Thermostabilization of enteroviruses by estuarine sediment.

The effect of estuarine sediment on the thermoinactivation of poliovirus type 1 and echovirus type 1 was evaluated. Poliovirus survival was prolonged at 24 and 37 degrees C but not at 4 degrees C in the presence of sediment over the time periods observed. Further inactivation studies were performed at 50 and 55 degrees C to maximize the thermal effects, and similar protection was observed. The supernatant fluid from a mixture of seawater and sediment lacked the protective effect against thermoinactivation, suggesting that prolonged virus survival in the presence of sediment was due to adsorption to particulates. From these observations, it appears that the adsorption of enteroviruses to estuarine sediments may play a significant role in protecting them against thermoinactivation.     

Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment.

This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.     

Influence of estuarine sediment on virus survival under field conditions.

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

Influence of estuarine sediment on virus survival under field conditions

The survival of poliovirus 1 (LSc) and echovirus 1 (Farouk) in estuarine water and sediment was studied in Galveston Bay, Texas. Viruses were suspended in estuarine water and sediment both in dialysis tubing and in chambers constructed with polycarbonate membrane walls. Virus inactivation rates in seawater were similar in both types of chambers. Virus adsorption to sediment greatly increased survival time. The time required to inactivate 99% (T-99) of poliovirus increased from 1.4 days in seawater alone to 6.0 days for virus adsorbed to sediment at a relatively nonpolluted site. At a more polluted site, poliovirus T-99 was increased from approximately 1 h to 4925 days by virus adsorption to sediment. This study demonstrates that under field conditions virus association with estuarine sediment acts to prolong its survival in the marine environment.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

Role of sediment in the persistence of enteroviruses in the estuarine environment.

The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.     

Role of sediment in the persistence of enteroviruses in the estuarine environment.

The survival of four enteroviruses commonly found in sewage effluents was examined when the viruses were adsorped to marine sediments in estuarine water and compared with virus survival in estuarine water alone. Echovirus 1, coxsackieviruses B3 and A9, and poliovirus 1 survived longer when associated with marine sediment. When the estuarine water was polluted with secondarily treated sewage effluent, virus survived for prolonged periods in sediments, but not in the overlaying estuarine water.     

Effects of beta-cyclodextrin-dextran polymer on stability properties of trypsin

Dextran modified with the mono-6-pentylene-diamino-6-deoxy-beta-cyclodextrin derivative was evaluated as a thermoprotectant additive for trypsin. The optimum temperature for trypsin activity was increased by 7 degrees C in the presence of this polymer. The enzyme thermostability was increased from 48.5 to 64 degrees C over 10 min of incubation, and the activation free energy of thermoinactivation at 50 degrees C was increased by 4.1 kJ/mol in the presence of the additive. Trypsin was 6-fold more resistant to autolytic inactivation at alkaline pH in the presence of the polymer.     

Use of microcosms to determine persistence of Escherichia coli in recreational coastal water and sediment and validation with in situ measurements

AIMS: To determine the persistence of the faecal indicator organism Escherichia coli in recreational coastal water and sediment using laboratory-based microcosms and validation with in situ measurements. METHODS AND RESULTS: Intact sediment cores were taken from three distinct coastal sites. Overlying estuarine water was inoculated with known concentrations of E. coli and decay rates from both overlying water and sediment were determined following enumeration by the membrane filtration method at fixed time intervals over a 28-day period. It was demonstrated that E. coli may persist in coastal sediment for >28 days when incubated at 10 degrees C. Escherichia coli survival was found to have an inverse relationship with temperature in both water and sediment. In general the decay rate for E. coli was greater in water than in sediment. Small particle size and high organic carbon content were found to enhance E. coli survival in coastal sediments in the microcosms. CONCLUSIONS: Results of this microcosm study demonstrated the more prolonged survival of E. coli in coastal sediments compared with overlying water, which may imply an increased risk of exposure because of the possible resuspension of pathogenic micro-organisms during natural turbulence or human recreational activity. SIGNIFICANCE AND IMPACT OF THE STUDY: A more accurate estimate of exposure risk has been described which may subsequently be used in a quantitative microbial risk assessment for recreational coastal waters.     

Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups

The incubation of glucoamylase from Aspergillus niger at 70 degrees C induced its rapid and irreversible inactivation. The covalent modifications of the protein structure involved in the thermoinactivation depended on the pH of the medium. We observed the formation of a low amount of disulfide-linked oligomers showing that disulfide exchange takes place at pH 5.5. Hydrolysis of peptide bonds at pH 3.5 and 4.5 was also detected. The chemical modification of carboxyl groups with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) decreased the rate of appearance of low-molecular-weight peptides at pH 3.5 and 4.5 upon heating at 70 degrees C. However, the rate of inactivation at such pH values was not modified. Modification of carboxyl groups with EDC in the presence of ethylenediamine leading to the transformation of three carboxyl groups to amino groups increased the thermostability of the enzyme for temperatures above the temperature of compensation, Tc, which is 60 degrees C.     

A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability

An alkalothermophilic Thermomonospora sp. producing high levels of xylanase was isolated from self-heating compost. The culture produced 125 IU/ml of xylanase when grown in shake flasks at pH 9 and 50 degrees C for 96 h. The culture filtrate also contained cellulase (23 IU/ml), mannanase (1 IU/ml) and beta-xylosidase (0.1 IU/ml) activities. The xylanase was active at a broad range of pH (5-9) and temperature (40-90 degrees C). The optimum pH and temperature were 7 and 70 degrees C, respectively. The enzyme was stable in the pH range 5-8 and was thermostable with half-lives of 8 and 4 h at 60 degrees C and 70 degrees C, respectively, but only 9 min at 80 degrees C. The effects of a variety of compounds to enhance the stability of xylanase at 80 degrees C was studied. Addition of sorbitol, mannitol and glycerol increased the thermostability of xylanase in proportion to the number of hydroxyl groups per polyol molecule. Glycine also offered protection against thermoinactivation. Xylan, trehalose, gelatin and trehalose-gelatin mixture had marginal effect on the thermostability of xylanase at 80 degrees C.     

Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity

Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2 degrees ADH) was optimally active near 90 degrees C displaying thermostability half-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80 degrees C, 90 degrees C, 92 degrees C, 95 degrees C, and 99 degrees C, respectively. Enzyme activity loss upon heating (90-100 degrees C) was accompanied by precipitation, but the soluble enzyme remaining after partial inactivation retained complete activity. Enzyme thermoinactivation was modeled by a pseudo-first order rate equation suggesting that the rate determining step was unimolecular with respect to protein and thermoinactivation preceded aggregation. The apparent 2 degrees ADH melting temperature (T(m)) occurred at approximately 115 degrees C, 20 degrees C higher than the temperature for maximal activity, suggesting that it is completely folded in its active temperature range. Thermodynamic calculations indicated that the active folded structure of the 2 degrees ADH is stabilized by a relatively small Gibbs energy (triangle upG(stab.)(double dagger) = 110 kJ mol(-1)). 2 degrees ADH catalytic activities at 37 degrees C to 75 degrees C, were 2-fold enhanced by guanidine hydrochloride (GuHCl) concentrations between 120 mM and 190 mM. These results demonstrate the extreme resistance of this thermophilic 2 degrees ADH to thermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity.     

Mercury(II) removal from aqueous solutions by nonviable Bacillus sp. from a tropical estuary

Use of microorganisms for removing mercury is an effective technology for the treatment of industrial wastewaters and can become an effective tool for the remediation of man-impacted coastal ecosystems with this metal. Nonviable biomass of an estuarine Bacillus sp. was employed for adsorbing Hg(II) ions from aqueous solutions at six different concentrations. It was observed that 0.2 g dry weight of nonviable biomass was found to remove from 0.023 mg (at 0.25 mg L(-1) of Hg(II)) to 0.681 mg (at 10.0 mg L(-1) of Hg(II)). Most of the mercury adsorption occurred during the first 20 min. It was found that changes in pH have a significant effect on the metal adsorption capacity of the bacteria, with the optimal pH value between 4.5 and 6.0 at 25 degrees C when solutions with 1.0, 5.0 and 10.0 mg L(-1) of Hg(II) were used.     

