Ernst Reinhard

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors.

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1−1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16′-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, α -acetyldigitoxin, and α-acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.

Biotransformation of β-methyldigitoxin by cell cultures ofDigitalis lanata in airlift and stirred tank reactors

SummaryDigitalis lanata cells were grown at dif-ferent dissolved-oxygen (DO) levels in 20-1 airlift reactors. A DO level of 30% saturation (using air for aeration) was found to be optimal for growth and the biotransformation ofβ-methyldigitoxin toβ-methyldigoxin. Product yield was further in-creased by using stirred tank reactors instead of the airlift reactor.

Synthesis of volatile oils in tissue cultures of Ruta graveolens

Abstract Callus cultures of Ruta graveolens were used to investigate the influence of several factors, independently, on volatile oil composition, in an attempt to explain observed differences in oil composition between the organs of this species. Autotrophic or heterotrophic conditions did not affect composition. However, drastic differences were found between light- and dark-grown callus cultures. The composition of light-grown cultures corresponded to that of leaf tissue. The effect of light does not depend on photosynthesis. Light-grown cultures placed in darkness altered their composition until, after 11 weeks, the volatile oils were similar to those in the roots of the plant.

Selective Uptake and Vacuolar Storage of Primary Cardiac Glycosides by Suspension-cultured Digitalis lanata Cells

Summary Cardenolide uptake by Digitalis cells and protoplasts was examined. The uptake of secondarycardiac glycosides (digitoxin, digoxin, β-methyldigitoxin, β-methyldigoxin) was not affected by ATPase inhibitors, uncoupling agents, or electron transport inhibitors and was thus inferred to result from simple diffusion of the relatively hydrophobic glycosides across the plasma-lemma. The uptake of primary glycosides (purpurea-glycoside A, lanatoside A, deacetyl-lanatoside C, lanatoside C), on the other hand, was inhibited reversibly by uncoupling agents and electron transport inhibitors. It was not affected by ATPase inhibitors. Cell cultures from plant species which do not synthesize cardenolides were unable to accumulate primary cardiac glycosides. Wash-out studies with Digitalis cells and protoplasts showed that secondary glycosides were easily released into the culture medium, while the primary glycosides were retained by the cells. To investigate the intracellular distribution of cardiac glycosides, vacuoles were prepared from cardenolide-containing Digitalis cells and analysed for their cardenolide content using an HPLC method. The primary glycosides were located almost exclusively in the vacuoles. A model for cardenolide uptake, transformation, and transport across the tonoplast is proposed.

Stability of biotransformation capacity in Digitalis lanata cell cultures after cryogenic storage

Suspension cultures of Digitalis lanata were stored at the temperature of liquid nitrogen. After thawing, viable cultures were recovered which showed a growth rate equal to that of untreated cells. The capacity of Digitalis cells to transform β-methyldigitoxin to β-methyldigoxin remained unchanged after cryostorage. This was shown in 300-ml shake cultures as well as in 20-1 bioreactors. From these results it is clear that cryostorage is a suitable method for preserving cell lines with specific capacities to produce compounds of medical and industrial importance.

Biotransformation of Cardiac Glycosides by Digitalis Cell Cultures in Airlift Reactors

The production of valuable plant products by biotransformation from inexpensive precursors which cannot be transformed effectively by chemical or microbial means is an interesting field for practical application of plant cell cultures (Reinhard and Alfermann 1980). Among the reactions observed biotransformation by cardiac glycosides is of special pharmaceutical importance because these are widely used in medicine for treatment of heart diseases. Digitoxin and digoxin and their derivatives which can be extracted from leaves of Digitalis lanata, are the most interesting ones. Nowadays, digitoxin is used in therapy to a lesser extent than digoxin due to special pharmacological characteristics. D. lanata plants, however, always contain substantial amounts of digitoxin, up to about one quarter as compared to the amount of digoxin. Digoxin differs from digitoxin only by an additional hydroxyl function at C-12 (Fig. 1). Undifferentiated cell cultures of Digitalis do not produce cardiac glycosides, however they are able to perform special biotransformation reactions on substrates added into the medium (Reinhard et al. 1975).

Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis, in cell cultures and plants of Digitalis lanata EHRH

SummaryThe in vitro transformation of pregnenolone into progesterone in Digitalis lanata tissues was shown to be catalyzed by a 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (3β-HSD). Product formation was monitored by HPLC. The enzyme could be partially characterized and 3β-HSD activities were measured in various Digitalis lanata tissues and in cell cultures of other plant species. Since no correlation was observed between biosynthetic competence of the tissue and 3β-HSD activity, it was concluded that this enzyme does not play a major role in regulating cardenolide biosynthesis.

Rapid Isolation of Vacuoles from Suspension-cultured Digitalis lanata Cells

Summary A rapid method of isolating vacuoles from suspension-cultured Digitalis lanata cells in less than 2.5 hours is described. A combination of 1 % purified Cellulase RS and 0.3 % Rhozyme HP resulted in optimum protoplast yield. High temperatures (32 °C) accelerated the release of protoplasts. Protoplasts were lysed by mild osmotic shock in an alkaline buffer (pH 8) containing EDTA. The vacuoles released were purified by floatation through a discontinuous Ficoll gradient. The yield of vacuoles was about 20%. Cytoplasmatic contamination was less than 10 % as determined by marker enzyme analysis. The potential use of the method with regard to kinetic studies is discussed.

Storage of cardiac glycosides in vacuoles of Digitalis lanata mesophyll cells

Abstract The intracellular distribution of cardenolides in mesophyll cells of Digitalis lanata was investigated. Plantlets were grown under axenic conditions. Protoplasts were isolated from sliced leaves and lysed to yield intact vacuoles which were subsequently purified by flotation centrifugation. The cardenolide contents of mesophyll cells, protoplasts and vacuoles were analysed by HPLC. Primary glycosides, e.g. the lanatosides A and C, were found to be stored exclusively in the vacuoles. Although present in quite high amounts in leaf tissue, secondary glycosides, such as α-acetyldigoxin or α-acetyldigitoxin, could not be detected in vacuoles isolated from mesophyll cells. It is concluded that in Digitalis leaves cardenolides are stored solely as primary glycosides, whereas secondary glycosides found in leaf extracts are either biosynthetic intermediates or degradation products.