Application of electrospun chitosan-based nanofibers as immobilization matrix for biomolecules.

Nanofiber meshes from electrospun chitosan, highly modified with biotin and arylazides, are well-suited for application as enzyme immobilization matrices. To test this, catalytically active biomolecules were immobilized onto photocrosslinked nanofibrous nonwovens consisting mainly of biotinylated fungal chitosan and a small amount (10 w%) of poly ethylene oxide. In this study, we show that over 10 µg eugenol oxidase per milligram dry polymer matrix can be loaded on UV-crosslinked chitosan nanofibers. We further demonstrate that bound enzyme activity can be fully retained for over 7 days of storage at ambient conditions in aqueous buffer. Samples loaded at maximum enzyme carrying capacity were tested in a custom-made plug-flow reactor system with online UV-VIS spectroscopy for activity determination. High wettability and durability of the hydrophilic chitosan support matrix enabled continuous oxidation of model substrate vanillyl alcohol into vanillin with constant turnover at flow rates of up to 0.24 L/h for over 6 h. This proves the above hypothesis and enables further application of the fibers as stacked microfluidic membranes, biosensors, or structural starting points for affinity crosslinked enzyme gels. KEY POINTS: • Biotinylated chitosan-based nanofibers retain enzymes via mild affinity interactions • Immobilized eugenol oxidase shows high activity and resists continuous washing • Nanofiber matrix material tolerated high flow rates in a continuous-flow setup.

G-type Halohydrin Dehalogenases Catalyze Ring Opening Reactions of Cyclic Epoxides with Diverse Anionic Nucleophiles.

Halohydrin dehalogenases are promiscuous biocatalysts, which enable asymmetric ring opening reactions of epoxides with various anionic nucleophiles. However, despite the increasing interest in such asymmetric transformations, the substrate scope of G-type halohydrin dehalogenases toward cyclic epoxides has remained largely unexplored, even though this subfamily is the only one known to display activity with these sterically demanding substrates. Herein, we report on the exploration of the substrate scope of the two G-type halohydrin dehalogenases HheG and HheG2 and a newly identified, more thermostable member of the family, HheG3, with a variety of sterically demanding cyclic epoxides and anionic nucleophiles. This work shows that, in addition to azide and cyanide, these enzymes facilitate ring-opening reactions with cyanate, thiocyanate, formate, and nitrite, significantly expanding the known repertoire of accessible transformations.

Position 123 of halohydrin dehalogenase HheG plays an important role in stability, activity, and enantioselectivity.

HheG from Ilumatobacter coccineus is a halohydrin dehalogenase with synthetically useful activity in the ring opening of cyclic epoxides with various small anionic nucleophiles. This enzyme provides access to chiral ß-substituted alcohols that serve as building blocks in the pharmaceutical industry. Wild-type HheG suffers from low thermostability, which poses a significant drawback for potential applications. In an attempt to thermostabilize HheG by protein engineering, several single mutants at position 123 were identified which displayed up to 14 °C increased apparent melting temperatures and up to three-fold higher activity. Aromatic amino acids at position 123 resulted even in a slightly higher enantioselectivity. Crystal structures of variants T123W and T123G revealed a flexible loop opposite to amino acid 123. In variant T123G, this loop adopted two different positions resulting in an open or partially closed active site. Classical molecular dynamics simulations confirmed a high mobility of this loop. Moreover, in variant T123G this loop adopted a position much closer to residue 123 resulting in denser packing and increased buried surface area. Our results indicate an important role for position 123 in HheG and give first structural and mechanistic insight into the thermostabilizing effect of mutations T123W and T123G.