Factors affecting lipase production in Syncephalastrum racemosum

Syncephalastrum racemosum grown as a static culture showed maximum lipase production at 30°C in 2d at pH 8.0. When the medium was supplemented with fructose, maximum production of lipase per unit of growth was achieved, followed by raffinose, sucrose, ribose, galactose, maltose, lactose, mannitol and glucose. Amongst the nitrogen sources tested, corn steep liquor at 8% (v/v) produced maximum enzyme; there was evidence of catabolite repression by glucose when groundnut protein, soybean meal, milk casein or wheat bran were the sources of nitrogen. Calcium, potassium and sodium citrates, each at 0.1% (w/v), increased the yield of lipase.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Correlations among the responses of suspensions of Dictyostelium Discoideum to pulses of 3′,5′-cyclic AMP: Transient turbidity decrease,the cyclic AMP signal,and variation in adenine mononucleotide levels

Aggregation-competent myxamoebae of the cellular slime mold Dictyostellium discoideum are known to exhibit two responses to extracellular pulses of 3′5′-cyclic AMP: an immediate chemotactic movement; and a delayed generation of intracellular cyclic AMP which is subsequently released into the medium. The mechanism of the latter, the so-called signalling response, may depend on alterations in intracellular metabolite levels and is the subject of this communication.Myxamoebae of the wild-type strain NC-4 of D. discoideum were suspended in an aerated, stirred 17 mM potassium phosphate buffer. pH 6.0, at a concentration of approx. 6 · 10^?7 cells/ml (8%, v/v) at 25°C and were pulsed with 1. 10^?8—1 · 10^?7 M cyclic AMP at 10–20-min intervals for periods of 3–5 h over incubation of 4–9 h. Suspensions were monitored continuously for transient turbidity decreases following the cyclic AMP pulses as an indication of the magnitude and duration of the cellular response to cyclic AMP. When the pattern of turbidity decrease indicated that a signalling response had developed, samples were withdrawn at 10–15-s intervals from the suspension, inactivated with perchloric acid, and analyzed for cyclic AMP, ATP, ADP, AMP, pyruvate, and glucose 6-phosphate. In separate experiments, steady-state oxygen tension was monitored along with turbidity to detect possible changes in respiratory rate.The following consistent patterns were observed after the added cyclic AMP pulse: a transient increase in the ADP level which reaches maximum between 0.7 and 1.7 min; transient decreases in ATP and pyruvate which concide with and approximately equal the magnitude of the increase in ADP; a later increase in glucose 6-phosphate which reaches maximum approx. 2 min after the ADP     

Comparison of polyvinyl chloride membrane electrodes sensitive to alkylphosphonium ions for the determination of the electrical difference (delta psi) of Streptococcus mutans and Lactobacillus casei

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP+), butyltriphenyl phosphonium ( bTPP +), and methyltriphenyl phosphonium ( mTPP +) ions have been compared for the determination of the electrical potential difference (delta psi) of the oral bacteria, Streptococcus mutans DR0001 /6 and Lactobacillus casei RB1014 . All three types of electrode proved suitable for determining delta psi, although the TPP+-sensitive electrode was particularly susceptible to interference by protonmotive force (delta p) dissipators known to inhibit sugar uptake by the bacteria. The mTPP +-sensitive electrode was the least affected. Similarly, both strains had a high nonspecific binding capacity for TPP+ and bTPP + ions, and this increased for all three ions when the bacteria were heated to 80 degrees C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% (v/v) toluene, 20 microM gramicidin, or 10 microM valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP + ion and corresponding electrode proved the most effective system, and delta psi values of -89 and -107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 mM Hepes-KOH buffer (pH 7.0), containing 1 mM dithiothreitol and 10 mM glucose. Although the delta psi of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to -119 mV in Tris-HCl buffer (pH 7.0).(ABSTRACT TRUNCATED AT 250 WORDS)     

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

Production of Extracellular Halo-alkaline Protease from a Newly Isolated Haloalkaliphilic Bacillus sp. Isolated from Seawater in Western India

