Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase and their role on hydrolysis of naringin solutions

The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

Purification and biochemical characteristics of pectinesterase from Malatya apricot (Prunus armeniaca L.)

Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH(4))(2)SO(4) precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60 degrees C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70 degrees C. Km and V(max) values were 0.77 mM and 1.75 micromol min(-1) mg(-1) for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na(2)CO(3), iodine, lauril sulphate, AgNO(3), EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol(-1) for the enzyme. Ascorbic acid, CaCl(2), and KCl showed activatory effect on the apricot PE enzyme.     

The varied responses of different F1-ATPases to chlorpromazine

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.     

Some properties of the extracellular protease produced by the psychotrophic bacterium Pseudomonas fluorescens strain AR-11.

The major extracellular protease from Pseudomonas fluorescens strain AR-11 has been partially purified by a factor of 300 by a combination of DEAE-cellulose ion-exchange chromatography and gel filtration. The enzyme had a molecular weight of 38 400 and exhibited optimum activity with isoelectrically precipitated casein substrate at pH 6.5 with Km - 0.13 mM. The protease was strongly inhibited by a number of heavy metal ions at the 10 mM level and also inhibited by thiol agents, while 10 mM EDTA led to slight activation. Optimum activity was retained, amounting to 33% of the maximum activity at 4 degrees C and 72% at 20 degrees C. Heat inactivation studies in which the isolated protease was heated at high temperature before subsequent incubation at 35 degrees C with substrate showed that for 50% inactivation 25 s heating at 130 degrees C or 17 s at 140 degrees C of 8.5 s at 150 degrees C was requried. The combination of high stability to heat treatments and retention of considerable activity at low incubation temperatures indicates that such a protease might have considerable significance in the processing and subsequent storage of food and other products.     

Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics.

The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.     

Temperature- and time-dependent inactivation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation of the enzyme

When pyrroline-5-carboxylate (PC) synthase activity in the membrane of mitochondria of rat small intestine mucosa was assayed in the presence of 0.5 mM ornithine, the time course of inactivation showed that the activity disappeared entirely by about 8 min at 30 degrees C, whereas there was no decrease in the activity at 15 degrees C. A prior incubation of the enzyme with ornithine at 30 or 37 degrees C in the presence of 50% sorbitol as a thermal stabilizer resulted in a marked loss of the activity, while that at 0 or 15 degrees C did not lose any. This suggests that PC synthase is inactivated by ornithine regardless of the presence of substrates. The inactivation at 30 degrees C proceeded gradually for about 7 h, until an equilibrium was attained. Extensive dialysis allowed the inactivated enzyme to regain about 60% of the original activity. These results suggest that the inactivation is reversible. The concentration of ornithine and the percentage of inactivation at equilibrium was correlated by the Hill equation and displayed a sigmoidicity with n = 1.47 and [S]50 = 0.036 mM. In the presence of sorbitol, the inactivation was prevented by 0.2 mM ATP or ADP. The role of the nucleotides in PC synthase regulation is discussed.     

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.     

Characteristics and application of immobilized papain in a continuous-flow reactor

Papain was immobilized on an anion-exchange resin by physical adsorption followed by cross-linking with glutaraldehyde. The activity and rate of heat inactivation of the enzyme was determined at 37 to 95 degrees C. Activity increased with an increase in temperature up to 85 degrees C, then decreased slightly at 95 degrees C. Activation of the enzyme by cysteine in 2 mM EDTA increased proportionally with an increase in concentration from 0.004 to 0.12 M. Levels of dissolved oxygen in solutions of EDTA containing cysteine in a closed system and with nitrogen purging in the absence of cysteine in an open system were monitored at 51 degrees C. Both the presence of cysteine and purging with nitrogen were effective in depleting dissolved oxygen. The final oxygen level was essentially independent of cysteine concentrations of 0.01, 0.02, and 0.10 M, and was reached in 95, 85, and 55 min, respectively. However, final levels of oxygen were dependent upon the flow rate of nitrogen from 1 to 10 SLPM and were reached within 15 min. Immobilized papain in a continuous-flow stirred-tank (300 rpm) reactor with a 4-liter reaction volume accompanied by nitrogen purging of 4 SPLM was used to hydrolyze a 1.5% casein solution in the presence of 0.01 M cysteine in 2 mM EDTA at 51 degrees C. The reaction rate was 1 liter/h and activity was measured based on the content of trichloroacetic acid-soluble protein in the outflow. Papain activity was not observed to decrease during a 30-h reaction period.     

