Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes.

Region-Directed Enzyme Immobilization through Engineering Protein Surface with Histidine Clusters.

Enzyme immobilization is a key enabling technology for a myriad of industrial applications, yet immobilization science is still too empirical to reach highly active and robust heterogeneous biocatalysts through a general approach. Conventional protein immobilization methods lack control over how enzymes are oriented on solid carriers, resulting in negative conformational changes that drive enzyme deactivation. Site-selective enzyme immobilization through peptide tags and protein domains addresses the orientation issue, but this approach limits the possible orientations to the N- and C-termini of the target enzyme.

Selective Magnetic Nanoheating: Combining Iron Oxide Nanoparticles for Multi-Hot-Spot Induction and Sequential Regulation.

The contactless heating capacity of magnetic nanoparticles (MNPs) has been exploited in fields such as hyperthermia cancer therapy, catalysis, and enzymatic thermal regulation. Herein, we propose an advanced technology to generate multiple local temperatures in a single-pot reactor by exploiting the unique nanoheating features of iron oxide MNPs exposed to alternating magnetic fields (AMFs). The heating power of the MNPs depends on their magnetic features but also on the intensity and frequency conditions of the AMF.

Solid-Phase Assembly of Multienzyme Systems into Artificial Cellulosomes.

We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads.

Microcompartmentalized Cell-Free Protein Synthesis in Hydrogel μ-Channels.

The rapid demand for protein-based molecules has stimulated much research on cell-free protein synthesis (CFPS); however, there are still many challenges in terms of cost-efficiency, process intensification, and sustainability. Herein, we describe the microcompartmentalization of CFPS of superfolded green fluorescent protein (sGFP) in alginate hydrogels, which were casted into a μ-channel device. CFPS was optimized for the microcompartmentalized environment and characterized in terms of synthesis yield.

Biocatalytic Protein-Based Materials for Integration into Energy Devices.

There is a current need to fabricate new biobased functional materials. Bottom-up approaches to assemble simple molecular units have shown promise for biomaterial fabrication due to their tunability and versatility for the incorporation of functionalities. Herein, the fabrication of catalytic protein thin films by the entrapment of catalase into protein films composed of a scaffolding protein is demonstrated.