<p>Lignocellulosic biomass represents a promising renewable resource for the production of valuable chemicals, attributed to its intricate chemical structure. Novozym 435 (N435), an immobilized lipase, acts as a biocatalyst in transesterification reactions, enhancing the compatibility of lignin in diverse industrial applications by selectively converting this substrate into high-value raw materials. The effective recycling of this biocatalyst fosters sustainability, both ecologically and economically, significantly reducing operational costs and environmental impacts. This study evaluated the efficacy of N435 as a biocatalyst in the glycerolysis of lignin to yield lignopolyols, particularly focusing on the influence of lignin concentrations on the biocatalyst’s performance. Findings indicate a notable decline in enzymatic activity at lignin levels exceeding 5 wt%. The investigation employs enzyme activity assays, optical microscopy, and Fourier Transform Infrared Spectroscopy (FTIR), revealing adverse interactions between lignin and the enzyme during several reaction cycles at higher concentrations. Nevertheless, lignopolyols derived from 5 wt% lignin displayed satisfactory performance, with a predictable decline in enzymatic activity, which can be anticipated under challenging reaction conditions, suggesting utility in subsequent batch processes. The results underscore the necessity of evaluating lignin concentration’s impact on enzymatic activity within transesterification, offering critical insights for the sustainable application of lignin in biotechnological processes. The pioneering study explores the reuse of N435 in transesterification reactions involving lignin, suggesting avenues for further research into its application in producing lignopolymers. Therefore, the enzymatic glycerolysis of lignin using N435 is an innovative and essential approach to maximize sustainability in the biochemical industry.</p>