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.

Abstract

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.

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