The influence of the microenvironment within enzyme-support complexes on the activity of Burkholderia cepacia lipase (BCL) immobilized on silica (SiO₂-BCL) remains insufficiently understood. Without data on these interactions, it is challenging to determine whether modifications to the support material or immobilization protocols are needed for optimal enzyme activity and stability. Besides evaluating catalytic performance in esterification reactions, enzyme-support interactions were characterized using ²⁹Si NMR, FTIR, BET, DSC, TGA, SEM-EDS, and molecular docking simulations. In this study, NMR²⁹Si-based structural analysis revealed significant protein-surface (pore wall) interaction networks. The immobilization resulted in 88.6% efficiency and a protein loading of 17.72 mg·g^-1, enabling further structural and functional characterization. Molecular docking elucidated the interaction mechanisms between silica functional groups (Qⁿ sites) and BCL binding residues. Docking simulations indicated that the Q3 group interacts with the catalytic residue Ser87, potentially hindering substrate access to the active site. Spectroscopic and morphological analyses confirmed this interaction and correlated it with a significant decrease in enzymatic activity. Experimental evidence and FTIR analyses demonstrated that the increase in the α-helix content of SiO₂-BCL correlated with the observed decrease in catalytic productivity. BCL exhibited a significantly higher esterification productivity (150.82 μmol·h^-1·mg^-1) than SiO₂-BCL (28.10 μmol·h^-1·mg^-1), confirming the importance of optimal enzyme conformation for catalytic efficiency. The results highlight that, beyond the support's composition, the spatial orientation and specific interactions with functional groups are critical determinants of catalytic efficiency. This integrative approach may guide the rational design of enzyme-based biocatalysts using mesoporous materials.

This study reports the development of a sustainable biocatalyst system for free fatty acid (FFA) production from cocoa bean shell (CBS) oil using Burkholderia cepacia lipase (BCL). CBS was explored as both a support material and a reaction substrate. Six immobilized systems were prepared using organic (CBS), inorganic (silica), and hybrid (CBS–silica) supports via physical adsorption or covalent binding. Among them, the covalently immobilized enzyme on CBS (ORG-CB) showed the most balanced performance, achieving a catalytic efficiency (Ke) of 0.063 mM⁻¹·min⁻¹ (18.6% of the free enzyme), broad pH–temperature tolerance, and over 50% activity retention after eight reuse cycles. Thermodynamic analysis confirmed enhanced thermal resistance for ORG-CB (Ed = 32.3 kJ mol⁻¹; ΔH‡ = 29.7 kJ mol⁻¹), while kinetic evaluation revealed that its thermal deactivation occurred faster than for the free enzyme under prolonged heating. In application trials, ORG-CB reached 60.1% FFA conversion from CBS oil, outperforming the free enzyme (49.9%). These findings validate CBS as a dual-function material for enzyme immobilization and valorization of agro-industrial waste. The results also reinforce the impact of immobilization chemistry and support composition on the operational and thermal performance of biocatalysts, contributing to the advancement of green chemistry strategies in enzyme-based processing.