Join the prestigious International Conference on Biological Inorganic Chemistry (ICBIC) in Long Beach, California in 2025. Discover the latest advancements in the field of biological inorganic chemist...

Hydrogenases allow microorganisms to consume and produce hydrogen gas (H2). Although most hydrogenases are oxygen (O2)-sensitive, recent studies show …

Ultrahigh-throughput methods such as flow cytometry are ideal tools for the directed evolution of enzymes by enabling the screening of up to 109 variants per day. In this study, we developed an assay based on fluorescence-activated cell sorting (FACS) for the detection and engineering of amidase activity in whole cells. The assay establishes a stable genotype–phenotype linkage by coupling coumarin-based hydrolysis products to intracellular glutathione via a recombinantly coexpressed glutathione S-transferase. To demonstrate the applicability of the FACS-based assay, we engineered an amidase from Sphingomonas alpina (SaAmd) by screening combinatorial libraries with multiple amino acid positions randomized simultaneously. SaAmd variants containing proximal double mutations exhibited not only almost 5-fold improved activity against structurally different amide substrates but also coevolved promiscuous carbamate- and ester-hydrolyzing activities, which exceeded the wildtype activity up to 6-fold. Importantly, triple variants featuring distal mutations in three highly flexible loop regions, displayed up to 16-fold enhanced specific activities toward small molecules containing highly stable N-aryl amide and carbamate bonds. These motifs are commonly used as protecting groups for amines in organic synthesis but can also be found in environmental contaminants like pesticides and plastic waste. Therefore, the developed FACS-assisted assay has great potential to accelerate the engineering of amidases for versatile biotechnological applications.

Explore the hidden forces shaping our world, from outer space to deep within the Earth. Dr. Staples reveals how solar storms from the Sun can dazzle with a…

Molecules known as stereoisomers possess identical numbers and types of atoms, which are oriented differently in space. Cis–trans isomerization of proline, a distinctive case of stereoisomerism in pep...

The R2-like ligand binding oxidase (R2lox) forms a novel tyrosine-valine crosslink upon O2 activation, reflecting an overall two-electron oxidation re…

The study of high-valent organometallic nickel compounds has gained considerable interest recently, primarily driven by the development of nickel-catalyzed alkyl–alkyl cross-coupling reactions that ar...