Complementary exploration of the action pattern of hyaluronate lyase from Streptococcus agalactiae using capillary electrophoresis, gel-permeation chromatography and viscosimetric measurements.

Hyaluronic acid (HA) was treated with hyaluronate lyase (GBS HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae strain 4755), and the products have been analyzed by capillary electrophoresis (CE-UV and online CE-ESIMS), gel-permeation chromatography (GPC) and viscosimetric measurements. The resulting electropherograms showed that the enzyme produced a mixture of oligosaccharides with a 4,5-unsaturated uronic acid nonreducing terminus. More exhaustive degradation of HA led to increasing amounts of di-, tetra-, hexa-, octa- and decasaccharides. Using CE, linear relationships were found between peak area of the observed oligosaccharides and reaction time. Determination of viscosity at different stages of reaction yielded an initial rapid decrease following Michaelis-Menten theory. A reaction time-dependent change in the elution position of the HA peak due to partial digestion of HA with GBS hyaluronate lyase has been observed by GPC. These results indicated that the HA lyase under investigation is an eliminase that acts in a nonprocessive endolytic manner, as at all stages of digestion a mixture of oligosaccharides of different size were found. For GBS HA lyase from Streptococcus agalactiae strain 3502, previously published findings reported an action pattern that involves an initial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharides. Our results suggest that differences between the two enzymes from distinct S. agalactiae strains (GBS strains 4755 and 3502) have to be considered.

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.

The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation.

The effects of hyaluronan and its degradation products on irradiation-induced lipid peroxidation were investigated. Liposomal skin lipid models with increasing complexity were used. Hyaluronan and its fragments were able to reduce the amount of lipid peroxidation secondary products quantified by the thiobarbituric acid (TBA) assay. The qualitative changes were studied by mass spectrometry. To elucidate the nature of free radical involvement electron paramagnetic resonance (EPR) studies were carried out. The influence of hyaluronan and its fragments on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the mucopolysaccharide's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) showed no concentration changes of the stable radical caused by hyaluronan. Hyaluronan was found to exhibit prooxidative effects in the Fenton assay in a concentration dependent manner. A transition metal chelation was proposed as a mechanism of this behavior. Considering human skin and its constant exposure to UV light and oxygen and an increased pool of iron in irradiated skin the administration of hyaluronan or its fragments in cosmetic formulations or sunscreens could be helpful for the protection of the human skin.