Standard electromotive force of the H2---AgCl;Ag cell in 30, 40, and 50 mass% dimethyl sulfoxide/water from −20 to 25 °C: pK2 and pH values for a standard “Bicine” buffer solution at subzero temperatures

The establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixtures at temperatures in the range −20 to 0 °C is reported. The buffer material selected was the ampholyte Bicine (N,N-bis(2-hydroxyethyl)glycine), and the reference standard consists of equal molal quantities of Bicine and its sodium salt. The assignment of pH* values rests on measurements of the emf of cells without liquid junction, Pt;H₂(g, 1 atm) ¦Bicine, Na Bicinate, NaCl ¦AgCl;Ag, and the pH* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Bicine) ^± (Bicinate)⁻ + H⁺. The standard emf in the DMSO/H₂O solvents at subzero temperatures was determined from emf measurements of the cell with solutions of HCl replacing the buffer-chloride mixture.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

The role of pH1 and buffer capacity in the recovery of function of smooth muscle cooled to −13 °C in unfrozen media

It has often been suggested that pH changes may be implicated in the injury sustained by biological systems during cooling. This particular mechanism of cryoinjury, however, has received little attention undoubtedly because of the difficulties encountered in making accurate pH measurements at low temperatures.New pH1 scales established for some mixtures of dimethyl sulfoxide and water at low temperatures are used in this study to assess the effect of pH1 and buffering ability upon the integrity of mammalian smooth muscle stored at ?13 °C in a variety of unfrozen solutions containing 30% ($\text{w}\text{v}$) Me₂SO. Smooth muscle, as a component of every organ, is a good model tissue intermediate between cells and organs. Furthermore, its overall function is conveniently tested by measuring isometric contractile responses to the drug histamine. In this way the function of strips of guinea pig taenia coli were examined at 37 °C before and after storage at ?13 °C in potassiumrich media containing a variety of zwitterionic buffers. Functional recovery depends markedly on the pH1 with a welldefined optimum at the surprisingly high pH1_?13 of 9.2. In medium containing TES buffer, which has a maximum buffer capacity at pH1_?13= 8.6, the cooled muscles recover 50% of their control contractility but in medium containing the buffer Tricine, which has a maximum capacity at the optimum pH1 for recovery, the contractile response upon rewarming improves to 70%.These data are the first to quantify the effect of pH in cryopreservation on a sound theoretical basis and some of the possible underlying mechanisms are discussed.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

Standard electromotive force of the H2-AgCl;Ag cell in 30, 40, and 50 mass% glycerol/water from −20 to 25 °C: pK2 and pH values for a standard “Mops” buffer in 50 mass% glycerol/water

Data are presented regarding the establishment of the pH (designated pH^*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from −20 to 25 °C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH^* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦ Mops, Na Mopsate, NaCl ¦ AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Mops)^±/ah (Mopsate)⁻ + H ⁺. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures.     

In vitro studies of the effect of Environmental Conditions on the anthacnose pathogen of bananas, Colletotrichum musae

In vitro studies were undertaken to determine the effect of pH, temperature, water availability and carbon dioxide (CO₂) concentration on germination and growth of Colletotrichum musae, the causal pathogen of anthracnose of bananas. The optimum pH for germination and growth varied between 4·0 and 5·0 depending on temperature. At low pH (< 3·0) and 15°C, both germination and growth were significantly reduced, with a marked increase in the lag time, in days, prior to growth. C. musae germinated and grew over a wide range of water activities (a_w; 0·995−0·94 and 0·995−0·92, respectively) at 20, 25 and 30°C. In all cases where germination occurred appresoria were subsequently produced. Optimum growth occurred at 30°C and 0·995 a_w, although this changed to 0·98 a_w at 35°C. Increasing CO₂ concentration to 15% or reducing oxygen concentration to 1% resulted in a significant (P < 0·05) reduction in growth, but did not inhibit growth completely.     

Aspects of the Cryobiology of the Intertidal Harpacticoid CopepodTigriopus brevicornis(O. F. Müller)

