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different salt concentrations for 48 hours;
  • the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;
  • FIG5 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different salt concentrations for 120 hours;
  • the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;
  • Figure 6 shows the complete degradation of scPET film catalyzed by dsPETase05; the reaction system in the test tube is 3 mL, the scPET is 3 mg, the reaction time is marked with the arrow on the left, and the enzyme concentration is marked below the enzyme name;
  • Figure 7 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different temperatures for 48 hours; the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution is used to represent The hydrolysis activity of PET is now revealed;
  • FIG8 shows the product concentrations of PET hydrolysis catalyzed by dsPETase05 at different temperatures for 120 hours;
  • the hydrolysis products detectable by HPLC are TPA and MHET, and the sum of the concentrations of TPA and MHET in the reaction solution represents the magnitude of PET hydrolysis activity;
  • Figure 9 is a comparison of the concentrations of PET hydrolysis products catalyzed by dsPETase05, HotPETase and FAST-PETase: A, activity comparison of dsPETase05 and FAST-PETase; B, activity comparison of dsPETase05 and FAST-PETase; No NaCl was added to the reaction systems of FAST-PETase-0 and HotPET-0, while 5.3 M NaCl was added to the reaction systems of dsPETase05, FAST-PETase-6 and HotPET-6.
  • the present invention screened from the deep-sea metagenome and obtained an enzyme, which was named dsPETase05.
  • the functional characteristics and catalytic activity of the enzyme were tested and verified by experiments.
  • GfPET (ES301445, Goodfellow, Huntingdon, England) was used to verify the function of dsPETase05, and a GfPET film with a diameter of 6 mm was prepared by a hole puncher as a substrate for the reaction.
  • scPET membrane Preparation of scPET membrane by solvent dissolution-evaporation to detect the hydrolysis activity of PET are shown in references (Liu et al., 2023; Cui et al., 2021).
  • the scPET membrane prepared in this example is as follows: GfPET (40 mg/mL) dissolved in 2 mL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) is uniformly coated on a flat glass plate with a diameter of 10 cm, HFIP is evaporated overnight at room temperature, incubated in 75% ethanol for 2 hours, and the resulting scPET membrane is peeled off the glass plate.
  • the present invention searched for its homologous proteins in GenBank by online BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and arranged them by sequence similarity. Table 1 lists the 30 sequences with the highest consistency with dsPETase05 in the GeneBank database, among which the consistency with the protein closest to dsPETase05 in evolution is 99.31%, indicating that the full-length sequence of the protein (SEQ ID No. 1) has not been publicly disclosed and is novel.
  • the whole gene was synthesized through the dsPETase amino acid sequence, and the codons were optimized for Escherichia coli expression.
  • the synthesized sequence is shown in SEQ ID No. 2.
  • the dsPETase05 gene fragment was amplified using DSP05-F and DSP05-R as primers.
  • pET32a-LIC as a template
  • 32a-F and 32a-R as primers
  • pET32a was linearly amplified.
  • the primer sequences are shown in Table 2.
  • the PCR amplification conditions are: 98°C for 10 minutes; 98°C for 10 seconds, 55°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C for 5 minutes.
  • the obtained dsPETase05 fragment was connected with the linearized pET32a vector fragment using a seamless cloning kit (ClonExpress II One Step Cloning Kit, Novozymes, Nanjing) to form the expression vector pDSP05.
  • the map is shown in Figure 2.
  • the expression strain Escherichia coli Rosset a-gami 2 (DE3) was transformed to obtain the expression strain RgDSP05.
  • the strain RgDSP05 expressed a fusion protein with a Trx-His6 tag at the N-terminus, and the N-terminal tag could be removed by TEV protease.
  • the protein-free dsPETase05 could be obtained by two Ni-NTA affinity chromatography for activity detection.
  • the collected bacteria were resuspended in 50 mL of pre-cooled lysis buffer and broken by ultrasonic disruptor in an ice bath.
  • the breaking program was 290 W, ultrasonic disruption for 4 s, and stop for 8 s.
  • the total ultrasonic duration was 15 min.
  • the supernatant was centrifuged at 4 ° C 13000g for 60 min to obtain the supernatant.
