Enzymatic recycling of thermoplastic polyurethanes: Synergistic effect of an esterase and an amidase and recovery of building blocks.

Biological recycling of polyurethanes (PU) is a huge challenge to take up in order to reduce a large part of the environmental pollution from these materials. However, enzymatic depolymerization of PU still needs to be improved to propose valuable and green solutions. The present study aims to identify efficient PU degrading enzymes among a collection of 50 hydrolases. Screenings based on model molecules were performed leading to the selection of an efficient amidase (E4143) able to hydrolyze the urethane bond of a low molar mass molecule and an esterase (E3576) able to hydrolyze a waterborne polyester polyurethane dispersion. Degradation activities of the amidase, the esterase and a mix of these enzymes were then evaluated on four thermoplastic polyurethanes (TPU) specifically designed for this assay. The highest degradation was obtained on a polycaprolactone polyol-based polyurethane with weight loss of 33% after 51 days measured for the esterase. Deep cracks on the polymer surface observed by scanning electron microscopy and the presence of oligomers on the remaining TPU detected by size exclusion chromatography evidenced the polymer degradation. Mixing both enzymes led to an increased amount of urethane bonds hydrolysis of the polymer. 6-hydroxycaproic acid and 4,4'-methylene dianiline were recovered after depolymerization as hydrolysis products. Such building blocks could get a second life with the synthesis of new macromolecular architectures.

Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid.

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.

Directed evolution of a 13-hydroperoxide lyase (CYP74B) for improved process performance.

The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand.