Application of a quinohaemoprotein alcohol dehydrogenase in bio-electrocatalysis: Immobilization of the enzyme and mediated electron transfer between the enzyme and an electrode

Summary Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni was immobilized on polypyrrole-coated track-etch and microporous membranes. On the track-etch membrane, 3.4 to 4.8 × 10^–3 Units of enzyme/cm² was immobilized whilst on the microporous membrane 0.05 U/cm² was immobilized. The track-etch membrane was then used in electrochemical studies using ferricyanide as a redox mediator giving a maximum catalytic current of 0.022 mA/cm² membrane with 1-pentanol as the substrate. The kinetic parameters (K_m and V_max) of the immobilized enzyme are of the same order of magnitude as those of the free enzyme.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Kinetic Characterization of Extracellular Catalases fromPenicillium piceum F-648 and Its Hydrogen Peroxide-Adapted Variants

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H₂O₂ was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H₂O₂ degradation (V _max), V _max/K _M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k _in, s^–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k _in ^*, s^–1) was determined at 45°C. In all samples, the specific activity and K _M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10^–4 mg/ml. The effective constants describing the rate of H₂O₂ degradation (k, s^–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H₂O₂. Catalases from variants 5 and 3 with high and low affinities for H₂O₂, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H₂O₂ can be used to obtain fungal variants producing extracellular catalases with improved properties.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

The enhancement by pervanadate of tyrosine phosphorylation on prostatic proteins occurs through the inhibition of membrane-associated tyrosine phosphatases

The Na⁺–K⁺ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na⁺–K⁺ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na⁺–K⁺ ATPase activity in the presence of different concentrations of MgATP or Na⁺ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in V_max and an increase in K_m for MgATP; however, K_a value for Na⁺ was decreased. Treatment of sarcolemma with H₂O₂ plus Fe²⁺, an hydroxyl and other radical generating system, increased MDA content but decreased both Na⁺–K⁺ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na⁺–K⁺ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H₂O₂ plus Fe²⁺ was associated with a decrease in V_max and an increase in K_m for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na⁺–K⁺ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na⁺–K⁺ ATPase activity may be due to a decrease in the affinity of substrate in the sarcolemmal membrane.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Maximal oxygen uptake, anaerobic threshold and running economy in women and men with similar performances level in marathons

Sex differences in running economy (gross oxygen cost of running, C_R), maximal oxygen uptake (VO_2max), anaerobic threshold (Th_an), percentage utilization of aerobic power (% VO_2max), and Th_an during running were investigated. There were six men and six women aged 20–30 years with a performance time of 2 h 40 min over the marathon distance. The VO_2max, Th_an, and CR were measured during controlled running on a treadmill at 1° and 3° gradient. From each subject's recorded time of running in the marathon, the average speed (v _M) was calculated and maintained during the treadmill running for 11 min. The VO_{2 max} was inversely related to body mass (m _b), there were no sex differences, and the mean values of the reduced exponent were 0.65 for women and 0.81 for men. These results indicate that for running the unit ml·kg^–0.75·min^–1 is convenient when comparing individuals with different m _b. The VO_2max was about 10% (23 ml·kg^–0.75·min^–1) higher in the men than in the women. The women had on the average 10–12 ml·kg^–0.75·min^–1 lower VO₂ than the men when running at comparable velocities. Disregarding sex, the mean value of C_R was 0.211 (SEM 0.005) ml·kg^–1·m^–1 (resting included), and was independent of treadmill speed. No sex differences in Th_an expressed as % VO_2max or percentage maximal heart rate were found, but Than expressed as VO₂ in ml·kg^–0.75·min^–1 was significantly higher in the men compared to the women. The percentage utilization of f _emax and concentration of blood lactate at v _M was higher for the female runners. The women ran 2 days more each week than the men over the first 4 months during the half year preceding the marathon race. It was concluded that the higher VO_2max and Th_an in the men was compensated for by more running, superior C_R, and a higher exercise intensity during the race in the performance-matched female marathon runners.     

Uptake of ethanolamine in neuronal and glial cell cultures

The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparentK _m of 6.7 M,V _max 41.4 pmol mg prot.^–1 min^–1 and glial cells aK _m of 119.6 M,V _max 3,917 pmol mg prot^–1 min^–1. The lower affinity of the transport and the 100 fold increase inV _max observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na⁺ ions dependent but was not affected by CN^– or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate.     

Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture

The uptake of ammonium, nitrate and phosphate by laboratory-grown young sporophytes of Laminaria abyssalis was measured in a perturbed system (batch mode) at 18 °C and 35 ± 5 µE m^–2 s^–1 photon flux density. Uptake of all appeared to follow saturation-type nutrient uptake kinetics. The NO _inf3 ^sup– (K _s = 14.0 µM, V _max = 5.0 µmol h^–1 g^–1 dry wt) and NH _inf4 ^sup+ (K _s = 4.6 µM, V _max= 2.0 µmol h^–1 g^–1 dry wt) were taken up simultaneously, although NH _inf4 ^sup+ was taken up more rapidly. Values of K ₃ and V _max for phosphate were, respectively, 2.21 µM and 0.83 µmol h^–1 g^–1 dry wt. Nitrate and phosphate were both consumed in similar rates (V _max /Ks 0.37) at low concentrations. NH _inf4 ^sup+ , thus, might be a more efficient form of N fertilizer if artificial enrichment of seawater is used.     

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Isolation of <Emphasis Type="BoldItalic">Serratia marcescens</Emphasis> as a chondroitinase-producing bacterium and purification of a novel chondroitinase AC

A strain of Serratia marcescens that produced chondroitinase was isolated from soil. It produced a novel chondroitinase AC, which was purified to homogeneity. The enzyme was composed of two identical subunits of 35 kDa as revealed by SDS-PAGE and gel filtration. The isoelectric point for the chondroitinase AC was 7.19. Its optimal activity was at pH 7.5 and 40 °C. The purified enzyme was active on chondroitin sulfates A and C and hyaluronic acid, but was not with chondroitin sulfate B (dermatan sulfate), heparin or heparan sulfate. The apparent K_m and V_max of the chondroitinase AC for chondroitin sulfate A were 0.4 mg ml^–1 and 85 mmol min^–1 mg^–1, respectively, and for chondroitin sulfate C, 0.5 mg ml^–1 and 103 mmol min^–1 mg^–1, respectively.     

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Cell-bound cholinesterase inTrichoderma harzianum

Cell-bound cholinesterase enzyme activity is reported for the first time in the mycelium ofTrichoderma harzianum. This enzyme hydrolyzes both the acetylcholine and the butyryl thiocholine esters. TheK _m andV _max for choline ester are 0.69 mM and 1.0 nmol acid released min^–1 g^–1 protein. However, the thiocholine ester has aK _m value of 2.2 mM andV _max value of 3.33 nmol product formed min.^–1 g^–1 protein. The enzyme is inhibited by eserine, a true classical cholinesterase inhibitor.     

Immobilization of catalase via adsorption onto metal-chelated affinity cryogels

A poly (acrylamide-allylglycidyl ether) [p(AAm-AGE)] cryogel was prepared by radical polymerization of acrylamide (AAm) and allylglycidyl ether (AGE). Cibacron Blue F3GA (CB) was covalently attached to the p(AAm-AGE) cryogel via the reaction between the chloride groups of the reactive dyes and the epoxide groups of the AGE. The CB-attached p(AAm-AGE) cryogel was chelated with Fe³⁺ ions. This immobilized metal ion affinity chromatography (IMAC) cryogel carrying 25.8 ± 2.0 μmol Fe³⁺ ions was used in adsorption studies to interrogate the effects of pH, protein initial concentration, flow rate, temperature and ionic strength on enzyme activity. Maximum adsorption capacities were found to be 75.7 ± 1.2 mg/g for p(AAm-AGE)-CB-Fe³⁺ cryogels and 60.6 ± 1.0 mg/g for p(AAm-AGE)-CB cryogels, respectively. The adsorbed amounts of catalase per unit mass of cryogel reached a plateau value at about 1.5 mg/mL at pH 6.0. The K_m values were found to be 0.73 ± 0.02 g/L for the free catalase and 0.18 ± 0.02 g/L for the immobilized catalase. The V_max value of free catalase (2.0 × 10³ U/mg enzyme) was found to be lower than that of the immobilized catalase (2.5 × 10³ U/mg enzyme). It was also observed that the enzyme could be repeatedly adsorbed and desorbed onto the p(AAm-AGE)-CB-Fe³⁺ cryogel.     

