Effect of gallic acid polydisulfide on activity and stability of catalase in various media

The effect of the active bioantioxidant polydisulfide of gallic acid (PDSG) on the catalytic activity and operational and thermal stability of catalase was studied in three media: distilled water (pH approximately 5.6), phosphate buffer, pH 7.4, and reversed micelles of Aerosol OT (AOT) in heptane of varied hydration degree w0. PDSG inhibited the catalase-induced decomposition of H2O2 by the mixed or noncompetitive mechanism: in various media the inactivation constant Ki varied in the range of (0.63-2.32).10-5 M. PDSG nearly twofold decreased the rate constant of interaction of the complex I of catalase with H2O2 (k2, M-1.sec-1) in water and reversed micelles of AOT and 3-5 times increased the effective rate constant of catalase thermal inactivation, k*in, sec-1, depending on the reaction medium. PDSG significantly decreased the rate constant of catalase inactivation during the enzymatic reaction, kin, sec-1, and thus increased the enzyme operational stability in water and reversed AOT micelles in heptane. The interaction of PDSG with catalase in water and in phosphate buffer was accompanied by significant changes in CD spectra in the far UV-region that indicated disturbances in the secondary structure of catalase subunits induced by the bioantioxidant; the latter was suggested to initiate the reaction of thiol--disulfide exchange with the enzyme. The problem of the compatibility of catalase with disulfide bioantioxidants is discussed.     

Inducible catalase in Pseudomonas fluorescens

The catalase activity of a non-proliferating suspension of Pseudomonas fluorescens doubled after six hours incubation in a 50 mM phosphate buffer medium (pH 7.3). The same effect was observed in a peptone medium. The increased activity was due to induced enzyme synthesis, and not to activation of preexisting catalase. Induced catalase was separated by electrophoresis from deuterium labelled constitutive catalase. The enzyme was also induced under anaerobic conditions in phosphate buffer or in culture when nitrate was supplied as an electron acceptor. Induction was considerably increased by the addition of various nucleotides and amino acids to the incubation medium.     

The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae

In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isoform (peroxisomal catalase), the mutant showed the same levels of total catalase activity as the control strain. Furthermore, in cells deficient in Ctt1, the reduced glutathione:oxidized glutathione ratio (GSH:GSSG) of dry cells was higher than that of the control strain, indicating a compensatory mechanism of defense in response to dehydration. Even so, desiccation tolerance of the ctt1 strain was significantly lower than in the control strain. Using a fluorescent probe sensitive to oxidation, we observed that cells of the ctt1 strain showed levels of intracellular oxidation 70% higher than those of control strain, suggesting that Ctt1 plays a role in the maintenance of the intracellular redox balance during dehydration and, therefore, in tolerance against a water stress.     

Alkali- and halo-tolerant catalase from Halomonas sp. SK1: overexpression in Escherichia coli, purification, characterization, and genetic modification

A catalase gene, ohktA, from an alkali- and halo-tolerant bacterium, Halomonas sp. SK1, on the pKK223-3, was expressed in the catalase-lacking Escherichia coli strain UM2. Highly purified catalase showing a single band on SDS-PAGE was obtained by two liquid chromatography steps on DEAE-Toyopear1 and Chelating-Sepharose Fast Flow. The enzyme, oHktA, shows high catalase activity with a pH optimum at 10, and the activity was stable in 4 M KC1. This enzyme is thermo-sensitive, showing a significant loss of activity within 5 minutes at 37 degrees C. To modify the stability of the catalase, the addition of domain II of the heat stable Mn catalase from Thermus thermophilus to the C-terminus was made. When coexpressed with a chaperone (PhFKBP29) gene product, peptidyl-prolyl cis-trans isomerase, from a thermophilic bacterium, a chimeric catalase was produced in the soluble fraction. The stability of this catalase in the range of 37 degrees -45 degrees C was improved and it was stable for more than 1 h at 37 degrees C.     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.     

Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films

Alpha-amylase was covalently immobilized onto maleic anhydride copolymer films preserving activity. The initial activity of the immobilized layers strongly depended on the immobilization solution, and on the physicochemical properties of the copolymer film. Higher enzyme loading (quantified by amino acid analysis using HPLC) and activity (measured by following starch hydrolysis) were attainable onto hydrophilic, highly swelling 3-D poly(ethylene-alt-maleic anhydride) (PEMA) copolymer films, while immobilization onto hydrophobic poly(octadecene-alt-maleic anhydride) (POMA) copolymer films resulted in low content enzyme layers and lower activity. No significant activity was lost upon dehydration/re-hydration or storage of enzyme containing PEMA copolymer layers in deionised water for up to 48 h. In contrast, α-amylase decorated POMA films suffered a significant activity loss under those conditions. The distinct behaviours may be attributed to the different intrinsic physicochemical properties of the copolymer films. The compact, hydrophobic POMA films possibly favours hydrophobic interactions between the hydrophobic moieties of the protein and the surface, which may result in conformational changes, and consequent loss of activity. Surprisingly, residual activity was found after harsh treatments of active α-amylase PEMA based layers revealing that immobilization onto the hydrophilic polymer films improved the stability of the enzyme.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.     

