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. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

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. In this work, we engineer the surface of two model dehydrogenases to introduce histidine clusters into flexible regions not involved in catalysis, through which immobilization is driven. By varying the position and the histidine density of the clusters, we create a small library of enzyme variants to be immobilized on different carriers functionalized with different densities of various metal chelates (Co2+, Cu2+, Ni2+, and Fe3+). We first demonstrate that His-clusters can be as efficient as the conventional His-tags in immobilizing enzymes, recovering even more activity and gaining stability against some denaturing agents. Furthermore, we find that the enzyme orientation as well as the type and density of the metal chelates affect the immobilization parameters (immobilization yield and recovered activity) and the stability of the immobilized enzymes. According to proteomic studies, His-clusters enable a different enzyme orientation as compared to His-tag. Finally, these oriented heterogeneous biocatalysts are implemented in batch reactions, demonstrating that the stability achieved by an optimized orientation translates into increased operational stability.

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. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.

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. Using a mixture of diluted colloids of MNPs we were able to generate a multi-hot-spot reactor in which each population of MNPs can be selectively activated by adjusting the AMF conditions. The maximum temperature reached at the surface of each MNP was registered using independent fluorescent thermometers that mimic the molecular link between enzymes and MNPs. This technology paves the path for the implementation of a selective regulation of multienzymatic reactions.

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. To extend the scope of this technology, the use of other biocompatible materials (collagen, laponite, and agarose) was explored. In addition, the diffusion of sGFP from the hydrogel microenvironment to the bulk was demonstrated, opening a promising opportunity for concurrent synthesis and delivery of proteins. Finally, we provide an application for this system: the CFPS of enzymes. The present design of the hydrogel µ-channel device may enhance the potential application of microcompartmentalized CFPS in biosensing, bioprototyping, and therapeutic development.

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. Extensive structural and functional characterization of the films provide evidence of the structural integrity, order, stability, catalytic activity, and reusability of the biocatalytic materials. Finally, these functional biomaterials are coupled with piezoelectric disks to fabricate a second generation of bio-inorganic generators. These devices are capable of producing electricity from renewable fuels through catalase-driven gas production that mechanically stimulates the piezoelectric material.