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.     

On the partial reactivation of inactivated pantothenase from Pseudomonas fluorescens.

Partial reactivation of inactivated pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) from Pseudomonas fluorescens was studied. After partial inactivation during storing, pantothenase activity is increased by 10-40% when incubated with, for instance, oxalate, oxaloacetate or pyruvate. Reactivation proceedes slowly; with oxaloacetate the stable level of enzyme activity is attained in 20-30 min. The same compounds also cause reactivation of thermally inactivated pantothenase when partial inactivation has occurred at 28-37 degrees C. The amount of the reactivating enzyme form is relatively greater the lower the temperature during inactivation, but it never exceeds 20% of the original amount of active enzyme. Also another, unstable form of pantothenase is formed in thermal inactivation. This form becomes inactivated in a few minutes after the heat treatment, at pH 6-8 and at temperatures between 0 and 10 degrees C. Reactivation causes special problems in enzyme kinetic measurements; for instance, curvature is found in the lines of Ki determination by the Dixon plot.     

A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.     

Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.     

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.     

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co2+ or Mn2+ (and Mg2+ to a lesser extent), the melting temperature [(Tm) approximately 100 degrees C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 degrees C and 114 degrees C. TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process. TNXI inactivation in the presence of 5 mm metal at 99-100 degrees C was slowest in the presence of Mn2+[half-life (t(1/2)) of 84 min], compared to Co2+ (t(1/2) of 14 min) and Mg2+ (t(1/2) of 2 min). While adding Co2+ to Mg2+ increased TNXI's t(1/2) at 99-100 degrees C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.     

Opposing effects of two osmolytes--trehalose and glycerol--on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 degrees C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t0.5 for thermal inactivation of 56+/-5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 degrees C. Indeed, when phosphofructokinase was incubated for 45 min at 50 degrees C in the presence of lower concentrations of glycerol (7.5-25%, v/v), the remaining activity was 2-4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules.     

Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara.

An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara. The enzyme has a molecular weight of approximately 26,000. It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities. The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide. It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0. The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM. The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature. One cell of T. thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C. An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T. thiopara.     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.     

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.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase

Processivity of T7 DNA polymerase relies on the coupling of its cofactor Escherichia coli thioredoxin (Trx) to gene 5 protein (gp5) at 1:1 stoichiometry. We designed a coexpression system for gp5 and Trx that allows in vivo reconstitution of subunits into a functional enzyme. The properties of this enzyme were compared with the activity of commercial T7 DNA polymerase. Examination of purified enzymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the thioredoxin subunit of the two enzymes did not comigrate. To our surprise, we identified a mutation (Phe102 to Ser) in the Trx component from the commercial T7 DNA polymerase (gp5/TrxS102) that was not in the enzyme from the coexpression system (wild type gp5/Trx). A comparison of polymerase activity of the T7 DNA polymerases shows that both enzymes possessed similar specific activity but they were different in their residual activity at 37 degrees C. The half-life of gp5/TrxS102 was 7 min at 37 degrees C and 12 min for gp5/Trx. gp5/TrxS102 polymerase activity was reduced by fourfold with 3'-5' exonuclease activity as the prominent activity detected after 10 min of heat inactivation at 37 degrees C. Supplementation of reaction mixtures containing gp5/TrxS102 with exogenous nonmutant thioredoxin restored the enzyme activity levels. Pulse proteolysis was used to demonstrate that TrxS102 unfolded at lower urea concentrations than wild type thioredoxin. Thus, Ser substitution at position 102 affected the structural stability of thioredoxin resulting in a reduced binding affinity for gp5 and loss of processivity.     

The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.