Doctoral Thesis
In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols.
By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines.
The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.
Cascade reactions are not only of interest to chemists and biotechnologists, but also to life in general, because every metabolic reaction resembles a cascade reaction. This principle of substrate/intermediate channeling was only adapted by scientists. That way especially one-pot reactions became very attractive as for this no isolation of intermediates is necessary. Furthermore, unstable or toxic intermediates are only produced in low amounts and directly transformed in situ. In this PhD thesis two previously established cascade reactions were subject of further optimization. In the first part, a cascade reaction established in a DFG-funded project (Bo1862/6-1)in cooperation with the Vienna Technical University (Austria) for the production of chiral lactones was further optimized and extended. Therefore, on the one hand the genes encoding the needed enzymes were cloned for co-expression into a single plasmid in different arrangements to be expressed in pseudo-operon mode, with the aim to lower the metabolic burden of the cascade host cell. One out of the welve created constructs showed a reasonable activity of 15.3 ± 1.2 U · gCDW-1. On the other hand, this cascade reaction was aimed to be extended by the use of a hydroxylating enzyme to enable the use of limonene as renewable and chiral precursor for the proposed production of chiral polymers. Therefore, the feasibility of cytochrome P450-monooxygenases was studied. These turned out to be not applicable due to their bad regioselectivity for the hydroxylation of limonene or due to the difficulties of activity reconstitution. As alternative system for an initial hydroxylation step the use of a Rhodococcus equi strain, which was isolated from Cellulosimicrobium cellulans EB-8-4 and which is capable of very regioselective limonene-hydroxylation, was investigated. Therefore, the dioxygenase cluster responsible for the desired reaction was identified and especially the recombinant expression in a suitable host (Pseudomonas putida S12) was further studied. The results from these experiments revealed that the recombinant expression needs to be further optimized to enable the use of the recombinant dioxygenase in combination with the other enzymes for cascade reactions. The third part of this PhD thesis dealt with the immobilization of an established cascade reaction for the synthesis of poly-[caprolactone] precursors. Therefore, the use of a rotating bed reactor (RBR) was investigated. Preliminary studies using single enzymes involved in the desired cascade reaction demonstrated the general feasibility of this reactor concept. Especially the reusability of the catalysts was highly improved, because the catalytic particles were protected very effectively from mechanical forces within the voids of the reactor. For further work-flow optimization the immobilization was transformed into an in situ process by the application of a gas-shear device, which leads to decreased capsule size and thereby to increased mass transfer inside the particles. The developed methods were applied for encapsulation of the cells containing the enzymes needed for the reaction. After additional improvement of the reaction parameters a conversion of 93% (based on substrate depletion) was reached using catalysts produced by the established encapsulation procedure. In summary, the described cascade reactions were successfully optimized by either co-expression, extension applying a dioxygenase or immobilization. Furthermore, the general feasibility of an RBR was demonstrated.
Oils and fats from natural origin are sustainable sources for a broad range of economically relevant products in food, feed, fuel, oleochemical, and cosmetic industries. Thereby, a huge variety of lipids or lipid-derived products exist which distinguish themselves by their unique physical properties making them suitable for their individual applications. To obtain such functional lipids in an environmentally friendly manner, enzymes can be employed. In that context, lipases have been proven to be valuable biocatalysts in lipid modification, which are broadly applied in industry. Even though they have been implemented successfully in the dairy, baking, and detergent industries, there is an increasing demand for the expansion of their utilization. New technologies like protein engineering and the implementation of process development are employed in solving this task. Within the enzymes in lipid modification, lipases are the most applied catalysts and in this thesis their utilization was expanded successfully to the implementation of novel separation processes and the production of improved drug delivery matrices.
