Discovery and Characterization of New Thermostable Nylon-Degrading Enzyme from Thermomicrobiales bacterium

Abstract

A crucial step in the biodegradation of solid polymers is their conversion to soluble oligomers or monomers. In the context of polyamide hydrolysis, NylB, one of the three types of nylonase, functions as an Ahx-oligomer exohydrolase. This thesis reports the characterization of a novel thermostable NylB from an unclassified Thermomicrobiales bacterium, dubbed TmbB, which was identified through targeted genome mining for thermostable polyamide-degrading enzymes. To confirm that our targeted genome mining led to the identification of a thermostable protein, melting point assays conducted with SYPRO Orange revealed TmbB to have a melting temperature of 59 °C. It exhibited a half-life of thermal inactivation of 1.89 hours at 60 °C and the optimal conditions for hydrolyzing Ahx oligomers were found to be pH 8.0 and 50 °C. The catalytic ability to hydrolyze amide bonds was first measured using p-nitrophenyl hexanamide as the amide substrate and the kinetic analysis demonstrated that TmbB has a KM of 0.4 ± 0.09 mM and a kcat of 0.08 ± 0.008 s-1 with this small molecule. To evaluate the hydrolytic activity of TmbB on nylon mimics and authentic nylon substrates, a UPLC-MS-based assay was used to characterize the degradation of several substrates harboring different amide bonds, including nylon-6 and nylon-6,6 polymers, the dimer of nylon-6 and related small molecule (6-hexanamido-N-hexylhexanamide), and laurolactam. TmbB demonstrated activity towards all linear amides but not laurolactam, suggesting that TmbB, like other NylBs, cannot catalyze the hydrolysis of cyclic amides. TmbB hydrolyzed 2.3 % of a 10 mM solution of nylon-6 dimer in 24 hours, whereas complete conversion was observed with 6-(hexanoyl amino) hexanoic acid. Although TmbB is unlikely to be efficient enough to make bio-based nylon recycling competitive with the production of fresh nylons, enzyme engineering and directed evolution could be used to develop variant TmbBs that are catalytically efficient enough to be employed as nylon-degrading biocatalysts.