WO2022160841A1 - 代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法 - Google Patents

代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法 Download PDF

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WO2022160841A1
WO2022160841A1 PCT/CN2021/128825 CN2021128825W WO2022160841A1 WO 2022160841 A1 WO2022160841 A1 WO 2022160841A1 CN 2021128825 W CN2021128825 W CN 2021128825W WO 2022160841 A1 WO2022160841 A1 WO 2022160841A1
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micro
nano
nano plastic
biodegradation
induced
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PCT/CN2021/128825
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French (fr)
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刘倩
黄秀
江桂斌
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中国科学院生态环境研究中心
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Priority to US18/274,244 priority Critical patent/US11952468B2/en
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2400/00—Characterised by the use of unspecified polymers
    • C08J2400/20—Polymers characterized by their physical structure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample

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  • the invention belongs to the field of biodegradation and its analysis, and more particularly relates to a method for inducing the biological metabolism of micro-nano plastics by metabolic enzymes and a method for product analysis thereof.
  • micro-nano plastics can cause adverse biological effects.
  • Bioaccumulation and magnification effects allow micro-nanoplastics to accumulate in organisms at different trophic levels, with serious consequences for the entire food web.
  • the accumulation of micro-nanoplastics in the body can also cause a strong inflammatory response.
  • other adverse health effects associated with micro-nanoplastics such as mortality and morbidity, remain unclear.
  • the purpose of the present invention is to provide a very common method for finding micro-nano plastic particles by efficiently degrading micro-nano plastics under mild conditions, which is considered to be inert in chemical properties and extremely difficult to be biodegraded.
  • the present application confirms that metabolic enzymes can induce the degradation of micro-nano plastics, and further elucidates the biodegradation pathways of micro-nano plastics.
  • the present invention proposes the following technical solutions:
  • a method for biodegradation of micro-nano plastic particles induced by metabolic enzymes comprising the following steps:
  • Step (1) take an appropriate amount of plastic products, cut into small pieces
  • Step (2) be placed in a ball mill, vacuum-ground in a ball mill;
  • Step (3) take by weighing the plastic sample after grinding, disperse with water and prepare a certain concentration of dispersion
  • Step (4) mixing the micro-nano plastic dispersion liquid with the metabolic enzyme solution to ensure that the mass ratio of the metabolic enzyme and the micro-nano plastic mixing is in the range of 0.00000001-10000, and placing the mixed liquid on a vortex shaker to mix;
  • the Metabolic enzymes include glutathione S-transferase;
  • micro-nano plastic in step (4) can be obtained by flotation of the ball-milled plastic products, and the flotation process needs to add surfactant and perform mechanical stirring.
  • the incubation time is set to 0-14 days, and the concentration range of the micro-nano plastic dispersion liquid is 0.0000001-1000 ⁇ g/mL.
  • glutathione S-transferase in step (4) is derived from animals or plants.
  • the incubation environment in step (5) is suitable for a dark or light environment, and the applicable temperature range is 15°C-37°C.
  • a product analysis method of a metabolic enzyme-induced biodegradation method for micro-nano plastic particles described in one of the preceding items comprising:
  • Step (6) take and incubate the mixture for a certain period of time and drop it on the MTP 384 stainless steel non-polishing target plate;
  • Step (7) without adding additional matrix, place in a fume hood, and volatilize naturally;
  • Step (8) after the sample is dried, the target plate is placed on the target holder of a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS), and the micro-nano plastic sample is directly analyzed by MALDI-TOF MS. Perform mass spectrometry.
  • MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometer
  • the mass spectrum range of the characteristic absorption and degradation products of the micro-nano plastic particles obtained in step (8) is within 0-2000 m/z.
  • the mass spectrum range of the characteristic absorption and degradation products of the micro-nano plastic particles obtained in step (8) is a small molecule region below 1000 m/z.
