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The focus of the first two articles was the engineering and application of enzymes for the conversion of the bio-based resources glycerol and its oxidation product glyceraldehyde for the production of the value added product glyceric acid. Article III focuses on the cloning, exploration and engineering of a polyol dehydrogenase, which later on was used as cofactor recycling system in order to produce ε-caprolactone from cyclohexanol as presented in arti-cle IV. The following paragraphs will give a short outline of each article. ARTICLE I: ASYMMETRIC SYNTHESIS OF D-GLYCERIC ACID BY AN ALDITOL OXIDASE AND DIRECTED EVOLUTION FOR ENHANCED OXIDATIVE ACTIVITY TOWARDS GLYCEROL. GERSTENBRUCH, S., WULF, H., MUßMANN, N., O’CONNELL, T., MAURER, K.-H. & BORNSCHEUER, U. T. (2012). Appl. Microbiol. Biotechnol. 96, 1243-1252. The alditol oxidase of Streptomyces coelicolor A3(2) (AldO) was used to catalyze the oxida-tion of glycerol to glyceraldehyde and glyceric acid. The enantioselectivity for the FAD-de-pendent glycerol oxidation was elucidated and different strategies were used to enhance the substrate specificity towards glycerol. Directed evolution by error-prone PCR led to an AldO double mutant with 1.5-fold improved activity for glycerol. Further improvement of activity was achieved by combination of mutations, leading to a quadruple mutant with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. In small-scale biotransformation concentrations up to 2.0 g•l-1 D-glyceric acid could be reached using whole cells. Investi¬gation of the effects of the introduced mutations led to a further identification of es¬sential amino acids with respect to enzyme functionality and structural stability. ARTICLE II: KINETIC RESOLUTION OF GLYCERALDEHYDE USING AN ALDEHYDE DEHYDROGENASE FROM DEINOCOCCUS GEOTHERMALIS DSM 11300 COMBINED WITH ELECTROCHEMICAL COFACTOR RECYCLING. WULF, H., PERZBORN, M., SIEVERS, G., SCHOLZ, F. & BORNSCHEUER, U. T. (2012). J. Mol. Catal. B Enzym. 74, 144-150. Two aldehyde dehydrogenases (ALDH) from Escherichia coli BL21 and Deinococcus geother-malis were cloned, characterized and evaluated according to their applicability for a bio-catalysis setup with electrolytic cofactor recycling. Both ALDHs turned out to have a sim¬ilar substrate scope and favor short to medium chain aldehydes and both oxidize glyceralde¬hyde to D-glyceric acid. The ALDH variant of D. geothermalis shows higher specific activity towards glyceraldehyde and has an elevated optimum temperature compared to the BL21 enzyme. Due to the higher specific activity of the ALDH of D. geothermalis, this enzyme was used to conduct a kinetic resolution of glyceraldehyde with electrolytic NAD+ recycling at a glassy carbon foam electrode with ABTS as redox mediator yielding in 1.8 g•l-1 glyceric acid. ARTICLE III: PROTEIN ENGINEERING OF A THERMOSTABLE POLYOL DEHYDROGENASE. WULF, H.*, MALLIN, H.*, BORNSCHEUER U.T. (2012). Enzyme Microb. Technol. 51, 217-224 (*equally contributed). The new enzyme polyol dehydrogenase PDH-11300 from D. geothermalis was extensively characterized regarding its temperature optimum and thermostability. A peptide stretch responsible for substrate recognition from the PDH-11300 was substituted by this particular stretch of a homolog enzyme, the galactitol dehydrogenase from Rhodobacter sphaeroides (PDH-158), resulting in a chimeric enzyme (PDH-loop). The substrate scopes were deter-mined and basically the chimeric enzyme represented the average of both wild-type en-zymes. A rather unexpected finding was the notably increased T5060, by 7°C to 55.3°C, and an increased specific activity against cyclohexanol. Finally, the cofactor specificity was suc¬cess-fully altered from NADH to NADPH by an Asp55Asn mutation, which is located at the NAD+ binding cleft, without influencing the catalytic properties of the dehydrogenase. ARTICLE IV: A SELF-SUFFICIENT BAEYER-VILLIGER BIOCATALYSIS SYSTEM FOR THE SYNTHESIS OF Ɛ-CAPROLACTONE FROM CYCLOHEXANOL. MALLIN, H. *, WULF, H. *, BORNSCHEUER U.T. (2013). Enzyme Microb. Technol., online, DOI: 10.1016/j.enzmictec.2013.01.007 (*equally contributed). The application of the engineered PDH-loopN mutant [1] (Article III) for the production of ε-caprolactone from cyclohexanol was investigated in a co-immobilization approach with the cyclohexanone monooxygenase from Acinetobacter calcoaceticus. Biotransformation with solubilized enzymes led to an isolated yield of 55% pure ε-caprolactone with no residual cy-clohexanol to be detected. During the immobilization experiments a higher enzyme ratio in favor of the CHMO led to higher reaction velocities. Similarly, the addition of soluble fresh CHMO during reuse of co-immobilization batches significantly increased the activity identi-fying the CHMO as the bottleneck in this reaction setup.
