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 O2 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 (O2 and NADH) on the activity. Intraparticle O2-sensing analysis revealed the superior performance of the enzyme immobilized at the outer surface in terms of effective supply of O2. Furthermore, the co-immobilization 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.
Highlights
Enzyme immobilization is a key enabling technology in synthetic, analytical, and environmental chemistry
We have optimized the immobilization of NADH oxidase (NOX) from a thermophilic organism on porous carriers to mitigate those substrate mass transport issues that limit the catalytic performance of an immobilized biocatalyst
We first engineered the spatial distribution of NOX across the porous surface using two different carriers based on agarose and acrylic materials
Summary
Enzyme immobilization is a key enabling technology in synthetic, analytical, and environmental chemistry. Fold, through selecting the hydrophilic carriers like agarose, locating the enzymes at the outer surface of the microbead carriers, operating the heterogeneous biocatalyst under optimal mixing conditions (magnetic stirring), and confining both enzymes and substrates within the same carrier particle This outstanding activity enhancement was possible through mitigating both oxygen and NADH mass transfer restrictions, giving rise to an immobilized NOX whose apparent specific activity is 11 ± 2 U/mg an effectiveness of roughly 100%. Compared with the free NOX measured under the same aeration, temperature, and pH conditions and using the same substrate concentrations
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