  • the actual sample to be tested in step (8) may be a plastic product in an environmental or biological sample
  • the characterization techniques include morphological characterization and molecular characterization by mass spectrometry.
  • the present invention proposes a new enzymatic method for removing micro-nano plastic pollution based on the special degradation ability of metabolic enzymes to micro-nano plastics.
  • micro-nano plastics can be metabolized and transformed by metabolic enzymes.
  • Various degradation products were found by mass spectrometry analysis.
  • the present invention reduces the co-crystallization process because no additional matrix is required, and also overcomes the hot spot problem often encountered in traditional matrices, improves the reproducibility of the analysis, and reduces the cost.
  • the present invention realizes rapid and high-throughput mass spectrometry detection of complex environmental samples.
  • the matrix-free detection method based on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry double-ion mode of the present invention is small in size, requires a very small amount of sample, can achieve high-sensitivity mass spectrometry detection of micro-nano plastics, and the detection limit can reach ppt is even lower than ppt level.
  • Figure 1 is an optical photo of micro-nano plastics incubated with glutathione S-transferase for different times using the metabolic enzyme-induced biodegradation method of micro-nano plastic particles and its product analysis method proposed in an embodiment of the present invention, from left to right Incubation time was 0h, 24h, 48h, 96h, respectively.
  • Figure 2 is an optical photograph of micro-nano plastics incubated with glutathione S-transferase for different times using the metabolic enzyme-induced biodegradation method of micro-nano plastic particles and its product analysis method proposed in an embodiment of the present invention, from left to right Incubation time was 0h, 12h, 96h, respectively.
  • Figure 4 shows the MALDI-TOF MS detection results of 1 ⁇ L of micro-nano plastic in a high-quality region using the metabolic enzyme-induced biodegradation method of micro-nano plastic particles and its product analysis method proposed in an embodiment of the present invention.
  • Figure 5 is a typical MALDI-TOF MS of 1 ⁇ L of micro-nano plastics incubated under the action of glutathione S-transferase for 12 h using the metabolic enzyme-induced biodegradation method of micro-nano plastic particles and its product analysis method proposed in an embodiment of the present invention mass spectrum.
  • Figure 6 is a typical MALDI-TOF MS in which 1 ⁇ L of micro-nano plastic was incubated for 96h under the action of glutathione S-transferase using the metabolic enzyme-induced biodegradation method of micro-nano plastic particles and its product analysis method proposed in an embodiment of the present invention mass spectrum.
  • Micro-nano plastics have characteristic molecular peaks in the low-mass region in MALDI-TOFMS, and the characteristic peaks of monomers and degradation products of micro-nano plastics are between 0 and 2000, especially in the small molecular region less than 1000.
  • micro-nano plastics can be metabolized and transformed by metabolic enzymes.
  • the present invention proposes a metabolic enzyme-induced biodegradation method for micro-nano plastic particles, comprising the following steps:
  • Step (2) be placed in a ball mill, vacuum-ground in a ball mill;
  • the present application also provides a product analysis method of a metabolic enzyme-induced micro-nano plastic particle biodegradation method, including:
  • Step (6) take and incubate the mixture for a certain period of time and drop it on the MTP 384 stainless steel non-polishing target plate;
  • Step (7) without adding additional matrix, place in a fume hood, and volatilize naturally;
  • the MALDI-TOF MS detection of the analytical method is carried out in reflection mode, and is applicable in both positive and negative ion modes.
  • the laser power of the mass spectrometer in the positive ion mode, is set to 15-90%, the frequency is set to 100-200, and the shots are set to 100-500; in the negative ion mode, the laser power of the mass spectrometer is set to 15 -90%, frequency is set to 100-200, shots are set to 100-500.
  • the grinding time of the plastic in the analysis method in the ball mill is set to 6h-24h, and the grinding method can be continuous or intermittent.
  • the micro-nano plastic of the analysis method can be obtained by flotation of the ball-milled plastic products, and the flotation process needs to add surfactant and perform mechanical stirring.