Cold-resistance studies of marine invertebrates have concentrated on intertidal sedentary organisms, which are often subjected to subzero air temperatures in winter. Mobile rock pool inhabitants have been rarely studied because such habitats are thought to buffer environmental variation. However, it is not uncommon for small upper-shore rock pools (2 by 1 cm) to become completely frozen. Such supralittoral habitats are subject to extreme physicochemical fluctuations especially in salinity (0 to 300‰) and temperature (−1 to +32°C) due to evaporation and dilution. The dominant invertebrate in such habitats is the harpacticoid copepodTigriopus brevicornis.Aspects of the cryobiology ofT. brevicorniswere investigated using differential scanning calorimetry (DSC). Thermograms obtained from DSC allowed determinations of freeze-onset (supercooling point, SCP), melt-onset, and melt-peak (melting point, MP) temperatures, together with estimation of the proportion of water freezing in the samples. The effects of acclimation salinity, temperature, starvation, and reproductive state on these cryobiological parameters were investigated. Acclimation to increasing salinity depressed the SCP, with the highest salinity (70‰) producing the lowest SCP, melt-onset, and MP temperatures at −27.5, −15.2, and −9.5°C respectively. The highest acclimation temperature (20°C) produced the lowest SCP (−23.4°C). Starvation significantly increased the SCP, melt-onset, and MP temperatures in comparison to fed individuals acclimated to the same salinity. The presence of eggs or ovaries in individual copepods elevated the SCP compared to nongravid females and males. LT₅₀studies showed that acclimation to high salinity improved the ability ofT. brevicornisto survive in frozen seawater. Seventy parts per thousand acclimated individuals had an LT₅₀of 64.9 h compared with just 1.4 h for 5‰ acclimated individuals in frozen seawater at −5°C. The study shows that the cold-resistance capabilities ofT. brevicorniscan be affected by several different factors, and the link between the osmoconforming nature of this species and its cold resistance is discussed.     

Properties and function of malate enzyme from Pseudomonas putida

Malate enzyme (l-malate : NADP⁺ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis ; monovalent ions like K⁺ and NH₄⁺ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35–80 °C showed broken Arrhenius plots with an inflexion at 65 °C. The enzyme halflife was 30s at 85 °C.The enzyme showed hyperbolic kinetics for both substrates with apparent K_m values of 4.0 × 10⁻⁴ M and 2.3 × 10⁻⁵ M for l-malate and NADP⁺ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.     

Physico-chemical and Catalytic Properties of Clostridium perfringens Hyaluronidase: An Update

Hyaluronidase (E.C. 4.2.2.1 hyaluronate lyase) or Mu toxin is one of the main components ofClostridium perfringens toxin complex. Although this enzyme has been studied for many years, data on its physico-chemical and catalytic characteristics are still quite contradictory and lack lucidity and completeness. In order to update knowledge of enzymatic properties of clostridial hyaluronidase, a chromatographically purified preparation from C. perfringens type A BP6K free of side phospholipase C (alpha toxin), neuraminidase (sialidase) and collagenase (kappa toxin) activities was obtained and characterized. The purification procedure included the following steps: processing the culture liquid with calcium phosphate gel, precipitation of the enzyme with acetone, ultrafiltration, and chromatography on Sephadex G-100 column. The purified hyaluronidase was homogenous as judged by rechromatography, SDS-PAGE and isoelectric focusing. Being a glycoprotein, the enzyme was most active at pH 5.7–6.2 (depending on the nature of the buffer used), at temperatures 37–45°C and at a relatively high ionic strength (0.15 and higher). The hyaluronidase was unstable when at pH values below 5.0 and above 9.0 as well as at temperatures below 30°C and above 50°C. The enzyme was most sensitive to Cu²⁺, Pb²⁺and Al³⁺ions, while the inhibitory effect of EDTA was moderate. Molecular mass of hyaluronidase was 96kDa as estimated by gel filtration and 48kDa when estimated by SDS-PAGE, suggesting that enzyme is composed of two subunits. The isoelectric point of the enzyme was 4.4. Substrate specificity of the enzyme was narrow (appart from hyaluronate, it slightly split chondroitin, but did not split heparin and various chondroitinsulphates). Moreover, unsplit glycosaminoglycans appeared to be competitive inhibitors with K_ivalues 5.3×10⁻², 4.9×10⁻², 4.5×10⁻²and 4.2×10⁻²mg/mL for heparin, chondroitinsulphates A, B and C, respectively. The Michaelis constant in regard to potassium hyaluronate was calculated to be (15.4±2.6)×10⁻²mg/mL.     

Purification and properties of an F420-dependent NADP reductase from methanobacterium thermoautotrophicum

The F₄₂₀-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F₄₂₀ as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent K_m of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH₂ nor FADH₂ could serve as electron donors; NAD was not utilized as electron acceptor.     

Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Phenotypic diversity amongst strains of Pleurotus sajor-caju: implications for cultivation in arid environments

In arid regions, biodiversity and biomass are limited by water availability, and this problem has been compounded by desertification associated with global climate change. The saprotrophic macrofungi that are indigenous to hot subtropical and tropical regions, such as Pleurotus spp., can play key roles in water sequestration, nutrient cycling, human nutrition, and bioremediation of waste materials. We studied 15 strains of Pleurotus sajor-caju, a widespread and phenotypically-diverse species, to establish variability in growth response and primordium development over a range of stress parameters: osmotic potential (−0.5 to −5 MPa), temperature (5–40 °C) and pH (2–12). The initiation of primordia precedes basidiome production and therefore represents a key stage in bioremediation strategies and fungi-driven nutrient cycles. Primordia were produced at low pH (4–6), at suboptimal growth temperatures (≤25 °C), and under moderate water stress (−0.5 to −3.5 MPa). Although the growth windows for different strains were similar, their maximum growth rates and the optimum conditions for growth varied. We discuss the phenotypic diversity of Pleurotus strains and discuss their potential for cultivation, bioremediation and ecological regeneration.     

Studies on preservation of schistosomula of Schistosoma mansoni and S. mattheei

Schistosomula were not damaged by exposure for 1 hr at room temperature to the cryoprotectant dimethylsulphoxide (DMSO) providing that concentrations greater than 10% were not used. Rapid dilution to remove the DMSO was less harmful to the organisms than was slow dilution. Schistosomula were not damaged by thermal shock (cooling in the absence of freezing) but were damaged by conditions produced by freezing. Although the freezing damage rendered schistosomula noninfective they retained flame cell activity and certain contractile properties in the oral sucker, gut, and musculature. The least damage was produced by slow cooling (at approximately 0.3 °C/min) and fast warming (approximately 300 °C/min). Schistosomula remained infective following freezing and slow cooling to −20 °C in DMSO (10%) and storage for 2 hr at this temperature but were damaged at temperatures below −26 °C and at −20 °C for longer time periods.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Characterizing the freezing behavior of liposomes as a tool to understand the cryopreservation procedures

Freezing behaviors of egg yolk l-α-phosphatidylcholine (EPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC) large unilamellar vesicles (LUV) were quantitatively characterized in relation to freezing temperatures, cooling rates, holding time, presence of sodium chloride and phospholipid phase transition temperature. Cooling of the EPC LUV showed an abrupt increase in leakage of the encapsulated carboxyfluorescein (CF) between −5 °C and −10 °C, which corresponded with the temperatures of the extraliposomal ice formation at around −7 °C. For the DPPC LUV, CF leakage started at −10 °C, close to the temperature of the extraliposomal ice formation; followed by a subsequent rapid increase in leakage between −10 °C and −25 °C. Scanning electron microscopy showed that both of these LUV were freeze-concentrated and aggregated at sub-freezing temperatures. We suggest that the formation of the extraliposomal ice and the decrease of the unfrozen fraction causes freeze-injury and leakage of the CF. The degree of leakage, however, differs between EPC LUV and DPPC LUV that inherently vary in their phospholipid phase transition temperatures. With increasing holding time, the EPC LUV were observed to have higher leakage when they were held at −15 °C compared to at −30 °C whilst leakage of the DPPC LUV was higher when holding at −40 °C than at −15 °C and −50 °C. At slow cooling rates, osmotic pressure across the bilayers may cause an additional stress to the EPC LUV. The present work elucidates freeze-injury mechanisms of the phospholipid bilayers through the liposomal model membranes.     

Chemical stability of prostacyclin (PGI2) in aqueous solutions

The rate constant for the hydrolysis of prostacyclin (PGI₂) to 6-keto-PGF_1α was measured by monitoring the UV spectral change, over a pH range 6 to 10 at 25°C and the total ionic strength of 0.5 M. The first-order rate constant (k_obs) extrapolated to zero buffer concentration follows an expression, k_obs = k_H+ (H+), where k_H+ is a second-order rate constant for the specific acid catalyzed hydrolysis. The value of k_H+ obtained (3.71 × 10⁴ sec⁻¹ M⁻¹) is estimated approximately 700-fold greater than a k_H+ value expected from the hydrolysis of other vinyl ethers. Such an unusually high reactivity of PGI₂ even for a vinyl ether is attributed to a possible ring strain release that would occur upon the rate controlling protonation of C₅. A Brønsted slope (α) of 0.71 was obtained for the acid (including H₃O+) catalytic constants, from which a pH independent first-order rate constant for the spontaneous hydrolysis (catalyzed by H₂O as a general acid) was estimated to be 1.3 × 10⁻⁶ sec⁻¹. An apparent activation energy (E_a) of 11.85 Kcal/mole was obtained for the hydrolysis at pH 7.48, from which a half-life of PGI₂ at 4°C was estimated to be approximately 14.5 min. when the total phosphate concentration is 0.165 M (cf. 3.5 min. at 25°C).     

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.