Lipophilic compounds in biotechnology--interactions with cells and technological problems.

Lipophilic compounds are of significant importance in modern biotechnology. Centerly of interest are the biodegradation as well as the biotransformation of such lipophilic and often water-immiscible substances. Both whole cells and/or enzymes are used for these processes. It is obvious that a wide range of problems arise in an application of such complex systems consisting of biocatalysts substrate(s), product(s), water, (in some cases water-immiscible organic solvents): (i) interactions between lipophilic compounds and the membranes resulting in the change of some physiological characteristics of the living system; (ii) problems in the transport of these compounds (substrates and/or products) within the complex structured reaction systems; (iii) the problem of increasing the solubility of the lipophilic and mostly water-immiscible compounds with a minimum of inhibition effects on the processes; (iv) the presence of lipophilic components may also cause changes of the transport processes within the system (e.g. immobilized cells) resulting in changed yield or activity of the biological system. These problems are critically discussed in this review in relation to the known modes of interaction of lipophilic compounds with membranes, the bioavailability of the substrates, and the cases of steroid biotransformations. An outlook of future aspects in research, development and application of such processes is given.

Formation, derivatization and applications of bacterial cellulose.

Acetobacter xylinum produces highly crystalline cellulose extracellularly using glucose as a carbon source. The polymer formed is free of other biogenic compounds, separable in a simple way and characterized by its high water-absorption capacity. Stepwise solvent exchange from water to unpolar solvents leads to a drastic decrease of the water content of the bacterial cellulose without decrease of the highly swollen and activated state. Heterogeneous as well as homogeneous derivatizations, e.g. carboxymethylation, silylation and acetylation, were performed on the wet or dried biopolymer. Furthermore, different methods for formation of hollow fibres during biosynthesis were investigated. Such tubes may have applications as biocompatible material in medicine.

Bioremediation of contaminated groundwater.

Contaminated groundwater from industrial areas in former East Germany was biologically treated using lab-scale solid-state reactors. The ability of bacterial strains of the autochroneous microflora to utilize representative pollutants was tested.

[Synthesis and pharmacologic properties of pranolium and its optical isomers]. / Synthese und pharmakologische Eigenschaften von Pranolium und seinen optischen Isomeren.

Propranolol is a well-known powerful betareceptor-blocking agent. Its quaternary dimethyl derivative, designated as pranolium was firstly prepared by Lucchesi. Compared to propranolol it possesses no betareceptor-blocking activity and no local anaesthetic properties but shows the same antiarrhythmic action as the starting material. The synthesis of pranolium and its optical isomers starting from the corresponding propranolol derivatives is described. Their pharmacological activities have been tested. No significant differences regarding the pharmacological action could be observed.

15 alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione using a hyphal fungus immobilized in calcium alginate gel beads.

Calcium alginate-entrapped mycelium of Penicillium raistrickii i 477 was used for the 15 alpha-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione. The system was tested in 10 batches during 40 days of operation, the catalytic activity could be completely regenerated by incubation in a nutrient medium between several batches.

Biosynthesis of phenylalanine and tyrosine in claviceps.

Some enzymes of the terminal steps of phenylalanine and tyrosine biosyntheses were investigated in various alkaloid-forming ergot strains. All strains studied utilize both arogenate and phenylpyruvate as an intermediate in L-phenylalanine synthesis. L-Tyrosine is preferentially or exclusively synthesized via the arogenate pathway. No feedback inhibition of arogenate pathway enzymes by aromatic amino acids was observed.

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra.

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.

[Effect of amitrol on Claviceps fusiformis, strain SD 58]. / Einfluss von Amitrol auf Claviceps fusiformis, Stamm SD 58.

The influence of amitrole (3-amino-1,2,4-triazole) on growth, alkaloid formation and on some aromatic as well as ergoline pathway specific enzymes were studied in Claviceps strain, SD 58. In a phosphate-rich medium the addition of amitrole can partially reverse the alkaloid-depressing effect of phosphate. Using a typical fermentation medium amitrole reduces the activities of ergoline specific enzymes but increases drastically tryptophan synthase activity and to a lesser extent transaminase of aromatic amino acids.

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Steroid hydroxylation with free and immobilized cells of Penicillium raistrickii in the presence of beta-cyclodextrin.

Free and Ca-alginate-immobilized cells of Penicillium raistrickii i 477 were used for 15 alpha-hydroxylation of 13-ethyl-gon-4-en-3,17-dione. The product formation of both free and immobilized cells was increased in the presence of beta-cyclodextrin, in comparison with reactions carried out in the presence of methanol. Application of beta-cyclodextrin led to increasing solubility of the steroid substrate. The fungus was able to utilize beta-cyclodextrin as a carbon source.

Studies on tissue cultures of the genus Cinchona L. : In vitro mass propagation through meristem-derived plants.

A procedure for the in vitro clonal mass propagation of shoots and plants of two Cinchona species derived from single shoot tips is described. Special attention is given to rooting problems of the shoots under in vitro conditions.

Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures.

Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to 0.9% (based on dry weight) or 6.35 mg/l have been obtained.

Investigations of the oxygen supply in Ca-alginate beads and microcapsules loaded with Penicillium raistrickii using a microelectrode.

The oxygen supply of free, Ca-alginate entrapped and microencapsulated mycelia of Penicillium raistrickii i 477 capable of 15 alpha-hydroxylation of 13-ethyl-gon-4-en-3,17-dione was investigated. Using an oxygen microelectrode distinct gradients of oxygen within the Ca-alginate beads as well as the microcapsules were detected. Slope and width of the gradients were investigated in dependence on the kind of immobilization, the culture age and the cell density on or in the carrier as well as the different forms of the oxygen supply in the medium. So it could be shown that large parts of immobilizates, approximately 96% of the diameter of both types, were oxygen-free. In comparison with free mycelia, the lower oxygen supply of the immobilized mycelia led to a metabolic shift to fermentative catabolism.

Titrostat: a reliable technique for continuous cultivation of microorganisms utilizing liquid or gaseous water insoluble substrates.

We describe a method for continuous cultivation of microorganisms utilizing liquid or gaseous water insoluble substrates as a single source of carbon and energy. The water insoluble substrate, which is also the growth-limiting factor, enters the cultivation space as a manually adjustable single-compound material flow. All nonlimiting nutrients (with the exception of oxygen) enter the cultivation space as ingredients of titrant solution which feed rate is reliably coupled to the rate of substrate addition by means of the system titrator. The method provides mild starting conditions appropriate for primary isolation of microorganisms utilizing substrates with growth-inhibiting properties such as BTEX (benzene, toluene, ethylbenzene, and xylene). The sound control over the microbial specific growth rate makes it suitable for precise kinetic studies as well. We provide a detailed description of both the principles of the method and the equipment used. The dependence of the systems operative range on the concentration of titrant solution is illustrated in the case of continuous cultivation of a mixed bacterial culture on toluene.