Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

Continuous acetone-butanol production with direct product removal

Summary To eliminate the product inhibition and increase the productivity of butanol formation, a continuously operated membrane bioreactor was connected to a four-stage mixer-settler cascade. Clostridium acetobutylicum was cultivated in this reactor. Butanol was selectively extracted with butyric acid saturated n-decanol from the cell-free cultivation medium, and the butanol-free medium was refed into the reactor. Due to the high boiling point of decanol, the recovery of butanol from the decanol solution is easy. The partition coefficient and selectivity of butanol in the cultivation medium-decanol-system is sufficiently high for removing it from the medium. Direct contact of the cells with the decanol phase causes cell damage. However, decanol is practically insoluble in the fermentation medium, thus the contact of the cell-free medium with the solvent phase does not influence of cell growth and product formation. At a dilution rate of D _z=0.1 h^-1, the butanol productivity was increased by removing butanol from the medium by a factor of four. A further increase was prevented by a contaminant of the technical decanol, which was identified by GC-MS-analysis as 1-,3-hexandiol.Symbols D dilution rate, h^-1 - D _eff effective dilution rate (Eq. 3), h^-1 - D _Ex extraction dilution rate (Eq. 3), h^-1 - D _g dilution rate of cell suspension in reactor-filter-system, h^-1 - E degree of extraction (Eq. 3), l - P product concentration in medium after extraction, g l^-1 - P _O product concentration in reactor, g l^-1 - R _P productivity and product formation rate, g l^-1 h^-1 - q _p S specific product formation coefficient with regard to the cell growth rate, l - V _F volume of cell suspension in filter module, l - V _g volume of the cell suspension in reactor and in filter module V _g =V _R +V _F , l - V _R volume of cell suspension in ractor, l - v _O cell free feed rate, l h^-1 - v ₁ flow rate of cell suspension leaves the reactor, l h^-1 - v _E flow rate of decanol through the extractor, l h^-1 - v _w flow rate of the cell free medium through the filter modul, l h^-1 - X cell mass concentration, g l^-1 - specific growth rate of the cells, h^-1 Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Reactive extraction of penicillin G in a pilot plant Karr-column II. Fermentation broths

Summary Penicillin G was extracted from mycelfree fermentation broths by means of the carrier (Amberlite LA-2) in n-butylacetate at pH 5 in a 7.6 m high pilot plant Karr-column with degrees of extraction E=98–99% and penicillin enrichments up to 3. The reextraction was carried out with phosphate buffer at pH-values above 7.5 with degree of extractions E=86–88% and penicillin enrichments up to 3. The penicillin and carrier losses were negligible. The influence of the process variables on the extraction degree was investigated. The penicillin extraction of the model medium and the fermentation broths were compared. Recommendations are given for the optimal penicillin recovery with reactive extraction.Symbols a specific interfacial area with regard to the volume of the continuous phase - cA concentration of carrier - cAHP,O concentration of complex in feed - cP,cP,O concentration of penicillin acid anion in theaqueous phase, in the feed - d ₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - V _aq throughput of the aqueous phase - V ₀ throughput of the organic phase - Z dimensionsless longitudinal coordinate of the column with regard to its active length (4m) - holdup of the organic phase     

The use of lignocellulosic wastes for production of cellulase by Trichoderma reesei

Summary The feasibility of cellulase production by Trichoderma reesei using inexpensive lignocellulosic material was examined. Sulfite pulp used as standard substrate yielded 3.7 IU/ml filter paper units (FPU) and 2.15 IU/ml -glucosidase. The yield was 185 FPU per gram total carbohydrate (CH) in the fermentation medium. Steam treated wheat straw (2%) gave 1.9 FPU/ml, 0.83 IU/ml -glucosidase and 151 FPU/g CH, whereas the spent fibres remaining after enzymatic hydrolysis of steamed wheat straw gave 2.4 FPU/ml, 1.55 IU/ml -glucosidase and 147 FPU/g CH. A good substrate (3%) was also the combustible fraction of municipal waste (BRAM) treated with NaOH, which gave 2.5 FPU/ml, 0.86 IU/ml -glucosidase and 130 FPU/g CH. A further increase in the final enzyme titer is obtainable by increasing the substrate concentration. In shake cultures 5% steamed wheat straw gave 3.8 FPU/ml and 1.95 IU/ml -glucosidase. Untreated wheat straw gave only low final enzyme titers and low yields of FPU/g CH. In the case of lignocellulosic substrates a constant pH-value of pH 6.0 during the fermentation gave optimal yields.     

Aldosterone biosynthesis and cytochrome P-45011 beta: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-45011 beta has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11 beta-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-45011 beta. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-45011 beta in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.     

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.     

Microcalorimetric investigation into the metabolic activity of rat caecal flora in the presence of different sugars and sugar substitutes

When adapting young rats to different sugar substitutes (sorbitol, PolydextroseR and PalatinitR), effects were seen in the caecal morphology and caecal content e.g. bacterial concentration, which did not occur when adapting rats to sugars (glucose, sucrose). For in vitro studies, anaerobic growth of caecal flora in thioglycollate medium with and without the respective substances was monitored by continuous measurement of heat production, optical density and pH. Additionally, biochemical analyses and light microscopic observations were performed in order to detect differences between adapted and non-adapted flora. In particular the microcalorimetric data furnished valuable information about alterations in bacterial metabolic activity after adaptation to sugars and sugar substitutes, and clearly indicated that all the substances tested influenced the metabolism of caecal flora.     

The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch

Cultures of the unicellular green alga Chlamydomonas reinhardii can be synchronized by light/dark cycling not only under photoautotrophic but also under mixotrophic growth conditions. We observed that cultures synchronized in the presence of acetate continue to divide synchronously for one cell-cycle period when transferred to heterotrophic growth conditions. This finding enabled us to investigate the differential effects of light on cell growth and cell division. When cells were exposed to continuous light at the beginning of the growth period they entered the division phase earlier than dark-grown cells as a consequence of an increased growth rate. Illumination at the end of the growth period, however, caused a considerable delay in cell division and an extended growth period. The light-induced delay in cell division was also observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. This finding demonstrates that cell division is directly influenced by a light/dard-responsive cell-cycle switch rather than by light/dark-dependent changes in energy metabolism. The importance of this light/dark control to the regulation of the Chlamydomonas cell cycle was investigated in comparison with other control mechanisms (size control, time control). We found that the light/dard-responsive cell-cycle switch regulates the transition from G1-to S-phase. This control mechanism is effective in cells which have attained the commitment to at least one round of DNA replication and division but have not attained the maximal cell mass which initiates cell division in the light.Abbreviations dCTP deoxycytidine 5-triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea