M. Mar Sojo

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa.

Rhodococcus erythropolis ATCC 25544 as a suitable source of cholesterol oxidase: cell-linked and extracellular enzyme synthesis, purification and concentration

BackgroundThe suitability of the strain Rhodococcus erythropolis ATCC 25544 grown in a two-liter fermentor as a source of cholesterol oxidase has been investigated. The strain produces both cell-linked and extracellular cholesterol oxidase in a high amount, that can be extracted, purified and concentrated by using the detergent Triton X-114.ResultsA spray-dry method of preparation of the enzyme inducer cholesterol in Tween 20 was found to be superior in both convenience and enzyme synthesis yield to one of heat-mixing. Both were similar as far as biomass yield is concerned. Cell-linked cholesterol oxidase was extracted with Triton X-114, and this detergent was also used for purification and concentration, following temperature-induced detergent phase separation. Triton X-114 was utilized to purify and to concentrate the cell-linked and the extracellular enzyme. Cholesterol oxidase was found mainly in the resulting detergent-rich phase. When Triton X-114 concentration was set to 6% w/v the extracellular, but not the cell-extracted enzyme, underwent a 3.4-fold activation after the phase separation process. This result is interpreted in the light of interconvertible forms of the enzyme that do not seem to be in equilibrium. Fermentation yielded 360 U/ml (672 U/ml after activation), 36% of which was extracellular (65% after activation). The Triton X-114 phase separation step yielded 11.6-fold purification and 20.3-fold concentration.ConclusionsThe results of this work may make attractive and cost-effective the implementation of this bacterial strain and this detergent in a purification-based industrial production scheme of commercial cholesterol oxidase.

Cyclodextrins as Diethylstilbestrol Carrier System: Characterization of Diethylstilbestrol-Cyclodextrins Complexes

AbstractPurpose. The in vitro formation of DES-cyclodextrins inclusion complexes was characterized using lipoxygenase as enzymatic system. Methods. DES-cyclodextrins complexes were obtained in aqueous solution. Results. The addition of cyclodextrins to the reaction medium had an inhibitory effect on DES oxidation by lipoxygenase due to the drugs complexation into the cyclodextrin cavity. This inhibitory effect depends on the complexation constant between DES and the cyclodextrins type used. In this case, β-, 2-hydroxypropyl-β- and γ-cyclodextrins have similar complexation constants and therefore produce the same inhibitory effect. Moreover, depending on the type of cyclodextrins used, the solubility of DES can be enhanced up to 956 times, while the lipoxygenase activity remains constant. Conclusions. These results suggest that the system described may be used as a controlled-release delivery system for DES, since it may diminish the local and systemic adverse side effects caused by high concentrations of the drug.

Oxidation of Hydrophobic O-Diphenols by Lipoxygenase in the Presence of Cyclodextrins

Cyclodextrins (CDs) are cyclic oligosaccharides composed of six to eight glucopyranose residues linked by α (l→4) glicosidic bonds in a cylinder-shaped structure. The most important feature of cyclodextrins is their ability to complex a variety of guest molecules into their hydrophobic cavities in aqueous solutions [2]. The physicochemical properties of these inclusion complexes have been extensively studied and several analytical techniques have been used to determine their formation constants [3]: spectrophotometry, fluorimetiy, calorimetry, circular dichroism, phase solubility, etc.