Reversible immobilization of catalase on fibrous polymer grafted and metal chelated chitosan membrane

Poly(itaconic acid) grafted and/or Fe(III) ions incorporated chitosan membranes were used for reversible immobilization of catalase (from bovine liver) via adsorption. The influences of pH and initial catalase concentration on the immobilization capacities of the CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membranes have been investigated in a batch system. Maximum catalase adsorption onto CH-g-poly(IA) and CH-g-poly(IA)-Fe(III) membrane were found to be 6.3 and 37.8 mg/g polymer at pH 5.0 and 6.5, respectively. The CH-g-poly(IA)-Fe(III) membrane with high catalase adsorption capacity was used in the rest of the study. The K_m value for immobilized catalase on CH-g-poly(IA)-Fe(III) (25.8 mM) was higher about 1.6-fold than that of free enzyme (13.5 mM). Optimum operational temperature was observed at 40 °C, a 5 °C higher than that of the free enzyme and was significantly broader. The optimum operational pH was same for both free and immobilized catalase (pH 7.0). Thermal stability was found to increase with immobilization. Free catalase lost all its activity within 20 days whereas immobilized catalase lost 23% of its activity during the same incubation period. It was observed that the same support enzyme can be repeatedly used for immobilization of catalase after regeneration without significant loss in adsorption capacity or enzyme activity. In addition, the CH-g-poly(IA)-Fe(III) membrane prepared in this work showed promising potential for various biotechnological applications.     

Inhibition of the Na+/Ca2+ antiport of heart mitochondria by diethylpyrocarbonate

Diethylpyrocarbonate inhibits Na⁺/Ca²⁺ antiport activity in isolated heart mitochondria. The inhibition is time-dependent with maximum activity developed after 5 min at 25°C. The reaction of diethylpyrocarbonate with the mitochondrial membrane is biphasic with 25–30 nmol mg^–1 reacting rapidly and an additional 30 nmol mg^–1 taken up slowly over a 30-min incubation. Inhibition of mitochondrial Na⁺/Ca²⁺ antiport by diethylpyrocarbonate decreases theV _max of the reaction, and the inhibition cannot be reversed by washing the mitochondria or addition of excess histidine. The inhibition occurs at levels of inhibitor that have little or no effect on Ca²⁺ uptake, Na⁺/H⁺ antiport, or succinate respiration. A portion of the Na⁺-dependent efflux of Ca²⁺ is insensitive to diethylpyrocarbonate and this component is abolished by diltiazem. The mechanism by which diethylpyrocarbonate inactivates Na⁺/Ca²⁺ antiport is still uncertain, but may involve the modification of an unprotonated histidine residue in the transporter.     

Biochemical and catalytic properties of endo-1,4-β-xylanases from Thermomyces lanuginosus (wild and mutant strains)

Endoxylanases from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 (cellulase free wild and mutant strains), were purified to homogeneity by anion-exchange and molecular-sieve chromatographic methods. The purified enzymes were monomers with molecular masses of 22 kDa (wild type) and 24 kDa (mutant), estimated by SDS-PAGE and gel filtration. As glycoproteins, the purified enzymes had 0.74% (wild type) and 11.8% (mutant) carbohydrate contents, and pI values of 5.8 and 6, respectively. The optimal pH and temperature values of wild type xylanase were determined to be pH 7 and 60 °C, whereas pH 6.7 and 70 °C, were optimal for the purified mutant enzyme (K _m and V _max values of 3.7 mg ml^–1 and 670 mol min^–1 xylose compared to the kinetic values of the purified wild type xylanase –5.1 mg ml^–1 and 385 mol min^–1 xylose). Inhibition studies suggested the possible involvement of histidine, tryptophan residues and carboxylic groups in the binding or catalysis.     

Substrate Inhibition of Nitric Oxide Synthase in Pulmonary Artery Endothelial Cells in Culture

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0–100 μM arginine increased NO production, detected as nitrite in the culture medium, in a dose-dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 μM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that theK_mof arginine as a substrate for NOS is 3–5 μM, theV_maxoccurred at 100 μM arginine, and substrate inhibition occurred at >100 μM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, and -arginine did not change NOS kinetics. These results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.