Chiral amines represent high-value fine chemicals serving as key intermediate products in pharmaceutical, chemical and agrochemical industries. In the past decades, application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones emerged to an environmentally benign and economically attractive alternative to transition metal-catalyzed asymmetric synthesis to afford optically pure amines at industrial scale. However, the restricted substrate scope of wild-type transaminases prohibited the conversion of particularly sterically demanding substrates, making protein engineering indispensable. The following thesis covers elaboration of a novel assay for transaminases (Article I) and identification and development of transaminase variants in order to achieve biocatalytic preparation of a set of pharmaceutically relevant model amines, ideally in optically pure form for both stereoisomers, preferentially using asymmetric synthesis and most preferably using isopropylamine as cost-efficient amine donor co-substrate (Article II-IV). The aforementioned target amines and the corresponding precursor ketones (see Scheme 4.1) were conceived and provided by the company F. Hoffmann-La Roche to attain suitable biocatalysts for a variety of potential intermediates for active pharmaceutical ingredients. Protein engineering of the transaminase scaffolds investigated in this thesis comprised: Initial screening for suitable starting enzyme scaffolds, structure-guided rational design of these scaffolds to enable bulky planar substrate acceptance, elaboration of a sequence motif, verification of the motif and preparative-scale asymmetric synthesis reactions (Article II). For non-planar and structurally different target substrates, namely spatially bulky or bi-cyclic bridged substrates, the transaminase variants were specifically refined and a different evolutionary route had to be pursued (Article III and Article IV). These results (Article II) represent not only the first successful endeavor to engineer a PLP-fold type I amine transaminase (commonly denoted as (S)-selective) for the conversion of highly sterically demanding substrates, but also generally expanded the scope of available fold type I amine transaminases by enzymes having a novel and exceptionally broad substrate spectrum. Aside from structure-guided rational protein engineering, as well non-rational methods, such as site-specific saturation mutagenesis or directed evolution, were applied for protein-engineering. In order to do so for all of the target compounds, a novel high-throughput solid phase activity assay for transaminases that was actually developed during the master thesis, was refined and published (Article I). In the context of this thesis, the same assay principle was as well adapted for quantification of specific activities in liquid phase (Article III). A comparison of different methodologies for developing agar plate assays and a detailed step by step protocol of our transaminase assay are illustrated in a book chapter.
