Enzymatische Decarboxylierung von Benzenpolycarbonsäuren : Isolierung bakterieller Benzenpolycarbonsäure-Decarboxylanten, vergleichende Untersuchungen des Mellitsäure-Katabolismus, Erstreinigung und Charakterisierung einer Mellitsäure-Decarboxylase sowie chemische Untersuchungen der Benzenpolycarbonsäuren

1. Ziel der Arbeit In diesem Dissertationsvorhaben sollten bakterielle Benzenpolycarbonsäure-Decarboxylanten isoliert und die zugehörigen Decarboxylasen untersucht werden. Hierfür war es zwingend erforderlich, Methoden zur Reinigung dieser bislang völlig unbekannten Enzyme zu etablieren. 2. Methoden Durch mikrobiologische Arbeitsschritte wurden Mikroorganismen isoliert und differenziert werden, welche über das gesuchte Enzym bzw. die gesuchten Enzyme verfügen. Darüber hinaus wurde die Verwertung verschiedener Benzencarbonsäuren (BCS) durch unterschiedliche Mikroorganismen vergleichend untersucht werden. In biochemischen Experimenten wurden Methoden entwickelt und angewendet, mit deren Hilfe eine solche BCS-Decarboxylase gereinigt und eindeutig nachgewiesen werden konnte. Die enzymatischen Decarboxylierungsschritte zur Einleitung des Abbaus verschiedener BCS wurden mittels hoch auflösender Flüssigchromatographie (HPLC) dokumentiert. 3. Ergebnisse und Schlussfolgerung Es gelang die Anzucht von insgesamt 22 selbst isolierten Bakterienstämmen mit Benzen-1,2,4,5-tetracarbonsäure (Pyromellitsäure) als einziger Kohlenstoff- und Energiequelle. Mit dem im Rahmen dieser Dissertation isolierten Bakterienstamm „Artern“ konnte eine neue Bakterienart entdeckt und erstmalig beschrieben werden, die wahrscheinlich eine neue Gattung innerhalb der alpha-Proteobakterien repräsentiert. Erstmals gelang der Nachweis des mikrobiellen Wachstums mit Benzenpentacarbonsäure als einziger Kohlenstoff- und Energiequelle durch den neu isolierten Stamm „Artern“ und andere Bakterien. Mittels HPLC wurde die Einleitung des Abbaus der Benzenpentacarbonsäure durch zweifache enzymatische Decarboxylierung nachgewiesen und dabei die intermediäre Bildung zweier unterschiedlicher Benzentetracarbonsäuren durch verschiedene Bakterienstämme beobachtet. Durch einen in dieser Arbeit entwickelten Enzymtest kann die enzymatische Decarboxylierung von Benzenpentacarbonsäure nun spektralphotometrisch quantifiziert werden. Mellitsäure konnte mit hoher Effizienz (ca. 50% d. Bindungsstellen) an EAH Sepharose 4B gekoppelt und somit ein neuartiges Material zur Liganden-Affinitätschromatographie (LAC) hergestellt werden. Mit Hilfe dieser neu entwickelten LAC gelang die weltweit erste Reinigung einer Benzenhexacarbonsäure- (Mellitsäure-) Decarboxylase (MDCase) aus dem selbst isolierten Bodenbakteriums LFG19a (Sinorhizobium morelense/Ensifer adhaerens). Durch eine neu entwickelte Aktivitätsfärbung konnte das mutmaßliche Monomer als aktive Form der MDCase nachgewiesen werden. Hierbei wurde außerdem endgültig bewiesen, daß dieses Enzym unabhängig von Cofaktoren arbeitet. Bei Anzucht des Bakterienstammes LFG19a mit Mellitsäure, Benzenpentacarbonsäure, Pyromellitsäure und Benzen-1,2,4-tricarbonsäure wird unabhängig von der Kohlenstoffquelle jeweils eine identische MDCase exprimiert. Mellitsäure und Benzenpentacarbonsäure werden in LFG19a durch dasselbe Enzym decarboxyliert. Für die Decarboxylierung der Pyromellitsäure wird in diesem Stamm eine weitere Decarboxylase benötigt; diese wird bei Wachstum mit Benzen-1,2,4-tricarbonsäure nicht exprimiert. Mit der MDCase aus LFG19a wurde das erste Enzym überhaupt isoliert, das zwei Carboxylgruppen von einem Aromaten abspaltet