Effect of shear on the inactivation kinetics of the enzyme dextransucrase

An inactivation model previously developed to characterize the rate of enzyme activity loss in unstirred solutions was extended to take into account orthokinetic interactions resulting from convective mixing. A synergistic relationship between shear rate and temperature was observed; the rate of inactivation of the enzyme dextransucrase was unaffected by the action of shear below 25 degrees C, but was increased by the shear rate at 30 degrees C. Shear rate does not appear to influence the equilibrium between native and denatured dextransucrase either directly in solution or indirectly by augmenting the turnover of the gas-liquid interface. However, a second-order plot of the inverse of relative activity (A(O)/A) versus Gt (shear rate x time) of dextransucrase at a constant temperature was linear because of the influence of shear on the coagulation of the denatured enzyme. The addition of 0.01 g L(-1) of polyethylene glycol (MW 20,000) blocked this coagulation reaction, thereby completely inhibiting the shear-induced inactivation of dextransucrase at 30 degrees C. (c) 1993 John Wiley & Sons, Inc.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase

The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.     

Chaperonin cpn60 from Escherichia coli protects the mitochondrial enzyme rhodanese against heat inactivation and supports folding at elevated temperatures.

The chaperonin protein cpn60 from Escherichia coli protects the monomeric, mitochondrial enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) against heat inactivation. The thermal inactivation of rhodanese was studied for four different states of the enzyme: native, refolded, bound to cpn60 in the form of a binary complex formed from unfolded rhodanese, and a thermally perturbed state. Thermal stabilization is observed in a range of temperatures from 25 to 48 degrees C. Rhodanese that had been inactivated by incubation at 48 degrees C, in the presence of cpn60 can be reactivated at 25 degrees C, upon addition of cpn10, K+, and MgATP. A recovery of about 80% was achieved after 1 h of the addition of those components. Thus, the enzyme is protected against heat inactivation and kept in a reactivable form if inactivation is attempted using the binary complex formed between rhodanese folding intermediate(s) and cpn60. The chaperonin-assisted refolding of urea-denatured rhodanese is dependent on the temperature of the refolding reaction. However, optimal chaperonin assisted refolding of rhodanese observed at 25 degrees C, which is achieved upon addition of cpn10 and ATP to the cpn60-rhodanese complex, is independent of the temperature of preincubation of the complex, that was formed previously at low temperature. The results are in agreement with a model in which the chaperonin cpn60 interacts with partly folded intermediates by forming a binary complex which is stable to elevated temperatures. In addition, it appears that native rhodanese can be thermally perturbed to produce a state different from that achieved by denaturation that can interact with cpn60.     

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family.

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.     

Inactivation of liver S-adenosylhomocysteine hydrolase in vitro of rats treated with erythro-9-(2-hydroxynon-3-yl)adenine.

S-Adenosylhomocysteine hydrolase activity decreased in vitro time-dependently in liver homogenates obtained from rats treated in vivo with erythro-9-(2-hydroxynon-3-yl)adenine, a potent inhibitor of adenosine deaminase. The inhibitor in itself had no effect on the stability of the hydrolase. The inactivation of S-adenosylhomocysteine hydrolase was irreversible, proceeded fairly rapidly at a low temperature (0 degrees C) and showed first-order reaction kinetics. Adenosine was found to accumulate in these tissue homogenates during storage. Several lines of evidence suggest that adenosine caused the observed suicide-like inactivation post mortem. Pre-incubation of purified S-adenosylhomocysteine hydrolase at 0 degrees C with adenosine showed a half-maximal inactivation rate at 33 microM substrate concentration; the rate constant of inactivation was 0.01 min-1. Inactivation during tissue preparation and storage complicates the assay of S-adenosylhomocysteine hydrolase activity in samples that contain an inhibitor of adenosine deaminase. These results also suggest that the decrease of S-adenosylhomocysteine hydrolase activity reported to occur in several disturbances of purine metabolism should be re-examined to exclude the possibility of inactivation of the enzyme in vitro.     

Kinetic and thermodynamic investigation on ascorbate oxidase activity and stability of a Cucurbita maxima extract

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 degrees C, as does the kidney enzyme at 42 degrees C (but not at 20 degrees C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (Tm approximately 45 degrees C) than does the kidney enzyme (Tm approximately 55 degrees C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 degrees C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Enhanced stability of cellulase-free xylanase from Chainia sp. (NCL 82.5.1)

Summary Chainia sp. (NCL 82.5.1) produces an extracellular, cellulase-free xylanase. The ready accessibility of the enzyme to cellulose pulp due to its small size and the absence of cellulase are advantageous features. The enzyme is stable at 40°C for 1h and in a pH range of 5–9 at 4°C. Improved stability of the enzyme at higher temperature and pH are desirable. Effect of a variety of compounds was studied to enhance stability. Glycerol, sorbitol, mannitol (10%) or glycine (1M) had marginal effect on thermostability. Addition of Ca⁺² or PEG (10mM) increased the half-life of the enzyme at 60°C. Cysteine (10mM) or Tween-80 (1%) showed 70% protection against thermal inactivation. Xylan (3%) offered complete protection against inactivation of the emzyme at 60°C and at pH 9.NCL Communication No. 5907