Increased drying rate lowers the critical water content for survival in embryonic axes of English oak (Quercus robur L.) seeds

The potential to cryopreserve embryonic axes of desiccation-sensitive (recalcitrant) seeds is limited by damage during the desiccation necessary for low temperature survival, but the basis of this injury and how to reduce it is not well understood. The effects of drying rate on the viability, respiratory metabolism and free radical-mediated processes were therefore investigated during dehydration of Quercus robur L. embryonic axes. Viability, assessed by evidence of germination and tetrazolium staining, showed a sharp decline at 0.27 and 0.8 g/g during rapid (<12 h) or slow (3 d) dehydration, respectively. Rapid dehydration therefore lowered the critical water content for survival. At any given water content rapid dehydration was associated with higher activities of the free radical processing enzymes, superoxide dismutase, catalase and glutathione reductase and lower levels of hydroperoxide and membrane damage. Rapid dehydration was also associated with lower malate dehydrogenase activity, and a reduced decline in phosphofructokinase activity and in levels of the oxidized form of nicotinamide dinucleotide. Ageing may have contributed to increased damage during slow dehydration, since viability declined even in hydrated storage after 3 d. The results presented are consistent with rapid dehydration reducing the accumulation of damage resulting from desiccation induced aqueous-based deleterious reactions.     

A rapid method for the purification of methanol dehydrogenase from Methylobacterium extorquens

Methanol dehydrogenase (MDH) is a water soluble quinoprotein that catalyzes the oxidation of methanol as an important carbon source in methylotrophic bacteria. A rapid method for the purification of MDH from Methylobacterium extorquens AM1 was developed using a single cation exchange chromatographic step, followed by ultrafiltration for final purification, enzyme concentration, and buffer exchange. MDH was obtained in an excellent overall yield with a final enzyme purity of greater than 97%. Storage at -80 degrees C in 20mM phosphate buffer, pH 7.0, showed only a negligible loss of enzyme activity after six months.     

The role of the glutathione system during dehydration of Boea hygroscopica

Plants of Boea hygroscopica F. Muell were subjected to dehydration for 25 days until the relative water content was 23%. The rate of water loss was very slow during the first 12 days of dehydration while it dramatically increased during the last 3 days of the treatment. On day 12 of dehydration total glutathione content was reduced to 24% of the control level and was mainly present in the oxidized form. At that time an oxidation of the sulfhydryl groups of soluble proteins was observed. A protection of glutathione against oxidation of the sulfhydryl groups of soluble proteins was established only after 22 days of desiccation, at the same time as glutathione began to accumulate. During the whole desiccation period enzyme activities related to glutathione utilization and regeneration and the activity of NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), which contains essential sulfhydryl groups, were maintained. The results of the present experiment suggest that during dehydration of Boea hygroscopica glutathione has above all, a primary role in the protection of the sulfhydryl groups of the thylakoid proteins, which were maintained in the reduced form during the whole dehydration period.     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium.

When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase. The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000. The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0. It has an isoelectric point of 4.0. This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm. Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect. The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity. The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate. Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme. Moreover, purification must be performed in the presence of 2 M NaCl. Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity. The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage. The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM. Thus, the mesohalic catalase purified from H. halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.     

微胶囊固定化过氧化氢酶的制取及对H_2O_2的分解作用

以乙基纤维素为膜材,用溶液干燥法制取了微胶囊固定化过氧化氢酶,探讨了制取过程中明胶的加入对微胶囊产率及固定化过氧化氢酶活性的影响,同时论述了存放时间、温度以及环境ｐＨ值对微胶囊固定化过氧化氢酶稳定性的影响．深入研究了微胶囊固定化过氧化氢酶对Ｈ２Ｏ２的分解作用,获得了十分有意义的结果     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.     

Sugar effects on membrane damage during desiccation of pea embryo protoplasts

Desiccation tolerance of protoplasts isolated from germinating pea (Pisum sativum L. cv. 'Alaska') embryonic axes depends, in part, on the osmotic strength and composition of the suspending medium. To determine the reason for this dependence and whether treatment with different solutions results in different types of damage, protoplast recovery and survival were assessed after dehydration to a range of water contents. Protoplasts were derived from germinating axes that had intermediate desiccation tolerance. Protoplasts were isolated and resuspended in buffers containing sucrose/raffinose (85:15, w/w) or sorbitol, which were isotonic or hypertonic to the cells of the embryonic axis, then were flash-dried to a range of water contents. Protoplasts were rehydrated and stained with fluorescein diacetate (FDA) to assess survival and to estimate two types of membrane injury: lysis and the loss of semipermeability. In all treatments, protoplast survival dropped sharply during the initial phase of dehydration due to lysis. Protoplast survival was greater in hypertonic sucrose/raffinose buffer than in isotonic sucrose/raffinose buffer, or in the latter made hypertonic by the addition of sorbitol. When sorbitol was substituted for sucrose/raffinose in either the isolation or desiccation buffer, or both, protoplast survival at intermediate and low hydrations decreased due to a loss of membrane semipermeability. The results indicate that additional sucrose/raffinose is beneficial for the desiccation tolerance of protoplasts, the benefit is not due to a simple osmotic effect, and the benefit is greatest at water contents less than 0.5 g g(-1) DW, where the presence of the sugars appears to protect membrane semipermeability.     

