Abstract

End-of-life vulcanized rubber tires constitute a highly complex material that currently lacks effective waste management strategies. Among their constituents, the vulcanizing additive N-(1,3-dimethylbutyl)-N '-phenyl-p-phenylenediamine is considered one of the most toxic components of vulcanized rubber elastomers and has attracted increasing scientific attention due to its widespread environmental occurrence and adverse effects on human health and ecosystems. In this context, the dioxygenase Latex clearing protein from Streptomyces sp. K30 is a key enzyme involved in the oxidative cleavage of poly(cis-1,4-isoprene), the main structural component of natural rubber; however, the influence of vulcanizing additives on its catalytic performance remains poorly understood. Here, the effect of the antioxidant 6PPD on LcpK30 activity was evaluated using vitro assays. The presence of 6PPD caused a clear concentration-dependent effect, reducing enzyme activity to from 5.34 +/- 0.38 U/mg to 2.51 +/- 0.16 U/mg at media concentrations representative of commercial tire formulations (6.33 mM) and an activity loss of up to 88% at a higher 6PPD concentration (63.34 mM). These experimental findings were consistent with molecular docking analyses, which revealed a high affinity of 6PPD for catalytically relevant regions of LcpK30, particularly the heme cofactor and key residues located in the E helix of the active site. Overall, the results indicate that 6PPD interferes with efficient LcpK30-substrate complex formation, limiting rubber oxidation and potentially restricting the biodegradation of vulcanized materials. These findings underscore the need to develop strategies for the pretreatment of vulcanized materials, to mitigate the effects of vulcanizing additives on enzymatic rubber degradation.