  • Glutathione S-transferases of animals and plants are suitable for the metabolic enzymes in the analysis method.
  • the analytical method can realize high-sensitivity mass spectrometry detection without adding a matrix during sample preparation.
  • micro-nano plastics obtained by the analysis method have characteristic monomer cluster peaks in MALDI-TOF MS, and the characteristic fingerprint peaks range from 0 to 2000 m/z, especially for small molecules below 1000 m/z area.
  • the incubation environment of the analysis method is suitable for dark and light environments, and the applicable temperature range is 15°C-37°C.
  • the degradation products of the micro-nano plastic particles of the analysis method are in the range of 0 to 2000 m/z, especially the small molecule region below 1000 m/z.
  • the degradation path of the micro-nano plastic particles in the analysis method includes an oxidation path and a nitridation path.
  • the analytical method can be used to characterize the mixed liquid sample obtained in step (5), such as optical photographs and mass spectrometry techniques.
  • the volume of the sample used in the analysis method is very small, and can be in the range of 1-10 ⁇ L.
  • the target plate is placed on the target holder of the matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS), and the micro-nano plastic sample is directly subjected to mass spectrometry detection by MALDI-TOF MS .
  • the instrument model used is BrukerDaltonicsAutoflex III Smartbean MALDI-TOF mass spectrometer, using 355nm Nd:YAG with a frequency of 200Hz, in the negative ion mode, the laser power is set to 70%, and the mass detection range is 0-3000; while in the positive ion mode , the laser power was set to 70%, and the mass detection range was 1-3000.
  • Figure 1 is the optical photo of the degradation of micro-nano plastics by metabolic enzymes. result.
  • Figure 2. Optical photos of the degradation of micro-nano plastics by metabolic enzymes. From left to right are the results of incubation with glutathione S-transferase for 0h, 12h and 96h, respectively.
  • Figure 3 is the MALDI- TOF MS detection results
  • Figure 4 is the MALDI-TOF MS detection results of the micro-nano plastics in the high-quality region
  • Figure 5 after adding glutathione S-transferase, the micro-nano plastics were incubated at 37°C for 12h in the dark environment MALDI -TOF MS test results.
  • Figure 6 is the MALDI- TOF MS detection results
  • micro-nano plastics can be biodegraded and metabolized under mild conditions under the induction of a biological metabolic enzyme - glutathione S-transferase.
  • Plastics are traditionally considered inert and resistant to biological digestion or degradation.
  • the present study finds that under mild conditions, micro-nano plastics can be degraded by glutathione S-transferase, and the process is proved by complementary multiple techniques.
  • a variety of degradation products were found by mass spectrometry, and thus the transformation mechanism of micro-nano plastic particles in vivo was proposed.