Enzyme sind bekannt als Biokatalysatoren, die spezifisch für ein oder wenige Substrate und ihre zu katalysierende Reaktion sind. Die Fähigkeit einiger Enzyme, mehr als nur eine bestimmte chemische Umsetzung zu katalysieren, bezeichnet man als Promiskuität. Einige Enzyme verfügen über ein breites Substratspektrum und können selbst strukturell verschiedene Substrate umsetzen. Man spricht hierbei von der Substratpromiskuität (substrate promiscuity) der Enzyme. Eine weitere Klasse der Promiskuität wird als Konditionspromiskuität (condition promiscuity) bezeichnet. Hierzu zählen Enzyme, die auch bei nicht-natürlichen Reaktionsbedingungen wie hohen Temperaturen, extremen pH-Werten oder in wasserfreiem Medium katalytische Aktivität aufweisen. Die katalytische Promiskuität (catalytic promiscuity) bildet die dritte Gruppe der Enzympromiskuität. Enzyme, die über diese Art der Promiskuität verfügen, zeichnen sich durch eine breite Reaktionsspezifität bei der Katalyse alternativer Reaktionen aus. Zudem lehren uns die Strukturen von über 30.000 Proteinen, dass die Natur nur von einem limitierten Repertoire von Proteingerüsten Gebrauch gemacht hat, um dennoch eine Vielzahl an verschiedensten Reaktionen herbeizuführen. Die Vielfältigkeit der Proteingerüste ist auf einige wenige Vorfahren zurückzuführen, deren Gerüst als Basis zur Generierung von Familien und Superfamilien diente. Die Überreste dieses Prozesses spiegeln sich in den ähnlichen Strukturen und katalytischen Resten der Familienmitglieder wieder. Über die Millionen von Jahren der Evolution haben sich jedoch die Sequenzähnlichkeiten der Mitglieder einer Familie stark verändert. Durch die Untersuchung der Beziehungen von Enzymen mit α/β-Hydrolasefaltung am Beispiel der Generierung von Epoxidhydrolaseaktivität in das Proteingerüst der Pseudomonas fluorescens Esterase (PFE) sollte die verwandtschaftliche Beziehung beider Enzyme näher dargestellt werden. Mit Hilfe der Methoden der positionsgerichteten Mutagenese und der gerichteten Evolution war es möglich eine Vielzahl von Mutanten zu kreieren. Zur Durchmusterung der Mutantenbliotheken kam sowohl ein neu entwickelter Agarplatten-Assay, als auch ein optimiertes Hochdurchsatz-Testsystem zum Einsatz. Mittels dieser Testformate konnten Mutanten der PFE identifiziert werden, die aktiv gegenüber Epoxiden sind. Des Weiteren erfolgte die genaue Charakterisierung der generierten Varianten.
Structure– and sequence–function relationships in (S)-amine transaminases and related enzymes
(2015)
Chiral primary amines are valuable building blocks for many biologically active compounds. Environmentally friendlier alternatives to the classical methods for α-chiral primary amine synthesis are highly desired. A biocatalytic alternative that recently proved beneficial for industrial applications is asymmetric synthesis utilising (S)-selective amine transaminases (S-ATAs). These enzymes can be utilized to transaminate a prochiral ketone with an amino donor (e.g. isopropylamine), to achieve a chiral amine and a carbonyl product (e.g. acetone). However, for several potential applications protein engineering is required to fit (S)-ATAS to the demands of an industrial process. Since no (S)-ATA crystal structure required for understanding the substrate recognition and thus protein engineering was available, we first aimed at obtaining structural data. Instead of solving crystal structures ourselves, we took advantage of structural genomics projects and discovered, that the protein data bank (PDB) already contained crystal structures of four enzymes with unknown function that we hypothesised to possess (S)-ATA activity. After developing a screening method, the four enzymes could be characterized as ω-amino acid:pyruvate transaminases (ωAA:pyr TAs). (S)-amine conversion was suggested to be a ‘substrate-promiscuous’ activity of these enzymes, as it is pronounced differently in the four investigated ones. By comparing the active sites of the highly and poorly active (S)-ATAs, the residues that determine the ability of amine conversion in these enzymes were discovered. Furthermore, the mechanism for dual substrate recognition, the binding of both, carboxyl and bulky hydrophobic substrates in the same active site, could be elucidated with the crystal structures. A flexible arginine side chain is able to adopt various positions thus enabling carboxylate binding and by ‘flipping’ out of the active site, to create space for amine binding. Then, a limitation of these enzymes, the restricted substrate scope caused by a small binding pocket was addressed. First, a rational protein engineering approach was set up to create more space. The tested mutations, however, destroyed most of the activity for both regular and more bulky substrates. We thus learned that the structural requirements for (S)-ATA activity are more complex than initially anticipated and a semi-rational approach was applied to broaden the substrate scope. By systematic