Transaminases for the synthesis of enantiopure beta-amino acids

Optically pure β-amino acids constitute interesting building blocks for peptidomimetics and a great variety of pharmaceutically important compounds. Their efficient synthesis still poses a major challenge. Transaminases (also known as aminotransferases) possess a great potential for the synthesis of optically pure β-amino acids. These pyridoxal 5'-dependent enzymes catalyze the transfer of an amino group from a donor substrate to an acceptor, thus enabling the synthesis of a wide variety of chiral amines and amino acids. Transaminases can be applied either for the kinetic resolution of racemic compounds or the asymmetric synthesis starting from a prochiral substrate. This review gives an overview over microbial transaminases with activity towards β-amino acids and their substrate spectra. It also outlines current strategies for the screening of new biocatalysts. Particular emphasis is placed on activity assays which are applicable to high-throughput screening

Alanins Wunderlampe : Vorkommen, Nutzung und Produktion nicht-kanonischer Aminosäuren

Als nicht-kanonische Aminosäuren (nkAS) fasst man alle Aminosäuren zusammen, die nicht über den genetischen Standardcode kodiert sind und entsprechend nicht über die zelluläre Proteinbiosynthese (Translation) in Proteine eingebaut werden. Die physiologisch und damit auch pharmakologisch bedeutendsten nkAS sind α‑l‑AS mit nicht genetisch kodierten Resten, α‑d‑AS sowie β‑AS. Prominente Beispiele sind das Parkinsonmedikament l‑DOPA, d‑Phenylglycinderivate als Seitenketten von Ampicillin und Amoxicillin (bei Kindern meistverschriebene Antibiotika), sowie β‑Phenylalanin in den meisteingesetzten Chemotherapeutika Taxol® und Taxotere® (u. a. gegen Brust- und Prostatakrebs). Die oft unbekannten Biosynthesewege für nkAS und die schwierige Übertragbarkeit auf standardisierte Verfahren mit etablierten Produktionsstämmen erwies sich in der Vergangenheit als große Herausforderung für die industrielle Herstellung. Eine Schlüsselrolle in der Produktion spielen mittlerweile Enzyme; also ausgerechnet Werkzeuge aus kanonischen Aminosäuren – und künftig vielleicht auch aus nicht-kanonischen

Origin and Evolution of Enzymes with MIO Prosthetic Group: Microbial Coevolution After the Mass Extinction Event

After major mass extinction events, ancient plants and terrestrial vertebrates were faced with various challenges, especially ultraviolet (UV) light. These stresses probably resulted in changes in the biosynthetic pathways, which employed the MIO (3,5-dihydro-5-methylidene-4H-imidazole-4-one)-dependent enzymes (ammonia-lyase and aminomutase), leading to enhanced accumulation of metabolites for defense against UV radiation, pathogens, and microorganisms. Up to now, the origin and evolution of genes from this superfamily have not been extensively studied. In this report, we perform an analysis of the phylogenetic relations between the members of the aromatic amino acid MIO-dependent enzymes (AAM), which demonstrate that they most probably have a common evolutionary origin from ancient bacteria. In early soil environments, numerous bacterial species with tyrosine ammonia-lyase genes (TAL; EC 4.3.1.23) developed tyrosine aminomutase (TAM; EC 5.4.3.6) activity as a side reaction for competing with their neighbors in the community. These genes also evolved into other TAL-like enzymes, such as histidine ammonia-lyase (HAL, EC 4.3.1.3) and phenylalanine ammonia-lyase (PAL; EC 4.3.1.24), in different bacterial species for metabolite production and accumulation for adaptation to adverse terrestrial environmental conditions. On the other hand, the existence of phenylalanine aminomutase (PAM; EC 5.4.3.10) and phenylalanine/tyrosine ammonia-lyase (PTAL; EC 4.3.1.25) strongly indicates the horizontal gene transfer (HGT) between bacteria, fungi, and plants in symbiotic association after acquiring the PAL gene from their ancestor

