Transformation of delta4-3-ketosteroids by free and immobilized cells of Rhodococcus erythropolis actinobacterium

9alpha-Hydroxy derivatives were prepared from 11 steroids ofandrostane and pregnane series using Rhodococcus erythropolis VKPM Ac-1740 culture with 0.5-20 g/l substrate concentration in the reaction mixture. 9alpha-Monohydroxylation proceeded regardless of the substituent structure at C17. However, the structure of the steroid molecule influenced the time of complete conversion of the substrate and the yield of the transformation product. 9alpha-Hydroxy-androstenedione was obtained in 35 h in a yield of 85% when the maximum concentration of androstenedione (AD) was 10 g/l. 9alpha-Hydroxy-AD was also formed by the actinobacterium cells entrapped in poly(vinyl alcohol) cryogel beads. Nine successive transformation cycles were carried out using immobilized cells at 4.0 g/l concentration of AD in the medium. The yield of 9alpha-hydroxy-AD formed during six cycles (from two to eight with the duration of each cycle for 22-24 h) was 98%.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

Development of a semi-closed underwater breathing apparatus, the "eOBA"

Nippon Sanso K.K developed a compact semi-closed underwater breathing apparatus, the eOBA. It consists of a mouthpiece, manifold with a purge valve, two spring-loaded flexible tubes, a small CO2 absorbent canister (net wt. = 190g), and two compact high pressure bottles (50ccx2: 190kg/cm2: 80%O2, 20%N2) with a regulator which supplies the gas at the constant flow rate of 1.5 l/min and lasts for 10 min. Thus, a counterlung is not incorporated. However, spring-loaded tubes act as a counterlung since its volume increases to 3.5 l when fully inflated. Dives to a depth of 5m are also recommended because of no bypass valve. This new eOBA was tested using the mechanical breathing machine and CO2 supply system to the circuit. For the various combinations of tidal volumes (0.5-2.5 l) and respiratory rates (10-20 breaths/min), the pressure at the mouthpiece, respiratory volume and the CO2 level were continuously monitored. The CO2 absorption rates were then calculated. The thin sloping P-V loops demonstrate that the eOBA is a flow dependent type of apparatus. It was found that the external work of breathing (0.1 kg.m/l at 30 l/min) were allowable. The CO2 absorption rates were sufficient when minute ventilation increased to 30 l/min. Thus, results show that the eOBA must be suitable for shallow and short dives.     

Effect of copper sulfate on the immune response and susceptibility to Vibrio alginolyticus in the white shrimp Litopenaeus vannamei

White shrimp Litopenaeus vannamei (Boone) held in 35 per thousand seawater were challenged with Vibrio alginolyticus at a dose of 3 x 10(5) colony-forming units (cfu) shrimp(-1), and then placed in water containing concentrations of Cu2+ at 0 (control), 1, 5, 10 and 20 mg l(-1). Mortality of shrimp in 5, 10 and 20 mg l(-1) Cu2+ was significantly higher than those in 1 mg l(-1) Cu2+ and the control solution after 24-96 h. In another experiment, L. vannamei which had been exposed to control, 1, 5, 10 and 20 mg l(-1) Cu2+ for 24, 48 and 96 h were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity and clearance efficiency to V. alginolyticus. Copper concentrations at 1 mg l(-1) or greater for 24h resulted in decreased THC, phenoloxidase activity, phagocytic activity and clearance efficiency, whereas copper concentration at 20 mg l(-1) caused significant increase in respiratory burst of L. vannamei. In conclusion, concentration of Cu2+ at 1 mg l(-1) or greater increased the susceptibility of L. vannamei to V. alginolyticus infection by a depression in immune ability. The release of superoxide anion by L. vannamei exposed to 20 mg l(-1) Cu2+ was considered to be cytotoxic to the host.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

