Effects of NH4 +, NO3 − and HCO3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK_a = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm²), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH₄ ⁺ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO₃ ⁻ was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO₃ ⁻, NO₃ ⁻ elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H⁺-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO₃ ⁻ provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging. Received: 26 August 1998 / Accepted: 4 May 1999     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Enhanced Solubility,Stability, and Transcorneal Permeability of Delta-8-Tetrahydrocannabinol in the Presence of Cyclodextrins

The purpose of this study was to investigate the effect of cyclodextrins (CDs) on aqueous solubility, stability, and in vitro corneal permeability of delta-8-tetrahydrocannabinol (Δ⁸-THC). Phase solubility of Δ⁸-THC was studied in the presence of 2-hydroxypropyl-β-cyclodextrin (HPβCD), randomly methylated-β-cyclodextrin (RMβCD) and sulfobutyl ether-β-cyclodextrin sodium salt (SβCD). Stability of Δ⁸-THC in 5% w/v aqueous CD solutions, as a function of pH, was studied following standard protocols. In vitro corneal permeation of Δ⁸-THC (with and without CDs) across excised rabbit cornea was also determined. Phase-solubility profile of Δ⁸-THC in the presence of both HPβCD and RMβCD was of the A_P type, whereas, with SβCD an A_L type was apparent. Aqueous solubility of Δ⁸-THC increased to 1.65, 2.4, and 0.64 mg/mL in the presence of 25% w/v HPβCD, RMβCD, and SβCD, respectively. Significant degradation of Δ⁸-THC was not observed within the study period at the pH values studied, except for at pH 1.2. Transcorneal permeation of Δ⁸-THC was dramatically improved in the presence of CDs. The results demonstrate that CDs significantly increase aqueous solubility, stability, and transcorneal permeation of Δ⁸-THC. Thus, topical ophthalmic formulations containing Δ⁸-THC and modified beta CDs may show markedly improved ocular bioavailability.     

Preformulation Studies of a Prodrug of Δ9-Tetrahydrocannabinol

Preformulation studies were performed on a hemiglutarate ester prodrug of Δ⁹-tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a T _g close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at −18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO.     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

Effects of hypercapnia on blood-gas and acid-base status in the white sturgeon, Acipenser transmontanus

The effect of environmental hypercapnia on respiratory and acid-base variables was studied in white sturgeon, Acipenser transmontanus. Blood PCO₂, PO₂, pH, hemoglobin concentration, and plasma lactate, glucose, catecholamines and cortisol were measured first under normocapnia (water PCO₂ < 0.5 Torr, 1 Torr = 133.32 Pa), then under hypercapnia (25–35 Torr) and a final return to normocapnia at 19 ± 0.5 °C. Acute (≤ 2h) hypercapnia significantly increased arterial PCO₂ (8-fold increase), ventilation frequency (2-fold increase), plasma HCO₃ ⁻ (2.3-fold) and decreased arterial pH (to 7.15 ± 0.02). After 24 h, norepinephrine, epinephrine and cortisol, were significantly increased, and arterial pH reached its nadir (7.10 ± 0.03). During the 72- and 96-h-periods, arterial PCO₂ (24 ± 4.4 Torr) and ventilatory frequency (105 ± 5 breaths min⁻¹) stabilized, HCO₃ ⁻ reached its apparent maximum (23.6 ± 0.0 mmol⁻¹), glucose decreased by 32%, and pH increased significantly to 7.31 + 0.03. The return to normocapnia completely restored arterial PCO₂ (2.5 ± 0.14 Torr), HCO₃ ⁻ (7.4 ± 0.59 mmol · l⁻¹), ventilation frequency (71 ± 7 breaths · min⁻¹), and pH (7.75 ± 0.04). Overall, hypercapnia produced a respiratory acidosis, hyperventilation, a transient norepinephrine “spike”, and increased plasma catecholamines, cortisol, and arterial PO₂. The respiratory acidosis was only partially compensated (35% pH restoration) 96 h after the onset of hypercapnia and resulted in a significantly decreased blood-O₂ affinity (Bohr effect), as determined by construction of in vitro blood O₂ equilibrium curves at 15 °C and 20 °C. Prolonged exposure to hypercapnia may lead to acid-base disturbances and negatively affect growth of white sturgeon. Accepted: 17 August 1997     

Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum

A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M _r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K _m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0. Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99–100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37°C, though it was active between pH 5.0 and pH 9.0 and up to 45°C. K _m and V _max values for recombinant P. aeruginosa lipase were found to be 151.5 ± 29 μM and 217 ± 22.5 μmol min⁻¹ mg⁻¹ protein, respectively.     

