Regioselective hydroxylation of cholecalciferol, cholesterol and other sterol derivatives by steroid C25 dehydrogenase.

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-ß-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min-1 mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Yarrowia lipolytica: the novel and promising 2-phenylethanol producer.

This is the first report on the ability of Yarrowia lipolytica strains to produce 2-phenylethanol (2-PE), which has not been identified for this species to date. 2-PE is a valuable aroma compound of rose-like odor. Its isolation from the other than microbial source-rose petals, is limited by the substrate availability. Thus, this chemical compound constitutes an attractive product for biotechnological conversions. To date, the ability to produce 2-PE has been described for such genera as Saccharomyces sp., Kluyveromyces sp., Geotrichum sp., and Pichia sp. This report provides evidence that Y. lipolytica is a novel 2-PE producer. Moreover, the titers of 2-PE obtained in Y. lipolytica NCYC3825 non-optimized cultures, nearly 2 g/l, are competitive to titers obtained by the other species.

Effect of O(2)/O(3) atmosphere on the rate of osmotic hemolysis of bovine erythrocytes in the presence of some antioxidants.

Purified red blood cells, exposed to an ozone atmosphere, show an increased rate of hemolysis on sudden osmotic stress. To determine this effect of ozone in the presence of natural antioxidants, bovine red blood cells, used as models, were suspended in blood plasma, or in physiological saline with one of the following antioxidants: albumin, glutathione, uric acid, glucose and a vitamin E analog (trolox). After exposure of the suspensions to oxygen and oxygen/ozone atmospheres the rates of osmotic hemolysis were measured, using a stop-flow technique, and compared with rates measured in air-exposed controls. Blood plasma, containing all natural antioxidants, caused a decreased rate of osmotic hemolysis of cells exposed to oxygen and also decreased the effect of ozone. Trolox cancelled the oxygen effects only. Albumin, glutathione and uric acid tended to protect the cells from the hemolytic effects of ozone. The antihemolytic effect of glucose, seen only in some samples, may depend on uncontrolled factors. The alteration of the rates correlates with an increased fluidity of red cell membranes exposed to ozone.

Critical cell volume and shape of bovine erythrocytes.

The relationship between erythrocyte shape and the critical cell volume was investigated. Agents able to increase the critical cell volume induced three main stable shapes of erythrocytes: discocytic, stomatocytic, and echinocytic. The absence of correlation between shape and critical cell volume under isoosmotic conditions suggests that relative differences between the surface areas of the inner and the outer leaflet of the cell membrane do not influence the critical volume of a cell.

The effect of arsenate and vanadate ions on the critical cell volume of bovine erythrocytes.

Arsenate, used as an inhibitor of glycolysis, decreases the critical cell volume of erythrocytes, whereas orthovanadate, used as an accelerator of dephosphorylation of phosphatidyl-inositol-4,5-biphosphate, exerts an opposite effect. The ATP-dependent changes of the critical cell volume do not depend on the phosphorylation state of phosphatidylinositol. An interaction of the membrane skeleton with the lipid-protein matrix has been proposed as a regulation mechanism of the critical cell volume of erythrocytes.

Effect of DIDS on osmotic properties of bovine erythrocytes.

The effect of DIDS on osmotic properties of bovine erythrocytes was studied. The isoosmotic volume, the amount of intracellular solutes, and the osmotically non-active volume were not influenced by DIDS. An increase of osmotic fragility of erythrocytes upon DIDS treatment was evident and identical both in NaCl and in NaCl + KCl hypotonic solutions. These results suggest that the critical cell volume decreases. The link between the effect of DIDS on the membrane skeleton extractability and on the osmotic fragility was postulated.

Alteration of Cl- transport in erythrocytes from patients with Huntington's disease.

Rate constants for Cl- transport across erythrocyte membranes were measured in experiments performed simultaneously on normal erythrocytes (control) and erythrocytes from patients with Huntington's disease (HD). The rate constants were observed to decrease in HD cells during aging in vitro from 12% higher to lower than normal values. Young HD cells, separated from blood, quickly decreased their permeability for anions by about 30%, while old cells seemed to increase it. Rate constants in control erythrocytes remained steady and equal in both fractions. A hypothesis has been proposed that there might be two populations of erythrocytes in HD blood, one abnormal, characterized by an initially high anion permeability, which decreases significantly during the cell life. A theoretical analysis of the results has suggested that the abnormal fraction of erythrocytes in HD blood may be associated with an increased population of stomatocytes observed by electron microscopy.