Antioxidant defense parameters as predictive biomarkers for fermentative capacity of active dried wine yeast

The production of active dried yeast (ADY) is a common practice in industry for the maintenance of yeast starters and as a means of long term storage. The process, however, causes multiple cell injuries, with oxidative damage being one of the most important stresses. Consequentially, dehydration tolerance is a highly appreciated property in yeast for ADY production. In this study we analyzed the cellular redox environment in three Saccharomyces cerevisiae wine strains, which show markedly different fermentative capacities after dehydration. To measure/quantify the effect of dehydration on the S. cerevisiae strains, we used: (i) fluorescent probes; (ii) antioxidant enzyme activities; (ii) intracellular damage; (iii) antioxidant metabolites; and (iv) gene expression, to select a minimal set of biochemical parameters capable of predicting desiccation tolerance in wine yeasts. Our results show that naturally enhanced antioxidant defenses prevent oxidative damage after wine yeast biomass dehydration and improve fermentative capacity. Based on these results we chose four easily assayable parameters/biomarkers for the selection of industrial yeast strains of interest for ADY production: trehalose and glutathione levels, and glutathione reductase and catalase enzymatic activities. Yeast strains selected in accordance with this process display high levels of trehalose, low levels of oxidized glutathione, a high induction of glutathione reductase activity, as well as a high basal level and sufficient induction of catalase activity, which are properties inherent in superior ADY strains.     

Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases

Cross-linked enzyme aggregates (CLEA^®) were prepared from laccases from three different sources: Trametes versicolor, Trametes villosa and Agaricus bisporus. The effect of the various parameters – nature of the precipitant, pH, temperature, glutaraldehyde concentration and cross-linking time – on the activity recovery and storage and operational stability of the resulting CLEAs was different. The laccase CLEAs exhibited the expected increased stability compared to the free enzyme but there was no direct correlation with the number of surface lysine residues in the latter. It is clearly not the only parameter influencing the properties of the CLEA. Co-aggregation with albumin did not improve the stability. The laccase CLEAs, in combination with the stable N-oxy radical, TEMPO, were shown to be active and stable catalysts for the aerobic oxidation of linear C₅–C₁₀ aliphatic alcohols, to the corresponding aldehydes, in aqueous buffer (pH 4). Rates were an order of magnitude higher than those observed with the corresponding free enzyme and the CLEAs could be recycled several times without appreciable loss of activity. The addition of water immiscible or water miscible solvents showed no further improvement in rate compared with reactions in aqueous buffer alone.     

Stability of the anti-oxidative enzymes in aqueous and detergent solution

Activities of the anti-oxidative enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase were studied in rat tissues to determine the ability of detergents both to solubilize the enzymes and also to stabilize enzyme activity. Rat brain, heart and liver were homogenized in 0.1M KCl, 0.1% sodium dodecyl sulfate, 0.1% lubrol, or 0.1% cetyl-trimethylammonium bromide. In general lubrol was more effective than the other solutions in solubilizing GPx and catalase. Lubrol and 0.1M KCl were equally effective in solubilizing SOD. The highest enzyme activities were (1) SOD: 2484 ng/mg (brain), 2501 ng/mg (heart), and 5586 ng/mg (liver); (2) GPx: 224 mU/mg (brain), 1870 mU/mg (heart), and 7332 mU/mg (liver); (3) catalase: 2.8 mU/mg (brain), 10.6 mU/mg (heart), and 309 mU/mg (liver). While cetyl trimethylammonium bromide is marginally better than sodium dodecyl sulfate in solubilizing active enzyme, neither ionic detergent has any advantage over lubrol or 0.1M KCl. For catalase and GPx, enzyme activity loss with time is biphasic. After initial, rapid activity loss (1–5 days for GPx and 7–10 days for catalase) the differences noted among the homogenizing solutions disappear and very little if any activity loss is noted over the next 2–3 weeks. For catalase and GPx, only baseline enzyme activity from t = 0 – 3 weeks is found in the most chaotropic solution, 0.1% sodium dodecyl sulfate while biphasic activity loss is most pronounced in 0.1% lubrol. These results may indicate active GPx and catalase species stabilized by a lipid-like environment. Correlatingin vitro catalase or GPx measurements within vivo anti-oxidative protection may underestimate tissue defences.