  • the supernatant was incubated with 3 mL of Ni-NTA resin at 4 ° C for 1 hour to allow the target protein to fully bind to the resin. It was rinsed with 15 mL of lysis buffer and 15 mL of wash buffer, and the target protein was eluted and collected with 5 mL of elution buffer.
  • the eluate was concentrated to 2.5 mL using an Ultracel-30K ultrafiltration tube.
  • the desalting column PD-10 was balanced with 25 mL of desalting buffer, 2.5 mL of concentrated protein solution was added to the desalting column PD-10, 3.5 mL of desalting buffer was added to collect the protein, and 10 ⁇ L of TEV protease with His6 tag was added to the collected protein solution and incubated at 4 ° C for 12 hours.
  • the Ni-NTA resin was balanced with 20 ml of desalting buffer, and 3 ml of Ni-NTA resin was added and incubated at 4°C for 1 hour to allow Trx-His6tag and TEV enzyme to fully bind to the resin.
  • the effluent was collected as unlabeled dsPETase05.
  • the dsPETase05 was concentrated to 1 ml using an Ultracel-30K ultrafiltration tube, and the absorbance at 280 nm was measured using a micro-spectrophotometer to determine the concentration of the purified protein. 5 ⁇ L of protein was taken for SDS-PAGE detection, and the detection results are shown in Figure 3. The remaining protein was quickly frozen with liquid nitrogen and stored at -80°C.
  • Wash buffer (1L): 3.03 g Tris, 8.77 g NaCl, 100 g glycerol, 2.72 g imidazole, pH 7.5.
  • dsPETase05 catalyzes the hydrolysis of GfPET at different salt concentrations
  • GfPET membrane with a diameter of 6 mm was used as the reaction substrate, and different concentrations of NaCl were added to 50 mMTris-HCl as the reaction buffer, and the pH of each buffer was adjusted to 9.0.
  • the concentrations of NaCl were: 0 M, 0.6 M, 1.2 M, 1.9 M, 2.8 M, 3.7 M, 4.5 M, 5.3 M, the total reaction volume was 500 ⁇ L, the final enzyme concentration was 50 nM, and the reaction temperature was 37 °C. IsPETase was reacted under the same conditions for activity comparison, and three replicates were set for each reaction.
  • the instrument model used for HPLC detection is Agilent Technologies 1220infinity LC, the analytical column model is ZORBAX SB-C18, and the column temperature is 30°C.
  • Mobile phase A is deionized water containing 0.1% trichloroacetic acid
  • mobile phase B is acetonitrile containing 0.1% TCA.
  • the flow rate is fixed at 1 mL/min.
  • PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0-5 minutes, 15% B; 5-20 minutes, 15%-100% B gradient; 20-25 minutes, 100%-15% B gradient; 25-30 minutes, 15% B.
  • the detection wavelength is 254 nm.
  • the concentrations of TPA and MHET produced are calculated based on standard samples.
  • dsPETase05 can show PET hydrolysis activity under all NaCl conditions, and the activity increases with the increase of NaCl concentration. It shows the highest activity when it reaches 5.3 M. At this time point, the activity shown is 5.4 times that of IsPETase.
  • dsPETase05 can show PET hydrolysis activity under all NaCl conditions, and the activity increases with the increase of NaCl concentration. It shows the highest activity when it reaches 5.3 M. At this time point, the activity shown is 16 times that of IsPETase.
  • dsPETase05 catalyzes the hydrolysis of scPET membranes at different salt concentrations
  • the PET hydrolysis activity of dsPETase05 was demonstrated by the complete hydrolysis of the homemade scPET film.
  • 3 mg of scPET film was placed in a glass test tube, and then 3 mL of reaction solution was added: IsPETase reaction solution contained 50 mM tris-HCl buffer (pH 9.0) and 500 nM enzyme; dsPETase05 reaction solution contained 50 mM tris-HCl buffer (pH 9.0), 5.3 M NaCl, and 500 nM enzyme.
  • IsPETase reaction solution contained 50 mM tris-HCl buffer (pH 9.0), 5.3 M NaCl, and 500 nM enzyme.
