Quantitative Liquid Chromatography‐Tandem Mass Spectrometry Analysis Reveals Product Selectivity and Substrate‐Accessibility Limits in Enzymatic Depolymerization of Nylon‐6,6, Nylon‐11, and Nylon‐12

Accurate quantification of reaction products from enzymatic nylon depolymerization is challenging due to the lack of strong intrinsic chromophores in the soluble products. In this work, we present a rapid LC-MS/MS-based method that enables sensitive, robust analysis of products from the enzymatic deconstruction of nylon-6,6, nylon-11, and nylon-12. Application of this method revealed distinct product selectivities across a range of nylonases, with maximum depolymerization extents of 0.64, 0.72, and 0.04 wt%, for nylon-6,6, nylon-11, and nylon-12, respectively. Reaction plateau experiments indicate that limited substrate accessibility, rather than enzyme deactivation or product inhibition, is a major factor constraining nylon-6,6, nylon-11, and nylon-12 depolymerization, as has been previously reported for nylon-6. The approach allows systematic evaluation of nylonase performance, providing a robust platform to support the development of nylon-active biocatalysts.