(129f) A Novel Approach to Enzyme Immobilization By Crosslinking Phase-Separated Biomolecular Condensates

Introduction: Enzymes are often immobilized to facilitate their industrial use, but existing immobilization techniques can impede the effectiveness of the biocatalyst. An alternative method of enzyme immobilization would be to utilize liquid-liquid phase separation (LLPS) of proteins. Intrinsically disordered regions can be appended to folded proteins to facilitate LLPS. When proteins are sequestered in biomolecular condensates, they would be in close enough proximity to be crosslinked. This could be implemented as a novel approach to enzyme immobilization. We show the feasibility of this method using several proteins fused to the intrinsically disordered RGG domain derived from the Caenorhabditis elegans LAF-1 protein.

Materials and Methods: All protein constructs were cloned into modified pET-29a(+) plasmids, expressed in BL21(DE3) Escherichia coli, lysed by sonication, purified by Ni-NTA affinity chromatography, and buffer exchanged. The RGG condensates were crosslinked using bis(sulfosuccinimidyl)suberate (BS3) according to the manufacturer’s (Thermo Fisher) protocol. BsADH-RGG activity was assayed at 60°C in an Agilent Cary 3500 UV-Vis spectrophotometer. The assay conditions were 50 mM sodium phosphate, 40 mM ethanol, and 5 mM NAD+.

Results: In the following experiments, we had four main goals: (1.) demonstrate that biomolecular condensates can be crosslinked into solid microparticles, (2.) achieve methods to control microparticle size, (3.) capture other proteins into microparticles using specific recruitment motifs, and (4.) directly immobilize enzymes with our novel method. After mixing phase-separated RGG-GFP-RGG and BS3, FRAP data showed that there was limited diffusion within the condensates, the crosslinked condensates had a significantly higher contact angle on untreated glass surfaces (p < 0.0001), and the crosslinked condensates no longer showed temperature-dependent phase behaviors. These results indicated that the crosslinking successfully solidified the biomolecular condensates. By varying the initial RGG-GFP-RGG concentration and allowing the liquid droplets to coalesce before crosslinking, the resulting particle size can be controlled (p < 0.0001). In our experiments, the median particle diameters ranging from 0.55 µm to 3.38 µm depending on the conditions used. RGG-SpyCatcher-RGG biomolecular condensates could also be crosslinked with BS3. When RGG-SpyCatcher-RGG microparticle condensates were incubated with either SpyTagged or non-SpyTagged GFP, SpyTagged GFP was greatly enriched in the crosslinked condensates compared to a non-SpyTagged GFP variant, even after successive washes. BsADH-RGG retained activity after crosslinking and was able to convert ethanol to acetaldehyde.

Conclusion: These experiments show the feasibility of immobilizing enzymes by crosslinking biomolecular condensates. The lack of fluorescence recovery, increase in contact angle, and loss of temperature-dependent phase behavior indicate that biomolecular condensates can be crosslinked to form solid microparticles. The size of the particles can be controlled to suit the user’s needs, such as decreasing their size to reduce diffusional limitations or increasing their size to improve their ease of handling. By encoding specific protein-protein interactions, the crosslinked condensates could be used as a carrier material for enzyme immobilization. Furthermore, this provides an opportunity to immobilize and purify enzymes in a single step, which would greatly streamline immobilization procedures. Crosslinked enzyme condensates show residual catalytic activity, this method could be utilized to directly immobilize enzymes. Future research would be directed towards optimizing this novel enzyme immobilization approach to maximize enzyme activity and immobilization yield.