saturation of active site positions, substantially improved mutants for bulkier amine synthesis could be obtained. As this study highlighted a lack of understanding of (S)-ATA, the functional important residues in the enzymes belonging to the class III TA family were surveyed. This family is defined by common sequence and structure features and besides (S)-ATAs mainly comprises TAs of various substrate scopes but also a few phospholyases, racemases and decarboxylases. To enable the comparison of active site residues among them, a commercial bioinformatics tool was used to create a family wide structure-based alignment of around 13,000 sequences. Based on statistical analyses of this alignment, structural inspections and literature evaluation, active site residues crucial for certain specificities within this family have been identified. By investigating the ingenious active site designs that enable such a plethora of reactions, and by identifying sets of functional important residues termed ‘active site fingerprints’, the understanding of catalysis in this enzyme family could be broadened. Furthermore, these functional important residues can on the one hand be applied to predict the specificity of uncharacterised enzymes, if a fingerprint is matched. On the other hand, if no fingerprint is matched, they can help to discover yet unknown activities or mechanisms to achieve a known specificity. We exemplified the latter case by functionally characterising a Bacillus anthracis enzyme with the crystal structure 3N5M, whose substrate specificity was unknown and could not be predicted. The 3N5M enzyme was found to possess ωAA:pyr TA and (S)-ATA activity even though it lacks the above-mentioned ‘flipping’ arginine. Based on molecular dynamics simulations we were able to propose an alternative mechanism for dual substrate recognition in the B. anthracis ωAA:pyr TA. By these findings the understanding of the requirements for (S)-ATA activity could be further broadened and a functional knowledge gap within the class III TA family was closed. The active site residue composition in 3N5M is now connected to enzymatic function and may be applied for future specificity predictions.
In this thesis, two novel assay systems had been developed, which allow a fast and easy screening for amine transaminase activity as well as the characterization of the amino donor and acceptor specificity of a given amine transaminase. The assays overcome some limitations of previously described assays but of course have some limitations themselves. The relatively low wavelength of 245 nm, at which the production of acetophenone is detected with the spectrophotometric assay, limits the amount of protein/crude extract that can be applied, which eventually results in a decreased sensitivity at higher enzyme loads due to an increased initial absorbance. Otherwise, this assay can be used very easily for the investigation of the amino acceptor specificity and both pH and temperature dependencies of amine transaminases. The conductometric assay is – by its very nature – limited to low-conducting buffers, a neutral pH and constant temperatures. In summary, the assays complement one another very well and the complete characterization of the most important enzyme properties can be accomplished quickly. Furthermore, we developed and applied a novel in silico search strategy for the identification of (R)-selective amine transaminases in sequence databases. Structural information of probably related proteins was used for rational protein design to predict key amino acid substitutions that indicate the desired activity. We subsequently searched protein databases for proteins already carrying these mutations instead of constructing the corresponding mutants in the laboratory. This methodology exploits the fact that naturally evolved proteins have undergone selection over millions of years, which has resulted in highly optimized catalysts. Using this in silico approach, we have discovered 17 (R)-selective amine transaminases. In theory, this strategy can be applied to other enzyme classes and fold types as well and for this reason constitutes a new concept for the identification of desired enzymes. Finally, we applied the seven most promising candidates of the identified proteins to asymmetric synthesis of various optical pure amines with (R)-configuration starting from the corresponding ketones. We used a lactate dehydrogenase/glucose dehydrogenase system for the necessary shift of the thermodynamic equilibrium. For all ketones at least one enzyme was found that allowed complete conversion to the corresponding chiral amine with excellent optical purities >99% ee. Bearing in mind that until last year there was only one (R)-selective amine transaminase commercially available and two microorganisms with the corresponding activity described, the identification of numerous enzymes is a breakthrough in asymmetric synthesis of chiral amines.
