New method of detection of certain bacterial oxidoreductases and transaminases by the Indicator reaction on agar

A method for detection of the following enzymes is described: glutamate oxalacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, 2. 6. 1. 1), lactate dehydrogenase (L-lactate: NAD oxidoreductase, 1. 1. 1. 27) and malate dehydrogenase (L-malate: NAD oxido-reductase, 1. 1. 1. 37), diffused from bacterial suspensions into agar, by means of a “sandwich” agar detector layer containing the specific substrates. The principle of the reaction: reduced nicotinamide-dinucleotide ? nicotinamide-adenine-dinucleotide (NADH₂?NAD) was used, in which fluorescence of NADH₂ (in u. v. light) disappears proportionately to the activity of the test enzyme in its diffusion zone.     

On the mechanism of action of tetracycline antibiotics

Oxytetracycline-sensitive and resistant strains of Staphylococci and ofListeria monocytogenes andPasteurella multocida were tested for differences in the reduction of triphenyltetrazolium (TTC) in the presence of glucose, acetate, lactate, pyruvate, glycerol, succinate, formate, malate, citrate, serine, glycine and asparagine. The sensitive staphylococci reduced TTC more actively than the resistant ones in the presence of glucose, acetate and serine. The resistant strains reduced TTC more actively in the presence of succinate, formate, glycine, pyruvate and malate. Oxytetracycline itself only inhibited the reducing activity greatly in the sensitive staphylococci. In the resistant ones, oxytetracycline only slightly decreased the TTC reduction. The insensitivity of the reducing activity of resistant staphylococci toward the effect of oxytetracycline indicates that this activity may be one of the sites of attack by this antibiotic. Ascorbic acid contained in the injection preparation of oxytetracycline interfered with the inhibitory effect of this antibiotic on the TTC reduction by staphylococci and actually increased the activity of reduction.     

Temperature profiles of cellular growth and exopolysaccharide synthesis by Botryococus braunii Kütz. UC 58

Temperature profiles (range 20–33 °C) were obtained for growth and exopolysaccharide (EPS) biosynthesis of the microalga Botryococcus braunii strain UC 58 under photoautotrophic conditions. The maximum temperature for growth was 32 °C and the temperature dependence of the specific growth rate was described by the Hinshelwood equation based on the Arrhenius relationship. The optimal range of temperatures for growth and extracellular EPS synthesis (25–30 °C) concurred and production of 4.5–5 g l⁻¹ of EPS was obtained routinely, leading to high broth viscosities. Below 23 °C EPS biosynthesis was negligible, although the specific growth rate maintained high values. At supraoptimal temperatures EPS biosynthesis decreased, accompanying the increase in doubling time. The polymers formed at temperatures within the optimal range for production, when dissolved in water, produced solutions (2 gl⁻¹) with the highest viscosity, suggesting that their molecular weight showed the highest values. The degree of polymerization of the EPS synthesized at suboptimal and supraoptimal temperatures was significantly below the values within the optimal range.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Enzymatic hydrolysis of inulin to fructose by glutaraldehyde fixed yeast cells

Inulin, a polyfruction, is found as the reserve carbohydrate in the roots and tubers of various plants (i.e. Jerusalem artichoke, chicory, and dahlia tubers). The beta-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. We have found that the inulase of K. fragilis can be immobilized in the yeast cells by glutaraldehyde treatment. These cells are resistant to physical and enzymatic destruction. Although the exact nature of the immobilization is not fully understood, the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. No reduction of enzyme activity was observed after glutaraldehyde treatment and glutaraldehyde concentration did not affect enzyme activity. A 96% hydrolysis of dahlia inulin was achieved in 10.5 h with a 9.5% (w/v) fixed enzyme suspension. A Jerusalem artichoke extract containing 16.8%polyfructan was completely hydrolyzed in 3.5 h with a 0.24% (w/v)fixed enzyme suspension. This is a time frame feasible for industrial consideration.     

Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity by prolonged periods of light

Summary Entrainment of the circadian rhythm in the pineal N-acetyltranferase activity by prolonged periods of light was studied in rats synchronized with a light:dark regime of 1212 h by observing phase-shifts in rhythm after delays in switching off the light in the evening or after bringing forward of the morning onset of light. When rats were subjected to delays in switching off the light of up to 10 h and then were released into darkness, phase-delays of the evening N-acetyltransferase rise during the same night corresponded roughly to delays in the light switch off. However, phasedelays of the morning decline were much smaller. After a delay in the evening switch off of 11 h, no N-acetyltransferase rhythm was found in the subsequent darkness. The evening N-acetyltransferase rise was phase-delayed by 6.2 h at most 1 day after delays. Phase-delays of the morning Nacetyltransferase decline were shorter than phasedelays of the N-acetyltransferase rise by only 0.7 h to 0.9 h at most. Hence, 1 day after delays in the evening switch off, the period of the high night N-acetyltransferase activity may be shortened only slightly. The N-acetyltransferase rhythm was abolished only after a 12 h delay in switching off the light.Rats were subjected to a bringing forward of the morning light onset and then were released into darkness 4 h before the usual switch off of light. In the following night, the morning N-acetyltransferase decline, but not the evening rise, was phase advanced considerably. Moreover, when the onset of light was brought forward to before midnight, the N-acetyltransferase rise was even phase-delayed. Hence, 1 day after bringing forward the morning onset of light, the period of the high night N-acetyltransferase activity may be drastically reduced. When rats were subjected to a 4 h light pulse around midnight and then released into darkness, the N-acetyltransferase rhythm in the next night was abolished.The data are discussed in terms of a two-component pacemaker controlling the N-acetyltransferase rhythm. It is suggested that delays in the evening switch off of light may disturb the N-acetyltransferase rhythm the next day only a little, as the morning component may adjust to phasedelays of the evening component almost within one cycle. On the other hand, bringing forward the morning onset of light may disturb the N-acetyltransferase rhythm heavily the next day, as the evening component not only does not adjust to phase-advances of the morning component, but it may even be phase-delayed when the light onset occurs before midnight.Abbreviations NAT N-acetyltransferase - PRC phase response curve - E evening component of the N-acetyltransferase rhythm or of its pacemaker - M morning component of the N-acetyltransferase rhythm or of its pacemaker - LD xy light dark cycle comprising x h of light and y h of darkness     

Heterologous transformation ofClaviceps purpurea

Summary A transformation system based on dominant selection markers was established for an industrialClaviceps purpurea strain. Transformants could be obtained by using plasmid pAN 7-1 carrying a bacterial gene for hygromycin (hph) resistance fused to a fungal promoter or by plasmid p3SR2 which carries the acetamidase gene (amdS) fromAspergillus nidulans.