聚对苯二甲酸乙二醇酯降解酶的研究进展

聚对苯二甲酸乙二醇酯(Polyethylene terephthalate,PET)因其良好的耐用性和可塑性,已在世界范围内的工业领域和日常生活中得到广泛应用。目前自然环境中大量PET使用废弃物的积累和迁移给全球生态系统带来了严重负担,因此PET的降解问题已成为全球性的热点问题。微生物酶降解法目前被认为是一种理想绿色PET降解方法,有希望应用于大规模降解PET废弃物降解处理。传统的PET降解酶主要包括脂肪酶、酯酶和角质酶等,但这些酶的PET降解活性相对不高。近期科学家从Ideonella sakaiensis细菌中分离了一种新型水解酶PETase,能够特异性高效降解PET。本文从结构生物学角度对多种PET降解酶进行梳理,重点总结了新近发现的PETase催化机制,为发展改造更有效的PET降解酶提供理论依据。     

聚对苯二甲酸乙二醇酯生物降解的研究进展

塑料广泛存在于人类的日常生活中，在给人们生活带来便利的同时，大量塑料废物也给环境带来很大压力。聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)是一种以石油为原料的高分子热塑性材料，因其具有耐用、透明度高、重量轻等特性，已成为世界上使用最广泛的塑料之一。由于PET具有结构复杂以及难降解的特性，可在自然界中长期存在，不仅对全球生态环境造成严重的污染，而且已经威胁到人类健康。如何对PET废弃物进行降解已成为全球的难题之一，相较于物理法和化学法，生物降解法是目前处理PET废弃物最为绿色环保的方法。本文分别介绍了微生物和生物酶对PET生物降解的研究现状、PET的生物降解途径、PET生物降解机制以及PET降解酶的分子改造等方面的研究，并对如何实现PET的高效降解、寻找和改造可降解高结晶度PET的微生物或酶进行展望，为PET的生物降解微生物或酶的有效开发应用提供理论依据。     

聚对苯二甲酸乙二醇酯(PET)降解酶的研究进展

聚对苯二甲酸乙二醇酯(Polyethylene terephthalate,PET)因其优越的物理化学性质,在各个领域尤其在包装产业得到了广泛的应用.然而,由于使用后的PET处置不当,对生态环境造成了严重威胁.目前生物降解尤其是酶促降解已成为极具可行性且环境友好的PET处理方式.本文集中梳理和总结了近年来已报道的PET...     

聚对苯二甲酸乙二醇酯水解酶检测方法的研究进展

聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)是应用最广泛的合成聚酯之一。由于PET不易降解,在环境中积累,对陆地、水生生态系统以及人类健康构成严重威胁。基于生物酶催化的生物降解策略为PET回收利用提供了一种绿色途径,在过去20年间,已发现了多种PET水解酶,并通过蛋白质工程等手段来改善这些酶的降解性能,但是目前仍未找到适合大规模工业应用的PET水解酶。利用传统的检测方法筛选PET水解酶是一个缓慢而复杂的过程。为了促进PET酶法回收的工业化应用,需要研发高效的检测方法。近年来,研究人员开发了多种表征PET水解酶的分析方法。本文总结了可用于筛选PET水解酶的检测方法,如高效液相色谱法、紫外吸光度法和荧光激活液滴分选法等,并对其在筛选PET水解酶的应用方面进行了展望。     

聚对苯二甲酸乙二醇酯水解酶研究进展

塑料自20世纪首次合成以来给人类生活带来了极大的便利。然而,塑料稳定的高分子结构导致了塑料废弃物的持续堆积,对生态环境和人类健康均造成严重威胁。聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]是产量最高的一种聚酯类塑料,近年来PET水解酶的相关研究展现出生物酶法对塑料进行降解、回收的巨大潜力,也为塑料生物降解机制研究建立了参考范例。本文综述了不同微生物来源的PET水解酶及其PET降解能力,阐述了最具代表性的PET水解酶—IsPETase降解PET的催化机理,并总结了近年来通过酶工程改造而获得的高效降解酶,为未来的PET降解机制研究、PET高效降解酶的进一步挖掘和改造提供参考。     

