Open Access

Promiscuous acyltransferases enable transesterification reactions in bulk water by preferentially catalyzing acyl transfer over hydrolysis. Until recently, only a small number of promiscuous acyltransferases have been described in the literature, exhibiting several limitations in terms of acyltransferase efficiency and applicability. This work focuses on the discovery of novel promiscuous acyltransferases and the engineering of promiscuous acyltransferases via rational design. Several promiscuous acyltransferases in the bacterial hormone-sensitive lipase family and family VIII carboxylesterases have been identified, demonstrating that promiscuous acyltransferase activity is not a rare phenomenon. Moreover, the efficiency and applicability of the enzymes could be improved via protein engineering in terms of acyltransferase activity, enantioselectivity, and substrate scope.

Free radicals are known to induce significant structural and functional modifications to the cell membrane and its components. Biophysical quantification of such changes using single molecule studies highlight the role of these individual biomolecules. In this PhD work, we focus on nitric oxide radical and try to understand how they influence interaction of different biomolecules with lipid membranes by using biomimetic systems. In specific we try to answer how cell membrane permeability and bilayer thickness would be influenced by the nitric oxide radical with different phospholipids compositions (i.e. on planar supported lipid bilayers). Later we tested, interaction of transmembrane protein integrin αiibβ3 incorporated into the bilayer (i.e. nanodiscs) with nitric oxide. Finally, how to overcome the negative effects encountered by the phospholipids and proteins using biopolymer coated gold nanoparticles as delivery system. The study involved use of atomic force microscopy and quartz-crystal microbalance with dissipation as primary investigation tools complemented with other relevant biophysical and biochemical techniques.

Unter promiskuitiver Acyltransferase-Aktivität versteht man die Eigenschaft bestimmter Hydrolasen, in wässriger Lösung bevorzugt Acyltransfer statt Hydrolyse zu katalysieren. Bis vor Kurzem waren nur wenige promiskuitive Acyltransferasen literaturbekannt. Dies führte zu der allgemeinen Annahme, dass diese Aktivität ein seltenes Phänomen in Hydrolasen ist. Diese Arbeit zeigt jedoch, dass promiskuitive Acyltransferase-Aktivität in der Familie der bakteriellen hormonsensitiven Lipasen und Carboxylesterasen der Familie VIII weit verbreitet ist. Detaillierte Struktur-Funktions-Analysen ermöglichen die sequenzbasierte Vorhersage und Optimierung der Acyltransferase-Aktivität in beiden Enzymfamilien. Insbesondere die Carboxylesterasen der Familie VIII überschreiten die Grenzen des bisher für möglich Gehaltenen, indem sie gute Enantioselektivität bei der kinetischen Racematspaltung sekundärer Alkohole zeigen und darüber hinaus die irreversible Bildung von Amiden und Carbamaten in Wasser katalysieren können. Die biokatalytische Acylierung von Zuckern in Wasser galt lange Zeit als unerreichtes Ziel der Biokatalyse. In dieser Arbeit wurde jedoch gezeigt, dass natürlich vorkommende und modifizierte Carboxylesterasen der Familie VIII die regioselektive Acetylierung von Glucose, Maltose und Maltotriose in Wasser mit hoher Effizienz katalysieren können.