  • photos were taken every 2 days to observe the degradation of the scPET film.
  • dsPETase05 at a concentration of 500 nM can see obvious degradation of the scPET film at 6 days, and it can remain active within 10 days, and can catalyze the degradation of most scPET within 10 days; while IsPETase, as a control, did not observe obvious degradation during the 10-day reaction process.
  • GfPET membrane with a diameter of 6 mm was used as the reaction substrate, 5.3 M NaCl was added to 50 mM Tris-HCl, and the pH of each buffer was adjusted to 9.0.
  • the total reaction volume was 500 ⁇ L, the final enzyme concentration was 50 nM, the reaction temperature was 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, and three replicates were set for each reaction. 100 ⁇ L samples were taken at 48 and 120 hours of reaction, 200 ⁇ L of methanol was added to terminate the reaction, and then the enzyme was inactivated by treatment at 100°C for 10 minutes. The supernatant was centrifuged and tested by HPLC.
  • the instrument model used for HPLC detection is Agilent Technologies 1220infinity LC, the analytical column model is ZORBAX SB-C18, and the column temperature is 30°C.
  • Mobile phase A is deionized water containing 0.1% trichloroacetic acid
  • mobile phase B is acetonitrile containing 0.1% TCA.
  • the flow rate is fixed at 1 mL/min.
  • PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0-5 minutes, 15% B; 5-20 minutes, 15%-100% B gradient; 20-25 minutes, 100%-15% B gradient; 25-30 minutes, 15% B.
  • the detection wavelength is 254 nm.
  • the concentrations of TPA and MHET produced are calculated based on standard samples.
  • the results of the 48-hour reaction are shown in FIG7 .
  • the amount of hydrolysis products catalyzed by dsPETase05 increases with increasing temperature from 25°C to 55°C and reaches a maximum at 55°C, and then decreases with increasing temperature from 55°C to 75°C.
  • the results of the 120-hour reaction are shown in FIG8 .
  • the amount of hydrolysis products catalyzed by dsPETase05 increases with increasing temperature from 25°C to 55°C and reaches a maximum at 55°C, and then decreases with increasing temperature from 55°C to 75°C.
  • the two most representative mutants of IsPETase are HotPETase (Bell et al., 2022) and FAST-PETase (Lu et al., 2022) has higher activity than the wild type and is also the best performing PET hydrolase mutant reported so far.
  • a 6mm diameter GfPET membrane was used as the reaction substrate to compare the activity of dsPETase05 and these two mutants.
  • the dsPETase05 reaction system was 50mMTris-HCl+5.3M NaCl, incubated at 55°C; the HotPETase reaction system was 50mMTris-HCl or 50mMTris-HCl+5.3MNaCl, incubated at 50°C; the FAST-PETase reaction system was 50mMTris-HCl or 50mMTris-HCl+5.3M NaCl, incubated at 40°C.
  • the final concentration of all enzymes was 50nM, and three replicates were set for each reaction.
  • the sampling time was set to 8 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours.
  • the instrument model used for HPLC detection is Agilent Technologies 1220infinity LC, the analytical column model is ZORBAX SB-C18, and the column temperature is 30°C.
  • Mobile phase A is deionized water containing 0.1% trichloroacetic acid
  • mobile phase B is acetonitrile containing 0.1% TCA.
  • the flow rate is fixed at 1 mL/min.
  • PET hydrolysis products TPA and MHET can be separated by the following gradient mobile phase: 0-5 minutes, 15% B; 5-20 minutes, 15%-100% B gradient; 20-25 minutes, 100%-15% B gradient; 25-30 minutes, 15% B.
  • the detection wavelength is 254 nm.
  • the concentrations of TPA and MHET produced are calculated based on standard samples.

