Following the initial screening, P450 BM3 variant (R48L_F82I_F88V_L189Q_E268V) was selected as the template for directed evolution. Amino acids for subsequent mutation were selected either on the basis of their proximity to the active site and substrate access channel, or from previous literature reports suggesting that they enhanced epoxidation activity. Over the course of one round of evolution, 24 positions were subjected to site-saturation mutagenesis, using NNK degenerate codons. Library members were evaluated in cell lysate, using a highthroughput ultra-performance liquid chromatography (UPLC)based assay. The most active (~1%) clones, identified on the basis of conversion to 3a, were then evaluated as purified proteins for improved activity and enantioselectivity. This process revealed 7 single-point mutations, in addition to the 5 already present in the parent enzyme, that were beneficial to both conversion and enantioselectivity: S73Q, A83V, R256L, R256M, R256V, L354V and K435G. Beneficial mutations identified were subsequently combined by DNA shuffling, generating a further 25 mutants. Pleasingly, this gave the triple mutant S73Q_R256V_K435G which was able to achieve conversions 9-fold higher than the original parent enzyme and with 98% enantioselectivity at 0.01 mol% loading on a 2 mM scale (Figure ). Residue 73 sits within the B'-helix and 256 in the I-helix of the haem domain, whilst 435 resides in the Hsheet of the protein (Figure , B). Although point mutations at all three positions have previously been shown to lead to improved performance in epoxidation reactions, the specific mutations identified here have not yet been reported. Time course studies revealed that the evolved enzyme could achieve turnover numbers (TON) >3000, and a turnover frequency (TOF) of more than 10 s -1 (Figure ). These experiments were conducted at low substrate concentration to ensure complete solubility throughout the reaction ([7a] = 0.2mM). Under such conditions, >90% conversion could be achieved in less than 5 mins at 0.1 mol% enzyme loading. This represents an order of magnitude increase relative to the parent enzyme.