S-adenosyl-L-methionine- (SAM) dependent methyltransferases (MTs) catalyse methylation of halide ions and the C, O, N, S, Se, and As atoms of biomolecules ranging from biopolymers to small molecules. They display different chemo-, regio- and stereoselectivity according to their specific functions. This thesis focuses on the engineering of O-methyltransferases (OMTs) and halide methyltransferases (HMTs) through rational design and directed evolution to study their structure-function relationship and to explore their catalytic promiscuity. The influence of substrate binding residues on the substrate scope and regioselectivity of a plant OMT against various phenolic substrates (Article I) and flavonoids (Article II) has been investigated. Article III describes the directed evolution of an HMT for the biocatalytic synthesis of diverse SAM analogues. With the evolved HMT, regioselective alkylation of phenolic compounds and flavonoids, as well as the SAM analogue regeneration, were achieved through an HMT-MT cascade reaction. Article I Specific residues expand the substrate scope and enhance the regioselectivity of a plant O-methyltransferase. It was reported in literature that an isoeugenol 4-OMT (IeOMT) can be engineered to a caffeic acid 3-OMT (CaOMT) by replacing three consecutive residues. In this article, we investigated the effect of these residues on substrate preference and regioselectivity of IeOMT. The triple mutant T133M/A134N/T135Q and the respective single mutants were constructed and tested against a series of phenolic compounds. The variant T133M had a universal effect to improve enzymatic activities against all tested substrates while the mutant A134N had enhanced regioselectivity. The triple mutant T133M/A134N/T135Q benefits from these two mutations, which not only expanded the substrate scope, but also enhanced the regioselectivity of IeOMT. On the basis of this work, regiospecific methylated phenolics can be produced in high purity by different IeOMT variants. Article II Influence of substrate binding residues on the substrate scope and regioselectivity of a plant O-methyltransferase against flavonoids Flavonoid OMTs (FOMTs), isoflavonoid OMTs (IOMTs) and phenylpropanoid OMTs (POMTs) display different substrate preferences. Sequence comparison showed that the substrate binding residues at positions 322 and 326 are different between these OMT groups and might be critical for the substrate discrimination. Residues at positions 322 and 326 in IeOMT (a POMT) were mutated to the commonly presented residues in FOMT and IOMT. The introduced mutants, in cooperation with the variant T133M, have improved or brought novel activities and regioselectivity against the tested flavonoids eriodictyol, naringenin, luteolin, quercetin, and also the isoflavonoid genistein compared to the wild-type IeOMT. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity. Article III Directed evolution of a halide methyltransferase enables biocatalytic synthesis of diverse SAM analogs Biocatalytic alkylations to obtain chemo‐, regio‐ and stereoselectively alkylated compounds can be achieved by MTs with the supply of SAM analogues. It was recently discovered that SAM can be directly synthesized from S adenosyl-L homocysteine (SAH) and methyl iodide, catalysed by an HMT. To explore the promiscuity of HMT in the synthesis of SAM analogues, we performed directed evolution of the Arabidopsis thaliana HMT based on a sensitive, colorimetric iodide assay. The identified variant V140T displayed activities against ethyl‐, propyl‐, and allyl iodides to produce the corresponding SAM analogues. With this HMT variant, regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde, as well as the SAM analogue regeneration, were achieved through this HMT-MT one-pot cascade reaction.

The synthesis of several bioactive compounds and active pharmaceutical ingredients relies on the development of general and efficient methods to prepare optically pure amines. Transaminases are industrially relevant enzymes and are useful for synthesizing a large number of compounds that contain a chiral amine functionality. Although the immense potential associated to the use of these biocatalysts, the equilibrium position is often unfavorable for amine synthesis. The use of an excess of amine donor, compared to the ketone substrate, combined with selective removal of the formed product, can help in overcoming this limitation. This work mainly focused on broadening the application of membrane-based in situ product recovery (ISPR) techniques for the transaminase-catalyzed synthesis of chiral amines. The overall work was designed around the implementation of amine donors, possessing considerably larger molecular ‘size’ compared to commonly used amine donors. To clearly distinguish these molecules from traditional donor amines, we designate them as High Molecular Weigh amine donors. With a molecular weight between 400 and 1500 g/mol, in contrast to traditional donor amines, HMW amine donors enable a size-based separation between amine donor and amine product molecules. HMW amines, provided in excess for thermodynamic equilibrium shifting can thus be simply retained by a size-exclusion mechanism by commercial membranes, while the smaller product amines are permeated. Therefore, a selective recovery of the desired chiral amine product is possible. The implementation of ISPR techniques using HMW amine donors can theoretically lead to (i) equilibrium shifting, (ii) alleviation of product inhibition, and (iii) a highly pure product stream. The feasibility of using HMW amine donors in aqueous, organic solvent and solvent-free media for the transaminase-catalyzed synthesis of 1-methyl-3-phenylpropylamine (MPPA) was proven in this thesis. The latter two approaches were investigated with the aim to achieve higher product concentrations. Along with that, we demonstrated two membrane-assisted ISPR proof of concepts. Specifically, nanofiltration was coupled with the enzymatic reaction performed in aqueous media (Article I), while liquid-liquid (L-L) extraction in a contactor was applied for transamination in organic solvent media (Article II). As an alternative to membrane-based strategies we also designed a spinning reactor concept for the integrated chiral amine synthesis (in organic solvent) and recovery (Article III).