Stable and Promiscuous Galactose Oxidases Engineered by Directed Evolution, Atomistic Design, and Ancestral Sequence Reconstruction.

Galactose oxidase (GOase) is a versatile biocatalyst with a wide range of potential applications, ranging from synthetic chemistry to bioelectrochemical devices. Previous GOase engineering by directed evolution generated the M-RQW mutant, with unprecedented new-to-nature oxidation activity at the C6-OH group of glucose, and a mutational backbone that helped to unlock its promiscuity toward other molecules, including secondary alcohols. In the current study, we have used the M-RQW mutant as a starting point to engineer a set of GOases that are very thermostable and that are easily produced at high titers in yeast, enzymes with latent activities applicable to sustainable chemistry.

In vitro evaluation of the influence of ultrasonic activation on physicochemical properties of three endodontic repair materials.

The influence of ultrasonic activation (UA) on the physicochemical properties of setting time (n = 5), volumetric stability (n = 10), alkalizing activity/calcium ions release (pH/Ca) (n = 10) and calcium/phosphate-rich substance formation in simulated body fluid (Hank's balanced salt solution; HBSS) of three cements was evaluated: white MTA-Angelus (MTA), MTA Repair HP (MTAHP) and Biodentine (BIO). It was observed that UA influenced the setting times (min) distinctly, delaying the initial setting time of MTA (40.8/64.

Enzyme Benchmarking with Polyethylene Furanoate Soluble Scaffolds for Directed Evolution of PEFases.

Plastic waste is a major threat in our industrialized world and is driving research into bioplastics. The success of biobased polyethylene furanoate (PEF) as a viable alternative to polyethylene terephthalate (PET) of fossil origin will depend on designing effective enzymes to break it down, aiding its recycling. Here, a panel of fungal and bacterial cutinases were functionally expressed in a tandem yeast expression system based on and .

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy.

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic HO. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O instead of toxic HO, and exhibits activity selective to the surface plasma membrane of both living and fixed cells.

Structural Insights and Reaction Profile of a New Unspecific Peroxygenase from Produced in a Tandem-Yeast Expression System.

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from (UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications.