To achieve the three main objectives:
• 7 work-packages and 24 tasks: scientific, management, training and dissemination were defined in the project proposal; • the tasks were implemented within the Laboratory of Applied and Computational Biocalalysis, Department of Chemical and Pharmaceutical Sciences, University of Trieste, Italy in collaboration with: Esteco S.p.a for the computational support and Department of Life Sciences of University of Trieste and Bioscience Research Center Srl, Orbetello, for the ecotoxicity evaluation studies.
Objective 1 (month 1-month 8)
In this respect computational studies were performed for a series of polyesters and enzymes by using three different computational approaches:
- for substrates modelling and simulation: • for 30 copolymers the physico-chemical properties were predicted using available platforms based on machine learning and softwares; •for 70 polyesters molecular descriptors from 3D Molecular Interaction Fields (MIFs) were generated and evaluated;
- for enzyme modelling: • MD studies were performed for 6 hydrolases at different temperature conditions.
- for enzyme-substrate modelling: • 2 docking methods were applied to the analysis of the selected substrates; • more than 40 copolymers were docked in the catalytic site of the 6 hydrolases using Lamarckian genetic algorithm search method and free energy estimation based on an empirical free-energy force field; • about 40 terpolymers were docked by using Monte Carlo search and rapid gradient-optimization conformational search coupled with a simple scoring function for free energy estimation;
• an automatic workflow using modeFrontier software was developed by integrating specific modelling software; • a series of bio-based monomers and enzymes were screened up and the computational studies results were correlated with experimental data
Objective 2 (month 8-month 13)
The second objective was achieved by implementing:
• the synthesis of 18 polyesters, by using the monomers and hydrolases selected from Objective 1; • experimental reactions in solvent less system, or novel sustainable solvents for 4 of the targeted products. Reaction yields were in the range of 86 and 96%; • complete characterization procedures for all polyesters;
Objective 3 (month 12-month 24)
The third objective was achieved by studying:
• the biodegradability in marine environment of 25 sample (starting bio-based monomers and the polyesters synthesized within Objective 2)
• the ecotoxicity of 18 samples consisting of starting bio-based monomers and the correspondent polyesters enzymatically synthesized within Objective 2. The ecotoxicity was evaluated in: • fresh water in the presence of microalgae and • marine water in the presence of bacteria and microalgae
• the hydrolysis of the synthesized polyesters by 2 commercially available enzymes, obtaining degradation yields up to ~ 90%.
• the recovery of the monomers at laboratory scale to demonstrate the circularity of the concept and strategy developed within the RenEcoPol project as a proof of concept. Two or three steps protocols were developed as a function of the monomers solubility in water. Monomers recovery yields were in the range of 76-98%.
DISSEMINATION
• PUBLICATIONS: 4 papers published in journals with impact factor as open access, cumulative impact factor WoS 2021: 23.575; 1 paper under revision at Sustainable Chemistry and Pharmacy; 2 manuscripts in preparation for publication.
• INTERNATIONAL CONFERENCES :7 prestigious international conferences in: Italy (2), Germany, Austria, Slovenia, Greece and France; 4 workshops organized in Italy, Romania (2) and one organized by ESAB - European Society of Applied Biocatalysis.
