Former approaches in our group to enhance the enantioselectivity of HheG in the ring opening of cyclic epoxides based on Rosetta analysis and active site mutagenesis only yielded single mutants with moderately increased enantioselectivity (e.g. HheG variant I104M achieved a product enantiomeric excess (eeP) of 72% in the azidolysis of cyclohexene oxide (1), unpublished data), which is in agreement with data recently published by Tian et al. As single point mutations on loop3947 of HheG turned out to influence the enantioselectivity of this enzyme as well, we shifted our focus to this loop for further protein engineering. To minimize our screening effort, positions T39 to G47 were initially only replaced by lysine, phenylalanine, cysteine and glutamate to incorporate a set of chemically diverse amino acids. Resulting HheG variants including wild type as well as a negative control (i.e. E. coli haboring an empty pET28a(+) vector) were produced in a 96-deep well plate yielding sufficient amounts of soluble enzyme (see Figure ). This reduced library (in form of cell-free extract, CFE) was then screened in the azidolysis of cyclohexene oxide (1) (Scheme S1), achieving moderate to high conversions with all variants within 2 h of reaction (Figure ). Interestingly, the corresponding eeP values revealed that enantioselectivity of HheG was mainly affected by mutations at loop positions 44 to 46. Thus, nearly all tested variants at those three positions displayed improvements in enantioselectivity compared to HheG wild type (Figure ). HheG M45F even formed azidoalcohol (1S,2S)-2a with a product enantiomeric excess of 96%. As positions T44, M45 and V46 turned out to exert the highest impact on HheG selectivity, completely randomized libraries only on those positions were investigated next. To this end, all possible single variants were generated separately, produced in a 96-deep well plate and screened in the ring opening of cyclohexene oxide (1) with azide (again using CFE). Indeed, almost all variants displayed improved enantioselectivity compared to wild-type HheG (Figure ), while conversion was always high (see Table for absolute values). This result is in line with our reduced library data, confirming the importance of those three positions for HheG enantioselectivity. Interestingly, variants carrying an aromatic residue at position 45 yielded highest product enantiomeric excesses of 86% (M45Y), 92% (M45W) and 96% (M45F) (Table ). This is striking as also aromatic residues at position 123 of HheG had the highest impact on the enzyme's stability and positively influenced enantioselectivity as well, presumably through regulation of the loop3947 dynamics. ) with azide as nucleophile as well as the ring opening of 1 with cyanide. Reactions were performed at 22 °C and 900 rpm in 1 ml 50 mM Tris•SO4, pH 7.0 using either 100 µL (in reactions with 3 and 5) or 200 µL CFE (in reactions with 1), 10 or 20 mM epoxide (1 and 3: 20 mM; 5: 10 mM) and 2 eq sodium azide or cyanide. Samples were taken after 2 h (azide) or 24 h (cyanide) for reactions with 1, after 10 min in case of reactions with 3 and after 30 min for reactions with 5. Samples were extracted and analyzed by achiral and chiral GC. Corresponding absolute eeP values as well as conversion data and E values are given in Tables S1-S6 in the supporting information.