Catalytic capsids: the art of confinement

Abstract

In the cell, enzymes are almost always spatially confined in crowded and tightly controlled cellular compartments. The entrapment of enzymes in artificial nanoreactors as biomimetic systems can be expected to contribute to the understanding of the activity and the interactions of enzymes in confined spaces. The capsid of the Cowpea Chlorotic Mottle virus (CCMV) represents such an artificial nanoreactor that can be used to encapsulate multiple proteins in its interior. Employing a controlled encapsulation process we are able to load a precise number of proteins (Pseudozyma antarctica lipase B and EGFP) into the CCMV capsid and to study their activity. In the case of the enzyme, our results indicate that the apparent overall reaction rate increases upon encapsulation and is almost independent of the number of enzymes in the capsid. These observation are the result of the extremely high confinement molarity of the enzyme inside the capsid (M conf ¼ $ 1 mM) leading to very rapid formation of the enzyme-substrate complex. These results highlight the importance of small volumes for efficient multi-enzyme cascade catalysis.

Enzyme Entrapped in Polymer‐Modified Nanopores: The Effects of Macromolecular Crowding and Surface Hydrophobicity

“…The loading of PCL per unit volume of the nanopores (L PCL , in mgcm À3 ), which was similar to the confinement molarity of an enzyme, as defined by Cornelissen and co-workers, [24] was evidently positively correlated with the polymer content (Table 1). This result is comparable to the increase in the degree of crowding in enzyme solution systems through the addition of crowding agents.…”

“…It has been demonstrated that macromolecular crowding could stabilize the folded (native) state of enzyme, restrain the dissociation of multiple enzyme, increase the effective concentrations of the enzyme molecules, and modulate the affinity between the enzyme and the substrate whilst inducing alteration in the enzyme structure. [16,[20][21][22][23][24] Abstract: Macromolecular crowding is an ubiquitous phenomenon in living cells that significantly affects the function of enzymes in vivo. However, this effect has not been paid much attention in the research of the immobilization of enzymes onto mesoporous silica.…”

“…However, the activity enhancement of macromolecular crowding is not tenable under all crowding conditions. As is known in protein solution systems, elevating the macromolecular crowding to a certain point causes the reaction velocity to be slowed, owing to diffusion resistance; [24] moreover, the high degree of crowding in the nanopores of PCL-PMA-4.8-MS could also cause serious diffusion limitations and lower the enzyme catalytic efficiency, as observed in the much lower pore parameters of PCL-PMA-4.8-MS than other samples (Table 1 and the Supporting Information, Figure S3). Tailoring the nanopores of mesoporous silica with PMA polymers can be described as killing two birds with one stone.…”

“…In our scenario, this result could be represented by an increase in PCL loading per unit volume of the nanopores (see above), which can shift the equilibrium towards the formation of the enzymeÀsubstrate complex, thereby accelerating the reaction velocity. [19,24,55] Moreover, Jiang and Guo reported that, in protein solution systems, macromolecular crowding could improve the intrinsic catalytic efficiency of an enzyme by enhancing its affinity for the substrates, as reflected by an observable decrease in the Michaelis-Menten constant (K m ). [20] This result could also contribute to increased enzyme activity in our heterogeneous system.…”

Controlling the loading of protein nanocages

“…[17] Although protein cages are not commonly considered as subcellular compartments, it surprisingly became more apparent that different organisms use protein cages as primitive organelles, [18,19] nanoreactors, [20] or storage compartments [8]. Furthermore, their combined roles in nature suggest that these compartments are permeable to substrate (and product) diffusion for their cargo.…”