Preparation of artificial gold metalloenzymes with enantioselective reductase-like activity by combining genetic engineering and protein chemistry

The conjugation of Au complexes with proteins and enzymes, generating new types of artificial metalloenzymes, has proven to be interesting and effective to obtain materials with improved properties such as higher stability, catalytic activity and selectivity. In this work, a novel method has been developed for the synthesis and design of artificial gold metalloenzymes at 50ºC in aqueous media, using two genetically modified variants of the alkalophilic lipase Geobacillus thermocatenulatus (GTL). The only difference between these two enzymatic variants is the possible coordination of the Au via active site (GTL-114) or Lid site (GTL-193). TEM analysis of the metalloenzymes revealed the formation of Au (0) nanoparticles with different structures (nanowires, nanorods, nanoshells, nanoclusters) and sizes depending on the mutant and the pH used during the synthesis. Characterisation by fluorescence spectroscopy demonstrated that conjugation of the enzyme to Au altered the tertiary structure of the protein. On the other hand, all metalloenzymes showed excellent reductase like activity. Finally, the selectivity of the enzyme-Au bioconjugates was tested in the asymmetric reduction of acetophenone to 1-phenylethanol in aqueous medium at room temperature. The protein environment played a key role in the reactivity and selectivity of the metalloenzymes, obtaining chiral nanoparticles with an enantiomeric excess of up to 39% towards (R)-1-phenylethanol after two hours of reaction using GTL-114 pH 10 as catalyst.