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Abstract

涉及一种深海来源的PET水解酶及其应用。提供了一种深海来源的PET水解酶dsPETase05，其氨基酸序列如SEQ ID No.1所示。还提供了所述PET水解酶dsPETase05的编码基因，其核苷酸序列如SEQ ID No.2所示。PET水解酶dsPETase05能够在高盐或高温的环境下展现出对PET的高水解活性。

Description

一种深海来源的PET水解酶dsPETase05及其应用 技术领域
本发明属于生物化学和酶工程技术领域，具体涉及一种深海来源的PET水解酶及其应用。
背景技术
塑料因其耐久性、延展性、可塑性、稳定性、透光性等优良属性已遍布人类生产生活的各个方面。而塑料在常温下的化学惰性使得大量的塑料废弃物造成的白色污染日益严重，危害生态环境与人类健康。PET是一类由对苯二甲酸和乙二醇为单体聚合而成的聚酯类塑料，在纺织产业、食品饮料包装等多个领域有着广泛应用。传统的PET处理手段如机械破碎、热塑再加工以及化学分解等方法成本昂贵，且会造成环境的二次污染。近年来，基于PET水解酶的生物酶法降解展现出巨大的应用潜力，如来自PET降解菌Ideonella sakaiensis 201-F6中的IsPETase(Yoshida et al.,2016)，以及来自于枝叶堆肥的宏基因组的LCC(Sulaiman et al.,2012)，均有着很好的PET水解活性，而利用它们的突变体进行PET水解、单体回收以及PET再合成，已展现出PET的循环经济在理论上的可行性(Lu et al.,2022；Tournier et al.,2020)。
PET水解酶的嗜盐性研究较少，来自嗜盐菌产期弧菌(Vibrio gazogenes)中的PET6被证明可耐受0～2.5M的NaCl，但其活性远远低于IsPETase(Weigert et al.,2022)，因此，高盐条件下能够表现出高PET水解活性的酶目前是一个空白。
发明内容
本发明的目的是解决现有的高PET水解活性的酶不能耐受高盐环境的问题，提供一种来自深海1199米处热泉的宏基因组中挖掘并鉴定的PET水解酶dsPETase05，该酶能够在高盐或高温的环境下展现出对PET的高水解活性。
本发明解决其技术问题采用的技术方案是：一种深海来源的PET水解酶dsPETase05，其氨基酸序列如SEQ ID No.1所示。
优选地，本发明还提供了编码所述PET水解酶dsPETase05的基因，其核苷酸序列如SEQ ID No.2所示。
优选地，本发明还提供了所述PET水解酶dsPETase05的载体，所述载体中 包含所述的基因。
优选地，所述的载体为真核载体或者原核载体。
优选地，所述的载体为质粒载体或者病毒载体。
优选地，本发明还提供了包含所述的载体的宿主细胞。
优选地，所述的宿主细胞为细菌。
优选地，本发明还进一步提供了所述的PET水解酶dsPETase05、所述的载体、所述的宿主细胞在高盐或高温条件下降解PET中的应用。
优选地，本发明还进一步提供了PET降解剂，该PET降解剂含有所述的PET水解酶dsPETase05、所述的表载体、所述的宿主细胞中的至少一种。
与现有技术相比，本发明具有如下有益效果：本发明提供的PET水解酶dsPETase05能够在各种高盐浓度或高温条件下，表现出高效的PET水解能力，生成单羟乙基对苯二甲酸(MHET)、对苯二甲酸(TPA)和乙二醇(EG)，从而实现PET的酶法降解。本发明的dsPETase05具有嗜盐性，且在高盐条件下能够展现出更高的活性与稳定性；同时，该酶嗜盐的特性使其更好的适用于高盐的工业废水、污水处理，因此本发明在环保、催化领域都有着重要意义。
附图说明
图1为dsPETase05在不同NaCl下催化PET水解；
图2为表达dsPETase05的pDSP05质粒图谱；
图3为纯化后的dsPETase05的SDS-PAGE分析；
图4为dsPETase05在不同盐浓度下催化PET水解48小时的产物浓度；HPLC可检测到的水解产物为TPA与MHET，以反应液中TPA与MHET的浓度之和表现PET水解活性的大小；
图5为dsPETase05在不同盐浓度下催化PET水解120小时的产物浓度；HPLC可检测到的水解产物为TPA与MHET，以反应液中TPA与MHET的浓度之和表现PET水解活性的大小；
图6为dsPETase05催化scPET膜的完全降解；试管中反应体系为3mL，scPET为3mg，反应时间见左侧箭头标注，酶浓度标注于酶名称的下方；
图7为dsPETase05在不同温度下催化PET水解48小时的产物浓度；HPLC可检测到的水解产物为TPA与MHET，以反应液中TPA与MHET的浓度之和表 现PET水解活性的大小；
