Enantioselectivity is often a major driver for application of enzyme-catalyzed reactions in chemical synthesis. Although a number of native enzymes have been described that generally display high to absolute enantioselectivity in their catalyzed reactions, in the majority of cases the selectivity of an enzyme varies from substrate to substrate. For this reason, protein engineering is often vital to adjust the enantioselectivity of a native enzyme (along with other enzyme characteristics) to fulfill the requirements of an industrial biocatalytic process. Halohydrin dehalogenases (HHDHs) have gained increasing attention in recent years for their application in the synthesis of a plethora of valuable products. Naturally, those enzymes catalyze the reversible dehalogenation of β-haloalcohols with formation of the corresponding epoxides. In the reverse reaction, a range of small C-, O-, Nand S-nucleophiles, such as azide, cyanide, nitrite, cyanate and thiocyanate (known as pseudohalogens), are also accepted for epoxide ring opening in addition to halide ions (Figure ). This enabled the preparation of enantioenriched β-nitroalcohols, β-cyanohydrins, oxazolidinones 15 and thiiranes amongst others, fueled by the recent discovery of many new HHDHs from public sequence databases. In most of these cases, however, protein engineering of the initially identified enzymes with highest activity was still necessary to achieve also high enantioselectivity. Apart from a few examples, most native HHDHs do not display high enantioselectivity as this would contradict their assumed natural function, which is presumably the detoxification and degradation of (naturally occurring) haloalcohols. Many examples for the protein engineering of selected HHDHs, such as HheC from Agrobacterium radiobacter AD1, have been reported in literature that do not only focus on enantioselectivity but also on activity and stability. Of these examples, the majority profited from the previously solved crystal structures of respective enzymes. Thus, crystal structures for members of almost all currently known HHDH subtypes A-G have been reported by now. One of them is HheG from Ilumatobacter coccineus. This enzyme was the first reported HHDH displaying unexpectedly high activity on cyclic as well as other sterically demanding internal epoxide substrates, owing to its much broader and more solvent-exposed active site compared to other HHDHs. Since its discovery in 2017, a few more G-type HHDHs have been characterized, all displaying similar substrate scopes as HheG. Despite this appealing activity towards bulky epoxides, HheG as well as other G-type members also come with some limitations including their insufficient stability for industrial application as well as an only moderate enantioselectivity. The first challenge, the poor (thermal) stability of HheG, was already addressed by us through protein engineering as well as immobilization. Thus, we could demonstrate that the exchange of residue T123 in HheG by aromatic amino acids or glycine resulted in variants with up to 14 K higher apparent melting temperature as well as increased activity, most likely through regulating the dynamics of an Nterminal loop spanning residues 39-47 (Figure ). Interestingly, a slight increase in the enantioselectivity of those HheG variants was observed as well. In a complementary approach to stabilize HheG for application, we recently prepared cross-linked enzyme crystals (CLECs) after crystal contact engineering of HheG to introduce defined cross-linking sites. This yielded highly stable and reusable enzyme preparations with remarkable resistance towards temperature, pH and the presence of organic solvents. Moreover, some of the HheG mutants generated during crystal contact engineering also displayed improved enantioselectivity. This time, respective mutations (M45C and V46K) were directly located on the flexible Nterminal loop, opposite to the catalytic triad of HheG (Figure ). This loop can occur in an open as well as closed conformation (Figure ), as demonstrated by X-ray analysis and MD simulations, thus confining the active site and impacting substrate access. Very recently, also the first studies on the enantioselectivity engineering of HheG have been reported. These yielded HheG mutants with high enantioselectivity in the synthesis of chiral aryl-and spiro-oxazolidinones as well as the azidolysis of cyclohexene oxide and cyclopentene oxide. In all cases, substrate docking based on the published crystal structure of HheG was used to identify relevant residues within the enzyme active site for subsequent sitesaturation mutagenesis. This way, even mutants displaying inverted enantioselectivity compared to HheG wild type could be identified, while the flexible N-terminal loop of HheG was not touched. As our preliminary data hinted at a potential impact of the flexible loop covering residues T39 to G47 (in the following abbreviated loop3947) on the enantioselectivity of HheG, we surmised that highly enantioselective HheG variants could also be accessed through systematic engineering of this loop. To this end, two libraries containing defined single point mutants were generated and screened in ring opening reactions of chemically diverse epoxide substrates in combination with different nucleophiles. This way, several HheG variants with largely increased or even inverted enantioselectivity as well as considerably improved activity towards the tested substrates could be identified.