Photooxidation of pterin in aqueous solutions: biological and biomedical implications

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.     

Bacterial sensors based on Acidithiobacillus ferrooxidans Part I. Fe2+ and S2O32- determination

An amperometric bacterial sensor with current response to Fe(2+) and S(2)O(3)(2-) ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O(2) flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe(2+) ions up to 2.5 mmol L(-1) with a detection limit of 9 x 10(-7) mol L(-1) and a sensitivity of 0.25 A L mol(-1). The response of the biosensor is 84 s for a determination of 2 x 10(-4) mol L(-1) Fe(2+). Optimizing the Fe(2+) determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 degrees C under 290 rpm solution stirring. S(2)O(3)(2-) concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L(-1). Sensitivity was 0.20 A L mol(-1) with a response time of 207 s for a 2 x 10(-4) mol L(-1) S(2)O(3)(2-) concentration.     

Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Latency to CNS oxygen toxicity in rats as a function of PCO2 and PO2

Central nervous system (CNS) oxygen toxicity can occur as convulsions and loss of consciousness, without any premonitory symptoms. We have made a quantitative study of the effect of inspired carbon dioxide on sensitivity to oxygen toxicity in the rat. Rats were exposed to four oxygen pressures (PO(2); 456, 507, 608 and 709 kPa) and an inspired partial pressure of carbon dioxide (PCO(2)) in the range 0-12 kPa until the appearance of the electroencephalograph first electrical discharge (FED) that precedes the clinical convulsions. Exposures were conducted at a thermoneutral temperature of 27 degrees C. Latency to the FED decreased linearly with the increase in PCO(2) at all four PO(2) values studied. This decrease, which is probably related to the cerebral vasodilatory effect of carbon dioxide, reached a minimal value that remained constant on further elevation of PCO(2). The slopes (absolute value) and intercepts of latency to the FED as a function of carbon dioxide decreased with the increase in PO(2). This log-linear relationship made possible the derivation of equations that describe latency to the FED as a function of both PO(2) and PCO(2) in the PCO(2) - dependent range: Latency (min) = e((5.19-0.0040)(P)(O(2)))-e((2.77-0.0034)(P)(O(2))) x PCO(2) (kPa), and in the PCO(2)-independent range: Latency(min) = e((2.44-0. 0009)(P)(O(2))). A PCO(2) as low as 1 kPa significantly reduced the latency to the FED. It is suggested that in closed-circuit oxygen diving, any accumulation of carbon dioxide should be avoided in order to minimize the risk of CNS oxygen toxicity.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

Direct electron transfer and enzymatic activity of hemoglobin in a hexagonal mesoporous silica matrix

The direct electrochemistry of hemoglobin (Hb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Hb and the HMS was investigated using UV-Vis spectroscopy, FT-IR, and electrochemical methods. The direct electron transfer of the immobilized Hb exhibited two couples of redox peaks with the formal potentials of -0.037 and -0.232 V in 0.1 M (pH 7.0) PBS, respectively, which corresponded to its two immobilized states. The electrode reactions showed a surface-controlled process with a single proton transfer at the scan rate range from 20 to 200 mV/s. The immobilized Hb retained its biological activity well and displayed an excellent response to the reduction of both hydrogen peroxide (H2O2) and nitrate (NO2-). Its apparent Michaelis-Menten constants for H2O2 and NO2- were 12.3 and 49.3 microM, respectively, showing a good affinity. Based on the immobilization of Hb on the HMS and its direct electrochemistry, two novel biosensors for H2O2 and NO2- were presented. Under optimal conditions, the sensors could be used for the determination of H2O2 ranging from 0.4 to 6.0 microM and NO2- ranging from 0.2 to 3.8 microM. The detection limits were 1.86 x 10(-9) M and 6.11 x 10(-7) M at 3sigma, respectively. HMS provided a good matrix for protein immobilization and biosensor preparation.     

Direct electrochemistry and electrocatalysis of myoglobin immobilized on a hexagonal mesoporous silica matrix

The direct electrochemistry of myoglobin (Mb) immobilized on a hexagonal mesoporous silica (HMS)-modified glassy carbon electrode was described. The interaction between Mb and HMS was investigated by using Fourier transfer infrared spectroscopy, nitrogen adsorption isotherm, and cyclic voltammetry. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the Mb intercalated in the mesopores and adsorbed on the surface of the HMS were observed with the formal potentials of -0.167 and -0.029V in 0.1M, pH 7.0, phosphate buffer solution, respectively. The electrode reaction showed a surface-controlled process with one proton transfer. The immobilized Mb displayed good electrocatalytic responses to the reduction of both hydrogen peroxide (H(2)O(2)) and nitrite (NO(2)(-)), which were used to develop novel sensors for H(2)O(2) and NO(2)(-). The apparent Michaelis-Menten constants of the immobilized Mb for H(2)O(2) and NO(2)(-) were 0.065 and 0.72mM, respectively, showing good affinity. Under optimal conditions, the sensors could be used for the determinations of H(2)O(2) ranging from 4.0 to 124microM and NO(2)(-) ranging from 8.0 to 216microM. The detection limits were 6.2x10(-8) and 8.0x10(-7)M at 3 sigma, respectively. The HMS provided a novel matrix for protein immobilization and the construction of biosensors via the direct electron transfer of immobilized protein.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Effects of oxygen concentration and oviductal epithelial tissue on the development of in vitro matured and fertilized bovine oocytes cultured in protein-free medium