Summary Haloalkaliphilic, gram positive, aerobic, coccoid Bacillus sp. Po2 was isolated from a seawater sample in Gujarat, India. On the basis of 16s rRNA gene homology, Po2 was 95% related to Bacillus pseudofirmus. A substantial level of extracellular alkaline protease was produced by Po2, which corresponded with the growth and reached a maximum level (264 U/ml) during the stationary phase at 24 h. The production thereafter remained nearly static at optimal level till 36 h. Po2 could grow in the range of 0–20% NaCl (w/v) and pH 7–9, optimally at 10% NaCl (w/v) and pH 8. The protease production was salt-dependent and optimum production required 15% NaCl (w/v) and pH 8. Among the organic nitrogen sources, optimum growth and protease production (260 U/ml) were supported by the combination of peptone and yeast extract. However, growth and protease production were highly suppressed by the inorganic nitrogen sources used; with the exception of potassium nitrate, which supported both growth and protease production to limited extent (24 U/ml). Strong inhibition of enzyme production was observed at above 1% glucose (w/v). Wheat flour served as both carbon and nitrogen source supporting growth and protease production.     

Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123

Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v) wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation. Received: 22 January 1999 / Received revision: 23 April 1999 / Accepted: 1 May 1999     

Some Factors Governing Respiration,Glucose Metabolism and Iodoacetate Sensitivity in Tetrahymena pyriformis*

SYNOPSIS. The physiologic response of Tetrahymena pyriformis W to glucose, as measured by the respiratory rate in a buffered suspension, was found to be altered by exposure of the culture to this carbohydrate in the peptone-based growth medium. Glucose-grown cells had an elevated respiratory rate in the presence of glucose in the Tris-buffered suspension, while cells not grown with the glucose supplement required sodium and potassium for the response. Orthophosphate elevated the rate of oxygen consumption independently of the glucose effect. The endogenous respiratory of glucose and non-glucose cultured cells were significantly diffe The 2 types of cells responded differently to the cations, sod and potassium, and were not identical in sensitivity to iodoacet Iodoacetate poisoning was found to be due to inhibition of “uncompetitive” type, potentiated by glucose, orthophosphate, the absence of potassium.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Gas phase composition effects on suspension cultures of Taxus cuspidata

The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. (c) 1995 John Wiley & Sons, Inc.     

Determination of calcium dobesilate in human plasma using ion-pairing extraction and high-performance liquid chromatography

A rapid, simple reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed for the determination of calcium dobesilate in human plasma. Sample processing is based on an ion-pairing extraction with tetra-n-butylammonium hydroxide as cationic pairing ion and dichloromethane. Separation of the investigated calcium dobesilate and 2,4-dihydroxybenzoic acid as internal standard was achieved on a Discovery RP-Amide C₁₆ analytical column with 50 mM, pH 2.5, potassium dihydrogenphosphate buffer–acetonitrile (75:25, v/v) mobile phase. The wavelength was set at 305 nm. The limit of quantitation is 100 ng/ml and the calibration curve is linear up to 50 μg/ml. Within-day and between-day precision expressed as the relative standard deviation is about 10% and the accuracy of the determination did not deviate from 100% by more than ±10%. The developed method was found to be suitable for application in human bioequivalence studies.     

Glucose-induced increase of potassium in pancreatic beta-cells associated with reduced mobilization of the ion

The potassium contents of beta-cell-rich pancreatic islets from ob/ob-mice were measured with an integrating flame photometer. After exposure to 5 mM glucose islet potassium increased by 17 +/- 2%, no additional effect being seen with increase of the sugar to 20 mM. Glucose counteracted the loss of islet potassium obtained on removal of the ion from the incubation medium, halving the initial disappearance rate. Whereas the effect of glucose in suppressing the mobilisation of potassium was mimicked by tolbutamide and quinine, it was antagonized by diazoxide. It is concluded that the glucose interference with the outward transport of K+ is sufficient to raise the beta-cell content of the ion.     