图8为dsPETase05在不同温度下催化PET水解120小时的产物浓度；HPLC可检测到的水解产物为TPA与MHET，以反应液中TPA与MHET的浓度之和表现PET水解活性的大小；
图9为dsPETase05、HotPETase和FAST-PETase催化PET水解产物浓度对比：A,dsPETase05与FAST-PETase的活性比较；B,dsPETase05与FAST-PETase的活性比较；FAST-PETase-0和HotPET-0反应体系里未添加NaCl，dsPETase05、FAST-PETase-6和HotPET-6反应体系里添加了5.3M NaCl。
具体实施方式
为了便于理解本研究，下面结合附图和具体实施例，对本研究进行更详细的说明。但是，本研究可以以许多不同的形式来实现，并不限于本说明书所描述的实施例。相反地，提供这些实施例的目的是使对本研究公开内容的理解更加透彻全面。
本发明从深海宏基因组中间进行筛选，筛选得到一个酶，将其命名为dsPETase05。以下通过实验对该酶进行功能特性及催化活性的测试和验证。
1.dsPETase05的底物的制备
采用GfPET(ES301445,Goodfellow,Huntingdon,England)验证dsPETase05功能，通过打孔器将制备成直径6mm的GfPET膜作为反应的底物。
scPET膜的制备：通过溶剂溶解—挥发的方式自制scPET膜来检测PET水解活性的报道见参考文献(Liu et al.,2023；Cui et al.,2021)。本实例中所制备的scPET膜方法为：将2mL 1,1,1,3,3,3-六氟-2-丙醇(HFIP)溶解的GfPET(40mg/mL)均匀涂膜在直径为10cm的平板玻璃板上，在室温下过夜蒸发HFIP，在75％乙醇中孵育2小时，将所得scPET膜从玻璃板上剥离。
2.dsPETase05基因与蛋白序列分析
为了确定dsPETase05氨基酸序列的新颖性，本发明通过在线BLAST(https://blast.ncbi.nlm.nih.gov/Blast.cgi)在GenBank中搜索它的同源蛋白并按序列相似性排列，如表1所列的为GeneBank数据库中与dsPETase05一致性最高的30个序列，其中与dsPETase05进化上最相近的蛋白的一致性为99.31％，说明该蛋白的全长序列(SEQ ID No.1)尚未公开披露过，具有新颖性。
表1.GeneBank中与dsPETase05一致性最高的30个蛋白序列
3.dsPETase05基因的克隆
通过dsPETase氨基酸序列进行全基因合成，并针对大肠杆菌表达做密码子优化，合成的序列见SEQ ID No.2。以DSP05-F及DSP05-R为引物对dsPETase05基因片段进行扩增。以pET32a-LIC为模板，32a-F及32a-R为引物对pET32a进行线性化扩增。引物序列如表2所示。PCR扩增条件为：98℃ 10分钟；98℃ 10秒,55℃ 30秒,72℃ 1分钟，循环35次；72℃ 5分钟。将获得的dsPETase05片段与线性化的pET32a载体片段利用无缝克隆试剂盒(ClonExpress II One Step Cloning Kit，诺唯赞，南京)进行连接形成表达载体pDSP05，图谱如图2所示， 并转化大肠杆菌表达菌株Escherichia coliRosseta-gami 2(DE3)，获得表达菌株RgDSP05。菌株RgDSP05表达N端带有Trx-His6tag标签的融合蛋白，并可通过TEV蛋白酶将N端标签切除，通过两次Ni-NTA亲和层析可获得无蛋白标签的dsPETase05以进行活性检测。
表2.dsPETase05克隆至pET32a-LIC的引物及其序列
4.dsPETase05的表达与纯化
挑取RgDSP05单克隆接到培养基中(含有50μg/mL氨苄青霉素和10μg/ml氯霉素双抗性)进行种子液培养，37℃220rpm摇床培养8-12h；扩大培养的比例为1:500～1:100，37℃220rpm摇床培养约4～6小时，当OD600达到0.6～0.8时加入IPTG开始诱导，IPTG终浓度可以为0.2～1mM。诱导条件为18～30℃诱导18-24h。将诱导结束的菌液在4℃3000～6000g条件下离心10分钟，收集菌体。
