Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis

In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l^?1 of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l^?1 were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0–8.0 and temperatures of 47–50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved.     

Transformation of wheat straw polysaccharides under dynamic conditions of subcritical autohydrolysis

Transformation of wheat straw polysaccharides under dynamic conditions of subcritical autohydrolysis has been examined at a pressure of 30 MPa in a temperature range of 150—290°C. The dependence of the yield of gaseous, liquid, and solid products on the process temperature has been studied. The formation of liquid products occurs mainly due to the hydrolysis of pentosans and partially of cellulose in a range of 150–200°C. The main components of liquid products are oligo- and monosaccharides. Xylose and arabinose comprise more than 65% of monosaccharides. A temperature rise in a range of 200–290°C is accompanied by a decrease in medium pH, cellulose hydrolysis, and intensive gasification. Glucose predominates in monosaccharides of liquid products isolated at 270°C; a portion of pentoses does not exceed 25% of the total of monosaccharides. The maximal yield of sugars is observed at 200°C and then decreases from 29.6 to 5.3% per straw oven-dry weight at 270°C. A decrease in reactivity of straw cellulose treated at 200°C to enzymatic hydrolysis has been found.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Preparation of Homogeneous Concanavalim A

Abstract

Concanavalin A (Con A), obtained either commercially or by affinity chromatography, was further purified by incubating at 6–8°C for 16–18 hr at pH 3.0–3.2 in 1 M MaCl, 0.08 M glycine and 3 mM each Ca⁺² and Mn⁺², heat treating at 45°C for 2 hr and centrifuqing. The supernatant was neutralized to pH 5 and stored in the cold. The overall yield was 70–80% Some of the properties of Con A at pH 5 are: The absorption coefficient of a 1 g/dl solution is 13.7 at 280 nm; the mean residue elliptic-ity at 224.5 nm is ?9,300° to ?9,800°; by sedimentation equilibrium, its molecular weight is 53,000 between pH 3.0 and pH 5.2. Con A solutions standing at room temperature at pH 7 for ten days lose through precipitation only 5–8% of the protein in 0.2 M NaCl and 15% of the protein in 0.1 M NaCI. In the solution conditions of SDS and urea-SDS gels, Con A not only unfolds slowly and incompletely, but it also forms high molecular weight aggregates. Thus, electrophoresis of Con A in such gels is unsuitable for tests of homogeneity. However, as judged by sedimentation equilibrium in 6.5 M quanidine at pH 8.1, purified Con A was monodisperse.     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

Ethanol fermentation characteristics of Thermoanaerobacter ethanolicus

Thermoanaerobacter ethanolicus is an extreme thermophilic non-spore forming ethanol-producing anaerobic bacterium. Minimum nutrient requirements and optimum growth conditions have been established. An optimum yeast extract-glucose ratio for ethanol yield has also been determined. Initial medium pH, optimally 7.5–8.0, significantly affected the amount of ethanol formed. Maximum specific growth rate was found to be 0.22 h^?1at pH 7.5 and 69°C. Ethanol concentration up to 11 g l^?1at pH 7.5 and 69°C was used to characterize ethanol inhibition. The growth kinetics of T. ethanolicus were characterized in terms of environmental parameters. Substrate utilization, ethanol formation and inhibition by both sugar and ethanol were also quantified.     

Purification and characterization of a novel endo-β-1,4-glucanase from the thermoacidophilic <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

An endo-β-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11, was purified 18-fold with 14% yield and a specific activity of 67 U mg⁻¹ protein. The optimal pH was 2 and the cellulase was stable from pH 2 to 5. The cellulase had a temperature optimum of 60°C measured over 30 min and retained more than 60% of its activity after heating at 70°C for 1 h. The molecular mass of the cellulase was about 25 kDa. Its activity was inhibited by 77% by Hg²⁺ (2 mM) and by 59% by Cu²⁺ (2 mM).     

Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788

The limiting amount of nitrogen required to trigger lipid accumulation in the oleaginous yeast Rhodosporidium toruloides ATCC 10788 was studied, batchwise, by subjecting washed mid-exponentially grown cells to nitrogen at levels of 10⁻² M down to 10⁻⁴ M per g l⁻¹ of lean cells (2–5% fat content) in a mineral medium where glucose was present at 35 g l⁻¹. The results showed that lipid accumulation always started sometime after nitrogen reached a level of 3 × 10⁻⁵ M and the specific initial lipid productivity was constant. Furthermore, the cells were subjected to nine combinations of temperature and pH, from (25° C, pH 4.5) to (35° C, pH 7.5) in the mineral medium supplemented with 0.5 g l⁻¹ of yeast extract and 1 g l⁻¹ (NH₄)₂SO₄. As was expected, lipid content in the cells was higher at 25° C, but pH around 6.0–7.5 slightly enhanced the effect of lower temperature. The effect of pH was also noticed to affect the size of changes in the temporal profiles of the oil's fatty acid distribution prior to nitrogen depletion, whereas no significant difference in the fatty acid composition of the oil was shown after exhaustion of nitrogen from the medium for all combinations of temperature and pH.     

Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH,temperature and substrate concentration

The effects of temperature, pH and xylose concentration on the fermentation parameters of Candida shehatae and Pichia stipitis were evaluated. The optimum pH was in the region of pH 4–5.5, with an optimum fermentation temperature of 30°C. Maximum fermentation rates were reached at 50 g l⁻¹ xylose. A maximum volumetric ethanol productivity of about 0.9 g (l h)⁻¹ was obtained with both yeast strains. The ethanol yield of C. shehatae decreased considerably when cultivated above 30°C or when the xylose concentration was increased. Xylitol accumulated concomitantly. Xylitol production by P. stipitis was observed only during cultivation at 36°C. Whereas the ethanol yield of C. shehatae was usually about 75% of the theoretical maximum, it was 85–90% with P. stipitis.     

The Influence of n-Propanol on the Growth and Conidiation of Pestalotia rhododendri

Pestalotia rhododendri was exposed to vapours from 1 ml propanol solution in water and linear growth, formation of aerial hyphae and production of conidia were determined. A special Petri dish technique was used and maximum stimulation of conidial formation was induced by the vapours from a propanol concentration of 3–4 % (v/v) at 25°C. When propanol was added directly to the medium, a concentration of 1.2 × 10^?2M was optimal for growth and sporulation at 30°C. Sporulation stimulated by propanol was observed at temperatures from 20–32°C, with an optimum at 30°C. Certain observations indicated that an exposure to propanol for 24 hours was enough to induce a stimulated spore production. The stimulation was noticed on different media at 25°C, and was more pronounced at 30°C. One exception was observed. Propanol did not promote sporulation when the fungus was grown on maltagar at 30°C. Propanol 3 ° (v/v) in combination with the standard medium containing (NH₄)₂-tartrate as sole nitrogen source, inhibited the linear growth at 15–20°C, was inactive at 22.5° and 25°C, and stimulated growth at 27.5–31°C. The stimulatory effect was maximal at 30°C. Other media were tested at 25° and 30°C. At both temperatures stimulations of linear growth caused by propanol were observed with a medium containing KNO₃ as sole nitrogen source, and inhibitions with maltagar and another medium containing l -asparaginc as sole nitrogen source. The linear growth could be either inhibited or stimulated while the sporulation was stimulated.     

Production,characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0–11.0) and moderate salinity (0–5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128?U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl₂?·?2H₂O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42?hr of incubation at 37°C. It was active over broad range of pH (7.0–12.0), temperature (15–100°C), and salinity (0–9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0–12.0 and salinities of 0–5% NaCl for 48?hr. Cu²⁺, Mn²⁺, Co²⁺, and Ca²⁺ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant–detergent-stable partially purified extracellular protease from A. caviae.     

Induction of an Increase in Nerve Growth Factor Content in the Cell-conditioned Medium of Mouse L-M Cells by Basic Peptides

Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu²⁺ , Hg²⁺, Zn²⁺, and Tris.     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Model estimates of methane emission from irrigated rice cultivation of China

A model developed by the authors ( Huang et al. 1998 ) was further validated against field measurements from various regions of the world and calibrated to estimate methane emission from irrigated rice cultivation of China. On the basis of available information on rice cultivated area, growth duration, grain yield, soil texture and temperature, methane emission from Chinese rice paddies was estimated for 28 rice cultivated provinces in mainland. The calculated daily methane emission rates, on a provincial scale, ranged from 0.15 to 0.86 g m^–2 with an average of 0.32 g m^–2. Five of the top six locations with higher daily methane emissions are located at a latitude between 28° and 31° N. A total amount of 9.66 Tg (1 Tg = 10¹² g) CH₄ per year, ranging from 7.19 to 13.62, was estimated to be released from Chinese rice paddy soils. Of the total, 45% is emitted from the single-rice growing season, and 19% and 36% are from the early-rice and the late-rice growing seasons, respectively. Approximately 70% of the total is emitted in the region located at latitude between 25° and 32° N. The emissions from rice fields in Sichuan and Hunan Province were calculated to be 2.85 Tg y^–1, accounting for ≈ 30% of the total. Comparisons of the estimated and the observed emission rates show that the estimates were, in general, close to the measurements at most locations.     