Synthesis of (S)- and (R)-β-Tyrosine by Redesigned Phenylalanine Aminomutase

Phenylalanine aminomutase from Taxus chinensis (TchPAM) is employed in the biosynthesis of the widely used antitumor drug paclitaxel. TchPAM has received substantial attention due to its strict enantioselectivity towards (R)-β-phenylalanine, in contrast to the bacterial enzymes classified as EC 5.4.3.11 which are (S)-selective for this substrate. However, the understanding of the isomerization mechanism of the reorientation and rearrangement reactions in TchPAM might support and promote further research on expanding the scope of the substrate and thus the establishment of large-scale production of potential synthesis for drug development. Upon conservation analysis, computational simulation, and mutagenesis experiments, we report a mutant from TchPAM, which can catalyze the amination reaction of trans-p-hydroxycinnamic acid to (R)- and (S)-β-tyrosine. We propose a mechanism for the function of the highly conserved residues L179, N458, and Q459 in the active site of TchPAM. This work highlights the importance of the hydrophobic residues in the active site, including the residues L104, L108, and I431, for maintaining the strict enantioselectivity of TchPAM, and the importance of these residues for substrate specificity and activation by altering the substrate binding position or varying the location of neighboring residues. Furthermore, an explanation of (R)-selectivity in TchPAM is proposed based on the mutagenesis study of these hydrophobic residues. In summary, these studies support the future exploitation of the rational engineering of corresponding enzymes with MIO moiety (3,5-dihydro-5-methylidene-4H-imidazole-4-one) such as ammonia lyases and aminomutases of aromatic amino acids

Enzymatic Synthesis of Alkyl Glucosides by β‐Glucosidases in a 2‐in‐1 Deep Eutectic Solvent System

Alkyl glycosides are biodegradable surfactants with excellent physico-chemical properties. Although their wide-range application is considered ecofriendly, their synthesis is not due to the need for toxic organic solvents and corrosive acids as catalysts. Moreover, chemical synthesis results in complex mixtures rather than defined compounds. To overcome both disadvantages as well as the limited solubility of sugars in organic solvents, a highly selective enzymatic synthesis was set up with a β-glucosidase condensing D-glucose and different fatty alcohols in a deep eutectic solvent

Computational-designed enzyme for β-tyrosine production in lignin valorization

Lignin is an underutilized sustainable source of aromatic compounds. To valorize the low-value lignin monomers, we proposed an efficient strategy, involving enzymatic conversion from trans-p-hydroxycinnamic acids to generate valued-added canonical and non-canonical aromatic amino acids. Among them, β-amino acids are recognized as building blocks for bioactive natural products and pharmaceutical ingredients due to their attractive antitumor properties. Using computational enzyme design, the (R)-β-selective phenylalanine aminomutase from Taxus chinensis (TchPAM) was successfully mutated to accept β-tyrosine as the substrate, as well as to generate the (R)-β-tyrosine with excellent enantiopurity (ee > 99%) as the unique product from trans-p-hydroxycinnamic acid. Moreover, the kinetic parameters were determined for the reaction of four Y424 enzyme variants with the synthesis of different phenylalanine and tyrosine enantiomers. In the ammonia elimination reaction of (R)-β-tyrosine, the variants Y424N and Y424C displayed a two-fold increased catalytic efficiency of the wild type. In this work, a binding pocket in the active site, including Y424, K427, I431, and E455, was examined for its influence on the β-enantioselectivity of this enzyme family. Combining the upstream lignin depolymerization and downstream production, a sustainable value chain based on lignin is enabled. In summary, we report a β-tyrosine synthesis process from a monolignol component, offering a new way for lignin valorization by biocatalyst modification