Examination was made of the effects of oxygen concentration and supplementation of bovine oviductal epithelial tissue (BOET) on the development of bovine in vitro matured and fertilized (IVM/IVF) oocytes in a protein-free medium. The IVM/IVF embryos were cultured in protein-free tissue culture medium 199 supplemented with or without BOET under 5% CO(2) in air (20% O(2)) or 5% CO(2), 5% O(2) and 90% N(2) (5% O(2)). We found that blastocyst development without BOET at 5% O(2) was the same as that with BOET at 20% O(2) (30 vs 33%); BOET suppressed blastocyst development at 5% O(2) (4%). Blastocysts cultured without BOET at 5% O(2) developed into normal fetuses after transfer to recipient heifers. Examination was also made of oxygen pressure in the medium cultured with or without BOET at 20% O(2) or 5% O(2) by a blood gas analyzer. Oxygen pressure in the medium cultured with BOET at 20% O(2) was lower than that without BOET (111.0 +/- 13.3 vs 149.2 +/- 1.3 mmHg). These results indicate that bovine IVM/IVF embryos can develop to the blastocyst stage in a protein-free medium without somatic cell support at low oxygen concentration (5%) and that the beneficial role of BOET for embryonic development may be to reduce oxygen concentration in the medium.     

Kinetics and activation energetics of death in Saccharomyces cerevisiae induced by sulfur dioxide

Death of Saccharomyces cerevisiae induced by sulfur dioxide (K(2)S(2)O(2) was used as the SO(2) source) followed saturation kinetics. The enthalpy of activation of death was not affected by concentration over the range tested (5-150) mg/L of (K(2)S(2)O(2) at pH 3.4) and averaged 3.6 x 10(4) cal/mol as compared with 8.5 x 10(4) cal/mol for DeltaH(double dagger) of thermal death. The entropy of activation of death was hyperbolic function of the sulfur dioxide concentration, extrapolated at zero concentration to DeltaS(0) (double dagger) = 36.8 cal mol(-1) K(-1) and tended to DeltaDeltaS(max) (double dagger) = 13.2 cal mol(-1) K(-1) at saturating concentration, yielding a dis0sociation constant of 5.8 x 10(-1) M sulfur dioxide. As was predicted from these results, In K(d) (the specific rate of death induced by sulfur dioxide) was hyperbolic function of concentration under isothermic conditions and extrapolated toa finite value at zero concentration. The Arrhenuis plots and the DeltaS(+/-) plot versus concentration revealed the occurrence of substrate inhabitation of the death effect at high concentrations (above 60 mg/L K(2)S(2)O(2) at pH 3.4). A model is presented involving two types of receptor sites for sulfur dioxide on the cell surface, on directly connected with the death process, the other modulating its entropy of activation.     

Effect of modified Fenton's reaction on microbial activity and removal of PAHs in creosote oil contaminated soil

This study describes the removal of polycyclic aromatic hydrocarbons (PAHs) from creosote oil contaminated soil by modified Fenton's reaction in laboratory-scale column experiments and subsequent aerobic biodegradation of PAHs by indigenous bacteria during incubation of the soil. The effect of hydrogen peroxide addition for 4 and 10 days and saturation of soil with H(2)O(2) on was studied. In both experiments the H(2)O(2) dosage was 0.4 g H(2)O(2)/g soil. In completely H(2)O(2)-saturated soil the removal of PAHs (44% within 4 days) by modified Fenton reaction was uniform over the entire soil column. In non-uniformly saturated soil, PAH removal was higher in completely saturated soil (52% in 10 days) compared to partially saturated soil, with only 25% in 10 days. The effect of the modified Fenton's reaction on the microbial activity in the soil was assessed based on toxicity tests towards Vibrio fischeri, enumeration of viable and dead cells, microbial extracellular enzyme activity, and oxygen consumption and carbon dioxide production during soil incubation. During the laboratory-scale column experiments, the toxicity of column leachate towards Vibrio fischeri increased as a result of the modified Fenton's reaction. The activities of the microbial extracellular enzymes acetate- and acidic phosphomono-esterase were lower in the incubated modified Fenton's treated soil compared to extracellular enzyme activities in untreated soil. Abundance of viable cells was lower in incubated modified Fenton treated soil than in untreated soil. Incubation of soil in serum bottles at 20 degrees C resulted in consumption of oxygen and formation of carbon dioxide, indicating aerobic biodegradation of organic compounds. In untreated soil 20-30% of the PAHs were biodegraded during 2 months of incubation. Incubation of chemically treated soil slightly increased PAH-removal compared to PAH-removal in untreated soil.     

Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell-associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia- or hyperoxia-treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion-exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.     

Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two-parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. pO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the pO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4-7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.     

