13 research outputs found
New approaches for flavoenzyme applications:Cofactor-mediated immobilization & in vitro production of human metabolites
New approaches for flavoenzyme applications:Cofactor-mediated immobilization & in vitro production of human metabolites
Dit proefschrift presenteert nieuwe benaderingen voor toepassingen van flavine-houdende enzymen als biokatalysatoren.Het eerste deel beschrijft een nieuwe methode van cofactor gemedieerde enzym immobilisatie. Door de flavine cofactor aan dragermateriaal te koppelen, kan een target prototype flavoenzyme geïmmobiliseerd worden. Dit resulteerde in een volledig actieve en zeer stabiele enzymformulering. Door de aard van de chemische koppelingen tussen het draagmateriaal en de flavine cofactor te variëren, maakt deze nieuwe methode het mogelijk om de oppervlaktebelading en afstand van de vaste fase naar het enzym (monolaag) af te stemmen. Om een betere toegang tot de vereiste chemisch gemodificeerde flavine cofactoren te verschaffen, werd ook een nieuw chemisch synthese protocol voor de bereiding van gealkyleerde flavine cofactoren ontwikkeld.Om de waarde van flavine-houdende enzymen als biokatalysatoren te demonstreren, wordt werk gepresenteerd bij het onderzoeken van een specifieke groep flavoenzymes: de flavin-bevattende monooxygenasen (FMO's). Ten eerste wordt een fysiologisch cruciale menselijke FMO, flavine-bevattende monooxygenase 3 (FMO3), uitgebreid besproken over de huidige kennis over de sequentie en (voorspelde) structurele eigenschappen. Biochemische studies op dit humane enzym worden belemmerd door de onzin tegen heterologe expressie van oplosbaar eiwit. Resultaten voor de productie van een actieve, zelfvoorzienende hFMO3 in bacteriële cellen worden gepresenteerd. Verder wordt aangetoond dat sequentie-gerelateerde microbiële enzymen kunnen worden gebruikt als humane FMO-mimics voor de synthese van geneesmiddelmetabolieten. Dergelijke enzymen blijken in staat zijn om enantio- en regioselectieve oxygenaties van (menselijke) geneesmiddelmoleculen te verrichten. Uit deze studie blijkt dat de momenteel beschikbare collectie thermostabiele microbiële flavoproteïne monooxygenasen een aantrekkelijk alternatief biedt voor het produceren en bestuderen van geneesmiddelmetabolieten.This thesis presents new approaches towards applications of flavin-containing enzymes as biocatalysts.The first part describes a novel method of cofactor-mediated enzyme immobilization. By coupling the flavin cofactor to carrier material, a target prototype flavoenzyme could be immobilized. This resulted in a fully active and highly stable enzyme formulation. By varying the type of chemical linkers between the carrier material and the flavin cofactor, this new method allows to tune the surface loading and distance from the solid phase to the enzyme (monolayer). To provide a better access to the required chemically modified flavin cofactors, a new chemical synthesis protocol for the preparation of alkylated flavin cofactors was also developed.To demonstrate the value of flavin-containing enzymes as biocatalysts, work is presented on the exploration of a specific group of flavoenzymes: the flavin-containing monooxygenases (FMOs). First, a physiologically crucial human FMO, flavin-containing monooxygenase 3 (FMO3), is extensively discussed concerning the current knowledge on its sequence and (predicted) structural properties. Biochemical studies on this human enzyme are hampered by its reluctance towards heterologous expression of soluble protein. Results on the production of an active, self-sufficient hFMO3 in bacterial cells are presented. Furthermore, it is demonstrated that sequence-related microbial enzymes can be used as human FMO mimics for the synthesis of drug metabolites. Such enzymes are shown to be capable of performing enantio- and regioselective oxygenations of (human) drug molecules. This study shows that the currently available collection of thermostable microbial flavoprotein monooxygenases provides an attractive alternative to produce and study drug metabolite
New approaches for flavoenzyme applications:Cofactor-mediated immobilization & in vitro production of human metabolites
Synthesis and characterisation of new metalloporphyrins for catalytic and biomedical applications
