Effects of ambient temperature and of temperature acclimation on nitrogen excretion and differential catabolism of protein and nonprotein resources in the intertidal snails,Littorina saxatilis (Olivi) and L. obtusata (L.)

Nitrogenous excretion in two snails, Littorina saxatilis (high intertidal) and L. obtusata (low intertidal) was studied in relation to temperature acclimation (at 4° and 21°C), including total N excretion rates, the fraction of urea in N excretion, corresponding O:N ratios and the partitioning of deaminated protein between catabolic and anabolic processes at 4°, 11° and 21°C. Aggregate N excretion rates in both species showed no significant compensatory adjustments following acclimation. Total weight specific N excretion rates at 21°C were higher in standard 3 mg L. saxatilis (739 ng N mg⁻¹ h⁻¹) than standard 5 mg L. obtusata (257 ng N mg⁻¹ h⁻¹) for snails acclimated to 21°C. Comparisons of Q₁₀ values of total weight specific N excretion to Q₁₀ values for weight specific oxygen consumption ({xxV}O₂) between 4° to 11 °C and 11° to 21°C indicated that, while total rates of catabolic metabolism ({xxV}O₂) and protein deamination in L. obtusata were essentially parallel, the relationship between N excretion and {xxV}O₂ in L. saxatilis revealed the partitioning of a larger share of deaminated protein carbon into anabolism at 4° and 21°C than at 11°C. Urea N accounted for a larger share of aggregate N excreted in L. saxatilis than in L. obtusata, but in both species urea N is a greater proportion of total N excreted when acclimated at 4°C (urea N: ammonia N ratio range: 1 to 2.15) than in snails acclimated to 21°C (urea N: ammonia N ratio range: 0.46 to 1.39). Molar O:N ratios indicate that the proportion of metabolism supported by protein catabolism is greater in L. saxatilis (O:N range: 2.5–8.4) than in L. obtusata (O:N range: 7.3–13.0). In both species, regardless of acclimation temperature, the O:N ratios are generally lowest (high protein catabolism) at 4°C and highest at 21°C.     

Kinetic comparisons of mesophilic and thermophilic aerobic biomass

Kinetic parameters describing growth and decay of mesophilic (30°C) and thermophilic (55°C) aerobic biomass were determined in continuous and batch experiments by using oxygen uptake rate measurements. Biomass was cultivated on a single soluble substrate (acetate) in a mineral medium. The intrinsic maximum growth rate (μ _max) at 55°C was 0.71±0.09 h⁻¹, which is 1.5 times higher than the μ _max at 30°C (0.48±0.11 h⁻¹). The biomass decay rates increased from 0.004 h⁻¹ at 30°C to 0.017 h⁻¹ at 55°C. Monod constants were very low for both types of biomass: 9±2 mg chemical oxygen demand (COD) l⁻¹at 30°C and 3±2 mg COD l⁻¹at 55°C. Theoretical biomass yields were similar at 30 and 55°C: 0.5 g biomass COD (g acetate COD)⁻¹. The observed biomass yields decreased under both temperature conditions as a function of the cell residence time. Under thermophilic conditions, this effect was more pronounced due to the higher decay rates, resulting in lower biomass production at 55°C compared to 30°C. Electronic Publication     

Enhanced Production and Partial Characterization of Thermostable α-galactosidase by Thermotolerant Absidia sp.WL511 in Solid-state Fermentation using Response Surface Methodology

Summary Response surface methodology was applied to optimize medium components for maximum production of a thermostable α-galactosidase by thermotolerant Absidia sp. WL511. First, the Plackett-Burman screening design was used to evaluate the effects of variables on enzyme production. Among these variables, MgSO₄ and soybean meal were identified as having the significant effects (with confidence level (90%). Subsequently, the concentrations of MgSO₄ and soybean meal were further optimized using central composite designs. The optimal parameters were determined by response surface and numerical analyses as 0.0503% (g/g) MgSO₄ and 0.406% (g/g) soybean meal and allowed α-galactosidase production to be increased from 4.4 IU g⁻¹ to 117.8 IU g⁻¹. The subsequent verification experiments confirmed the validity of the model. The optimum pH of enzymatic activity was 7.5 and the enzyme was stable at pH values ranging from 5.0 to 9.0. The optimum temperature was 73 °С. The enzyme was fairly stable at temperatures up to 60 °С and had 87% of its full activity at 65 °С after 2 h of incubation.     

