The Core–Shell Structure, Not Sugar, Drives the Thermal Stabilization of Single-Enzyme Nanoparticles

Abstract

Trehalose is widely assumed to be the most effective sugar for protein stabilization, but exactly how unique the structure is and the mechanism by which it works are still debated. Herein, we use a polyion complex micelle approach to control the position of trehalose relative to the surface of glucose oxidase within cross-linked and noncross-linked single-enzyme nanoparticles (SENs). The distribution and density of trehalose molecules in the shell can be tuned by changing the structure of the underlying polymer, poly (N-[3-(dimethylamino)propyl] acrylamide (PDMAPA). SENs in which the trehalose is replaced with sucrose and acrylamide are prepared as well for comparison. Isothermal titration calorimetry, dynamic light scattering, and asymmetric flow field-flow fraction in combination with multiangle light scattering reveal that two to six polymers bind to the enzyme. Binding either trehalose or sucrose close to the enzyme surface has very little effect on the thermal stability of the enzyme. By contrast, encapsulation of the enzyme within a cross-linked polymer shell significantly enhances its thermal stability and increases the unfolding temperature from 70.3 °C to 84.8 °C. Further improvements (up to 92.8 °C) can be seen when trehalose is built into this shell. Our data indicate that the structural confinement of the enzyme is a far more important driver in its thermal stability than the location of any sugar.

Synthesis and Application of Trehalose Materials

“…The same trends could be found when measuring enzyme activity after exposure to 60 °C for 60 min, with remaining activity increasing from 25% to 65% after cross-linking and to 85% when encapsulated within a trehalose shell. 74 …”

“…To form SENs, the polymers were mixed with the enzyme glucose oxidase (GOx) and then cross-linked by a radical process using trehalose acrylate and bis(acrylamide). 74 Moving beyond RAFT polymerization, atom transfer radical polymerization (ATRP), and specifically activators generated by electron transfer (AGET) ATRP, has been employed by our group and others to prepare poly(trehalose) polymers. For example, an insulin−poly(trehalose) conjugate was synthesized by installing a nitrophenyl carbonate-activated ATRP initiator at a lysine residue (LysB29), HPLC purifying the singly modified insulin, and using AGET ATRP to "graft from" the protein (Figure 8b).…”

“…The same trends could be found when measuring enzyme activity after exposure to 60 °C for 60 min, with remaining activity increasing from 25% to 65% after cross-linking and to 85% when encapsulated within a trehalose shell. 74 Other recent examples of trehalose-based hydrogels have shown expanded uses for skin burn treatment 105 and for cryopreservation and to act as a cell scaffold. 22,106 Recently, microgels were used as soft matrices for 3D cell culture 99 or in microfluidic microchambers.…”

Structurally analogous trehalose and sucrose glycopolymers – comparative characterization and evaluation of their effects on insulin fibrillation

“…These glycomonomers were synthesized precisely by employing a trimethylsilyl protecting group strategy (Schemes S1 and S2 †) described in our recent reports. 47,48 The details and a short discussion of this synthesis, as well as the relevant 1D and 2D NMR spectra (Fig. S1-S16 †), are given in the ESI.…”

Immobilization-Stabilization of β-Glucosidase for Implementation of Intensified Hydrolysis of Cellobiose in Continuous Flow Reactors

“…Theirs is a synergistic action with depolymerizing enzymes to avoid or mitigate product inhibition over the diverse enzymatic routes leading to glucose. Curiously, we find in nature β-glucosidases that are devoid of the classical cellulose binding domain (CBM) typical in exoglucanases for example, but others have this binding domain [7], suggesting that they can immobilized near the source of cellobiose and cellooligosaccharides of low molecular weight that are their substrates [8] while possibly benefitting from the stabilizing environment created by the confinement near the surface of the reacting solid substrate [9,10].…”