Design of the Enzyme–Carrier Interface to Overcome the O₂ and NADH Mass Transfer Limitations of an Immobilized Flavin Oxidase

Abstract

Understanding how the immobilization of enzymes on solid carriers affects their performance is paramount for the design of highly efficient heterogeneous biocatalysts. An efficient supply of substrates onto the solid phase is one of the main challenges to maximize the activity of the immobilized enzymes. Herein, we apply advanced single-particle analysis to decipher the optimal design of an immobilized NADH oxidase (NOX) whose activity depends both on O 2 and NADH concentrations. Carrier physicochemical properties and its functionality along with the enzyme distribution across the carrier were implemented as design variables to study the effects of the intraparticle concentration of substrates (O 2 and NADH) on the activity. Intraparticle O 2 -sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O 2 . Furthermore, the coimmobilization of NADH and NOX within the tuned surface of porous microbeads increases the effective concentration of NADH in the surroundings of the enzyme. As a result, the optimal spatial organization of NOX and its confinement with NADH allow a 100% recovery of the activity of the soluble enzyme upon the immobilization process. By engineering these variables, we increase the NADH oxidation activity of the heterogeneous biocatalyst by up to 650% compared to NOX immobilized under suboptimal conditions. In conclusion, this work highlights the rational design and engineering of the enzyme−carrier interface to maximize the efficiency of heterogeneous biocatalysts.

Dual Valorization of Lignin as a Versatile and Renewable Matrix for Enzyme Immobilization and (Flow) Bioprocess Engineering

“…This effect might be the result of a faster incubation with the enzyme (1 h) that promotes only the ionic adsorption of the enzyme on the outer surface of the PEI‐lignin. These results are in agreement with previously reported studies where enzymes immobilized predominantly on the exposed surface of the support (as opposed to those that diffuse through the porous matrix) display a higher activity due to better mass transfer ability from the bulk to the catalyst [33–35] . However, more sophisticated studies would be needed to decipher the exact effect observed.…”

“…To avoid undesired hydrophobic interactions during enzyme immobilization, several additives (ethanol, SDS, glycerol, triton) were trialed during the immobilization process [35,36] . Nevertheless, no significant improvements over the resulting activity (U g −1 ) of the biocatalyst were detected (Figure S7).…”

“…These results are in agreement with previously reported studies where enzymes immobilized predominantly on the exposed surface of the support (as opposed to those that diffuse through the porous matrix) display a higher activity due to better mass transfer ability from the bulk to the catalyst. [ 33 , 34 , 35 ] However, more sophisticated studies would be needed to decipher the exact effect observed. Nevertheless, the particle size of the TALD‐lignin is very similar to the acrylic microbeads as observed by microscopy, which facilitates comparison with previous work (Figure S4B).…”

“…We hypothesized that the homogeneous enzyme distribution and associated lower recovered activity may be an effect of the hydrophobic interactions between the aromatic structure of the lignin and the hydrophobic residues of the enzyme, as described for other immobilized biocatalysts. [35] To test this, the aldehyde groups of lignin were reduced to avoid covalent binding of the Gs‐Lys6DH (Figure S5), and the enzyme was then left in contact with the support which resulted in more than 90 % of immobilization yield, clearly showing a strong hydrophobic affinity. The interactions between the enzyme and the lignin were shown to be reversible by SDS‐PAGE analysis (Figure S5).…”

“…To avoid undesired hydrophobic interactions during enzyme immobilization, several additives (ethanol, SDS, glycerol, triton) were trialed during the immobilization process. [ 35 , 36 ] Nevertheless, no significant improvements over the resulting activity (U g −1 ) of the biocatalyst were detected (Figure S7). Additionally, lignin was functionalized with glucosamine to increase the hydrophilicity of the support following a modified protocol reported before for functionalization of acrylic resins.…”