收集的菌体用50mL预冷的lysis buffer重悬，在冰浴中利用超声破碎仪对菌体进行破碎，破碎程序为功率为290W，超声破碎4s，停止8s，超声总时长为15min。在4℃13000g 60min条件下离心取上清，上清液与3mL Ni-NTA树脂在4℃的下孵育1小时使目标蛋白与树脂充分结合，先后用15mL lysisbuffer和15mL wash buffer冲洗，用5mL elution buffer洗脱收集目标蛋白，用Ultracel-30K的超滤管对洗脱液浓缩至2.5mL。将脱盐柱PD-10用25mL desalting buffer平衡，将2.5mL浓缩蛋白液加入到脱盐柱PD-10中，加入3.5mL的desalting buffer收集蛋白，向收集到的蛋白液加入10μL的带有His6tag的TEV蛋白酶4℃孵育12小时。Ni-NTA树脂用20ml desalting buffer平衡，加入3mL Ni-NTA树脂在4℃的下孵育1小时Trx-His6tag以及TEV酶与树脂充分结合，收集流出液即为无标签的dsPETase05。用Ultracel-30K的超滤管将dsPETase05浓缩至1mL，用微量分光光度计检测280nm的吸光值以测定纯化蛋白质的浓度，取5μL蛋白用于SDS-PAGE检测，检测结果如图3所示。剩余蛋白用液氮速冻并保存于-80℃。
上述蛋白纯化-各缓冲液配方：
Lysis buffer(1L)：3.03g Tris、8.77g NaCl、100g甘油，1.36g咪唑，pH 7.5。
Wash buffer(1L)：3.03g Tris、8.77g NaCl、100g甘油，2.72g咪唑，pH 7.5。
Elution buffer(1L)：3.03g Tris、8.77g NaCl、100g甘油，10.2g咪唑，pH 7.5。
Desalting buffer(1L)：3.03g Tris、8.77g NaCl、100g甘油，pH 7.5。
5.dsPETase05在不同盐浓度下催化GfPET水解反应
直径6mm的GfPET膜为反应底物，50mMTris-HCl中加入不同浓度的NaCl作为反应缓冲液，调整各缓冲液的pH至9.0。NaCl的浓度为：0M，0.6M，1.2M，1.9M，2.8M，3.7M，4.5M，5.3M，反应总体积500μL，酶终浓度50nM，反应温度37℃。IsPETase以相同条件反应以进行活性比较，每个反应设三个重复。反应48和120小时分别取100μL样品，加入200μL甲醇终止反应之后用100℃处理10min使酶失活，离心取上清液进行HPLC检测。
HPLC检测所用仪器型号为Agilent Technologies 1220infinity LC，分析柱型号ZORBAX SB-C18，柱温30℃。流动相A为含0.1％三氯乙酸的去离子水，流动相B为含0.1％TCA的乙腈，流速固定为1mL/min。PET水解产物TPA和MHET可通过如下梯度的流动相分离：0～5分钟，15％B；5～20分钟，15％～100％B梯度；20～25分钟，100％～15％B梯度；25～30分钟，15％B。检测波长为254nm。产生的TPA和MHET的浓度根据标准样品进行计算。
反应结果如图4、图5所示。从结果可见，IsPETase在不加NaCl的条件下表现出最高活性，因此在该条件下的IsPETase催化的PET水解反应作为参照。
48小时反应结果如图4所示，dsPETase05在所有NaCl条件下均能表现出PET水解活性，且活性随着NaCl浓度的增加而提高，当达到5.3M时表现出最高的活性，在该时间点下表现出的活性是IsPETase的5.4倍。
120小时反应结果如图5所示，dsPETase05在所有NaCl条件下均能表现出PET水解活性，且活性随着NaCl浓度的增加而提高，当达到5.3M时表现出最高的活性，在该时间点下表现出的活性是IsPETase的16倍。
6.dsPETase05在不同盐浓度下催化scPET膜的水解反应
dsPETase05的PET水解活性可通过对自制scPET膜的完全水解来展现。称 取3mg scPET薄膜放入玻璃试管中，随后加入3mL反应液：IsPETase反应液中包含50mM tris-HCl缓冲液(pH 9.0)和500nM酶；dsPETase05反应液中包含50mM tris-HCl缓冲液(pH 9.0)，5.3M NaCl，以及500nM酶。在总计为10天的反应时间里每2天进行拍照观察scPET膜的降解情况。结果如图6所示，dsPETase05在500nM浓度下在6天时能看到明显的scPET膜的降解，且在10天内能够一直保持活性，10天内能够催化大部分scPET发生降解；而作为对照的IsPETase，在10天的反应过程中并未观察到明显的降解现象。
以上结果表明dsPETase05在高盐条件下具有更高活性的同时也具有很高的稳定性。
7.dsPETase05在不同温度度下催化GfPET水解反应