Role of lysosomotrophic reagents in glucocorticoid hormone action

Administration of (10 mg/200 g) methylamine or chloroquine to adrenalectomized rats for 2 days followed by a single injection of either cortisol (2.5 mg/200 g) or dexamethasone (0.5 mg/200 g) resulted in a significant enhancement of the tyrosine aminotransferase enzymatic activity in rat liver versus rats given a single injection only of either steroid. Lysosomotrophic reagents were unable to induce tyrosine aminotransferase when administered alone. Cytosols from rat liver treated with lysosomotrophic reagents in vivo had approx. 20-30% more specific binding to [3H]dexamethasone as compared to the control, untreated rats. This enhanced binding was due to an increase in the concentration of the receptor rather than a change in the affinity of the hormone for the receptor. Rat livers perfused with and homogenized in 10 mM Tris-HCI/0.25 M sucrose buffer (pH 7.5) containing about 5 mM lysosomotrophic reagents showed optimum stabilization of the steroid unbound glucocorticoid receptor in vitro at both 4 degrees C and 25 degrees C. These reagents had no effect on in vitro transformation of [3H]dexamethasone-receptor complex or on the binding of the thermally transformed receptor to the nuclei. It is concluded from these studies that lysosomotrophic reagents enhance tyrosine aminotransferase induction by glucocorticoids and stabilize unbound glucocorticoid receptor both in vivo and in vitro without any effect on in vitro transformation of the steroid-receptor complex.     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Some characteristics of human adrenal microsomal 21-hydroxylase activity

The following general characteristics of 21-hydroxylase activity were determined using pooled microsomes obtained from three glands. Enzyme activity exhibited a broad pH dependence, being optimal between pH 7.4-pH 7.8, and was maximal with NADPH in the range 2 to 4.75 X 10(-4)mol/l. No microsomal 21-hydroxylase activity was detected in the absence of NADPH or substrate and when heat denatured microsomes were employed. Enzyme activity was depressed by greater than 75% in the presence of 100% oxygen or nitrogen. In a second set of experiments, microsomal fractions were prepared individually from 7 glands. In the presence of 17 alpha-hydroxy progesterone (2.0 X 10(-7) and 2.0 X 10(-6)mol/l) product formation was linear with time for up to 90 s when the microsomal protein concentration was 5, 10 and 20 micrograms/ml. Between 5 and 30% of the substrate was converted during the first 60 s. In 5/7 of the glands the addition of the autologous cytosol (20 micrograms protein/ml) was without effect, and enzyme activity (using a 60 s reaction and either 2.0 X 10(-7) or 2 X 10(-6)mol/l 17 alpha-hydroxy progesterone was directly proportional to the microsomal protein concentration (range 0-20 micrograms/ml). With the other 2 adrenals 21-hydroxylation was not proportional to the same range of microsomal protein concentrations, although it became so upon the addition of cytosol, which significantly augmented activity. There was considerable variation in enzyme activity between glands from different individuals (Vmax ranging from 2.6 to 16.6 X 10(-9) mol/min/mg protein) and in the apparent Km's (from 0.22 to 1.1 X 10(-6)mol/l). In the two preparations sensitive to cytosol, the Vmax increased 2-fold, and the Km was 3 times lower. Cytosol was without effect upon the kinetic characteristics of the other 5 microsomal preparations. Ascorbic acid (1 X 10(-3) mol/l) depressed enzyme activity by 25-43% whereas oxidised and reduced glutathione (1 X 10(-3) mol/l) showed a slight and variable effect upon 21-hydroxylation.     

Conversion of phytosterols into androstenedione by <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

Optimum conditions for transformation of phytosterols by Mycobacterium neoaurum, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Dissolved oxygen and the scleroglucan fermentation process

The effect of culture dissolved oxygen (D.O.) upon biomass, scleroglucan and oxalate formation bySclerotium glucanicum was examined in a stirred tank fermenter by oxygen enrichment. Controlling culture D.O. at 5 or 10% saturation led to increased biomass formation and decreased scleroglucan formation. The mechanism by which this occurred probably involved a non-specific diversion of C source (sucrose) away from product and towards biomass synthesis. This is at variance with the reported stimulatory effect of limiting D.O. levels upon scleroglucan synthesis. Controlling the culture at higher levels (20 and 30%) involved increases in impeller speed. The results of such changes were distinct from those of D.O., thus demonstrating that attempts to examine D.O. effects by means of impeller speed changes are inappropriate.     

Efficient production of protopectinases by Bacillus subtilis using medium based on soybean flour

We have developed a culture system for efficient production of protopectinases (PPases) by Bacillus subtilis. PPase shows the pectin-releasing activity and is expected to be utilized in the enzymatic cotton scouring. B. subtilis IFO3134 was cultivated using defatted soybean flour as a main component of culture media. This strain produced three different types of PPases, namely, PPase-C, -N and -R performing endo-arabinase activity, pectate-lyase activity and pectin-lyase activity, respectively. The effects of alkaline solubilization and autoclave treatments to extract nutrients from soybean flour and initial soybean flour concentration (20-80g/l) on production of PPases in batch fermentation were investigated. Alkaline solubilization of soybean flour with NaOH remarkably reduced enzyme productivity. In addition, a higher initial concentration of soybean flour reduced the enzyme productivity of cells. The pectin-releasing activity was the largest and reached up to 2200-2400U/ml, when the culture medium containing an initial soybean flour concentration of 40g/l was autoclaved for 45-60min without alkaline solubilization treatment.     