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Abstract

提供了一种代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法，包括以下步骤：步骤(1)、取适量的塑料制品，剪成小块；步骤(2)、置于球磨罐中，在球磨机中真空研磨；步骤(3)、称取研磨后的塑料样品用水分散并配制一定浓度的分散液；步骤(4)、将微纳塑料分散液与代谢酶溶液混合，将混合液置于涡旋振荡器混匀；步骤(5)、将混合液置于隔水式培养箱中孵育。提供了一种微纳塑料在温和的条件下在生物代谢酶——谷胱甘肽S-转移酶的诱导下被生物降解和代谢的方法。

Description

代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法 技术领域
本发明属于生物降解及其分析领域，更具体地涉及一种代谢酶诱导微纳塑料生物代谢的方法及其产物分析方法。
背景技术
不易生物降解的塑料垃圾已经引发了众多的环境问题。全球每年大概有3.59亿吨塑料被生产，其中1.5-2亿吨会很快变成垃圾直接暴露于自然界中。大块塑料在受到太阳辐射、机械力和微生物作用后可以降解成微尺度或纳米尺度的碎片。而微纳塑料被认为是一种新兴的环境污染物，并且广泛存在于自然环境中。微塑料是指颗粒大小为5mm的塑料碎片、颗粒、纤维或泡沫，小于1μm的则被称为纳米塑料。大量不可降解的微小塑料碎片已经被排放到了环境中，海洋中的微塑料更是被誉为“海洋中的PM2.5”，最终会随着海洋生物的吞食进入食物链和生态圈。微/纳米塑料可在生物体内积累，对海洋生态系统和人类健康构成威胁。此外，在陆地生态系统中也发现了微纳塑料。研究表明，微纳塑料也可以在土壤中积累，在蚯蚓和鸡等生物中赋存。
大量累积的微纳塑料会造成不良的生物学效应。生物累积和放大效应使得微纳塑料可以在不同营养级别的生物体中积累，对整个食物网造成严重影响。已有研究表明生物体摄入微纳塑料可引起多种效应，包括摄食活动减少和能量储备耗尽，甚至导致个体的死亡。微纳塑料在体内的积累也可引起强烈的炎症反应。然而，与微纳塑料相关的其他不利健康影响，如死亡率和发病率等仍不清楚。因此，研究生物体内微纳塑料的赋存、分布及代谢过程对评估维纳塑料的安全性和追溯它们的归驱具有重要的意义。据我们所知，在这之前大家都认为微纳塑料是惰性的，在生物体内特别是在人体内是不会被降解的。
为了准确监测生物体内微纳塑料的归驱，研究人员一直试图寻找可获得的、标准化的分析方法来识别和量化复杂介质中的微纳塑料。基于微、纳米塑料的聚合物性质和高分子量，具有软电离和宽质量检测范围的MALDI-TOF MS为其的表征提供了一种有效的工具。在本研究中，我们使用MALDI-TOF MS监测了微塑料的降解过程，并研究了降解机理。
发明内容
本发明的目的在于针对目前认为微纳塑料的化学性质惰性，极难被生物降解的问题，提供一种非常常见的代谢酶在温和条件下高效降解发现微纳塑料颗粒的方法。本申请通过多种表征，特别是质谱表征，确认了代谢酶可以诱导微纳塑料的降解，并深入阐明微纳塑料生物降解的途径。
为实现上述目的，本发明提出以下技术方案：
一种代谢酶诱导微纳塑料颗粒生物降解方法，包括以下步骤：
步骤(1)、取适量的塑料制品，剪成小块；
步骤(2)、置于球磨罐中，在球磨机中真空研磨；
步骤(3)、称取研磨后的塑料样品用水分散并配制一定浓度的分散液；
步骤(4)、将微纳塑料分散液与代谢酶溶液混合，保证代谢酶与微纳塑料混合的质量比范围为0.00000001-10000，将混合液置于涡旋振荡器混匀；其中，所述代谢酶包括谷胱甘肽S-转移酶；
步骤(5)、将混合液置于隔水式培养箱中孵育。
进一步地，塑料在球磨机中研磨时间设置为6h-24h。
进一步地，步骤(4)中的微纳塑料可通过对球磨后的塑料制品进行浮选得到，浮选过程需加入表面活性剂并进行机械搅拌。
进一步地，孵育时间设置为0-14天，微纳塑料分散液浓度范围为0.0000001-1000μg/mL。
进一步地，步骤(4)中所述谷胱甘肽S-转移酶来自动物或植物。
进一步地，步骤(5)中孵育环境适用于黑暗或光照环境，温度适用范围为15℃-37℃。
另一方面，前述之一所述的一种代谢酶诱导微纳塑料颗粒生物降解方法的产物分析方法，包括：
步骤(6)、取孵育一定时间的混合液滴加在MTP 384不锈钢非抛光靶板上；
步骤(7)、不额外添加基质，置于通风橱中，自然挥发；
步骤(8)、样品干燥后，将所述靶板放置于基质辅助激光解吸离子化飞行时间质谱仪(MALDI-TOF MS)的靶托上，通过MALDI-TOF MS对所述微纳塑料样品直接进行质谱检测。