Oxygen effect on lactose catabolism by a Leuconostoc mesenteroides strain: modeling of general O2-dependent stoichiometry

Lactose metabolism of a Leuconostoc mesenteroides strain was studied in batch cultures at a pH of 6.5 and 30 degrees C in 10 L of a modified MRS (De Man, Rogosa, Sharp) broth. The end products of this heterolactic bacterium were D-lactate, acetate, ethanol, and carbon dioxide. To test the effect of oxygen on their synthesis, the medium was sparged with different gases: nitrogen, air, and pure oxygen. When oxygen was available, oxygen uptake occurred, which caused a modification in acetate and ethanol production but not in lactate or carbon dioxide production; acetate plus ethanol together were produced in constant amounts, which were independent of the level of aeration. The influence of oxygen on end-product formation could be summed up by the general equation: lactose + x O(2) --> 2 D-lactate + (x + 0.1) acetate + (2 - x) ethanol + 2 CO(2). Maximal oxygen uptake (x = 2) was reached under a 120 L/h flow rate of pure oxygen. In addition, this equation provided useful information on the possible pathway of galactose catabolism by a heterofermentative microorganism. (c) 1996 John Wiley & Sons, Inc.     

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION, PRELIMINARY IDENTIFICATION, AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K(2)HPO(4)?·?3H(2)O, and MnSO(4)?·?H(2)O were identified as significant components influencing pentocin LPL1-5 production using the Plackett-Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO(4)?·?H(2)O 0.84, K(2)HPO(4)?·?3H(2)O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO(4)?·?7H(2)O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65?±?27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.     

Whole animal and gill tissue oxygen uptake in the Eastern oyster, Crassostrea virginica: Effects of hypoxia, hypercapnia, air exposure, and infection with the protozoan parasite Perkinsus marinus(1)

The Eastern oyster, Crassostrea virginica, lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus. The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was -0.060x10(-3) (+/-0.01x10(-3) S.E.M.; n=20; low and high CO(2) treatments combined) in oysters uninfected with P. marinus and -0.056x10(-3) (+/-0.01x10(-3) S.E.M.; n=16; low and high CO(2) treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia (pCO(2)=6-8 Torr; pH 7) was 9.10 μmol O(2) g ww(-1) h(-1) +/-0.62 S.E.M. (n=9), significantly different from oxygen uptake under normocapnia (pCO(2) 相似文献   

Two roads diverged: the structure of hydroxymandelate synthase from Amycolatopsis orientalis in complex with 4-hydroxymandelate

The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation.     

Studies on the mechanism of alkaline peroxide delignification of agricultural residues

Alkaline solutions of hydrogen peroxide partially delignify wheat straw and other lignocellulosic materials, leaving a cellulosic residue that is highly susceptible to enzymatic digestion by cellulase. The delignification reaction is strongly dependent upon the pH of the reaction mixture, with an optimum at pH 11.5-11.6, pKa for the dissociation H(2)O(2) right harpoon over left harpoon H(+) + HOO(-). The data are consistent with a mechanism in which H(2)O(2) decomposition products such as .OH and O(2) (-)., rather than H(2)O(2) or HOO(-), are the primary lignin oxidizing species. During the course of the delignification reaction, O(2) is evolved from the reaction mixture indicating active H(2)O(2) decomposition. At a given concentration of H(2)O(2), the rate of O(2) evolution is proportional to the amount of lignocellulosic substrate present in the reaction mixture. However, the total amount of O(2) evolved is inversely proportional to the amount of substrate present, indicating that some of the peroxide oxygen becomes incorporated into lignin degradation products. The amount of peroxide oxygen incorporated can range as high as 2 O(2) per lignin C(9) unit, depending upon the initial concentration of lignocellulosic substrate.     

Improvement in enzyme productivities from mold cultivations using the liquid-phase oxygen supply strategy

Cultivations of Aspergillus niger cells in which oxygen was provided through the liquid-phase oxygen supply strategy (which involves the need-based decomposition of H2O2 pulses to yield the necessary oxygen) were studied. Concentrations of H2O2 in the range of 1 to 5 mM were found to be suitable for use in cultivations. The growth rate constant of 0.17 h(-1) obtained in the H2O2-based cultivation, was comparable to that obtained in the cultivation with aeration; however, the maximum cell concentration in the H2O2-based cultivation was 124% of that obtained in cultivation with aeration. Maximum concentrations of catalase, protease and glucose oxidase obtained in the H2O2-based cultivation, were 240%, 172%, and 124% respectively, of those obtained in the cultivation with aeration. Further, the specific enzyme levels (units per gram cell) of catalase and protease obtained in the H2O2-based cultivation were 172% and 156% of those obtained in the cultivation with aeration, whereas, the specific glucose oxidase levels were comparable. In addition, the oxygen profiles inside the pellets ofA. niger during both the modes of oxygen supply were described using mathematical models. Studies to elucidate the mechanism of oxygen availability showed that the decomposition of the H2O2 supplied in the extracellular space occurred intracellularly, as well as extracellularly, to yield oxygen. Also, the proton motive force (PMF) was found to be involved in the process of oxygen availability from H2O2 to A. niger cells.