Minimal levels of cold-adaptation in postsynaptic currents of a subantarctic teleost, <Emphasis Type="Italic">Notothenia rossii</Emphasis> (Perciformes: Notothenioidei: Nototheniidae)

As a part of the ICEFISH04 project on the RVIB Nathaniel B. Palmer, miniature end plate currents (MEPCs) were recorded from the extraocular muscles of Notothenia rossii captured at King Edward Point, South Georgia. A total of 1,176 MEPCs were recorded from the inferior oblique extraocular muscles of four specimens, over a temperature range of 1–12°C. The MEPCs were normal in form, with a rapid quasi-linear increase in inward current (typically <500 μs), followed by a slower exponential decay of the inward current to baseline. Exponential decay rates were calculated for individual MEPCs by linear regression of the log-transformed data, and converted to exponential time constants (τ). Only those MEPCs that fit the exponential model well, with r ² ≥ 0.95 (or in some cases r ² ≥ 0.99) were used for further calculations. At temperatures between 1 and 2°C, τ ranged from about 2,000 to 4,000 μs, similar to values extrapolated for temperate teleosts at the same temperature, but significantly longer than τ from MEPCs of high-latitude Antarctic nototheniids. Between 11 and 12°C, τ values for the N. rossii MEPCS were mainly between 1,100 and 1,700 μs, giving a Q ₁₀ of 2.05. An Arrhenius plot and linear regression were used to describe the effect of changing temperature on the decay phase of the N. rossii MEPCs: −ln τ = 27.887−6078/K, yielding an Arrhenius temperature coefficient (μ or apparent E _a) of −50.5 ± 2.9 (95% CL) kJ mol⁻¹ deg⁻¹. When compared with other nototheniids, these results showed that the neuromuscular junctions of N. rossii are compensated for low temperature, but not to the same degree as those of high Antarctic species. The ICEFISH Cruise (International Collaborative Expedition to collect and study Fish Indigenous to Sub-antarctic Habitats) was conducted on board the RVIB Nathaniel B. Palmer in May to July 2004. For further information, please visit .     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Phytoplankton production after the collapse of the Larsen A Ice Shelf,Antarctica

Part of the Larsen A Ice Shelf (64°15′S to 74°15′S) collapsed during January 1995. A first oceanographic and biological data set from the newly free waters was obtained during December 1996. Typical shelf waters with temperatures near and below the freezing point were found. A nutrient-rich water mass (max: PO₄ ³⁻ 1.80 μmol L⁻¹ and NO₃ ⁻ 27.64 μmol L⁻¹) was found between 70 and 200 m depth. Chlorophyll-a (Chl-a) values (max 14.24 μg L⁻¹) were high; surface oxygen saturation ranged between 86 and 148%. Diatoms of the genera Nitzschia and Navicula and the prymnesiophyte Phaeocystis sp. were the most abundant taxa found. Mean daily primary production (Pc) estimated from nutrient consumption was 14.80 ± 0.17 mgC m⁻³ day⁻¹. Pc was significantly correlated with total diatom abundance and Chl-a. Calculated ΔpCO₂ (difference of the CO₂ partial pressure between surface seawater and the atmosphere) was –30.5 μatm, which could have contributed to a net CO₂ flux from the atmosphere to the sea and suggests the area has been a CO₂ sink during the studied period. High phytoplankton biomass and production values were found in this freshly open area, suggesting its importance for biological CO₂ pumping.     