Separation of Cyclic Dipeptides (Diketopiperazines) from Their Corresponding Linear Dipeptides by RP-HPLC and Method Validation

Simple, rapid, sensitive, precise, and accurate methods for detection and separation of seven diketopiperazines (DKPs), cyclo(Gly-Gly), cyclo(dl-Ala-dl-Ala), cyclo(l-Asp-l-Phe), cyclo(l-Asp-l-Asp), cyclo(Gly-l-Phe), cyclo(l-Pro-l-Tyr), and cyclo(l-Arg-l-Arg), from their corresponding linear dipeptides and related amino acids l-Phe and l-Tyr by reversed-phase high-performance liquid chromatography (RP-HPLC) were established. Moreover, for the racemic DKP cyclo(dl-Ala-dl-Ala) and dipeptide dl-Ala-dl-Ala, separation of the diastereomers was achieved. All methods can be performed within 15 min. For all DKPs, dipeptides, and amino acids, linear ranges with correlation coefficients R2 greater than 0.998 were determined. Lowest limits of detection were found to be between 0.05 and 10 nmol per 10 μL injection, depending on the substance. For all tested substances intrarun and interrun precision ranged from 0.5 to 4.7% and 0.7 to 9.9% relative standard deviation, and accuracy was between −4.2 and 8.1% relative error. Short-term and freeze-thaw stabilities were 93% or greater for all substances. Recovery rate after heat treatment was determined to be at least 97%. These methods will be useful for quantitative determination of DKPs and their potential biodegradation products: dipeptides and amino acid

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

Improving protein stability is an important goal for basic research as well as for clinical and industrial applications but no commonly accepted and widely used strategy for efficient engineering is known. Beside random approaches like error prone PCR or physical techniques to stabilize proteins, e.g. by immobilization, in silico approaches are gaining more attention to apply target-oriented mutagenesis. In this review different algorithms for the prediction of beneficial mutation sites to enhance protein stability are summarized and the advantages and disadvantages of FoldX are highlighted. The question whether the prediction of mutation sites by the algorithm FoldX is more accurate than random based approaches is addressed

Separation of Cyclic Dipeptides (Diketopiperazines) from Their Corresponding Linear Dipeptides by RP-HPLC and Method Validation

Simple, rapid, sensitive, precise, and accurate methods for detection and separation of seven diketopiperazines (DKPs), cyclo(Gly-Gly), cyclo(dl-Ala-dl-Ala), cyclo(l-Asp-l-Phe), cyclo(l-Asp-l-Asp), cyclo(Gly-l-Phe), cyclo(l-Pro-l-Tyr), and cyclo(l-Arg-l-Arg), from their corresponding linear dipeptides and related amino acids l-Phe and l-Tyr by reversed-phase high-performance liquid chromatography (RP-HPLC) were established. Moreover, for the racemic DKP cyclo(dl-Ala-dl-Ala) and dipeptide dl-Ala-dl-Ala, separation of the diastereomers was achieved. All methods can be performed within 15 min. For all DKPs, dipeptides, and amino acids, linear ranges with correlation coefficients R2 greater than 0.998 were determined. Lowest limits of detection were found to be between 0.05 and 10 nmol per 10 μL injection, depending on the substance. For all tested substances intrarun and interrun precision ranged from 0.5 to 4.7% and 0.7 to 9.9% relative standard deviation, and accuracy was between −4.2 and 8.1% relative error. Short-term and freeze-thaw stabilities were 93% or greater for all substances. Recovery rate after heat treatment was determined to be at least 97%. These methods will be useful for quantitative determination of DKPs and their potential biodegradation products: dipeptides and amino acid