Celem pracy była synteza i charakterystyka spektroskopowa, fotochemiczna i elektrochemiczna nowych metaloporfiryn w kontekście ich potencjalnych zastosowań biomedycznych i katalitycznych. Dokonano syntezy kompleksów mezo-tetra-(2,6-difluoro-5-N-metylo-sulfonamidofenylo)porfiryny (TDFPPSO2NHCH3) z metalami: kobaltem, miedzią, manganem (paramagnetyki) oraz cynkiem (diamagnetyk). Obecność podstawników fluorowych sprawia, iż badane związki posiadają wysokie potencjały utlenienia (E >1 V), natomiast podstawniki sulfonamidowe zwiększają ich hydrofilowość. Nowe metaloporfiryny wykazują wysokie molowe współczynniki absorpcji w zakresie widzialnym (rzędu 105 M¬-1s-1) oraz niskie wydajności kwantowe fluorescencji, przy czym manganowa pochodna praktycznie nie fluoryzuje. Badane pochodne charakteryzują się dużą fotostabilnością i są fotokatalizatorami rozpadu azuru B (z wyjątkiem MnTDFPPSO2NHCH3). ZnTDPFFSO2NHCH3 jest obiecującym fotosensybilizatorem, gdyż posiada długie czasy życia stanu trypletowego i wysoką wydajność tworzenia tlenu singletowego (ΦΔ=0,92). Ponadto wykazano, że po adsorpcji na TiO2 generuje on fotoprąd poprzez przeniesienie elektronu z porfiryny na pasmo przewodnictwa dwutlenku tytanu. Przebadane pochodne mogą być w przyszłości zastosowane w katalizie (CoTDPFFSO2NHCH3, ZnTDPFFSO2NHCH3), terapii fotodynamicznej (ZnTDPFFSO2NHCH3), diagnostyce fotoakustycznej (MnTDPFFSO2NHCH3) czy fotowoltaice (ZnTDPFFSO2NHCH3).The aim of this thesis was the synthesis and spectroscopic, photochemical and electrochemical characterization of new metalloporphyrins in the context of their potential biomedical and catalytic applications. New complexes of meso-tetra-(2,6-difluoro-5-N-methylsulfamylophenyl)-poprhyrin with following metals: cobalt, copper, manganium (paramagnetic) and zinc (diamagnetic) were synthetized. The presence of fluorine substituents increases their oxidation potentials (E > 1 V) and sulfonamide substituents increase their hydrophilicity. New metalloporphyrins have high molar absorption coefficients in the visible range (105 M¬-1s-1 for Soret band) and low or negligible (manganese derivative) fluorescence quantum yield. The studied derivatives are enough photostable and are good photocatalysts in the azur B degradation (except MnTDFPPSO2NHCH3). ZnTDPFFSO2NHCH3 is a promising photosensitizer, because it has a long triplet lifetimes and high quantum yield of singlet oxygen generation (ΦΔ = 0.92). In addition, it is demonstrated that after adsorption on TiO2 it generates photocurrent by electron transfer from porphyrin to the conduction band of titanium dioxide. All these results lead to conclusion that presented metalloporphyrin derivatives might be applied in the future in catalysis (CoTDPFFSO2NHCH3, ZnTDPFFSO2NHCH3), photodynamic therapy (ZnTDPFFSO2NHCH3), photoacoustic diagnostics (MnTDPFFSO2NHCH3) and photovoltaics (ZnTDPFFSO2NHCH3)
High-density cholesterol and apolipoprotein AI as modifiers of plasma fibrin clot properties in apparently healthy individuals
Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites
Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites
Influence of Centrifugation and Shaking on the Self-Assembly of Lysozyme Fibrils
Protein self-assembly into fibrils and oligomers plays a key role in the etiology of degenerative diseases. Several pathways for this self-assembly process have been described and shown to result in different types and ratios of final assemblies, therewith defining the effective physiological response. Known factors that influence assembly pathways are chemical conditions and the presence or lack of agitation. However, in natural and industrial systems, proteins are exposed to a sequence of different and often complex mass transfers. In this paper, we compare the effect of two fundamentally different mass transfer processes on the fibrilization process. Aggregation-prone solutions of hen egg white lysozyme were subjected to predominantly non-advective mass transfer by employing centrifugation and to advective mass transport represented by orbital shaking. In both cases, fibrilization was triggered, while in quiescent only oligomers were formed. The fibrils obtained by shaking compared to fibrils obtained through centrifugation were shorter, thicker, and more rigid. They had rod-like protofibrils as building blocks and a significantly higher β-sheet content was observed. In contrast, fibrils from centrifugation were more flexible and braided. They consisted of intertwined filaments and had low β-sheet content at the expense of random coil. To the best of our knowledge, this is the first evidence of a fibrilization pathway selectivity, with the fibrilization route determined by the mass transfer and mixing configuration (shaking versus centrifugation). This selectivity can be potentially employed for directed protein fibrilization
Covalent immobilization of a flavoprotein monooxygenase via its flavin cofactor
A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 degrees C) when compared to PAMO in solution (no activity detected after 1 h at 60 degrees C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier. (C) 2015 Elsevier Inc. All rights reserved