Strategies for designing biocatalysts with new functions.

The engineering of natural enzymes has led to the availability of a broad range of biocatalysts that can be used for the sustainable manufacturing of a variety of chemicals and pharmaceuticals. However, for many important chemical transformations there are no known enzymes that can serve as starting templates for biocatalyst development. These limitations have fuelled efforts to build entirely new catalytic sites into proteins in order to generate enzymes with functions beyond those found in Nature. This bottom-up approach to enzyme development can also reveal new fundamental insights into the molecular origins of efficient protein catalysis. In this tutorial review, we will survey the different strategies that have been explored for designing new protein catalysts. These methods will be illustrated through key selected examples, which demonstrate how highly proficient and selective biocatalysts can be developed through experimental protein engineering and/or computational design. Given the rapid pace of development in the field, we are optimistic that designer enzymes will begin to play an increasingly prominent role as industrial biocatalysts in the coming years.

Biocatalysis: landmark discoveries and applications in chemical synthesis.

Biocatalysis has become an important tool in chemical synthesis, allowing access to complex molecules with high levels of activity and selectivity and with low environmental impact. Key discoveries in protein engineering, bioinformatics, recombinant technology and DNA sequencing have contributed towards the rapid acceleration of the field. This tutorial review explores enzyme engineering strategies and high-throughput screening approaches that have been applied for the discovery and development of enzymes for synthetic application. Landmark developments in the field are discussed and have been carefully selected to highlight the diverse synthetic applications of enzymes within the pharmaceutical, agricultural, food and chemical industries. The design and development of artificial biocatalytic cascades is also examined. This tutorial review will give readers an insight into the landmark discoveries and milestones that have helped shape and grow this branch of catalysis since the discovery of the first enzyme.

Utilising a visual image quality metric to optimise spectacle prescriptions for eyes with keratoconus.

PURPOSE: To compare optical performance, visual performance, and patient-perceived quality of vision with: (1) spectacles determined using subjective refraction and (2) spectacles determined using an objective optimisation method based on wavefront aberration data for eyes with keratoconus. METHODS: Thirty-seven eyes (20 subjects) with keratoconus underwent both subjective refraction and uncorrected wavefront aberration measurement. Wavefront aberration data were used to objectively identify a sphero-cylindrical refraction that optimised the visual image quality metric visual Strehl ratio (VSX). The two refractions were assembled in trial frames and worn by the subject in a random order. High-contrast visual acuity (VA), letter contrast sensitivity (CS), and the patient's short-term subjective preference were recorded for each prescription. RESULTS: Median magnitude of the dioptric difference (a measure of similarity between the subjective and objective refractions) was 2.77 D (range = 0.21-20.44 D, first quartile = 1.02 D, third quartile = 4.36 D). Sixty-eight per cent of eyes had better VA with the objective refraction and 32% of eyes gained more than one line of VA. Monocularly, objective refraction was preferred 68% of the time when looking at a distant acuity chart and 76% of the time when viewing a real-world dynamic scene. CONCLUSIONS: Objective refraction based on visual image quality derived from wavefront aberration data can be valuable in the determination of monocular spectacle refractions for individuals with keratoconus.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2 ) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization.

Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 °C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylCK variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, Tm = 87.4 °C, 16.4 °C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

GeneORator: An Effective Strategy for Navigating Protein Sequence Space More Efficiently through Boolean OR-Type DNA Libraries.

Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N ( Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structure-containing regions (276 out of a total 495 amino acids) to screen for improvements in kcat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 1010-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein's sequence space. OR-type libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.

Managing hyperglycemia and diabetes in patients receiving enteral feedings: A health system approach.

Evidence of poor outcomes in hospitalized patients with hyperglycemia has led to new and revised guidelines for inpatient management of diabetes. As providers become more aware of the need for better blood glucose control, they are finding limited guidance in the management of patients receiving enteral nutrition. To address the lack of guidelines in this population, Duke University Health System has developed a consistent practice for managing such patients. Here, we present our practice strategies for insulin use in patients receiving enteral nutrition. Essential factors include assessing the patients' history of diabetes, hyperglycemia, or hypoglycemia and timing and type of feedings. Insulin practices are then designed to address these issues keeping in mind patient safety in the event of abrupt cessation of nutrition. The outcome of the process is a consistent and safe method for glucose control with enteral nutrition.