Biomimetic Strategy To Reversibly Trigger Functionality of Catalytic Nanocompartments by the Insertion of pH-Responsive Biovalves

Abstract

We describe an innovative strategy to generate catalytic compartments with triggered functionality at the nanoscale level by combining pH-reversible biovalves and enzyme-loaded synthetic compartments. The biovalve has been engineered by the attachment of stimuli-responsive peptides to a genetically modified channel porin, enabling a reversible change of the molecular flow through the pores of the porin in response to a pH change in the local environment. The biovalve functionality triggers the reaction inside the cavity of the enzyme-loaded compartments by switching the in situ activity of the enzymes on/off based on a reversible change of the permeability of the membrane, which blocks or allows the passage of substrates and products. The complex functionality of our catalytic compartments is based on the preservation of the integrity of the compartments to protect encapsulated enzymes. An increase of the in situ activity compared to that of the free enzyme and a reversible on/off switch of the activity upon the presence of a specific stimulus is achieved. This strategy provides straightforward solutions for the development of catalytic nanocompartments efficiently producing desired molecules in a controlled, stimuli-responsive manner with high potential in areas, such as medicine, analytical chemistry, and catalysis.

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics

“…The relative fluorescence intensity of the resorufin‐like product inside individual E‐GPMVs when the encapsulated AOs were equipped with OmpF (Figure C), and when deficient of OmpF (Figure D), allowed the functionality of the MFs to be established. Amplex UltraRed was chosen as an enzymatic substrate to evaluate whether AOs preserved their functionality inside MF cavities because it can readily diffuse through their lipid membrane, but not through the polymersome membranes, unless they are equipped with OmpF pores . Permeabilization of AOs by OmpF allowed the nonfluorescent substrate Amplex UltraRed to reach the encapsulated enzyme HRP within the cavity where it was converted to a fluorescent resorufin‐like product in the presence of H 2 O 2 .…”

“…Amplex UltraRed was chosen as an enzymatic substrate to evaluate whether AOs preserved their functionality inside MF cavities because it can readily diffuse through their lipid membrane, but not through the polymersome membranes, unless they are equipped with OmpF pores . Permeabilization of AOs by OmpF allowed the nonfluorescent substrate Amplex UltraRed to reach the encapsulated enzyme HRP within the cavity where it was converted to a fluorescent resorufin‐like product in the presence of H 2 O 2 . As the in situ enzymatic activity inside polymersomes was preserved in physiological media, such as human serum, the activity of AOs is not affected by their encapsulation inside E‐GPMVs.…”

“…[29,51] Permeabilization of AOs by OmpF allowed the nonfluorescent substrate Amplex UltraRed to reach the encapsulated enzyme HRP within the cavity where it was converted to a fluorescent resorufin-like product in the presence of H 2 O 2 . [51] As the in situ enzymatic activity inside polymersomes was preserved in physiological media, such as human serum, [30] the activity of AOs is not affected by their encapsulation inside E-GPMVs. Therefore, the reaction rate limiting step is no longer determined by enzymatic affinity, but by the diffusion of H 2 O 2 through the lipid membrane of the MFs.…”

Biomolecule–polymer hybrid compartments: combining the best of both worlds

“…77 OmpF can also be chemically modified prior to the insertion to obtain pores that open upon pH change 82 or biovalves able to control the opening and closing of the pore. 83 Interestingly, the successful incorporation of membrane proteins can be achieved even if the polymeric membrane is thicker than biological lipid membranes and thicker than the hydrophobic part of the protein, which suggests a conformational adaptation between the polymer and the protein. 34 However, this phenomenon of adaption requires specific properties of the polymeric membrane: high flexibility of block-copolymers is essential to achieve membrane fluidity that is similar to natural phospholipidic bilayers.…”

Toward Functional Synthetic Cells: In‐Depth Study of Nanoparticle and Enzyme Diffusion through a Cross‐Linked Polymersome Membrane

“…Thus, possibly interesting enzymes and nanoparticles are simply too large to diffuse through the narrow channels. Furthermore, biopores/channels are able to induce only a passive membrane transport, apart from one very recent achievement in integrating pH‐responsive biovalves in a polymersome membrane . As a consequence, alternative approaches for the membrane transport of nanometer‐sized biomacromolecules (enzymes, proteins, protein complexes, etc.)…”