Mammary glucose 6-phosphate dehydrogenase. Molecular weight studies

Glucose 6-phosphate dehydrogenase was isolated from lactating rat mammary glands by a procedure extended and modified from one previously described. The sedimentation coefficient, S_20,W, was 10.3 in 0.01 m potassium phosphate, pH 6.9, containing 0.1 m NaCl at three protein concentrations between 0.51 and 1.45 mg/ml. The partial specific volume, v?, was 0.735 ml/g as determined by equilibrium sedimentation centrifugation in H₂O and D₂O containing buffers at pH(D) 6.5 containing 0.01 m potassium phosphate and 0.1 m NaCl. In the same buffer, but with 2.0 m NaCl, the apparent partial specific volume, φ′, was 0.756 ml/g. Equilibrium sedimentation of the enzyme at an initial concentration of 0.8 mg/ml was performed in 0.01 m potassium phosphate, pH 6.5, containing 1.0 mm EDTA, 7.0 mm mercaptoethanol, and various concentrations of NaCl between 0 and 2.0 m and with or without 0.1 mm NADP⁺. Weight-average and Z-average molecular weights were calculated and, from these values, the molecular weights of the monomer and dimer were derived. Under these conditions, the enzyme existed principally as a dimer, of molecular weight approximately 235,000, at low salt concentration, and as a monomer, of molecular weight approximately 120,000 in 1.0 m and 2.0 m NaCl. The subunit molecular weight was found to be 64,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Equilibrium sedimentation in 6 m guanidine hydrochloride gave a subunit molecular weight of 62,000 (assuming v? was unaltered) or 58,000 or 54,000 (assuming v? is decreased by 0.01 or 0.02, respectively, in 6 m guanidine). We conclude that rat mammary glucose 6-phosphate dehydrogenase has a molecular weight similar to that of glucose 6-phosphate dehydrogenases isolated from various other mammalian sources with the notable exception of human erythrocyte glucose 6-phosphate dehydrogenase which, like the microbial glucose 6-phosphate dehydrogenases thus far examined, has a significantly lower molecular weight.     

Effect of glucose on pH and potassium ion balance in suspensions of ascites tumor cells subjected to x-irradiation

Glucose (2.14 microM) was introduced into nonirradiated and irradiated (6 and 60 Gy) ascites tumor cell suspension. Irradiation was shown to suppress the consumption of potassium by cells having no influence on the rate of protoxidation of the suspension, induced by glucose, and on the passive release of potassium from cells.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.     

Strategy for pH control and pH feedback-controlled substrate feeding for high-level production of l-tryptophan by Escherichia coli

Optimum production of l-tryptophan by Escherichia coli depends on pH. Here, we established conditions for optimizing the production of l-tryptophan. The optimum pH range was 6.5–7.2, and pH was controlled using a three-stage strategy [pH 6.5 (0–12 h), pH 6.8 (12–24 h), and pH 7.2 (24–38 h)]. Specifically, ammonium hydroxide was used to adjust pH during the initial 24 h, and potassium hydroxide and ammonium hydroxide (1:2, v/v) were used to adjust pH during 24–38 h. Under these conditions, NH₄ ⁺ and K⁺ concentrations were kept below the threshold for inhibiting l-tryptophan production. Optimization was also accomplished using ratios (v/v) of glucose to alkali solutions equal to 4:1 (5–24 h) and 6:1 (24–38 h). The concentration of glucose and the pH were controlled by adjusting the pH automatically. Applying a pH-feedback feeding method, the steady-state concentration of glucose was maintained at approximately 0.2 ± 0.02 g/l, and acetic acid accumulated to a concentration of 1.15 ± 0.03 g/l, and the plasmid stability was 98 ± 0.5 %. The final, optimized concentration of l-tryptophan was 43.65 ± 0.29 g/l from 52.43 ± 0.38 g/l dry cell weight.     

Variation in the components of palm wine during fermentation

Palm sap from Raphia vinifera was autofermented to palm wine. The pH of the sap fell from 7.0 to 4.5 in 120 h. During this autofermentation the components of the palm sap varied. Quantitative analyses indicated that fresh unfermented sap contained 3.92% (w/v) glucose, 10 mg/ml Vitamin C, 4 mg/ml protein and 0% (w/v) alcohol.     

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

Summary Using ion-specific electrodes, the potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely near physiological conditions. Upon small additions of isotonic sucrose solution to a suspension of red cells in physiological saline the passive potassium efflux increases proportionally to the chloride ratio. The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of a penetrating substance like glucose to the cell suspension. Adding water or hypertonic sodium chloride solution shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as it can be interpreted as a pure ionic strength effect, this result supports the hypothesis of independence of potassium efflux and intracellular concentrations. The results of this investigation together with other studies show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for large membrane potentials. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions.