Engineering Enantioselectivity in Enzyme‐Catalyzed Reactions

Validating computer simulations of enantioselective catalysis; reproducing the large steric and entropic contributions in Candida Antarctica lipase B

“…The empirical Kazlauskas rule has been used to assess the most likely binding mode of the fast‐reacting enantiomer. That is, while the rule suggests a clear preference for the (R)‐enantiomer of a secondary alcohol, which seems confirmed by experiment, it makes modeling the (S)‐enantiomer almost impossible, as its' large phenyl ring cannot fit in the postulated stereoselectivity pocket, providing no significant conformational changes are involved, while for the R‐enantiomer the large group would point toward bulk solvent.…”

“…The productive and unproductive binding modes proposed for the binding of secondary alcohols to CalB. While the binding mode shown on the left can easily accommodate both substituents, the binding mode on the right cannot accommodate the large substituent L in the postulated pocket, thus making it the slow‐reacting enantiomer according to Kazlauskas …”

“…Optimizing enzymes to catalyze selective enantioselective reactions has a major potential in biotechnology, including in the generation of biocatalysts for efficient synthesis of enantiomerically pure chiral molecules for the production of drugs by the pharmaceutical industry …”

A selection assay for haloalkane dehalogenase activity based on toxic substrates

“…Directed evolution is one option for protein engineering and can be used to generate custom-made enzymes with desired properties for industrial applications (Bornscheuer 2013). In contrast to rational protein design, where an extensive knowledge of the structure and the catalytic mechanism is needed and which only covers a small sequence space, directed evolution can be performed without this information, but it requires suitable high-throughput screening or selection methods (Arnold 1998;Bornscheuer et al 2012;Kazlauskas 2009). …”

“…Alternatives like tryptophane quenching (Tang et al 2003) or the use of isothermal titration calorimetry (Arnold and Volkov 1999) provide a hint of substrate binding and are thus extremely sensitive, but cannot be used for the analysis of mutant libraries as purified enzyme is required. One selection system for dehalogenases was established, but this is restricted to Pseudomonas strains able to grow on long chain alcohols released (Kazlauskas 2009) and was also limited to a small number of substrates (Kawasaki et al 1995). We here describe a concept for the selection of active haloalkane dehalogenase variants, which can be derived from directed evolution approaches or metagenome libraries using typical substrates of this enzyme class.…”