The use of an in situ immobilization procedure for the immobilization of hyperhalophilic alcohol dehydrogenase in a metal organic framework material is described. The easy and rapid in situ immobilization process enables retention of activity over a broad range of pH and temperature together with a decrease in the halophilicity of the enzyme. The catalytic activity of the immobilized enzyme was studied in nonaqueous solvent mixtures with the highest retention of activity in aqueous solutions of methanol and acetonitrile. The approach demonstrates that this immobilization method can be extended to hyperhalophilic enzymes with enhancements in activity and stability.

An alcohol dehydrogenase from the halophilic archaeon Haloferax volcanii (HvADH2) has been engineered by rational design to broaden its substrate scope towards the conversion of a range of aromatic substrates, including flurbiprofenol, that is an intermediate of the non-steroidal anti-inflammatory drug, flurbiprofen. Wild-type HvADH2 showed minimal activity with flurbiprofenol (11.1 mU/mg). A homology model of HvADH2 was built and docking experiments with this substrate revealed that the biphenyl rings of flurbiprofenol formed strong interactions with residues F85 and F108, preventing its optimal binding in the active site. Mutations at position 85 however did not increase activity. Site directed mutagenesis at position F108 allowed the identification of three variants showing a significant (up to 2.3-fold) enhancement of activity towards flurbiprofenol, when compared to wild-type HvADH2. Interestingly, F108G variant did not show the classic inhibition in the presence of (R)-enantiomer when tested with rac-1-phenylethanol, underling its potential in racemic resolution of secondary alcohols.