Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

β-Amylase from Clostridium thermocellum SS8 - a thermophilic, anaerobic, cellulolytic bacterium

The extracellular amylase produced by Clostridium thermocellum strain SS8 on starch was characterized as a β-amylase based on blue value reduction test and the production of maltose from starch. The enzyme had a temperature and pH optima of 60°C and 6.0, respectively. Of the metal ions tested, Ca^{2 +} and Mg^{2 +} had little effect on enzyme activity, but their presence increased its thermal stability. Ca^{2 +} displayed a higher stabilizing effect and at 10 mmol 1^-1 Ca^{2 +}, the enzyme retained 86% activity even after exposure at 70°C for 30 min. The amylase was induced on starch or maltose but was repressed strongly by glucose.     

Interaction of triacontanol with gibberellin in control of lettuce seed germination

Triacontanol at concentrations from 2.3 × 10^-9 M to 2.3 × 10^-7 M did not affect the germination of lettuce ( Lactuca sativa L., cv. Grand Rapids) seeds in darkness, stimulated by light at 25°C or by benzyladenine at 31°C. Stimulation of seed germination by gibberellin A₃ (10^-5 M ) was significantly inhibited by triacontanol; the most effective concentration was 4.6 × 10^-8 M. Pulse experiments demonstrated that triacontanol was ineffective when applied later than gibberellin, whereas an inverse sequence of treatment caused an inhibition comparable to that resulting from continuous treatment of seeds with both factors. Possible interaction of triacontanol with gibberellin receptor is discussed.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH ≥ 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12° and 16°C and pH values between 5·2 and 5·5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5·5 and incubated at 16°C, corresponding to a probability of growth from a single spore within 14 d of 1·6 × 10^-5. The probability of growth in 28 d in the remaining conditions was <9 × 10^-6. After transfer of inoculated media from 12° to 30°C growth occurred at pH 5·2–5·5 within 19 d. After transfer of inoculated media from 12° to 20°C growth occurred at pH 5·5 and 5·4 but not at pH 5·3 or 5·2 in 40 d. Growth at pH 5·2–5·5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

Sequestration of 125I-Labeled p Nerve Growth Factor by Embryonic Sensory Neurons

Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 × 10^-11 M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 × 10^-9M. These two receptors are not a result of negative cooperatively, but apparently are different receptors. At 22°C the rate of association is 1 × 10⁷ M^-1 S^-1 and the rates of dissociation are 6.5 × 10^-4 s^-1 (type I) and 3.2 × 10^-2 s^-1'(type II). After binding, a time-dependent process occurs that makes the NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.     

Enzymes involved in ammonia assimilation in the fungus Acremonium persicinum

Abstract Acremonium persicinum grown in batch culture with ammonium tartrate as the nitrogen source possessed an NADP⁺-dependent glutamate dehydrogenase and a glutamine synthetase. Glutamate synthase was not detected under the culture conditions used. Kinetic studies of the NADP⁺-dependent glutamate dehydrogenase at 25°C and pH 7.6 revealed an apparent K _m of 3.2 × 10⁻⁴ M for 2-oxoglutarate and an apparent K _m of 1.0 × 10⁻⁵ M for ammonium ions, with corresponding apparent V _max values of 0.089 and 0.13 μmol substrate converted/min/mg of protein, respectively. Glutamine synthetase was measured by the γ-glutamyl transferase reaction at 30°C and pH 7.55. This transferase reaction of glutamine synthetase had a higher rate at 30°C than at 25°C or 37°C.     

Thermal resistance of partially purified proteinase of Pseudomonas fluorescens P-26

Heat resistance of the Pseudomonas fluorescens P-26 proteinase in terms of D -value was studied in whole milk, skim milk, whey and 0.05 mol 1^-1 phosphate buffer at 72.5, 130, 135, 140, 145 and 150°C subsequent to its partial purification through (NH₄)₂ SO₄ precipitation (45–65% saturation) and solvent fractionation with 1.0 to 2.0 volumes of isopropanol. The D -value was maximum for the proteinase at all temperatures when determined in whole milk ( D ₁₅₀= 0.088).     

Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Characterization of L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.)

B.R. MOHAPATRA, R.K. SANI AND U.C. BANERJEE. 1995. The bacterial flora associated with an intertidal marine alga ( Sargassum sp.) were screened for the presence of extracellular L-asparaginase; one out of five Bacillus strains was found positive. The maximum L-asparaginase activity was found at 37°C and pH 8.0. The optimum NaCl concentration for enzyme activity was found to be 2% (w/v). The enzyme activity was not affected by the addition of different metal ions (Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺and Ni²⁺) at 10 mmol 1^-1, but was strongly inhibited by EDTA.     