进一步地，步骤(8)得到的微纳塑料颗粒特征吸收和降解产物的质谱范围在0～2000m/z内。
进一步地，步骤(8)得到的微纳塑料颗粒特征吸收和降解产物的质谱范围在1000m/z以下的小分子区域。
进一步地，步骤(8)得到微纳塑料颗粒的降解路径包括氧化路径和氮化路径。
进一步地，步骤(8)中实际待测样品可为环境或生物样本中的塑料制品，表征技术包括形态学表征和质谱的分子表征。
本发明提出的一种代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法具有以下有益效果：
1.本发明发现球磨过程可能会改变微纳塑料的表面性质，从而促进其降解。
2.本发明基于代谢酶对微纳塑料的特殊降解能力，提出了一种新的去除微纳塑料污染的酶法。
3.本发明发现在温和的条件下，微纳塑料可以被代谢酶代谢转化。通过质谱分析发现了多种降解产物。这些发现更新了我们过去对微纳塑料生物命运的认识。
4.本发明在样品配置时不需要另外添加基质，就可以实现高灵敏度的质谱检测，从而简化了操作步骤、节省了样品准备时间且大大提高了检测效率。
5.本发明由于不需要额外的添加基质，减少了共结晶过程，也克服了传统基质中常遇到的热点问题，提高了分析的重现性，减少了成本。
6.本发明实现了复杂环境样品的快速且高通量的质谱检测。
7.本发明的基于基质辅助激光解吸离子化飞行时间质谱双离子模式的免基质的检测方法体积极小，所需样品量极少，可以实现微纳塑料的高灵敏度质谱检测，检测限可达到ppt甚至低于ppt级别。
8.本发明的基于基质辅助激光解吸离子化飞行时间质谱双离子模式的免基质的检测方法具有快速、高效、灵敏、信号稳定、高通量、准确的特点，并且具有对微纳塑料进行定量的可能。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法对微纳塑料在谷胱甘肽S-转移酶孵育不同时间的光学照片，从左至右孵育时间分别为0h、24h、48h、96h。
图2是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法对微纳塑料在谷胱甘肽S-转移酶孵育不同时间的光学照片，从左至右孵育时间分别为0h、12h、96h。
图3是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法对1μL微纳塑料在低质量区域的MALDI-TOF MS检测结果。
图4是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法对1μL微纳塑料在高质量区域的MALDI-TOF MS检测结果。
图5是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析 方法对1μL微纳塑料在谷胱甘肽S-转移酶作用下孵育12h的典型MALDI-TOF MS质谱图。
图6是采用本发明一实施例提出的代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法对1μL微纳塑料在谷胱甘肽S-转移酶作用下孵育96h的典型MALDI-TOF MS质谱图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本申请发明人在研究中发现：
(1)微纳塑料在MALDI-TOFMS中在低质量区域有特征的分子峰，微纳塑料的单体和降解产物特征峰在0-2000之间，尤其是在小于1000的小分子区域。
(2)在温和的条件下，微纳塑料可以被代谢酶代谢转化。
(3)球磨过程可能会改变微纳塑料的表面性质，从而促进其降解。
因此，本发明提出了一种代谢酶诱导微纳塑料颗粒生物降解方法，包括以下步骤：
步骤(1)、取适量的塑料制品，剪成小块；
步骤(2)、置于球磨罐中，在球磨机中真空研磨；
步骤(3)、称取研磨后的塑料样品用水分散并配制一定浓度的分散液；
步骤(4)、将微纳塑料分散液与代谢酶溶液混合，置于涡旋振荡器混匀；
步骤(5)、将混合液置于隔水式培养箱中孵育。
另一方面，本申请还提供一种代谢酶诱导微纳塑料颗粒生物降解方法的产物分析方法，包括：
步骤(6)、取孵育一定时间的混合液滴加在MTP 384不锈钢非抛光靶板上；
步骤(7)、不额外添加基质，置于通风橱中，自然挥发；
步骤(8)、样品干燥后，将所述靶板放置于基质辅助激光解吸离子化飞行时间质谱仪(MALDI-TOF MS)的靶托上，通过MALDI-TOF MS对所述微纳塑料样品直接进行质谱检测。
进一步地，本申请发明人进行了相关实验验证：
