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

Spectrophotometric determination of bacitracin in bulk drug as dabsyl derivative in a range of visible light

A fast spectrophotometric method has been developed for bacitracin identification and determination after condensation reaction with dabsyl chloride. In addition, determination of dye stability of sulfonamide derivative and identification of the molar ratio of reagents was done at various time-points. The developed method has a good linearity with very broad spectrum, correlation coefficient of r = 0.9972, good precision (RSD = 1.54 ± 0.11%), and recovery at three different levels of concentration was found between 98.33% and 103.47%. Usefulness of the method was demonstrated by positive results obtained during determination of bacitracin concentration in bulk drug

Photostability of triazole antifungal drugs in the solid state

The publication is devoted to photostability assessment of four triazole antifungal drugs: fluconazole, itraconazole, posaconazole and voriconazole. The compounds were exposed in the solid state using the whole spectrum of UV-Vis radiation. The analyses were performed using high performance thin layer chromatography (HPTLC) technique with densitometric detection. The results indicates considerable degradation of structurally similar itraconazole and posaconazole which could be clinically significant. After 72 hours of itraconazole irradiation there remain less than 25%, and 60% in case of posaconazole. To a lesser extent photodegradation concern two other compounds with a separate chemical structure: fluconazole and voriconazole. After 72 hours of irradiation there left 75% and 82% of these substances, respectively. The strict dependence between compound photostability and its chemical structure was observed

Determination of azole antifungal medicines using zero-order and derivative UV spectrophotometry

This paper presents a new methodology of quantitative determination of seven azole antifungal medicines widely used in therapy. Analyses were performed directly by using zero-order (fluconazole), first derivative (bifonazole, clotrimazole, econazole, itraconazole, miconazole) and second derivative (ketoconazole) UV spectrophotometry. Validation of all methods confirms their proper precision (%RSD = 0.47 - 2.86), recovery (98.7 - 101.4) and linearity (r coefficient over 0,999) in concentrations under investigation. The parameters received enable the developed procedure to be used in quantitative and as auxiliary in qualitative pharmaceutical analysis

Determination of Se(IV), Cd(II) and Pb(II) ions in homeopathic drugs by inversion voltammetry method

The conditions for identification and quantification of Se(IV), Cd(II) and Pb(II) ions in homeopathic drugs by inversion voltammetry method with the use of EAGRAPH software were established. The studies proved that the method was of high sensitivity in established conditions. The detection limits were 0.66 μg/mL, 0.08 μg/mL and 0.12 μg/mL for Se(IV), Pb(II) and Cd(II) ions, respectively. This method was characterized by repeatability of measurements, a wide range of linearity and satisfactory percent recovery