直径6mm的GfPET膜为反应底物，50mMTris-HCl中加入5.3M的NaCl，调整各缓冲液的pH至9.0。反应总体积500μL，酶终浓度50nM，反应温度为25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃，每个反应设三个重复。反应48和120小时分别取100μL样品，加入200μL甲醇终止反应之后用100℃处理10min使酶失活，离心取上清液进行HPLC检测。
HPLC检测所用仪器型号为Agilent Technologies 1220infinity LC，分析柱型号ZORBAX SB-C18，柱温30℃。流动相A为含0.1％三氯乙酸的去离子水，流动相B为含0.1％TCA的乙腈，流速固定为1mL/min。PET水解产物TPA和MHET可通过如下梯度的流动相分离：0～5分钟，15％B；5～20分钟，15％～100％B梯度；20～25分钟，100％～15％B梯度；25～30分钟，15％B。检测波长为254nm。产生的TPA和MHET的浓度根据标准样品进行计算。
48小时反应结果如图7所示，dsPETase05催化的水解产物量在25℃到55℃下随着温度的升高而增加，并且在55℃达到最高，之后55℃到75℃随着温度的升高而降低。
120小时反应结果如图8所示，dsPETase05催化的水解产物量在25℃到55℃下随着温度的升高而增加，并且在55℃达到最高，之后55℃到75℃随着温度的升高而降低。
8.dsPETase05催化活性与高活性突变体HotPETase、FAST-PETase的比较
IsPETase的两个最具代表性的突变体HotPETase(Bell et al.,2022)和 FAST-PETase(Lu et al.,2022)具有比野生型更高的活性，也是目前报道的表现最好的两个PET水解酶突变体。本实例以直径6mm的GfPET膜为反应底物，对比dsPETase05与这两个突变体的活性。dsPETase05反应体系为50mMTris-HCl+5.3M NaCl，55℃孵育；HotPETase反应体系为50mMTris-HCl或50mMTris-HCl+5.3MNaCl，50℃孵育；FAST-PETase反应体系为50mMTris-HCl或50mMTris-HCl+5.3M NaCl，40℃孵育。所有酶终浓度均为50nM，每个反应设三个重复，取样时间设置为8小时、24小时、48小时、72小时、96小时、120小时。分别取20μL样品，加入120μL甲醇和40μL去离子水终止反应之后用100℃处理10min使酶失活，离心取上清液进行HPLC检测。
HPLC检测所用仪器型号为Agilent Technologies 1220infinity LC，分析柱型号ZORBAX SB-C18，柱温30℃。流动相A为含0.1％三氯乙酸的去离子水，流动相B为含0.1％TCA的乙腈，流速固定为1mL/min。PET水解产物TPA和MHET可通过如下梯度的流动相分离：0～5分钟，15％B；5～20分钟，15％～100％B梯度；20～25分钟，100％～15％B梯度；25～30分钟，15％B。检测波长为254nm。产生的TPA和MHET的浓度根据标准样品进行计算。
反应结果如图9所示，在5.3MNaCl存在下，HotPETase和FAST-PETase失去了大部分活性，只有在不加NaCl的缓冲体系中才能正常表现活性。而dsPETase05的酶活性在各个时间点均高于HotPETase和FAST-PETase，120小时时总产物的量为HotPETase的2.5倍，为FAST-PETase的4.5倍。本实例证明了dsPETase05酶的嗜盐和嗜温的特性，且在高盐、高温下表现出比IsPETase酶工程改造后的突变体更好的催化能力。

Claims (10)

  1. 一种深海来源的PET水解酶dsPETase05，其特征在于：其氨基酸序列如SEQ ID No.1所示。
  2. 编码如权利要求1所述的PET水解酶dsPETase05的基因，其特征在于：其核苷酸序列如SEQ ID No.2所示。
  3. 包含如权利要求2所述的基因的载体。
  4. 根据权利要求3所述的载体，其特征在于：所述的载体为真核载体或者原核载体。
  5. 根据权利要求3所述的载体，其特征在于：所述的载体为质粒载体或者病毒载体。
  6. 包含如权利要求3-5任一项所述的表达载体的宿主细胞。
  7. 根据权利要求6所述的宿主细胞，其特征在于：所述的宿主细胞为细菌
  8. 如权利要求1所述的PET水解酶dsPETase05、权利要求3-5任一项所述的载体或权利要求6-7任一项所述的宿主细胞在降解PET中的应用。
  9. 根据权利要求8所述的应用，其特征在于：在高盐或高温条件下降解PET。
