Protein engineering provides a means to alter protein structure leading to new functions. Much work has focused on the engineering of enzyme active sites to enhance catalytic activity, however there is an increasing trend towards engineering other aspects of biocatalysts as these efforts can also lead to useful improvements. This tutorial discusses recent advances in engineering an enzyme's local chemical and physical environment, with the goal of enhancing enzyme reaction kinetics, substrate selectivity, and activity in harsh conditions (e.g., low or high pH). By introducing stimuli-responsiveness to these enzyme modifications, dynamic control of activity also becomes possible. These new biomolecular and protein engineering techniques are separate and independent from traditional active site engineering and can therefore be applied synergistically to create new biocatalyst technologies with novel functions.

Supramolecular assembly of enzymes into metabolon structures is thought to enable efficient transport of reactants between active sites via substrate channeling. Recombinant versions of porcine citrate synthase (CS), mitochondrial malate dehydrogenase (mMDH), and aconitase (Aco) were found to adopt a homogeneous native-like metabolon structure in vitro. Site-directed mutagenesis performed on highly conserved arginine residues located in the positively charged channel connecting mMDH and CS active sites led to the identification of CS(R65A) which retained high catalytic efficiency. Substrate channeling between the CS mutant and mMDH was severely impaired and the overall channeling probability decreased from 0.99 to 0.023. This work provides direct mechanistic evidence for the channeling of reaction intermediates, and disruption of this interaction would have important implications on the control of flux in central carbon metabolism.

The performance of immobilized enzyme systems is often limited by cofactor diffusion and regeneration. Here, we demonstrate an engineered enzyme capable of utilizing the minimal cofactor nicotinamide mononucleotide (NMN⁺ ) to address these limitations. Significant gains in performance are observed with NMN⁺ in immobilized systems, despite a decreased turnover rate with the minimal cofactor.

Nature's reliance on proteins to carry out nearly all biological processes has led to the evolution of biomolecules that exhibit a seemingly endless range of functions. Much research has been devoted toward advancing this process in the laboratory in order to create new proteins with improved or unique capabilities. The protein-engineering field has rapidly evolved from pioneering studies in engineering protein stability and activity to an application-driven powerhouse on the forefront of emerging technologies in biomedical engineering and biotechnology. A classic protein-engineering technique in the medical field has focused on manipulating antibodies and antibody fragments for various applications. New classes of alternative scaffolds have recently challenged this paradigm, and these structures have been successfully engineered for applications including targeted cancer therapy, regulated drug delivery, in vivo imaging, and a host of others. This review aims to capture recent advances in the engineering of nonimmunoglobulin scaffolds as well as some of the applications for these molecular recognition elements in the biomedical field.