Before crystallization and cross-linking, these mutants were characterized regarding their specific activity, enantioselectivity and thermal stability for comparison with the wild-type enzyme (Table ). This analysis revealed a similar activity and enantioselectivity of HheG D114C for formation of azidoalcohol 2b and a similar melting temperature (Tm) as determined for HheG wild type. In contrast, variant M45C displayed significantly reduced specific activity, while product 2b was obtained with higher enantiomeric excess. As replacement of M45 by cysteine significantly altered the biochemical characteristics of this variant compared to wild-type HheG, this variant was not investigated further.