The aim of this thesis was to validate a method called OSCARR for One-pot, Simple Cassette Randomization and Recombination for focused directed evolution, which had been developed by Dr. Hidalgo. It is based upon the megaprimer PCR method using outer primers differing in TM and including asymmetric cycles before the addition of the forward primer to generate more mutated megaprimer. As mutation-carrying primers, spiked oligonucleotides are employed. These spiked oligonucleotides are designed using an algorithm and have strictly defined composition of nucleotides at each position. An OSCARR library of the Pseudomonas fluorescens esterase I (PFE I) of approximately 8000 clones was generated and screened for altered chain-length selectivity. Two mutants with higher activity towards medium chain length p-nitrophenyl esters were identified, both carried the mutation F126I, which causes the substrate entrance tunnel to be widened, thus facilitating access of bulkier substrates to the active site. One mutant carried the additional mutation G120S which completes a catalytic tetrad which is observed mainly in proteases. F126I had a stronger influence on chain-length specificity, so the further amino acids which form the “bottleneck” to the active site were mutated to further widen the entrance, and mutants with improved activity were found. The bottleneck mutants which consist of single, double, triple and quadruple mutants which are mostly combinations of F126L, F144L, F159L and I225L were then assayed for altered enantioselectivity against chiral acids and secondary alcohols. For substrates 1-phenyl-1-propyl acetate (2), 1-phenyl-2-propyl acetate (3) and 1-phenyl ethyl acetate (4), mutants with increased enantioselectivity were found. I225L plays a crucial role, as it is vital for enantioselectivity against 3, but destroys selectivity against 2, both facts obvious from the comparison of the triple mutant without I225L (mutant T3) and the corresponding quadruple mutant including I225L (mutant Q). However, the single mutant I225L alone does not possess high selectivity against 3, so synergistic effects play an important role. The PFE I wild type already possesses a good enantioselectivity in the hydrolysis of 4, but all mutants which were analyzed in detail surpass the wild type. The program YASARA was then used to calculate docking solutions for both enantiomers of 2 and 3 into the wild type and the best mutant. The results revealed that the mutants’ widened bottleneck allows the phenyl moiety of the substrates to point towards the access tunnel, while only (R)-2 does so in the wild type. Residues 126 and 144 do not come very close to the substrate and are more likely to influence substrate diffusion. Another goal was to find a way to confer promiscuous amidase activity upon the PFE I. In the search for structural homologues, a close structural neighbour with amidase activity was found. The --lactamase from Aureobacterium sp. was named after its activity toward the Vince lactam 2-azabicyclo[2.2.1]hept-5-en-3-one. Biocatalysis experiments with the PFE I and its mutants revealed an excellent enantioselectivity against the ( )-lactam. Specific activities were determined for purified proteins, and the activity of some mutants was within the same order of magnitude as lactamase’s activity.
This thesis deals with the characterisation and engineering of new thermophilic PET hydrolases as potential candidates for an eco-friendly biocatalytic recycling approach for the upcycling or downcycling of polyethylene terephthalate (PET) on industrial scale. Furthermore, high-throughput screening methods are described that detect the products of PET hydrolysis. The high demand of PET in the packaging and textile industries with a global production of 82 million metric tons per year has significantly contributed to the global solid waste stream and environmental plastic pollution after its end-of-life. Although PET hydrolases have been identified in various microorganisms, only a handful of benchmark enzymes have been engineered for industrial applications. Therefore, the identification of new PET hydrolases from metagenomes or via protein engineering approaches, especially thermophilic PET hydrolases with optimal operating temperatures (i.e., increased thermostability and activity) near the glass transition temperature of the polymer PET, is a crucial step towards a bio-based circular plastic economy. Article I demonstrates that metagenome-derived thermophilic PET hydrolases can be significantly improved using different engineering approaches to achieve a similar activity level as the well-established leaf-branch-compost cutinase (LCC) F243I/D238C/S283C/Y127G variant (LCC ICCG). In Article II, thermostable variants of a mesophilic enzyme (PETase from Ideonella sakaiensis) were identified from a mutant library and characterised against PET substrates in various forms. Articles III and IV describe the application of high-throughput methods for the identification of novel PET hydrolases by directly assaying terephthalic acid (TPA), one of the monomeric building blocks of PET. Furthermore, Article IV describes the possibility of a one-pot conversion of the TPA-based aldehydes produced to their diamines as example for an open-loop upcycling method.
