Robbmelendez6936

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.For decades, polyurethanes (PUR) have mainly been synthesized for long-term applications and are therefore highly persistent in the environment. Proper waste disposal approaches, including recycling techniques, must be developed to limit the accumulation of PUR in the environment. Evaluation of enzymatic polyurethane degradation is needed for the development of enzymatic recycling. A series of techniques has been carefully implemented to monitor the biotic and abiotic degradation of PUR. Both the degraded polymer and the degradation products are analyzed to obtain a complete overview of the degradation.In the past years, several serine hydrolases such as cutinases, esterases and lipases have shown the ability to degrade not only natural polymers but also synthetic polyesters, even aromatic representatives like polyethylene terephthalate (PET). Hence, cutinases and related ester hydrolases have become very important to be applied in the biocatalytic plastic recycling as green alternative to chemical recycling as well as to the functionalization of polyester surfaces in order to change superficial properties like hydrophobicity or hydrophilicity. Sorption characteristics of the enzymes to the polymers have turned out to be a crucial process for efficient polymer hydrolysis. Hence, special attention was paid on tuning the sorption of the enzymes to the hydrophobic polymers. Engineering of the enzyme surface, fusion of hydrophobic substrate-binding domains or truncation of domains hindering the access of the polymer to the enzyme has led to significant improvement of sorption processes and consequently increased activity on the bulky substrate. Finally, the combination of engineering approaches has proved that they can bring additional advantages in improving the enzyme activity when used in a synergistic manner.Resource stewardship and sustainable use of natural resources is mandatory for a circular plastic economy. The discovery of microbes and enzymes that can selectively degrade mixed-plastic waste enables to recycle plastics. Knowledge on how to achieve efficient and selective enzymatic plastic degradation is a key prerequisite for biocatalytic recycling of plastics. Wild-type natural polymer degrading enzymes such as cellulases pose often selective non-catalytic binding domains that facilitate a targeting and efficient degradation of polymeric substrates. Recently identified polyester hydrolases with synthetic polymer degrading activities, however, lack in general such selective domains. Inspired by nature, we herein report a protocol for the identification and engineering of anchor peptides which serve as non-catalytic binding domains specifically toward synthetic plastics. The identified anchor peptides hold the promise to be fused to known plastic degrading enzymes and thereby enhance the efficiency of biocatalytic plastic recycling processes.Microplastic (plastic smaller than 5mm in size) is ubiquitous around the world both in the ocean and the freshwater system. Due to their potential serious negative impact on marine organisms and human beings, marine microplastics have attracted worldwide attention in the past decade. Information and knowledge of the spatial and temporal distribution of marine microplastics are crucial for accurately assessing our current and future environmental health conditions. This is also important for developing mitigation plans and measures to protect our environment. Since the measured microplastic pollution level is closely related to the sampling methods and identification techniques, it is important to employ standardized sampling and analysis operation procedures for cross-comparison. In this chapter, we present the basic sampling, sample pretreatment and microplastic identification techniques involved in microplastic pollution assessment and discuss the adaptability of different sampling and pretreatment methods. The pros and cons of different techniques are also discussed.Biocatalysis has recently emerged as a powerful and eco-friendly technology in waste plastic recycling, especially for the widely used polyethylene terephthalate (PET). So far, however, a high-throughput screening assay specifically toward PET-hydrolyzing activity has rarely been applied. This hinders the identification of new polyester hydrolases and their variants with adequate activities fulfilling the requirements for industrial applications. Darovasertib price This chapter describes the detailed procedure for assaying terephthalate as a major product of enzymatic PET hydrolysis in a 96-well microtiter plate format. Using PET nanoparticles derived readily from waste food packaging as a substrate, an active thermophilic PET hydrolase was clearly distinguished from an inactive variant by a Fenton chemistry-mediated fluorimetric detection. The assay uses enzymes in crude cell lysates, obtained by a simple freeze-thaw protocol. The experimental work validates the applicability of this method for screening mutant libraries of novel PET hydrolases and will thus facilitate the identification of promising variants useful for effective plastic waste recycling.The biocatalytic degradation of polyethylene terephthalate (PET) by thermophilic microbial enzymes has recently emerged as an option for a future eco-friendly recycling process for plastic waste, as it occurs under mild conditions and requires no harmful additives. In this chapter, we present a brief overview of solution and solid-state nuclear magnetic resonance (NMR) spectroscopic methods for the characterization of composition and chemical microstructure of PET and also associated chain dynamics over multiple time scales. Such detailed information provides an understanding of the enzymatic PET degradation mechanism by polyester hydrolases at the molecular level.