Miroslav Flieger

Biodegradable plastics from renewable sources.

Plastic waste disposal is a huge ecotechnological problem and one of the approaches to solving this problem is the development of biodegradable plastics. This review summarizes data on their use, biodegradability, commercial reliability and production from renewable resources. Some commercially successful biodegradable plastics are based on chemical synthesis (i.e. polyglycolic acid, polylactic acid, polycaprolactone, and polyvinyl alcohol). Others are products of microbial fermentations (i.e. polyesters and neutral polysaccharides) or are prepared from chemically modified natural products (e.g., starch, cellulose, chitin or soy protein).

Ergot alkaloids — Sources, structures and analytical methods

Natural sources,i.e. fungal strains and species producing ergot alkaloids (EA), are surveyed together with the chemical structures of EA and a list of new natural EA discovered in the last three decades. Other topics include new efficient chromatographic methods (HPLC) for the separation and isolation of new natural EA and also immunological methods of EA detection.

Chemoraces and habitat specialization of Claviceps purpurea populations

ABSTRACT We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), anEcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 μm long, the conidia of G2 isolates were 7 to 10 μm long, and the conidia of G3 isolates were 10 to 12 μm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.

Sequence Analysis and Heterologous Expression of the Lincomycin Biosynthetic Cluster of the Type Strain Streptomyces lincolnensis ATCC 25466

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to ≈1–3 % of the production in the ATCC 25466 strain.

High-throughput analysis of tetracycline antibiotics and their epimers in liquid hog manure using Ultra Performance Liquid Chromatography with UV detection

Antibiotics contained in animal manure can contaminate soil, groundwater and eventually surface and drinking water. To reduce the usage of antibiotics in livestock industry the EU banned their application as growth promoters in 2006. Even though the antibiotics are still used for this purpose and therefore it is necessary to control their applications. An Ultra Performance Liquid Chromatography method (UPLC) with UV detection for determination of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) including their epimers in the liquid hog manure was developed. The antibiotics were extracted with ethyl acetate and separated on UPLC BEH Shield RP18 column. The validated method was selective for all analytes and system suitability was assessed. Calibration curves ranged from 7.8 to 250.0mugmL(-1) with determination coefficient of 0.9999. The method limits of quantification ranged from 0.9 to 1.6mgkg(-1). Recoveries were 52.4+/-3.8%, 72.4+/-5.0%, 83.8+/-5.7% and 95.9+/-4.7% for TC, OTC, CT, and DOX, respectively. The method was used for the determination of TC, OTC, CT, and DOX in liquid hog manure samples.

Enantioseparation of semisynthetic ergot alkaloids on vancomycin and teicoplanin stationary phases

The macrocyclic antibiotics, vancomycin and teicoplanin, were used as chiral stationary phase selectors for the enantioselective separation of semisynthetic ergot alkaloids in reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic behavior of the ergot preparations was investigated in order to obtain a deeper insight into the enantiodiscriminative process. A variety of factors, including mobile phase parameters such as the nature and concentration of the organic modifier, buffer concentration and pH, were examined. Conditions for the enantioseparation of real pharmaceutical preparations, i.e. lisuride, terguride and nicergoline, were found. Differences in the chiral stationary phases are presented and the interaction mechanism is discussed.

Analysis of ergot alkaloids by high-performance liquid chromatography : I. Clavinces and simple derivatives of lysergic acid

Abstract A method of high-performance liquid chromatography has been developed for the separation and quantitative analysis of a mixture of ergot alkaloids on MicroPak NH2 columns using isocratic and gradient elution. The mobile phase is diethyl ether-ethanol. Ultraviolet detection is employed at various wavelengths, and the ergot alkaloids are determined using the method of internal normalization.

High-performance liquid chromatography study of the enantiomer separation of chrysanthemic acid and its analogous compounds on a terguride-based stationary phase

The direct enantioseparation of chrysanthemic acid [2,2-dimethyl-3-(2-methylpropenyl)-cyclopropanecarboxylic acid] and its halogen-substituted analogues was systematically studied by HPLC using a terguride-based chiral stationary phase in combination with a UV diode array and chiroptical detectors. Isomers with (1R) configuration always eluted before those with (IS) configuration. The elution sequence of cis- and trans-isomers was strongly affected by mobile phase pH, whereas the enantioselectivity remained the same. Conditions for the separation of all the enantiomers were also examined. This method was used for monitor the hydrolytic degradation products of Cyfluthrin (Baythroid) in soil under laboratory conditions.

Direct resolution of optically active isomers on chiral packings containing ergoline skeletons: II. Enantioseparation of carboxylic acids

Structurally different carboxylic acids and dansyl derivatives of amino acids were examined on a chiral stationary phase for high-performance liquid chromatography. The packing was prepared by bonding to silica gel an aminopropyl derivative of the ergot alkaloid (+)-terguride. Optimization of the enantioseparations was attained through the study of the influence of the organic modifier content and the pH and ionic strength of the buffer in the eluent. Electrostatic and hydrophobic interactions between the ergot alkaloid and the analyte contribute to a large extent to the retention and chiral discrimination. The selector was shown to be effective for the resolution of dicarboxylic acids, 2-arylcarboxylic acids and amino acid derivatives.

Valine Dehydrogenase from Streptomyces fradiae: Purification and Properties

Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The Mr of the native enzyme was determined to be 218,000 and 215,000, by equilibrium ultracentrifugation and size-exclusion high-performance liquid chromatography, respectively. The enzyme is composed of 12 subunits of Mr 18,000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. The Michaelis constants (Km) were 1 mM for L-valine and 0.029 mM for NAD+. Km values for reductive amination were 0.80 mM for 2-oxoisovalerate, 0.050 mM for NADH and 22 mM for NH4+.