Sten Ohlson

High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigens.

High Performance Liquid Afinity Chromatography (HPLAC) and its Application to the Separation of Enzymes and Antigens

High-performance liquid affinity chromatography of nucleosides, nucleotides and carbohydrates with boronic acid-substituted microparticulate silica

High-Performance Liquid Affinity Chromatography of Nucleosides, Nucleotides and Carbonhydrates with Boronic Acid-Substituted Microparticulate Silica

High-Performance Liquid Affinity Chromatography of Proteins on Cibacron Blue F3G-A Bonded Silica

Abstract The reactive triazine dye, Cibacron Blue F3G-A, has been covalently attached to microparticulate porous silica and used for the resolution of a number of complementary proteins by high-performance liquid affinity chromatography (HPLAC). Cibacron Blue F3G-A was converted to its 6-aminohexyl derivative by reaction with 1,6-diaminohexane and coupled to γ-glycidoxypropyltrimethoxysilane-activated porous silica (5 μm) to generate an adsorbent containing 5.5–6.7 μmol dye/g silica. Cibacron Blue F3G-A silica columns, in conjunction with on-line monitoring of protein concentration and enzyme activity, may be exploited for the high speed fully automated resolution of dehydrogenases, isoenzymes, kinases and other proteins such as pancreatic ribonuclease A from simple or complex mixtures. The examples demonstrate the versatility of HPLAC on silica-immobilised Cibacron Blue F3G-A.

Steroid Transformation by Living Cells Immobilized in Calcium Alginate

SummaryWhole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-Δ1-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The Δ1-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-Δ1-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.

Denitrification of Water Using Immobilized Pseudomonas denitrificans Cells

SummaryPreparations of living Pseudomonas denitrificans cells immobilized in alginate gel were used in the denitrification of water. In the presence of an exogenous carbon source the entrapped microorganisms reduced nitrate and nitrite to gaseous products and to achieve complete reduction, carbon to nitrogen ratios of over two were required. The effects on denitrification of particle size and the number of bacteria in the gel were investigated. Apparent Km values for nitrate and nitrite reduction were calculated for free and immobilized cells. When the immobilized cells were incubated in nutrient media, an increase in reduction rate was observed and this was shown to be caused by the growth of cells within the gel particles. Immobilized P. denitrificans cells retained 75% of their initial nitrate reduction capacity after 21 days of storage at +4°C. The operational stability of the alginate-immobilized cells was studied both in batch and in a column which was operated continuously. A column (45 g of alginate-cell fibers in 80 ml) denitrified a high nitrate drinking water (100 mg NO3/l) with a rate of 300 ml of nitrate and nitrite free water/day/g of gel. The half life for nitrate reduction was estimated to be 30 days.

Columnar denitrification of water by immobilized Pseudomonas denitrificans cells

SummaryWhole cells of Pseudomonas denitrificans, immobilized in alginate gel, were used for columnar denitrification of ground water. Ethanol was selected as a suitable carbon source and the C/N-ratio necessary for satisfactory nitrate reduction was established (1.6 mg ethanol-C/mg nitrate-N). The course of the reaction and the diffusional limitations were investigated during columnar denitrification. The mechanical integrity of the gel matrix, as judged from leakage of cells was studied. The release of cells into the effluent was effectively inhibited (<102 cells/ml) by the use of different filter devices. The operational characteristics were determined by studying a column operating for nearly four months. Theoretically, the alginate gel column should, from high nitrate drinking water (22 mg NO3−-N/1), produce 3 1 of denitrified water/kg gel/h (wet wt.) during a period of two months. The regeneration of nitrate reduction activity by means of activation in nutrient media proved a useful tool for restoring initial activity, the gel column having shown no loss in activity at the end of the operation period.

Steroid Hydroxylation Using Immobilized Spores of Curvularia lunata Germinated in situ

SummarySpores of Curvularia lunata were immobilized in polyacrylamide granules and in calcium alginate beads (2–3 mm in diam.). Germination of the spores, initiated by the addition of nutrients, resulted in an even distribution of mycelium throughout the beads after 48 h. Such beads were used for the conversion of cortexolone to cortisol by steroid-11β-hydroxylation. In order to improve the steroid transforming ability several parameters were studied. It was found that preparations based on calcium alginate gave the best results.The possible merits of immobilizing spores rather than vegetative cells, followed by in situ germination are discussed also for other microorganisms and immobilization processes.

Immobilized cells in microbial nitrate reduction.

The microorganismPseudomonas denitrificans was immobilized in alginate. These immobilized cells were capable of reducing 0.8 mg NO3-/min/g wet weight of cells.

Steroid Conversion Using Immobilized Living Microorganisms

In steroid transformations some of the advantages of immobilized microorganisms seem to be especially important, since the manufacture of steroid drugs often involves very expensive intermediates and products (1). Therefore, we have been studying two corticosteroid transformations. The first reaction is an 11-β-hydroxylation catalyzed by free or polyacrylamide immobilized Curvularia lunata, producing cortisol from Reichstein’s S (cortexolon) (1, 2). The second reaction is a 3-ketosteroid-Δ1-dehydrogenation of cortisol to predniso’one catalyzed by free or polyacrylamide immobilized Anthnobacten simplex (3, 4).