Biocatalysis is an emerging and important scientific filed for the asymmetric synthesis of pharmaceuticals and fine chemicals that has accomplished astonishing growth in industrial sector in recent years. For individual biocatalytic synthesis to reach its full potential, the process must be explored from the aspect of various scientific fields, including protein engineering, organic chemistry, reaction engineering, and more. The focus of this PhD thesis are halohydrin dehalogenases (HHDHs), promising and yet quite unexplored group of enzymes, and their potential for industrial-scale synthesis. Through extensive research of the existing literature, a serious lack of kinetic data in biotransformations with enzymes from HHDH group was determined. Therefore, one of the objectives of the thesis was to gain insight into kinetic characteristics of HHDH from Agrobacterium radiobacter AD1 (HheC) and develop a mathematical model in synthesis of important building blocks, since this approach may lead to the discovery of enzyme kinetic limitations and process bottlenecks, and, more importantly, enable the enhancement of process outcome through model-based simulations. The investigated reactions were kinetic resolutions for the synthesis of optically pure, fluorinated β-substituted alcohols and epoxides that represent valuable building blocks in pharmaceutical and fine chemicals industries. Ring-opening reactions of fluorinated styrene oxide derivatives, catalyzed by wild-type HheC and its variants W249P and ISM-4, were explored. Fluorinated derivatives of styrene oxide with substituents in para-position were found to be most convenient substrates for HheC based on the activity, enantioselectivity and hydrolytic stability, whereby 2-[4-(trifluoromethyl)phenyl]oxirane stood out as the best option. Enzyme variant W249P, with exchanged Trp and Pro amino acids on position 249, displayed higher substrate affinity in comparison to the wild type. Hence, it was selected for further kinetic investigation. The synthesis of (R)-2-azido-1-[4-(trifluoromethyl)phenyl]ethanol was described by double substrate Michaelis-Menten kinetics, with the presence of enzyme inhibitions with reacting substrate (R)-2-[4-(trifluoromethyl)phenyl]oxirane, opposite enantiomer (S)-2-[4-(trifluoromethyl)phenyl]oxirane, product (R)-2-azido-1-[4-(trifluoromethyl)phenyl]ethanol, by-product rac-2-[4-(trifluoromethyl)phenyl]-1,2-ethanediol, and co-solvent DMSO. Apart from numerous inhibitions and substantial hydrolysis effect, operational stability decay was found to contribute greatly to the synthesis outcome on higher concentration scale, since deactivation constant is directly correlated to the initial concentration of the substrate, 2-[4-(trifluoromethyl)phenyl]oxirane. The mathematical model was developed, and process simulations were employed in process optimization. Significant improvements in process metrics were achieved by modifying reactor set-up and selecting suitable initial conditions. The optimized biocatalytic system in repetitive batch reactor led to high reaction yield and optical purity (Y = 95%, ee > 99 %). However, process should be optimized further in order to increase reaction productivity that meets the target for industrial synthesis. The existence of various enzyme inhibitions and concentration-dependent enzyme deactivation, as well as low solubility and hydrolytic instability of the substrate, makes the system convenient for the switch from aqueous to alternative media. In the second part of the thesis, the focus was on the investigation of the influences of organic solvents (OSs) on HheC catalytic and structural performances. The stability of HheC in presence of dimethyl sulfoxide (DMSO), the most used co-solvent in HHDH-catalyzed biotransformations, was found to be preserved in cases when DMSO volume ratio does not exceed 30% (v/v). In higher DMSO content, HheC is not able to retain native structure and is completely and rapidly inactivated at 50% (v/v) co-solvent. This was confirmed by the combination of experimental studies, including monitoring enzyme stability and protein size distributions during incubation with different DMSO content, together with molecular dynamic studies (MD). DMSO also proved to be a mixed-type inhibitor of HheC in the reactions of para-nitro-2-bromo-1-phenylethanol (PNSHH) dehalogenation and para-nitro styrene oxide (PNSO) ring-opening with bromide ions. The inhibitory behavior was detected by kinetic Lineweaver-Burk analysis and confirmed by MD. Likewise, DMSO was found to be inhibitor of W249P variant in (R)-2-[4-(trifluoromethyl)phenyl]oxirane azidolysis during kinetic investigation. Wild-type HheC also displayed inadequate catalytic properties in presence of other tested water-miscible co-solvents, specifically dimethylformamide, methanol, isopropanol, acetonitrile, and tetrahydrofuran. In case of hydrophobic solvents, a direct correlation between HheC activity and logP value was found in PNSHH ring-closure reaction. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. However, this trend was not reported for PNSO ring-opening reaction. From hydrophobic OSs, tested alkanes (cyclohexane, n-hexane, n-heptane) were found to be compatible with HheC activity and stability during incubation, indicating the preservation of high enzyme structural integrity in these biphasic systems. Chloroform and toluene displayed inhibitory properties, especially in ring-opening reaction, which is more valuable from the synthetic point of view. In comparison to the wild type HheC, thermostable variant ISM-4 performed better in presence of OSs in terms of activity, stability, and enantioselectivity. In other words, the link between thermal stability and resistance to the action of OSs was established. These results revealed that ISM-4 has excellent potential for biotransformations in organic media, and as such should be explored for future implementations.