Effect of nifedipine on the ion channels formed by α1 subunits of the cardiac and vascular muscle L-type Ca channels

We investigated the voltage dependence of nifedipine sensitivity of the ion channels formed by α₁ subunits of the cardiac and smooth muscles (CM and SM, respectively) L-type Ca²⁺ channels stably expressed in Chinese hamster ovary (CHO) cells. Equilibrium inhibition of the α₁ subunits, directing Ba²⁺ current (I _α1), by different concentrations of nifedipine was measured at the holding potentials (V _h ) of −100 mV and −50 mV. AtV _h =−100 mV, the SM α₁ subunit was found to be 6-fold more sensitive for nifedipine than the subunit (K ₋₁₀₀=8.3 and 50.4 nM, respectively). Depolarization to −50 mV resulted in about sevenfold increase in the nifedipine potency for both subunits (K ₋₅₀=1.25 and 6.95 nM, respectively). The voltage dependence of steady-state inactivation could be fitted by a sum of two Boltzmann’s equations with slope factors of about 12 and 5 mV. The midpoints of both components in the CM α₁ subunit (−75.6 and −42.8 mV) were more negative than those in the SM subunit (−63.7 and −37.7 mV). The relative contribution of the less sloped component in the control was rather low, being less pronounced in the CM (0.15) than in the SM (0.34) subunits. Nifedipine shifted the midpoints of inactivation curves to more negative potentials. The shift was more pronounced for the SM α₁ subunit (−24.8 mV compared with −11.8 mV for the CM subunit in the presence of 10 nM nifedipine). Nifedipine differentially affected the two Boltzmann components of inactivation curves, more effectively inhibiting the steeper component. In the presence of 10 nM nifedipine, this component completely disappeared in the SM subunit, while its relative contribution in the CM subunit decreased from 0.85 to 0. 57, resulting in an apparent decrease in the steepness. These results are inconsistent with the receptor modulated hypothesis and suggest the existence of two mechanisms of inactivation characterized by different voltage dependence.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Amino acid transport inRhodotorula glutinis

The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10⁻⁶ m l-lysine and 1500∶1 for 10⁻⁶ m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically. The influx is saturable with an apparentK _m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was 13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed a powerful “trans” inhibitory effect on the uptake of amino acids.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

A maltooligosaccharide-forming α-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of α-amylase in KCC103 was not catabolite-repressed. The α-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified α-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65–70 °C, it was rapidly deactivated at 70 °C with a half-life of 7 min and at 50 °C, the half-life was 94 min. The K _m and V _max for starch hydrolysis were 2.6 mg ml⁻¹ and 909 U mg⁻¹, respectively. Ca²⁺ did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg²⁺, Ag²⁺, and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The α-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2–D4) were formed more predominantly than larger maltooligosaccharides (D5–D7). This maltooligosaccharide forming endo-α-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.     

Lack of Alpha-Synuclein Modulates Microglial Phenotype In Vitro

Alpha (α)-synuclein neuronal effects are continually being defined although its role in regulating glial phenotypes remains unclear. An ability to regulate microglial activation was investigated using primary cultures from wild type and α-synuclein deficient mice (Snca ^−/−). Snca ^−/− microglia demonstrated increased secretion of the cytokine tumor necrosis factor-alpha (TNF-α), impaired phagocytic ability, elevated prostaglandin levels, and increased protein levels of key enzymes in lipid-mediated signaling events, cytosolic phospholipase (cPLA₂), cyclooxygenase-2 (Cox-2) and phospholipase D2 (PLD2) when compared to wild type cells. Increased cytokine secretion and cPLA₂ and Cox-2 levels in Snca ^−/− microglia were partially attenuated by inhibiting PLD-dependent signaling with n-butanol treatment.     

Simultaneous production and characterization of medium-chain-length polyhydroxyalkanoates and alginate oligosaccharides by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> NK-01

When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L⁻¹ medium-chain-length polyhydroxyalkanoate (PHA_MCL) and 0.57 g L⁻¹ alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHA_MCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T _m and T _g values for the PHA_MCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was 700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Variation in leaf physiology of <Emphasis Type="Italic">Salix arctica</Emphasis> within and across ecosystems in the High Arctic: test of a dual isotope (Δ<Superscript>13</Superscript>C and Δ<Superscript>18</Superscript>O) conceptual model