Increased ethanol production during growth of electric-field stimulated Kluyveromyces marxianus IMB3 during growth on lactose-containing media at 45°C

Summary Following treatment of the thermotolerant ethanol producing yeast strain Kluyveromyces marxianus IMB3 with single electric field pulses of 2.4kV/cm, ethanol production was found to increase from 25 to 35% of the theoretical maximum yield within 20 hours, during growth on lactose-containing media (4% [w/v]). Following treatment with electric fields and addition of 1mM MnCl₂ to lactose-containing media, ethanol production almost doubled within a 30 hour period. In addition, low levels of functional -galactosidase activity could be detected in the extracellular culture filtrates, presumably as a result of electropermeabilization.     

Lithoautotrophic growth of the freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402

The freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402 was shown to be capable of lithoautotrophic growth in a batch culture under microaerobic conditions at O2 concentrations in the medium of no higher than 0.5 mg/l. The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l. A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells. The high rate of CO2 assimilation (112-139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91-92% of which was accounted for by CO2 carbon, were close to those typical of autotrophic bacteria. Thiosulfate was oxidized almost completely to sulfate, and the fraction of elemental sulfur in the final products did not exceed 0.2-1.7% of the thiosulfate sulfur. The cell membrane fraction contained cytochromes (b + o) and two cytochromes c with M(r) of 23 and 26 kDa; the soluble fraction contained cytochrome c with M(r) of 12 kDa.     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

Purification and properties of a hyperthermoactive α-amylase from the archaeobacterium Pyrococcus woesei

The cultivation of the hyperthermophilic archaeobacterium Pyrococcus woesei on starch under continuous gassing (80% H₂:20% CO₂) caused the formation of 250 U/l of an extremely thermoactive and thermostable -amylase. In a complex medium without elemental sulphur under 80% N₂ and 20% CO₂ atmosphere enzyme production could be elevated up to 1000 U/l. Pyrococcus woesei grew preferentially on poly-and oligosaccharides. The amylolytic enzyme formation was constitutive. Enzyme production was also observed in continuous culture at dilution rates from 0.1 to 0.4 h^-1. A 20-fold enrichment of -amylase was achieved after adsorption of the enzyme onto starch and its desorption by preparative gel electrophoresis. The -amylase consisted of a single subunit with a molecular mass of 70 000 and was catalytically active at a temperature range between 40°C and 130°C. Enzymatic activity was detected even after autoclaving at a pressure of 2 bars at 120°C for 5 h. The purified enzyme hydrolyzed exclusively -1,4-glycosidic linkages present in glucose polymers of various sizes. Unlike many -amylases from anaerobes the enzyme from P. woesei was unable to attack short chain oligosaccharides with a chain length between 2 and 6 glucose units.     

退热1号口服液抗4种病毒的实验研究

采用病变抑制法和血凝试验在鸡胚和2种细胞培养上测定了中成药退热1号口服液对4种病毒的抑制作用。试验结果表明,退热1号口服液在浓度为1:2(原液浓度为每100毫升含生药量245克)以下时,对鸡胚和细胞无毒性作用。浓度在1:4—1:32时,对流感病毒、呼吸道合胞病毒和巨细胞病毒有较强的抑制作用,但对腺病毒无抑制作用。     

Efficient production of desulfurizing cells with the aid of expert system

An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 5l jar fermentor with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20mmolkg-drycells(-1)h(-1) and of 45g-drycellsl(-1), respectively. At 100h of cultivation, 1g/l of dibenzothiophene (DBT) was converted to 2HBP within 20h, i.e., 10mmolkg-drycells(-1)h(-1) of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment.     

Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes

GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb₁ and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb₁ after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb₁ was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.Abbreviations ABTS 2,2-Azino-bis(3-ethylbenzthiazoline-6-sulfonate) - CDNB I-Chloro-2,4-dinitrobenzene - DCNB 1,2-dichloro-4-nitrobenzene; DEX, dexamethasone - DMSO dimethylsulfoxide - GST glutathione Stransferase - MC methylcholanthrene - N, NIC nicotinamide - -NF -naphthoflavone - PB phenobarbital - PBS phosphate buffered saline