(1)所述分析方法的MALDI-TOF MS检测在反射模式下进行，并且在正离子模式和负离子模式下都适用。
(2)所述分析方法在正离子模式下，质谱的激光功率设置为15-90％，频率设置为100-200，shots设置为100-500；在负离子模式下，质谱的激光功率设置为15-90％，频率设置 为100-200，shots设置为100-500。
(3)所述分析方法配制的微纳塑料分散液浓度范围为0.0000001-1000μg/mL。
(4)所述分析方法的塑料在球磨机中研磨时间设置为6h-24h，研磨方式可为连续或者间断进行。
(5)所述分析方法的微纳塑料可通过对球磨后的塑料制品进行浮选得到，浮选过程需加入表面活性剂并进行机械搅拌。
(6)所述分析方法的孵育时间设置最长设置为0-14天，代谢酶与微纳塑料混合的质量比范围为0.00000001-10000。
(7)所述分析方法中的代谢酶适用于动物和植物的谷胱甘肽S-转移酶。
(8)所述分析方法在样品配置时不需要另外添加基质，就可以实现高灵敏度的质谱检测。
(9)所述分析方法得到的微纳塑料在MALDI-TOF MS中有特征的单体团簇峰，特征指纹峰的范围在0～2000m/z内，特别是在1000m/z以下的小分子区域。
(10)所述分析方法的孵育环境适用于黑暗和光照环境，温度适用范围为15℃-37℃。
(11)所述分析方法的微纳塑料颗粒的降解产物范围在0～2000m/z内，特别是在1000m/z以下的小分子区域。
(12)所述分析方法中的微纳塑料颗粒的降解路径包括氧化路径和氮化路径。
(13)所述分析方法实际待测样品可为环境或生物样本中的塑料制品。
(14)所述分析方法可用于表征步骤(5)得到的混合液样品，如光学照片和质谱技术。
(15)所述分析方法所用样品体积极小，在1-10μL范围内即可。
实施例
取适量的PET塑料制品，剪成小块，将其置于球磨罐中，在球磨机中真空研磨。研磨后的塑料样品用水分散并配制一定浓度的分散液，然后将微纳塑料分散液与代谢酶溶液混合，置于涡旋振荡器混匀，将混合液置于隔水式培养箱中孵育。孵育一段时间后，用移液枪吸取1μL的混合液滴加在商业化的MTP 384不锈钢非抛光靶板上，不额外添加基质，置于通风橱中，自然挥发。样品干燥后，将所述靶板放置于所述基质辅助激光解吸离子化飞行时间质谱仪(MALDI-TOF MS)的靶托上，通过MALDI-TOF MS对所述微纳塑料样品直接进行质谱检测。使用的仪器型号为BrukerDaltonicsAutoflex III Smartbean MALDI-TOF质谱仪，使用频率为200Hz的355nm Nd:YAG，在负离子模式下，激光功率设置为70％，质谱检测范围为0-3000；而在正离子模式下，激光功率设置为70％，质谱检测范围为1-3000。测定结果如图1-图6所示，其中图1为代谢酶降解微纳塑料的光学照片，从左至右为分别为与谷胱甘肽S- 转移酶孵育0h、24h、48h、96h的结果。图2代谢酶降解微纳塑料的光学照片，从左至右为分别为与谷胱甘肽S-转移酶孵育0h、12h、96h的结果，图3为微纳塑料在低质量区域的MALDI-TOF MS检测结果，图4为微纳塑料在高质量区域的MALDI-TOF MS检测结果，图5加入谷胱甘肽S-转移酶后，微纳塑料置于黑暗环境37℃孵育12h后的MALDI-TOF MS检测结果。图6加入谷胱甘肽S-转移酶后，微纳塑料样品置于黑暗环境37℃孵育96h后的MALDI-TOF MS检测结果。从图1-2可以看出，加入谷胱甘肽S-转移酶后，微纳塑料随着时间延长逐渐减少，证明谷胱甘肽S-转移酶使微纳塑料发生了代谢转化。从图3-4可以看出，在无基质的情况下，无论是在低质量区域还是高质量区域，我们发现所测微纳塑料在MALDI-TOFMS中有特征的分子峰团簇，证明本实施例的分析方法在样品配备时，无需加入基质，既可直接用于微纳塑料样品的分析。从图5-6可以看出，MALDI-TOF MS质谱进一步证明了谷胱甘肽转移酶诱导了微纳塑料发生了代谢转化。转化产物主要分为两类，分别为氧化产物和氮化产物。这些发现表明塑料在生物体内，甚至是人体内是能够被降解的。人体对塑料具有一定的清除能力。
从以上实施例可以看出，本申请研究发现微纳塑料在温和的条件下可以在生物代谢酶——谷胱甘肽S-转移酶的诱导下被生物降解和代谢。塑料传统上被认为是惰性的，可以抵抗生物消化或降解。本发明研究发现在温和的条件下，微纳米塑料可以被谷胱甘肽S-转移酶降解，并通过互补的多重技术证明了这一过程。在本发明中，通过质谱发现了多种降解产物，并由此提出了微纳塑料颗粒在生物体内的转化机理。这些发现更新了现有技术对微纳塑料生物命运的认识。基于代谢酶对微纳塑料的特殊降解能力，提供了一种新的去除微纳塑料污染的酶法。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。