Leaf carbon isotope discrimination (Δ¹³C) varies with the balance between net photosynthesis (A) and stomatal conductance (g _s ). Inferences that can be made with Δ¹³C are limited, as changes could reflect variation in A and/or g _s . Investigators have suggested that leaf δ¹⁸O enrichment above source water (Δ¹⁸O) may enable differentiation between sources of variation in Δ¹³C, as leaf Δ¹⁸O varies with transpiration rate (E), which is closely correlated with g _s when leaves experience similar leaf to air vapor pressure differences. We examined leaf gas exchange of Salix arctica at eight sites with similar air temperatures and relative humidities but divergent soil temperatures and soil water contents near Pituffik, Greenland (76°N, 38°W). We found negative correlations at the site level between g _s and Δ¹⁸O in bulk leaf tissue (r ² = 0.62, slope = −17.9‰/mol H₂O m⁻² s⁻¹, P = 0.02) and leaf α-cellulose (r ² = 0.83, slope = −11.5‰ mol H₂O m⁻² s⁻¹, P < 0.01), consistent with the notion that leaf water enrichment declines with increasing E. We also found negative correlations at the site-level between intrinsic water-use efficiency (iWUE) and Δ¹³C in bulk leaf tissue (r ² = 0.65, slope = −0.08‰/μmol CO₂ /mol H₂O, P = 0.02) and leaf α-cellulose (r ² = 0.50, slope = −0.05 ‰/[μmol CO₂ /mol H₂O], P = 0.05). When increasing Δ¹³C was driven by increasing g _s alone, we found negative slopes between Δ¹³C and Δ¹⁸O for bulk leaf tissue (−0.664) and leaf α-cellulose (−1.135). When both g _s and A _max increased, we found steeper negative slopes between Δ¹³C and Δ¹⁸O for bulk leaf tissue (−2.307) and leaf α-cellulose (−1.296). Our results suggest that the dual isotope approach is capable of revealing the qualitative contributions of g _s and A _max to Δ¹³C at the site level. In our study, bulk leaf tissue was a better medium than leaf α-cellulose for application of the dual isotope approach.     

Biosorption of Ni (II) by Schizosaccharomyces pombe: kinetic and thermodynamic studies

The potential of the dried yeast, wild-type Schizosaccharomyces pombe, to remove Ni(II) ion was investigated in batch mode under varying experimental conditions including pH, temperature, initial metal ion concentration and biosorbent dose. Optimum pH for biosorption was determined as 5.0. The highest equilibrium uptake of Ni(II) on S. pombe, q _e, was obtained at 25 °C as 33.8 mg g⁻¹. It decreased with increasing temperature within a range of 25–50 °C denoting an exothermic behaviour. Increasing initial Ni(II) concentration up to 400 mg L⁻¹ also elevated equilibrium uptake. No more adsorption took place beyond 400 mg L⁻¹. Equilibrium data fitted better to Langmuir model rather than Freundlich model. Sips, Redlich–Peterson, and Kahn isotherm equations modelled the investigated system with a performance not better than Langmuir. Kinetic model evaluations showed that Ni(II) biosorption process followed the pseudo-second order rate model while rate constants decreased with increasing temperature. Gibbs free energy changes (ΔG°) of the system at 25, 30, 35 and 50 °C were found as −1.47E + 4, −1.49E + 4, −1.51E + 4, and −1.58E + 4 J mol⁻¹, respectively. Enthalpy change (ΔH°) was determined as −2.57E + 3 J mol⁻¹ which also supports the observed exothermic behaviour of the biosorption process. Entropy change (ΔS°) had a positive value (40.75 J mol⁻¹ K⁻¹) indicating an increase in randomness during biosorption process. Consequently, S. pombe was found to be a potential low-cost agent for Ni(II) in slightly acidic aqueous medium. In parallel, it has been assumed to act as a separating agent for Ni(II) recovery from its aqueous solution.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Purification and characterization of caprine kidney uricase, possessing novel kinetic and thermodynamic properties

Purified uricase from a caprine kidney, possessed K _m and V _max values of 1.1 mg ml⁻¹ and 3512 IU (mg protein)⁻¹ for uric acid hydrolysis, respectively. The optimum temperature and pH for catalytic activity were 40 °C and 8.5, respectively. The activation energy for formation of ES complex was 13.6 kJ mol⁻¹. Enthalpy (ΔH*), entropy of activation (ΔS*) and Gibbs free energy demand of uricase inactivation were 62.8 kJ mol⁻¹, −102 J mol⁻¹ K⁻¹ and 104.3 kJ mol⁻¹, respectively. Gibbs free enrgy demand for substrate binding and transition state stabilization were also determined which were comparable with those for themostable enzymes.