Enzymatic Bioconjugation: A Perspective from the Pharmaceutical Industry.

Enzymes have firmly established themselves as bespoke catalysts for small molecule transformations in the pharmaceutical industry, from early research and development stages to large-scale production. In principle, their exquisite selectivity and rate acceleration can also be leveraged for modifying macromolecules to form bioconjugates. However, available catalysts face stiff competition from other bioorthogonal chemistries. In this Perspective, we seek to illuminate applications of enzymatic bioconjugation in the face of an expanding palette of new drug modalities. With these applications, we wish to highlight some examples of current successes and pitfalls of using enzymes for bioconjugation along the pipeline and try to illustrate opportunities for further development.

Evolution of a highly active and enantiospecific metalloenzyme from short peptides.

Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ~ 106 M-1 s-1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.

Droplet confinement and leakage: Causes, underlying effects, and amelioration strategies.

The applicability of droplet-based microfluidic systems to many research fields stems from the fact that droplets are generally considered individual and self-contained reaction vessels. This study demonstrates that, more often than not, the integrity of droplets is not complete, and depends on a range of factors including surfactant type and concentration, the micro-channel surface, droplet storage conditions, and the flow rates used to form and process droplets. Herein, a model microfluidic device is used for droplet generation and storage to allow the comparative study of forty-four different oil/surfactant conditions. Assessment of droplet stability under these conditions suggests a diversity of different droplet failure modes. These failure modes have been classified into families depending on the underlying effect, with both numerical and qualitative models being used to describe the causative effect and to provide practical solutions for droplet failure amelioration in microfluidic systems.