Claims (12)

  1. 一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，包括以下步骤：
    步骤(1)、取适量的塑料制品，剪成小块；
    步骤(2)、置于球磨罐中，在球磨机中真空研磨；
    步骤(3)、称取研磨后的塑料样品用水分散并配制一定浓度的分散液；
    步骤(4)、将微纳塑料分散液与代谢酶溶液混合，保证代谢酶与微纳塑料混合的质量比范围为0.00000001-10000，将混合液置于涡旋振荡器混匀；其中，所述代谢酶包括谷胱甘肽S-转移酶；
    步骤(5)、将混合液置于隔水式培养箱中孵育。
  2. 根据权利要求1所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，塑料在球磨机中研磨时间设置为6h-24h。
  3. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(4)中的微纳塑料可通过对球磨后的塑料制品进行浮选得到，浮选过程需加入表面活性剂并进行机械搅拌。
  4. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，孵育时间设置为0-14天，微纳塑料分散液浓度范围为0.0000001-1000μg/mL。
  5. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(4)中所述谷胱甘肽S-转移酶来自动物或植物。
  6. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(5)中孵育环境适用于黑暗或光照环境，温度适用范围为15℃-37℃。
  7. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(5)孵育后的微纳塑料颗粒的降解率为50％-100％。
  8. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(5)孵育后得到的微纳塑料颗粒特征吸收和降解产物的质谱范围在0～2000m/z内。
  9. 根据权利要求8所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(5)孵育后得到的微纳塑料颗粒特征吸收和降解产物的质谱范围在1000m/z以下的小分子区域。
  10. 根据权利要求1-9之一所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(5)孵育后得到微纳塑料颗粒的降解路径包括氧化路径和氮化路径。
  11. 根据权利要求1或2所述的一种代谢酶诱导微纳塑料颗粒生物降解方法，其特征在于，步骤(1)中的塑料制品可为环境或生物样本中的塑料制品，表征技术包括形态学表征和质谱的分子表征。
  12. 根据权利要求1-11之一所述的一种代谢酶诱导微纳塑料颗粒生物降解方法的产物分析方法，其特征在于，包括：
    步骤(6)、取孵育一定时间的混合液滴加在MTP 384不锈钢非抛光靶板上；
    步骤(7)、不额外添加基质，置于通风橱中，自然挥发；
    步骤(8)、样品干燥后，将所述靶板放置于基质辅助激光解吸离子化飞行时间质谱仪(MALDI-TOF MS)的靶托上，通过MALDI-TOF MS对所述微纳塑料样品直接进行质谱检测。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103170089A (zh) * 2012-06-13 2013-06-26 中国矿业大学(北京) 一种谷胱甘肽-s转移酶(gst)蛋白在降解多种农药中的应用
US20140275436A1 (en) * 2011-10-12 2014-09-18 Molecon (Suzhou) Novel Materials Co., Ltd. Fast degradable polyester polymer and preparation method and use thereof
CN109900774A (zh) * 2018-12-13 2019-06-18 中国科学院生态环境研究中心 通过高温处理提高maldi-tof-ms对聚苯乙烯微/纳米颗粒检测灵敏度的方法