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.
Within this thesis the protein engineering, immobilization and application of enzymes in organic synthesis were studied in order to enhance the productivity of diverse biotransformations. Article I is a review about Baeyer-Villiger monooxygenases (BVMO) and provides a detailed overview of the most recent advantages in the application of that enzyme class in biocatalysis. Protein engineering of a former uncharacterized polyol-dehydrogenase (PDH) identified in the mesothermophilic bacterium Deinococcus geothermalis 11300 is described in Article II. Article III covers the combination of one PDH mutant with a BVMO in a closed-loop cascade reaction, thus enabling direct oxidation of cyclohexanol to ε-caprolactone with an internal cofactor recycling of NADP(H). Article IV and Article V report a process optimization for transamination reactions due to a newly developed immobilization protocol for five (S)- and (R)-selective aminotransferases (ATA) on chitosan support. Furthermore, the immobilized ATAs were applied in asymmetric amine synthesis. In Article VI, an ATA immobilized on chitosan, an encapsulated BVMO whole cell catalyst and a commercially available immobilized lipase were applied in a traditional fixed-bed (FBR) or stirred-tank reactor (STR), and were compared to a novel reactor design (SpinChem, SCR) for heterogeneous biocatalysis.
In ersten Teil der Arbeit wurde versucht, in einer α/β-Hydrolase (Esterase) durch den Austausch einzelner Aminosäuren die Aktivität eines anderen Mitgliedes zu erzeugen (Haloalkan-Dehalogenase (HLD)). Als Modellenzym diente die Arylesterase PFE aus Pseudomonas fluorescens, welche wie die HLDs eine cap-Domäne aus &alhpa;-Helices aufweist. Bei den HLDs wurde sich an den Unterfamilien HLD-I und II orientiert, deren Mitglieder Unterschiede in ihren katalytischen Triaden und in einigen weiteren, an der Katalyse beteiligten Aminosäuren zeigen. Sie katalysieren jedoch die gleiche Reaktion und unterscheiden sich nicht in ihrem Mechanismus. Mit Hilfe von Sequenz- und Strukturalignments einiger HLDs und der PFE wurde nach konservierten Bereichen innerhalb der HLDs gesucht. Neben der katalytischen Triade mussten die für HLD-Aktivität essenziellen Halogen-stabilisierenden Reste in der PFE eingefügt werden. Sowohl in der katalytischen Triade als auch bei den Halogen-stabilisier enden Resten gibt es Unterschiede zwischen den Unterfamilien HLD-I und HLD-II, woraus sich zwei unterschiedliche Sätze von hotspots ergaben. Mit Hilfe der error-prone PCR wurden auf Basis einer einfachen Mutante 4.000 Varianten erstellt und analysiert. Ein weiterer Ansatz zur Generierung von HLD-Aktivität beruhte auf einer Sättigungsmutagenese der Halogen-stabilisierenden Reste. Insgesamt wurden in diesem Experiment 765 Varianten erstellt und untersucht. Keine der mittels rationalem Design und gerichteter Evolution entwickelten Mutanten zeigte Aktivität. Das Fehlen von HLD-Aktivität liegt vermutlich im variabelsten Teil der HLDs begründet: dem loop nach dem β6-Strang. Die Aminosäuren aus diesem loop sind an der Bildung der Tunnel zum aktiven Zentrum beteiligt und haben