基于磷脂酶水解圈定向筛选高效聚对苯二甲酸乙二醇酯降解酶

【目的】目前自然环境中聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)废弃物的积累严重威胁生态健康,因此PET的降解问题已成为全球性的热点问题。生物酶法降解PET技术以其绿色环保而备受关注,但天然PET降解酶的催化活性普遍偏低,亟待进一步定向改造。现阶段定向进化为快速提高PET降解酶催化性能提供了可能,其中筛选方法是成功获得高性能突变体的关键所在。本研究旨在提出一种新型高效灵敏的筛选方法并应用于褐色喜热裂孢菌(Thermobifida fusca)来源角质酶Tfu-0883的定向改造,以期快速获得PET降解活性提高的突变体。【方法】基于易错PCR构建突变体文库,涂布于卵黄磷脂平板,以水解圈的大小作为筛选指标获得PET降解活性提高的突变体;对突变体进行酶学定性并筛选出潜在的分子改造位点,最终获得高性能突变体。【结果】从卵黄磷脂平板中挑取水解圈直径最大的单菌落,即突变体H10(N2D/D94H/A149E),其PET降解能力是野生型的1.5倍,最适温度与pH分别为60℃和8.0。突变体H10中第2位和第149位氨基酸残基远离底物结合凹槽,其突变会导致酶蛋白稳定性下降;第94位氨基酸残基则位于底物结合凹槽附近,由负电荷氨基酸Asp突变为正电荷氨基酸His,有利于吸附在带负电荷的PET表面,是突变体H10降解能力提升的关键因素;随后将野生型的第94位氨基酸残基Asp分别突变为His及同为正电荷且空间位阻更小的Lys和Arg,突变体D94H、D94K和D94R对PET降解能力均有提升,其中,突变体D94K降解PET能力是野生型的3.6倍。【结论】本研究基于磷脂酶水解圈构建了一种新的PET降解酶定向筛选方法,以此获得了降解活性提高的突变体,并证实角质酶Tfu-0883第94位氨基酸残基位点具有提升其PET降解活性的潜在能力。     

木霉属真菌的生物降解及生物转化作用研究进展

木霉（Trichoderma spp．）属于半知菌亚门、丝孢纲、丝孢目,粘孢菌类,是一类具有很大应用生产潜力的真菌,目前国际上已查明并命名的木霉属真菌,共计60种和2个变型,国内正式发表的木霉菌有10种,记录种名20个。木霉属真菌的生物降解、转化功能与分泌纤维素酶、葡聚糖酶、几丁质酶、脂肪酶、木聚糖酶等酶的能力相关,综述了木霉属真菌生物降解和生物转化底物及其他方面的研究进展。     

聚乳酸的生物降解

聚乳酸(polylactic acid, PLA)因其良好的理化性能、生物相容性和生物降解性而备受关注，已被认为是石油基塑料最具潜力的替代者，但在实际应用中仍然存在降解缓慢循环周期长的问题，因此对PLA的生物降解深入研究对于解决塑料垃圾污染和缓解能源危机至关重要。近年来，有关微生物(放线菌、细菌和真菌)和酶(蛋白酶、脂肪酶、酯酶和角质酶)降解PLA的研究已经取得了一定的进展。本文从降解微生物、降解酶和降解机制等方面综述了PLA生物降解的研究进展，并展望了PLA生物降解研究未来的发展趋势。     

来源于嗜热氢化杆菌的新型对苯二甲酸双(羟乙)酯水解酶的表达纯化与酶学性质

石化来源的聚对苯二甲酸乙二酯(polyethylene terephthalate,PET)被广泛用于矿泉水瓶、食品包装和纺织品等领域,因其在自然界中不易分解,大量使用后的PET废弃物造成了严重的环境污染与资源浪费。使用生物酶法对PET废弃物进行解聚,并对解聚产物进行升级循环利用是进行塑料污染治理的重要方向之一,其中关键的是PET水解酶的解聚效率。对苯二甲酸双(羟乙基)酯(bis(hydroxyethyl)terephthalate,BHET)是PET生物酶解的中间产物,其累积是限制PET水解酶催化效率的一个重要因素,BHET水解酶和PET水解酶的联用能提升PET的整体水解效率。来源于嗜热氢化杆菌(Hydrogenobacter thermophilus)的双烯内酯酶(HtBHETase)对BHET有显著水解效果,将该酶在大肠杆菌(Escherichia coli)中进行重组表达并纯化后,对其酶学性质进行了研究。结果显示,HtBHETase对短碳链的酯类如对硝基苯酚乙酸酯催化活性较高,HtBHETase以BHET为底物时的最适反应pH值和最适反应温度分别为5.0和55℃;该酶有较好的热稳定性,经80℃的条件处理1 h仍能保持80%以上活性,显示出了良好的热稳定性,HtBHETase有在PET塑料生物解聚中使用的潜力,本研究为推动生物酶法降解PET提供了新的参考。     

评论：塑料结合模块促进聚对苯二甲酸乙二醇酯的酶法降解

塑料的大量生产和无节制的使用已造成严重的环境污染。为了减少塑料废物对环境的影响,近年来塑料酶法降解已成为国内外研究者关注的热点。例如,通过蛋白质工程策略提高塑料降解酶催化活性和热稳定性,进一步提高酶法降解的效率。另外,通过融合酶策略将塑料结合模块与塑料降解酶融合,也可以促进塑料降解。近期发表在期刊Chem Catalysis的一项研究表明,采用碳水化合物结合模块融合策略可以在低浓度(<10 wt%)的底物聚对苯二甲酸乙二醇酯[poly(ethylene terephthalate),PET]中提高塑料降解酶的活性。但是在高浓度底物(10 wt%−20 wt%)中,该策略无法提高PET的酶法降解。该项研究对于采用塑料结合模块促进酶法降解塑料具有重要的指导意义。     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Active Site Flexibility as a Hallmark for Efficient PET Degradation by I. sakaiensis PETase

Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.     

Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis

Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETase^S93M, IsPETase^W159F, and IsPETase^N241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/β-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.     

Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SP_MalE:IsPETase and pET22b-SP_LamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.     

A high-throughput expression and screening platform for applications-driven PETase engineering

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.     

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three‐fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.     

Glacier as a source of novel polyethylene terephthalate hydrolases

Polyethylene terephthalate (PET) is a major component of microplastic contamination globally, which is now detected in pristine environments including Polar and mountain glaciers. As a carbon-rich molecule, PET could be a carbon source for microorganisms dwelling in glacier habitats. Thus, glacial microorganisms may be potential PET degraders with novel PET hydrolases. Here, we obtained 414 putative PET hydrolase sequences by searching a global glacier metagenome dataset. Metagenomes from the Alps and Tibetan glaciers exhibited a higher relative abundance of putative PET hydrolases than those from the Arctic and Antarctic. Twelve putative PET hydrolase sequences were cloned and expressed, with one sequence (designated as GlacPETase) proven to degrade amorphous PET film with a similar performance as IsPETase, but with a higher thermostability. GlacPETase exhibited only 30% sequence identity to known active PET hydrolases with a novel disulphide bridge location and, therefore may represent a novel PET hydrolases class. The present work suggests that extreme carbon-poor environments may harbour a diverse range of known and novel PET hydrolases for carbon acquisition as an environmental adaptation mechanism.     

Thermophilic whole-cell degradation of polyethylene terephthalate using engineered Clostridium thermocellum

Polyethylene terephthalate (PET) is a mass-produced synthetic polyester contributing remarkably to the accumulation of solid plastics waste and plastics pollution in the natural environments. Recently, bioremediation of plastics waste using engineered enzymes has emerged as an eco-friendly alternative approach for the future plastic circular economy. Here we genetically engineered a thermophilic anaerobic bacterium, Clostridium thermocellum, to enable the secretory expression of a thermophilic cutinase (LCC), which was originally isolated from a plant compost metagenome and can degrade PET at up to 70°C. This engineered whole-cell biocatalyst allowed a simultaneous high-level expression of LCC and conspicuous degradation of commercial PET films at 60°C. After 14 days incubation of a batch culture, more than 60% of the initial mass of a PET film (approximately 50 mg) was converted into soluble monomer feedstocks, indicating a markedly higher degradation performance than previously reported whole-cell-based PET biodegradation systems using mesophilic bacteria or microalgae. Our findings provide clear evidence that, compared to mesophilic species, thermophilic microbes are a more promising synthetic microbial chassis for developing future biodegradation processes of PET waste.     

来源于海洋宏基因组塑料降解酶Ple629的耐热性提升改造

石化来源的聚酯类塑料如聚对苯二甲酸乙二醇酯(polyethylene terephthalate,PET)以及聚己二酸/对苯二甲酸丁二醇酯(polybutylene adipate terephthalate,PBAT)等已被广泛使用,但由于它们在自然界中难以降解或生物降解周期较长导致了严重的环境污染,因此对这些塑料废弃物的处理是亟待解决的问题之一。从循环经济的角度考虑,利用生物酶法对聚酯类塑料如PET或PBAT等的废弃物进行解聚,再将解聚产物进行循环利用,是一个很有潜力的研究方向。探究近年来关于聚酯塑料降解酶的报道发现,高活性且耐高温的降解酶会有更大的潜在优势。来自海洋微生物宏基因组的中温塑料降解酶Ple629,在常温下对聚酯类塑料PET和PBAT均有较好的降解活力,但由于不耐受高温,限制了其潜在应用。在前期获得Ple629三维结构的基础上,本研究基于结构比对及能量设计,找到了一些潜在提升其热稳定性的位点进行改造设计,并对突变体进行了表达纯化和热稳定性测定。突变体V80C和D226C/S281C的熔点温度(Tm)值分别提升了5.2℃和6.9℃,突变体D226C/S281C的活性也比野生型酶提高了1.5倍,为后续对Ple629的进一步改造提供了思路和依据。