Structural insight and engineering of a plastic degrading hydrolase Ple629.

Polyethylene terephthalate (PET) is one of the most abundantly produced synthetic polyesters. The vast number of waste plastics including PET has challenged the waste management sector while also posing a serious threat to the environment due to improper littering. Recently, enzymatic PET degradation has been shown to be a viable option for a circular plastic economy, which can mitigate the plastic pollution. While protein engineering studies on specific PET degradation enzymes such as leaf-branch compost cutinase (LCC), Thermobifida sp. cutinases and Ideonella sakaiensis PETase (IsPETase) have been extensively published, other homologous PET degrading enzymes have received less attention. Ple629 is a polyester hydrolase identified from marine microbial consortium having activity on PET and the bioplastic polybutylene adipate terephthalate (PBAT). In order to explore its catalytic mechanism and improve its potential for PET hydrolysis, we solved its crystal structure in complex with a PET monomer analogue, and validated its structural and mechanistic similarity to known PET hydrolases. By structural comparisons, we identified some hot spot positions described in previous research on protein engineering of PET hydrolases. We substitute these amino acid residues in Ple629, and obtained variants with improved activity and thermo-stability. The most promising variant D226A/S279A exhibited a more than 5.5-fold improved activity on PET nanoparticles than the wild-type enzyme, suggesting its potential applicability in the biotechnological plastic recycling.

[Identification and characterization of a novel polyamide hydrolase].

The widespread use of non-naturally degradable plastics is causing increasingly serious harm to the environment. Reducing plastic pollutants has become the core of ecological and environment management. Biological methods such as enzymes demonstrate advantages in depolymerizing plastics with mild reaction conditions and recycling of depolymerization products. However, there are few reports on the biological depolymerization of polyamide plastics. In this study, by using 4-nitropropionanilide as the model substrate, we screened against our plastic depolymerase library and obtained a Meiothermus ruber-derived enzyme (MrABH) that can hydrolyze the polyamide bond. We expressed this enzyme in Escherichia coli and purified the protein by affinity chromatography. Furthermore, we investigated the catalytic properties, enzymatic properties, and catalytic products of this enzyme with polyamide as the substrate. MrABH had good stability at pH 8.0-10.0, with the optimal performance at pH 9.0 and 30 ℃. The catalytic performance of this enzyme for ester bonds and amide bonds was similar. MrABH can catalyze the depolymerization of PA6 and PA66 to produce monomers and oligomers, demonstrating the potential to be used in the depolymerization and recycling of polyamide.

[Engineering the plastic degradation enzyme Ple629 from marine consortium to improve its thermal stability].

Petrochemical-derived polyester plastics such as polyethylene terephthalate (PET) and polybutylene adipate terephthalate (PBAT) have been widely used. However, the difficulty to be degraded in nature (PET) or the long biodegradation cycle (PBAT) resulted in serious environmental pollution. In this connection, treating these plastic wastes properly becomes one of the challenges of environment protection. From the perspective of circular economy, biologically depolymerizing the waste of polyester plastics and reusing the depolymerized products is one of the most promising directions. Recent years have seen many reports on polyester plastics degrading organisms and enzymes. Highly efficient degrading enzymes, especially those with better thermal stability, will be conducive to their application. The mesophilic plastic-degrading enzyme Ple629 from the marine microbial metagenome is capable of degrading PET and PBAT at room temperature, but it cannot tolerate high temperature, which hampers its potential application. On the basis of the three-dimensional structure of Ple629 obtained from our previous study, we identified some sites which might be important for its thermal stability by structural comparison and mutation energy analysis. We carried out transformation design, and performed expression, purification and thermal stability determination of the mutants. The melting temperature (Tm) values of mutants V80C and D226C/S281C were increased by 5.2 ℃ and 6.9 ℃, respectively, and the activity of mutant D226C/S281C was also increased by 1.5 times compared with that of the wild-type enzyme. These results provide useful information for future engineering and application of Ple629 in polyester plastic degradation.