Einfluss auf die Positionierung der Reste in der α4-Helix, welche z.T. das aktive Zentrum dieser Enzyme bilden. Der zweite Teil dieser Arbeit beschäftigte sich mit der Entwicklung eines enzymatischen Verfahrens zum Abbau von 3-Chlor-1,2-Propandiol (3-MCPD) und seinen Estern, welche in Lebensmitteln bei deren Herstellung entsehen können. Das Verfahren basiert auf einer Enzymkaskade aus Lipase, Haloalkohol-Dehalogenase (HHD) und Epoxydhydrolase (EH). Die Lipase setzt zunächst den Ester um und somit das 3-MCPD frei, welches im nächsten Schritt von der HHD zu Glycidol umgesetzt wird. Das ebenfalls toxische Glycidol wird schließlich durch eine EH zum nicht-toxischen Glycerin hydrolisiert. Im konkreten Fall wurden die Lipase A aus Candida antarctica (CAL-A), die HHD HheA aus Arthrobacter sp. AD2 und die EH EchA aus Agrobacterium radiobacter AD1 verwendet. Als Fettsäurekomponente für die Umsätze mit einem 3-MCPD-Ester wurde die Ölsäure gewählt. Im wässrigen System konnten innerhalb von 3,5 h 75% des 3-MCPD umgesetzt werden, wobei nach Beendigung der Reaktion das Zwischenprodukt Glycidol nicht mehr detektiert werden konnte. Im 2-Phasen-System mit dem 3-MCPD-Ölsäureester als Substrat war die Lipase CAL-A in der Lage, den Ester nahezu quantitativ aus dem Reaktionsgemisch zu entfernen und die entsprechenden Mengen an 3-MCPD freizusetzen, welches dann in der wässrigen Phase durch die HHD und die EH umgesetzt wurde. Dabei konnte der Wassergehalt im System ohne Einbußen in der Effektivität bis auf 5% gesenkt werden. Im letzten Teil dieser Arbeit wurde eine neue HLD (DppA) aus Plesiocystis pacifica SIR-1 identifiziert und charakterisiert. Sequenz- und Strukturanalysen erlaubten die Einordnung des Proteins in die HLD-Unterfamilie I. Nach Untersuchung der Substratspezifität von DppA wurde das Enzym der Spezifitätsgruppe SSG-I zugeordnet. Im Unterschied zu den anderen Mitgliedern dieser Gruppe setzte DppA allerdings keines der getesteten chlorierten Substrate um. Bei der Suche nach strukturellen Erklärungen für diese Tatsache fiel eine Lücke im Sequenzalignment von DppA und dessen nächsten Verwandten DhlA auf. DppA fehlt hier ein Bereich von 11 Aminosäuren. Der Bereich liegt in der cap-Domäne und es wurde postuliert, dass das betreffende Segment sich durch die Anpassung eines Vorfahren heutiger Dehalogenasen an das Substrat 1,2-Dichlorethan entwickelte. In einer darauffolgenden Arbeit konnte gezeigt werden, dass DhlA nach Entfernen einer Kopie der Sequenzwiederholungen nicht mehr in der Lage war, sein natürliches Substrat 1,2-Dichlorethan umzusetzen, dafür aber immer noch Aktivität gegenüber 1,2-Dibromethan zeigte. Die Aminosäurereste der Wiederholungen in DhlA haben Einfluss auf die Positionierung sowohl von Tunneln als auch von Resten im aktiven Zentrum. Ein Alignment der Strukturen von DhlA und DppA zeigte, dass ein Teil der Sequenz die Postion des slot-Tunnels aus DppA blockiert. Dementsprechend befindet sich dieser Tunnel in DhlA auch an anderer Stelle. Dem Enzym fehlen die mit der Fähigkeit zur Umsetzung von 1,2-Dichlorethan in Verbindung gebrachten Sequenzwiederholungen und dementsprechend setzt es insbesondere dieses Substrat nicht um.