Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a “strategic” hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 β-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.

Enzyme catalysis is used to generate approximately 50,000 tons of value-added chemical products per year. Nearly a quarter of this production requires a stoichiometric cofactor such as NAD+/NADH. Given that NADH is expensive, it would be beneficial to regenerate it in a way that does not interfere with the enzymatic reaction. Water electrolysis could provide the proton and electron equivalent necessary to electrocatalytically convert NAD+ to NADH. However, this form of electrocatalytic NADH regeneration is challenged by the formation of inactive NAD2 dimers, the use of high overpotentials or mediators, and the long-term electrochemical instability of the enzyme during electrolysis. Here, we show a means of overcoming these challenges by using a bioelectrocatalytic palladium membrane reactor for electrochemical NADH regeneration from NAD+. This achievement is possible because the membrane reactor regenerates NADH through reaction of hydride with NAD+ in a compartment separated from the electrolysis compartment by a hydrogen-permselective Pd membrane. This separation of the enzymatic and electrolytic processes bypasses radical-induced NAD+ degradation and enables the operator to optimize conditions for the enzymatic reaction independent of the water electrolysis. This architecture, which mechanistic studies reveal utilizes hydride sourced from water, provides an opportunity for enzyme catalysis to be driven by clean electricity where the major waste product is oxygen gas.

Synthesis of sensitive coumarin α-GalNAc glycosides as substrates for droplet-based screening of GalNAcases.

Modified 1,5-dideoxy-1,5-imino- d -xylitol analogues with different substitution patterns involving position C-1 and/or the ring nitrogen were prepared, which were designed to serve as precursors for the preparation of iminoxylitol-based ligands and tools for the elucidation and modulation of human lysosomal β -glucocerebrosidase. Biological evaluation of the synthesized glycomimetics with a series of glycoside hydrolases revealed that these substitution patterns elicit excellent β -glucosidase selectivities. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00706-019-02427-1) contains supplementary material, which is available to authorized users.

A magnetic nano-adsorbent was synthesized via the radical polymerization of methyl acrylate on modified Fe3O4 MNPs, followed by amidation of the methyl ester groups using pentaethylenehexamine, to create active sites for adsorption of anionic dyes.