Biocatalytic oxyfunctionalizations, especially of non - activated hydrocarbons, attract considerable attention for synthetic organic chemistry thanks to their high chemo-, regio-, and stereoselectivity, which difficult to achieve by chemical means. Many enzymatic oxyfunctionalizations have been described. However, there are many hurdles towards large scale applications, such as low activity and stability of enzymes, substrate toxicity, overoxidation, oxygen mass transfer, etc. Therefore, the aim of this thesis is to setup robust and scalable biocatalytic oxyfunctionalizations. Chapter 1 gives a general introduction on enzymatic C-H oxyfunctionalizations highlighting the great potential of heme - iron peroxygenases. Peroxygenases do not rely on expensive NAD(P)H cofactors and catalyze a variety of useful synthetic transformations utilizing H2O2 as an oxidant. However, the practical applicability of heme - peroxygenases is limited by their low stability towards H2O2. To avoid the inactivation of the enzymes, we have developed two alternative catalytic approaches for the controlled in situ H2O2 generation from O2. General applicability of the proposed methods has been demonstrated for various peroxygenase - based biotransformations in Chapters 2, 3, 4. Thus, in Chapter 2 a photocatalytic approach for in situ H2O2 generation has been applied for the CPO (chloroperoxidase from Caldariomyces fumago) catalyzed thioanisole sulfoxidation. The enzyme stability has been drastically improved; however, the productivity of the system was severely limited by low solubility and evaporation of the substrate. Therefore, the photocatalytic approach was demonstrated at preparative - scale using a surfactant - stabilized two – liquid phase system (2 LPS). Both, initial rate and robustness of the system could be enhanced significantly leading to an increase of the final product concentration by more than one order of magnitude in comparison with monophasic set - up. In Chapter 3 the proposed photocatalytic in situ generation of H2O2 proved to be a suitable approach for AaeAPO (Agrocybe aegerita aromatic peroxygenase) catalyzed epoxidation and hydroxylation reactions. High productivities and excellent enantiomeric excesses (>97%) were obtained with a broad range of substrates. Furthermore, preliminary results indicate that hundreds of thousands of turnovers can be achieved after reaction engineering, demonstrating the high potential of AaeAPO for oxyfunctionalization catalysis. In Chapter 4 an alternative in situ H2O2 generation method has been developed using a synthetic nicotinamide cofactor mimic and flavin. This method has been applied for the specific ?- or ?-hydroxylation of fatty acid catalyzed by the cytochrome P450 peroxygenases. The cytochrome P450 peroxygenases P450bs? from Bacillus subtilis and P450cl? from Clostridium acetobutylicum belong to a unique group of P450s which consume H2O2 and therefore do not require additional electron transfer proteins and NAD(P)H cofactor. Using the new method for in situ H2O2 generation the final productivity of P450 peroxygenases could be enhanced due to higher enzyme stability under operation conditions. In addition to peroxygenases, various oxyfunctionalizations can be performed by oxygenases. However, the application of isolated oxygenases, relies on efficient cofactor regeneration techniques. Formate dehydrogenase (FDH) is widely applied for NAD(P)H regeneration. However, classical FDH -based reaction systems are limited to mainly aqueous media, wherein the majority of substrates are poorly soluble. In order to circumvent this limitation we propose in Chapter 6 using formic acid esters as ‘hydrophobized’ formic acid equivalents, which simultaneously can serve as an organic phase in 2LPS reaction and as source of reducing equivalents. The concept was demonstrated using 2-hydroxybiphenyl-3-monooxygenase (HbpA)-catalyzed specific ortho-hydroxylation of phenols as model reaction. In addition, chemical methods for NAD(P)H regeneration have been employed as an alternative to enzymatic methods. The organometallic compound [Cp*Rh(bpy)(H2O)]2+ has emerged as a catalyst of choice. However, the mutual inactivation between [Cp*Rh(bpy)(H2O)]2+ and enzymes is usually encountered. To overcome this issue, in Chapter 5, we propose using artificial transfer hydrogenases (ATHs) wherein the biotin - coordinated transition metal complex is sterically shielded by complexation with streptavidin. Thereby, the mutual inactivation of the regeneration catalysts and the production enzyme(s) can be efficiently circumvented. The applicability of the concept was illustrated by combining the ATHase with HbpA, demonstrating that the general catalytic properties of ATHase and enzyme are preserved. In conclusion, this thesis addresses some major challenges of oxyfunctionalization catalysis including enzyme stability, cofactor dependency and substrate supply. The proposed methods to overcome these issues have been implemented to various enzymatic oxyfunctionalizations. Furthermore, their practical applicability has been demonstrated at preparative - scale using a two liquid phase approach. Promising results in terms of productivity and selectivity have been obtained, thereby paving the way to preparative biocatalytic oxyfunctionalization. Nevertheless, further studies, e.g. combining enzyme immobilization techniques with reaction and process engineering, are needed to substantiate the full potential of biocatalytic oxyfunctionalizations.