  10. PET降解剂，其特征在于：含有权利要求1所述的PET水解酶dsPETase05、权利要求3-5任一项所述的载体、权利要求6-7任一项所述的宿主细胞中的至少一种。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120118883A (zh) * 2025-05-15 2025-06-10 山东大学 一种深海来源的非特异性核酸酶dsNuc354及用途
CN120118885A (zh) * 2025-05-15 2025-06-10 山东大学 一种深海来源的非特异性核酸酶dsNuc925及用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120118884B (zh) * 2025-05-15 2025-07-18 山东大学 一种深海来源的非特异性核酸酶dsNuc392及用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108467857A (zh) * 2018-03-14 2018-08-31 四川大学 Pet水解酶突变体及其应用
WO2022043545A1 (en) * 2020-08-28 2022-03-03 Cyclezyme Ab Ideonella sakaiensis pet-hydrolase variant with increased thermostability.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108467857A (zh) * 2018-03-14 2018-08-31 四川大学 Pet水解酶突变体及其应用
WO2022043545A1 (en) * 2020-08-28 2022-03-03 Cyclezyme Ab Ideonella sakaiensis pet-hydrolase variant with increased thermostability.

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 17 April 2017 (2017-04-17), "alpha/beta hydrolase [Halopseudomonas pachastrellae]", XP093258568, Database accession no. WP_083728668.1 *
DATABASE GenPept 13 July 2018 (2018-07-13), "MAG: alpha/beta hydrolase [Pseudomonadales bacterium]", XP093258570, Database accession no. MAB42154.1 *
SHI LI-XIA, GAO SONG-FENG; ZHU LEI-LEI: "Research Advance in Polyethylene Terephthalate Hydrolytic Enzymes", BIOTECHNOLOGY BULLETIN, vol. 36, no. 10, 9 June 2020 (2020-06-09), CN, pages 226 - 236, XP093258579, ISSN: 1002-5464, DOI: 10.13560/j.cnki.biotech.bull.1985.2020-0336 *
ZHAO ZHIYI, ZHANG GUOQIANG; LIU KUN; LI SHENGYING: "Advances in poly(ethylene terephthalate)hydrolases", CHINESE JOURNAL OF BIOTECHNOLOGY, 25 May 2023 (2023-05-25), CN , pages 1998 - 2014, XP093258574, ISSN: 1000-3061, DOI: 10.13345/j.cjb.220915 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120118883A (zh) * 2025-05-15 2025-06-10 山东大学 一种深海来源的非特异性核酸酶dsNuc354及用途
CN120118885A (zh) * 2025-05-15 2025-06-10 山东大学 一种深海来源的非特异性核酸酶dsNuc925及用途

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