US20190345472A1 (en) * 2016-12-16 2019-11-14 Carbios Improved plastic degrading proteases
CN112903805A (zh) * 2021-01-27 2021-06-04 中国科学院生态环境研究中心 代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102675712B (zh) * 2012-04-27 2013-10-09 谷尚昆 生物降解塑料及其生产方法
CN107794252B (zh) * 2016-10-18 2021-07-23 电子科技大学 降解pet塑料的基因工程菌
MX2020006359A (es) * 2017-12-21 2020-08-17 Carbios Nuevas proteasas y usos de las mismas.

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140275436A1 (en) * 2011-10-12 2014-09-18 Molecon (Suzhou) Novel Materials Co., Ltd. Fast degradable polyester polymer and preparation method and use thereof
CN103170089A (zh) * 2012-06-13 2013-06-26 中国矿业大学(北京) 一种谷胱甘肽-s转移酶(gst)蛋白在降解多种农药中的应用
US20190345472A1 (en) * 2016-12-16 2019-11-14 Carbios Improved plastic degrading proteases
CN109900774A (zh) * 2018-12-13 2019-06-18 中国科学院生态环境研究中心 通过高温处理提高maldi-tof-ms对聚苯乙烯微/纳米颗粒检测灵敏度的方法
CN112903805A (zh) * 2021-01-27 2021-06-04 中国科学院生态环境研究中心 代谢酶诱导微纳塑料颗粒生物降解方法及其产物分析方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LUO WEI: "Progress on Co-metabolism of Biorefractory Pollutants", CHINESE JOURNAL OF SOIL SCIENCE, vol. 43, no. 6, 1 December 2012 (2012-12-01), pages 1 - 7, XP055954674, DOI: 10.19336/j.cnki.trtb.2012.06.041 *
SAMAK NADIA A.; JIA YUNPU; SHARSHAR MOUSTAFA M.; MU TINGZHEN; YANG MAOHUA; PEH SUMIT; XING JIANMIN: "Recent advances in biocatalysts engineering for polyethylene terephthalate plastic waste green recycling", ENVIRONMENT INTERNATIONAL., PERGAMON PRESS., US, vol. 145, 25 September 2020 (2020-09-25), US , XP086301339, ISSN: 0160-4120, DOI: 10.1016/j.envint.2020.106144 *
SHEHU DAYYABU, ALIAS ZAZALI: "Dechlorination of polychlorobiphenyl degradation metabolites by a recombinant glutathione S-transferase from Acidovorax sp. KKS102", FEBS OPEN BIO, ELSEVIER, US, vol. 9, no. 3, 18 November 2017 (2017-11-18), US , pages 408 - 419, XP055954671, ISSN: 2211-5463, DOI: 10.1002/2211-5463.12405 *

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