Susanne Flygare

A general method for the immobilization of cells with preserved viability

SummaryMicrobial, algal, plant and animal cells have been immobilized, with preserved viability, by entrapment in various matrices according to a new bead polymerization technique. The cell polymer/monomer mixture is kept suspended in a hydrophobic phase such as soy, paraffin, or silicon oil, tri-n-butylphosphate, or dibutyphtalate, which is compatible with the cells. The various monomers or polymers tested include agarose, agar, carrageenan, alginate, fibrin, and polyacrylamide. Furthermore, by adjustment of the stirring speed of the suspension, beads of desired diameter can easily be obtained. The entrapped cells are fully viable and biosynthetically active.

Magnetic aqueous two-phase separation: A new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles

A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-micron iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.

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.

Stabilization ofd-amino acid oxidase from yeastTrigonopsis variabilis used for production of glutaryl-7-aminocephalosporanic acid from cephalosporin C

AbstractThe studies to improve the production of glutaryl-7-ACA from cephalosporin C are described in this paper.During the conversion of cephalosporin C to keto-adipyl-7-aminocephalosporonic acid by d-amino acid oxidase (d-AAO), with the simultaneous production of equimolar amount of hydrogen peroxide, an incomplete nonenzymatic conversion of the keto form into the glutaryl form occurs, where cephalosporin C as well asd-AAO are partly destroyed in the presence of hydrogen peroxide. d-AAO was immobilized to different carriers in order to achieve better enzyme stability. The activity of immobilizedd-AAO on manganese oxide remained above 100% during the first 9 h of a semicontinuous conversion of cephalosporin C. The presence of catalase coimmobilized with D-AAO and coupled to CNBr-activated Sepharose 4B improved the operation stability ofd-AAO.An additional approach for the continuous transformation of cephalosporin C used whole cells ofTrigonopsis variabilis, containingd-AAO, immobilized to magnetic iron oxide particles.

Affinity precipitation of dehydrogenases

Affinity precipitation, a novel technique closely related to immunoprecipitation and affinity chromatography, has been evaluated in systems comprised of dehydrogenases and a bifunctional NAD derivative, Bis-NAD. Lactate dehydrogenase and glutamate dehydrogenase were easily precipitated whereas yeast alcohol dehydrogenase required the presence of salt to enhance the affinity precipitation. Liver alcohol dehydrogenase did not precipitate, probably because most of the affinity complexes formed were composed of only two enzyme molecules. Affinity precipitation was carried out on a preparative scale for the isolation of ox heart lactate dehydrogenase from a crude extract. The yield and purity of the enzyme and the general properties of the procedure are considered very satisfactory.

Magnetic aqueous two-phase separation in preparative applications

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331-339). It is based on the addition of magnetically susceptible material (e.g. 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.

Steroid transformation in aqueous two-phase systems: side-chain degradation of cholesterol by Mycobacterium sp.

Abstract We report on the use of aqueous two-phase systems for the side-chain degradation of cholesterol into androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) by Mycobacterium sp. A problem encountered at the start of this investigation, extensive aggregation of the cells, was solved by careful choice of the polymer and detergent composition of the phase system. The systems showing the best properties were composed of polyethylene glycol, dextran and Brij 35 or polyvinylpyrrolidone, dextran and Brij 35 (or Tween 40). The bacterium maintained in the upper polyethylene glycol-rich (or polyvinylpyrrolidone-rich) phase was found to have high activity. The substrated partitioned to the upper phase or the interface (if present as crystals), while the products had partition coefficients around 2.

Steroid transformation using magnetically immobilized Mycobacterium sp.

Abstract Mycobacterium sp. ( NRRL-B 3683 ) has been immobilized by adhesion of magnetic materials of submicron size to the bacterial surface. Preparations based on laboratory-prepared magnetic oxide that had been derivatized with hydrophobic octyltrichlorosilane showed the best properties. The magnetically immobilized bacteria were used for side-chain degradation of cholesterol into androsta-1,4-diene-3,17-dione. The magnetic bacteria behaved as free cells in the transformation media and no mass transfer limitations were observed. The magnetic bacteria could be used repeatedly without any cell loss, the cells being retrieved at the end of each transformation cycle by a magnet .

Affinity precipitation of enzymes.

Lactate dehydrogenase has been purified by precipitation with a bis-ligand. The precipitating agent in this case was Bis-NAD. This approach of affinity precipitation is also applicable to other enzymes.

Magnetically Enhanced Aqueous Two-Phase Separation

A new technique to increase the rate of phase separation in aqueous two-phase systems is described. The technique is based on the use of magnetically susceptible additives, ferrofluids or iron oxide particles. In a magnetic field these additives will induce a faster phase separation. When dextran-covered ferrofluid or iron oxide particles were mixed with an aqueous two-phase system (PEG and dextran), it was found that the magnetic additive was totally distributed to the dextran phase. After mixing of the two-phase system, it was possible to reduce the time for phase separation by a factor of 35 (ferrofluid) or 70 (iron oxide particles) by applying a magnetic field (0.3 Tesla) to the system. It was found that neither the activity, nor the partitioning of the enzymes tested, was influenced by the addition of magnetically susceptible material. When ferrofluid was used it was possible to keep the dextran-phase stationary in a column system, while the PEG-phase could be pumped through the column.