Nature has provided a fantastic array of enzymes that are responsible for essential biochemical functions but not usually suitable for technological applications. Not content with the natural repertoire, protein engineering holds promise to extend the applications of improved enzymes with tailored properties. However, engineering of robust proteins remains a difficult task since the positive mutation library may not cooperate to reach the target function in most cases owing to the ubiquity of epistatic effects. The main demand lies in identifying an efficient path of accumulated mutations. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to improve the robustness of a PETase from Ideonella sakaiensis. A systematic clustering analysis combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the redesign of a variant, DuraPETase, which exhibits an apparent melting temperature that is drastically elevated by 31 °C and a strikingly enhanced degradation toward semicrystalline poly(ethylene terephthalate) (PET) films (30%) at mild temperatures (over 300-fold). Complete biodegradation of 2 g/L microplastics to water-soluble products under mild conditions is also achieved, opening up opportunities to steer the biological degradation of uncollectable PET waste and further conversion of the resulting monomers to high-value molecules. The crystal structure revealed the individual mutation match with the design model. Concurrently, synergistic effects are captured, while epistatic interactions are alleviated during the accumulation process. We anticipate that our design strategy will provide a broadly applicable strategy for global optimization of enzyme performance.

Mixing oppositely charged hydrophilic polyelectrolytes is the simplest path to constructing a polyampholyte gel that is useful as a soft tissue scaffold for binding enzymes in their native state. The swelling and viscoelastic properties of such a synthetic polyampholyte gel coacervate, constructed from polyions of different charge density, are reported in water with various amounts of NaCl salt. When constructed, this coacervate is roughly 70% water and 15% of each polyion, nearly charge balanced. If salt is removed from the surrounding supernatant, the gel swells owing to the weak charge imbalance because small amounts of salt screen electrostatic repulsions. If instead more salt is added to this coacervate, the gel behaves as any polyampholyte gel, swelling as salt is added because the excess salt screens the electrostatic attractions and eventually this leads to redissolving the coacervate. The amount of salt needed to redissolve the coacervate increases with polyion molecular weight. To our surprise, we discovered that the small charge imbalance within the coacervate grows with the molecular weight of the more strongly charged polyion.

Viscoelastic, dielectric, and rheo-optical behavior was examined for miscible blends of high-M cis-polyisoprene (PI) and poly(p-tert-butylstyrene) (PtBS). The slow dielectric relaxation of the blends was exclusively attributed to the global motion of the PI chains having the type-A dipoles. The PI and PtBS chains behaved as the fast and slow (low- and high-friction) components and were well entangled with each other. The dynamics of these chains changed significantly with temperature T. At high T, the blend exhibited two-step entanglement plateau of the storage modulus G′(ω), and the plateaus at high and low angular frequencies (ω) were attributed, with the aid of the dielectric data, to the entanglement among all component chains and that between the PtBS chains, respectively. The entanglement length a characterizing the high-ω plateau was well described by a simple mixing rule based on the number fraction n of the Kuhn segments of the components, a = n PI a PI bulk + n PtBS a PtBS bulk. This result was consistent with the current molecular picture relating the entanglement density to the packing length p (≅a/20). The complex moduli G* of the blends in the high-ω plateau zone were well described by a simple blending law combined with this mixing rule of a, which was consistent with the rheo-optical data. At low T, the blend exhibited the Rouse-like power-law behavior of storage and loss moduli, G′ = G′′∝ ω1/2, in the range of ω where the high-ω plateau was supposed to emerge. This lack of the high-ω plateau was attributed to retardation of the Rouse equilibration of the PI chain over the entanglement length a due to the hindrance from the slow PtBS chains: The PI and PtBS chains appeared to be equilibrated cooperatively/simultaneously at a rate essentially determined by PtBS. The Rouse equilibration time, evaluated from the G* data of the blend, was just moderately shorter than the dielectrically determined relaxation time of PI. Thus, the high-ω plateau zone was too narrow to be resolved experimentally, and the PI chains relaxed almost immediately after their Rouse equilibration (retarded by PtBS). This PI relaxation activated the constraint release (CR) relaxation of PtBS to dilate the entanglement mesh for PtBS. A simple model considering the Rouse equilibration and CR/dilation processes described the G* data of the blend surprisingly well, lending support to the molecular picture of the cooperative/simultaneous Rouse equilibration of the PI and PtBS chains. The model calculation was consistent with the rheo-optical data, which lent further support to this molecular picture.