Improved Bioavailability of Poorly Water-Soluble Drug Curcumin in Cellulose Acetate Solid Dispersion

Curcumin (Cur), one of the most widely used natural active constituents with a great variety of beneficial biological and pharmacological activities, is a practically water-insoluble substance with a short biologic half-life. The aim of this study was to develop a sustained-release solid dispersion by employing water-insoluble carrier cellulose acetate for solubility enhancement, release control, and oral bioavailability improvement of Cur. Solid dispersions were characterized by solubility, in vitro drug release, Fourier transform infrared spectroscopy, X-ray diffractometry, and differential scanning calorimetry studies. The in vivo performance was assessed by a pharmacokinetic study. Solid-state characterization techniques revealed the amorphous nature of Cur in solid dispersions. Solubility/dissolution of Cur was enhanced in the formulations in comparison with pure drug. Sustained-release profiles of Cur from the solid dispersions were ideally controlled in vitro up to 12 h. The optimized formulation provided an improved pharmacokinetic parameter (C _max = 187.03 ng/ml, t _max = 1.95 h) in rats as compared with pure drug (C _max = 87.06 ng/ml, t _max = 0.66 h). The information from this study suggests that the developed solid dispersions successfully enhanced the solubility and sustained release of poorly water-soluble drug Cur, thus improving its oral bioavailability effectively.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

The Relative Rates of Thiol–Thioester Exchange and Hydrolysis for Alkyl and Aryl Thioalkanoates in Water

This article reports rate constants for thiol–thioester exchange (k _ex), and for acid-mediated (k _a), base-mediated (k _b), and pH-independent (k _w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k _a = 1.5 × 10⁻⁵ M⁻¹ s⁻¹, k _b = 1.6 × 10⁻¹ M⁻¹ s⁻¹, and k _w = 3.6 × 10⁻⁸ s⁻¹. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange between S-methyl thioacetate and 2-sulfonatoethanethiolate is k _ex = 1.7 M⁻¹ s⁻¹. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions (pH, temperature, pK _a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude.     

Bioluminescence induction response and survival of the bioreporter bacterium Pseudomonas fluorescens HK44 in nutrient-deprived conditions

Pseudomonas fluorescens HK44 is a bioluminescent bioreporter synthesizing light in the presence of naphthalene or salicylate. Upon immobilization, HK44 is useful as an in situ or on-line biosensor of bioavailable naphthalene and salicylate in waste streams or contaminated fields. The bioreporting efficacy of alginate/SrCl₂-immobilized HK44 was investigated in simulated groundwater with different pH regimes. When induced with complex (salicylate plus auxiliary energy supplements) and simple (salicylate as the sole energy supplement) inducer solutions, the specific light response was steadier at pH 6 than at pH 7 in a 35-day study. There was no bioluminescence response from cells incubated in groundwater samples with pH below 6. The rate of the luminescence reaction was stable at pH 6 irrespective of the type of inducer solution, indicating the robust physiological status of the bioreporter bacteria. In addition, the quantity of light synthesized was at least one order of magnitude higher with complex inducer solution than with simple inducer solution. The numbers of viable and cultivable cells remained constant in groundwater at pH 6 and 7 (approx. 10⁷ g⁻¹ beads). The numbers decreased by four orders of magnitude (10⁷ to 10³) to zero in groundwaters with pH below 6. This study suggested that HK44 is useful for long-term biosensor applications in moderately acidic to neutral groundwater conditions. Received: 6 August 1996 / Received revision: 12 December 1996 / Accepted: 4 January 1997     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

On the Interaction of Vanadium Species with the Monoisoamyl Ester of <Emphasis Type="Italic">Meso</Emphasis>-2,3-Dimercaptosuccinic Acid

The interaction of the VO²⁺ cation with the monoisoamyl ester of meso-2,3-dimercaptosuccinic acid (MiADMSA) was investigated by electron absorption spectroscopy in aqueous solutions at different pH values. The spectral behavior, complemented with a spectrophotometric titration, shows the generation of a [VO(MiADMSA)₂]⁴⁻ complex in which the oxocation interacts with two pairs of deprotonated –SH groups of the ester. Besides, MiADMSA rapidly reduces VO₃⁻ to VO²⁺, which might be chelated by an excess of the ester, and also produces relatively rapid reduction of V₂O₅ suspensions at pH = 6.5. The results of this study suggest that MiADMSA might be a potentially useful detoxification agent for vanadium.     

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column. Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Cell-free extract(s) of Pseudomonas putida catalyzes the conversion of cyanides,cyanates, thiocyanates,formamide, and cyanide-containing mine waters into ammonia

 Our isolate, Pseudomonas putida, is known to be capable of utilizing cyanides as the sole source of carbon (C) and nitrogen (N) both in the form of free cells and cells immobilized in calcium alginate. In the present study, the cell-free extract(s) were prepared from the cells of P. putida grown in the presence of sodium cyanide. The ability of enzyme(s) to convert cyanides, cyanates, thiocyanates, formamide and cyanide-containing mine waters into ammonia (NH₃) was studied at pH 7.5 and pH 9.5. The kinetic analysis of cyanide and formamide conversion into NH₃ at pH 7.5 and pH 9.5 by the cell-free extract(s) of P. putida was also studied. The K _m and V _max values for cyanide/formamide were found to be 4.3/8 mM and 142/227 μmol NH₃ released mg protein^-1 min^-1 respectively at pH 7.5 and 5/16.67 mM and 181/434 μmol NH₃ released mg protein^-1 h^-1 respectively at pH 9.5. The study thus concludes that the cell-free extract(s) of P. putida is able to metabolize not only cyanides, cyanates, thiocyanates, and formamide but also cyanide-containing mine waters to NH₃. Received: 10 April 1995/Received revision: 24 July 1995/Accepted: 22 August 1995     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.