High-throughput strategies for the discovery and engineering of enzymes for biocatalysis

Abstract

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Microbioreactor Systems for Accelerated Bioprocess Development

“…Typically, relevant performance indicators (PIs) are determined, including biomass and product titers ( c X , c P ), specific rates for biomass growth ( μ ), substrate consumption ( q S ), and product formation ( q P ) as well as specific yields relating substrate conversion and product formation to biomass growth ( Y X/S , Y P/X ). These PIs are usually obtained by fitting experimental data to single mathematical expressions or small process models, preferably in an automated way . By comparing such PIs with reference values or defined benchmark settings, new strain variants and/or cultivation conditions can be found (see Figure ).…”

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

“…Enzyme screening campaigns are frequently the bottleneck of drug, biomarker, and biocatalyst discovery programs, making high‐throughput enzyme activity assays essential to accelerating research in these areas . Such screens often rely on fluorogenic or chromogenic enzyme substrate analogues, coupled assays, or biosensors, which—while high‐throughput—are unsuitable for monitoring many important enzymatic transformations …”

Recent Trends for Discovery and Enhancement of Enzyme Function: A Review

“…10 A). Recently, different efficient HTS methods were developed such as microfluidic and fluorescence activated cell sorting (FACS) devices that have the ability to screenup to 10 7 events per hour (Jacques et al, 2017). In microfluidic devices, enzymatic reaction is carried out in nano-to picoliter reaction volumes, and the compounds to be screened are injected consecutively into the microfluidic capillary that is connected with a proper detector (Wohlgemuth et al, 2015).…”

“…In microfluidic devices, enzymatic reaction is carried out in nano-to picoliter reaction volumes, and the compounds to be screened are injected consecutively into the microfluidic capillary that is connected with a proper detector (Wohlgemuth et al, 2015). On the other hand, the flow cytometry-based screening system is based on detection of fluorescent product in a compartment such as cells-aqueous/oil/aqueous emulsions (Pitzler et al, 2014 andJacques et al, 2017). FACS based screening system with an in vitro compartmentalization in double emulsion was developed to screen proteases variants libraries generated by directed evolution approach for improvement of the enzyme resistance to various proteases inhibitors (Tu et al, 2011).…”