OLISTHODISCUS LUTEUS (CHRYSOPHYCEAE) I. EFFECTS OF SALINITY AND TEMPERATURE ON GROWTH. MOTILITY AND SURVIVALS1, 2

The effect of salinity and temperature on Olisthodiscus luteus Carter has been examined to across the relative importance of these factory on dynamics of natural population. A salinity range 2–50% was observed with increased tolerance to low salinity (<5%.) at higher temperature (20–30°C). Slinities at 4–5%. Had densities of 10³ cells/ml^?1, and growth >0.5 division day^?1 at temperature of 15–30°C higher salinities (5–50%.) variable but distinct optima for density, growth and motility were observed 5, 10 and 30°C. Density and motility showed no clear optima from 10–10%.15–25°C where maximum growth rates >1.0 division/day^?1 were common. Temperature increased from (0.5–1.9 division. Day^?1) and increases of three orders of magnitude (10^2?10³) for maximum densities. Temperature optima 20°C for growth 5–35%. And 25°C for >40%. were observed. The implications of these findings to natural populations of O. luleus are discussed.     

Analysis of the calcium-dependent interaction of calmodulin with bovine serum albumin

An air-driven ultracentrifuge has been used to investigate the calcium-dependent association between calmodulin and bovine serum albumin. Procedures were described which allowed the interaction to be analyzed to yield the equilibrium constant. At low ionic strength (25 mm Tris-HCl, pH 7.5, pCa 6.68, 9°C) the equilibrium constant for the interaction was estimated to be 2.1 × 10⁴m⁻¹, while at high ionic strength (25 mm Tris-HCl, pH 7.5, 150 mm KCl, pCa 6.68, 9°C) the value was 4.5 × 10³m⁻¹. Under similar conditions, calmodulin was also found to interact with β-lactoglobulin A and gelatin, but no detectable association was observed with ovalbumin.     

PHOSPHOLIPASE ACTIVITY IN SQUID AND FROG AXONS

Evidence for the presence of phosphatide acylhydrolase activity (EC 3.1.1) in centrifuged homogenate supernatants and extracts of squid giant axons and centrifuged homogenate supernatants of frog sciatic nerve bundles is reported. The enzyme was assayed by measurement of the rate of deacylation of [U-¹⁴C]phosphatidyl choline. The deacylation activity in the nerve homogenate supernatants exhibits: a pH maximum at 7.2–7.4 (25°C); a calcium ion maximum at 12–13 mM-CaCl₂(aq); a K_m value of 3.4 × 10^?4 M (25°C); and a temperature maximum at 37°C. The activation energy over the range 8–37°C is 5.7 ± 0.2kcal-mol^?1.     

Fermentative Production of Piperazinium Dilactate

In order to test whether piperazinium dilactate can be produced by fermentation in its exact stoichiometric composition without losses of yield, the kinetics of cell growth and lactate production were investigated in the batch cultivation of Lactobacillus paracasei, using piperazine as a neutralizer in pH control. It was found that piperazine dilactate is formed in its stoichiometric composition at about pH 5.0, and lactic acid fermentation occurred with yields of about 90% under these conditions. Piperazine at concentrations less than or equal to 50 g/l did not affect growth and product formation. The presence or absence of piperazine did not produce any significant differences in either the maximum specific growth rate or the maximum specific lactate formation rate when piperazine was present or absent (0.65 h^—1 compared to 0.68 h^—1 and 3.86 g/g × h compared to 3.63 g/g × h, respectively). The Luedeking‐Piuret relationship between the two quantities was also not changed significantly when piperazine was added. To estimate the optimum parameters for cell growth and lactate formation in the presence of piperazine, a factorial experiment was designed and carried out under consideration of the parameter ranges 5.0 ≤ pH ≤ 7.0 and 30 °C ≤ T ≤ 36 °C. In this way, three‐dimensional models of the specific growth rate, the specific lactate formation rate and the lactate yield were obtained.     

Heterologous expression and characterization of a novel halotolerant,thermostable, and alkali-stable GH6 endoglucanase from <Emphasis Type="Italic">Thermobifida halotolerans</Emphasis>

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462^T, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.     

Stability of snakehead (Ophiocephalus striatus) rhabdovirus under different environmental conditions

Snakehead rhabdovirus stored in cell culture medium was found to be relatively temperature resistant with ≥ 99.9% reduction in infectivity only occurring after 6 months at 15°C, 6 weeks at 25°C and 10 days at 35°C. At 25°C, virus stability was greatest in the range pH 5–7. Infectivity was less well retained at pH9 and rapidly lost at pH3 and pH11. Stability was poor in serum-free media with a 99.9% reduction in infectivity occurring in less than 48 hours at 25°C in de-ionized water, tap water, balanced salt solutions and artificial seawater.