Interaction of photoperiod and gibberellin on growth and photosynthesis of high-latitude Poa pratensis

The relative growth rate of pot-grown plants of Poa pratensis L. cv. Holt, origin 69s°N, was increased by 20–40% by photoperiod extension with low intensity incandescent light from 8 to 24 h at 9–21°C. The main increase occurred over the 14 to 18 h photoperiod range. The true photoperiodic nature of the response was demonstrated by the effectiveness of night interruption in stimulating growth. Fortnightly sprayings with gibberellic acid (GA₃) (3 × 10^-6 to 3 × 10^-5 M ) mimicked all the effects of long days, whereas (2-chloroethyl)-trimethylammonium chloride (CCC) counteracted the effects of long days. Both growth substances exhibited pronounced interactions with photoperiod, GA₃ being most effective in short days and CCC in long days. The growth stimulation, whether caused by long days or GA₃, was exerted mainly through increases in individual and total leaf area. This was associated with a reduction in CO₂, exchange rate and a parallel fall in specific leaf weight. Proportionally, however, the increase in leaf area was greater than the fall in CO₂ exchange rate, resulting in a 38 to 118% increase in photosynthesis per leaf. No evidence was found of any direct and promotive effect of transition to long days on the CO₂ exchange rate of already expanded leaves.     

General properties of extracellular bacterial inulinase

The extracellular inulinase system of a strain of Arthrobacter sp. consists of a β -fructofuranosidase active on inulin raffinose and sucrose with a relative rate inulin/sucrose (I/S) of 0.2.
Crude enzyme preparations were obtained by fractionation of the liquid culture at stationary phase of growth with ammonium sulphate. Purification was carried out by DEAE cellulose chromatography and ultrogel ACA 34. Only one protein band was observed by electrophoresis. The enzyme was stable at high temperatures and was active at neutral or slightly alkali pH. Fructose is liberated as the sole reaction product of inulin hydrolysis, suggesting that the enzyme was an exoinulinase. The Michaelis constant (calculated at 40°C and pH 6) was 0.25 × 10^-2 mol/l for the inulin and 0.12 × 10^-2 mol/l for sucrose.
The enzyme was suitable for fructose production from root extracts of plants rich in polyfructosans or sucrose.     

Thermophilic glucoamylase from Talaromyces flavus

The heat-resistant mold, Talaromyces flavus , was found to produce a thermophilic glucoamylase that exhibited the highest activity at 50°C and in the pH range of 4.0–4.8. The K _m and V _max values of the crude enzyme for amylopectin were 0.21% and 16.7 mg glucose 1^-1, min^-1, respectively. The molecular weight of the enzyme as estimated by the gel filtration method was 42 kDa.     

Nickel and rubidium uptake by whole oat plants in solution culture

Nickel and rubidium uptake by oat plants ( Avena sativa L. cv. Victory) were examined in relation to solution temperature, solution concentrations, metabolic inhibitors, anaerobic root conditions, transpiration and time. Over a 4-h period, uptake rates for both Ni²⁺ and Rb⁺ remained constant at 23°C. Decreasing temperatures to 2°C, 20 μ M concentrations of 2,4-dinitrophenol (DNP), or anaerobic root conditions decreased Ni²⁺ and Rb⁺ uptake rates by 97 to 86% in whole plants. Treatment of excised roots with 20 μ M DNP decreased Ni²⁺ uptake by 93%. Nickel and Rb⁺ uptake rates measured as a function of the external solution concentration followed a typical parabolic curve. K_m (0.012 m M ) and V_max [2.72 μmol (g dry weight)^-1 h^-1] values for Ni²⁺ were nearly 7 times lower than those for Rb⁺ [0.09 m M and 19.2 μmol (g dry weight)^-1 h^-1]. In all experiments, Ni²⁺ and Rb⁺ showed qualitatively similar uptake patterns, but Rb⁺ uptake was quantitatively more sensitive than Ni²⁺ to experimental manipulations.     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Cultural conditions for production of glucoamylase from Lactobacillus amylovorus ATCC 33621

Lactobacillus amylovorus ATCC 33621 is an actively amylolytic bacterial strain which produces a cell-bound glucoamylase (EC 3.2.1.3). Conditions of growth and glucoamylase production were investigated using dextrose-free de Man-Rogosa-Sharpe (MRS) medium in a 1.5 I fermenter, with varying dextrin concentration (0.1–1.5% (w/v)), pH (4.5–6.5) and temperature (25–55°C). Cell extracts were prepared by subjecting cells to treatment with a French Pressure cell in order to release intracellular proteins. Glucoamylase activity was then assayed. The effects of pH (4.0–9.0), temperature (15–85°C) and substrate (dextrin and starch, 0–2% w/v) concentration on crude enzyme activity were investigated. Optimal growth was obtained in MRS medium containing 1% (w/v) dextrin, at pH 5.5 and 37°C. Glucoamylase production was maximal at the late logarithmic phase of growth, during 16–18 h. Crude enzyme had a pH optimum of 6.0 and temperature optimum of 60°C. With starch as the substrate, maximal activity was obtained at a concentration of 1.5% (w/v). The effects of ions and inhibitors on glucoamylase activity were also investigated. Enzyme activity was not significantly influenced by Ca²⁺ and EDTA at 1 mmol 1⁻¹ concentration; however Pb²⁺ and Co²⁺ were found to inhibit the activity at concentrations of 1 mmol 1⁻¹. The crude enzyme was found to be thermolabile when glucoamylase activity decreased after about 10 min exposure at 60°C. This property can be exploited in the brewing of low calorie beers where only mild pasteurization treatments are used to inactivate enzymes. The elimination of residual enzyme effect would prevent further maltodextrin degradation and sweetening during long-term storage, thus helping to stabilize the flavour of beer.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.