Effect of the thermal variation on the molecular weight estimation of the acid phosphatase from Physarum polycephalum

A Binding Protein (BP) of nearly 95,000 daltons interacts with the acid phosphatase at the permissive temperature, elevating its molecular weight from 48,000 +/- 2,000 to 136,000 +/- 7,000 in CL and CHR strains of Physarum polycephalum plasmodia. The same molecule has not been observed in the extracts of M3C VIII and K strains. 28-31 degrees C is the critical temperature range for the breaking of enzyme-BP association in CL strains. This critical temperature is higher than 39 degrees C for the extract of CHR strains. CHR BP recognizes and binds CL acid phosphatase as strongly as CHR acid phosphatase. Km determination at 32 degrees C and thermoinactivation patterns at 66 degrees C indicate that the BP has no apparent regulatory role for the acid phosphatase activities. The presence or the absence of the BP in some of the strains may be used as a marker in the genetical studies of this organism.     

Sweetness of sweet protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions

Thermostability of thaumatin and mechanisms of thermoinactivation were examined at 80 degrees C in the pH range from 2 to 10. The sweetness of thaumatin disappeared on heating at pH above 7 for 15 min, but the sweetness remained even after heating at 80 degrees C for 4 h at pH 2. This indicated that the sweet protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions. Prolonged heating of thaumatin under acid conditions slowly reduced sweetness, and produced a heterogeneous population of molecules, all of which was soluble and monomeric. The resultant molecules were clearly distinct from those generated by heating at pH above 7. Hydrolysis of peptide bonds and other irreversible chemical reactions slowly took place in the molecule heated under acid conditions, and it would be, in part, a cause of thermoinactivation of thaumatin under acid conditions. The thermostability of thaumatin and the mechanism of thermoinactivation were largely dependent on pH.     

Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.     

Effect of temperature on structure and functional properties of horseradish peroxidase

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.     

RNase A effects on sedimentation and DNA binding properties of dexamethasone-receptor complexes from HeLa cell cytosol

Dexamethasone-receptor complexes from HeLa cell cytosol sediment at 7.4S in low salt sucrose gradients, and at 3.8S in high salt gradients. If cytosol is heated at 25 degrees C, receptor complexes sediment at 6.9S in low salt, and at 3.6S in high salt gradients. RNase A treatment at 25 degrees C, instead, results in receptor complexes which sediment in low salt gradients as two major forms at 6.5 and 4.8S. Receptor complexes from RNase A-treated cytosols sediment as their counterparts from untreated cytosols in high salt gradients. Although the shift in sedimentation properties of receptor complexes at 2 degrees C is induced by RNase A, and not by other low molecular weight basic proteins or RNase T1, the effect can be also obtained by inactive RNase A. The catalytically active enzyme, however, is required to observe 6.5 and 4.8S complexes after cytosol incubations at 25 degrees C. Placental ribonuclease inhibitor prevents the appearance of RNase A-induced receptor forms at 25 degrees C, but not at 2 degrees C. Moreover, this inhibitor can prevent the 7.4 to 6.9S shift in sedimentation coefficient of receptor complexes caused by cytosol heating. Dexamethasone-receptor complexes from HeLa cell cytosol show low levels of binding to DNA-cellulose, and heating at 25 degrees C is required to observe a six-fold increase in DNA binding levels. RNase A treatment of cytosols at 2 degrees C does not result in significant enhancement in receptor complex binding to DNA. If RNase A treatment is carried out at 25 degrees C, however, DNA binding levels of receptor complexes increased by 25% over the values observed with control heated cytosol. This effect cannot be observed if RNase T1 substitutes for RNase A. Placental ribonuclease inhibitor can prevent the temperature-dependent increase in DNA binding properties of dexamethasone-receptor complexes either in the presence or absence of exogenous RNase A. These findings indicate that exogenous RNases can perturb the structure of dexamethasone-receptor complexes without being involved in the transformation process.     

Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water.

The fate of Ralstonia solanacearum bv. 2, the causative agent of brown rot in potato, in aquatic habitats of temperate climate regions is still poorly understood. In this study, the population dynamics and the physiological response of R. solanacearum bv. 2 were tested in sterile pure water and in agricultural drainage water obtained from waterways near potato cropping fields in The Netherlands. The behaviour of five different biovar 2 isolates in drainage water at 20 degrees C was very similar among strains. One typical isolate with consistent virulence (strain 1609) was selected for further studies. The effects of temperature, light, canal sediment, seawater salts, and the presence of competing microorganisms on the survival of strain 1609 were assessed. Moreover, the impacts of the physiological state of the inoculum and the inoculum density were analyzed. The population dynamics of strain 1609 in sterile pure water were also characterized. In sterile pure water, the fate of R. solanacearum 1609 cells depended strongly on temperature, irrespective of inoculum density or physiological state. At 4 degrees C and 44 degrees C, strain 1609 CFU numbers showed declines, whereas the strain was able to undergo several cell divisions at 12 degrees C, 20 degrees C, and 28 degrees C. At 20 degrees C and 28 degrees C, repeated growth took place when the organism was serially transferred, at low inoculum density, from grown water cultures into fresh water devoid of nutrients. Both at low and high cell densities and regardless of physiological state, R. solanacearum 1609 cells persisted as culturable cells for limited periods of time in drainage water. A major effect of temperature was found, with survival being maximal at 12 degrees C, 20 degrees C, and 28 degrees C. Temperatures of 4 degrees C, 36 degrees C, or 44 degrees C induced accelerated declines of the culturable cell numbers. The drainage water biota had a strong effect on survival at 12 degrees C, 20 degrees C, and 28 degrees C, as the persistence of strain 1609 was significantly enhanced in sterile drainage water systems. Furthermore, there was a negative effect of incident light, in a light:dark regime, on the survival of R. solanacearum 1609 in natural drainage water. Also, levels of seawater salts realistic for drainage water in coastal areas were detrimental to strain survival. Ralstonia solanacearum 1609 showed considerable persistence in canal sediment saturated with drainage water, but died out quickly when this sediment was subjected to drying. Evidence was obtained for the conversion of R. solanacearum 1609 cells to nonculturable cells in water microcosms kept at 4 degrees C, but not in those kept at 20 degrees C. A substantial fraction of the cells found to be nonculturable were still viable, as evidenced by the direct viable count and by staining with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. The potential occurrence of viable-but-nonculturable cells in natural waters poses a problem for the detection of R. solanacearum by cultivation-based methods.     

Sorbitol-fermenting bifidobacteria as indicators of diffuse human faecal pollution in estuarine watersheds

Sorbitol fermenting bifidobacteria were evaluated as indicators of non-point source human faecal pollution to three sub-estuaries with elevated faecal coliform densities. Human-specific bifidobacteria correlated with identifiable human sanitary deficiencies in feeder streams to estuarine creeks in two of three watersheds examined, one rural and one moderately developed. Sorbitol-fermenting bifidobacteria were recovered at densities ranging from 1 to 90 colony-forming-units 100 ml-1 in 11 of 258 water samples but were undetected in sediment (n = 68) and scat from resident wildlife (deer, muskrat and raccoon, n = 20). Failure to detect sorbitol-fermenting bifidobacteria in water samples during the summer months was consistent with laboratory microcosm results showing non-recoverability of Bifidobacterium adolescentis after 5-9 d in membrane-filtered estuarine water at 23 and 30 degrees C, but persistence for 4 weeks at 10 degrees C. Persistence of sewage-derived bifidobacteria in membrane-filtered freshwater at 15 degrees C was also observed. Recovery of sorbitol-fermenting bifidobacteria was complicated by high background levels of Gram-positive rods and cocci. Use of propionic acid and reduced pH (pH = 5.0), or use of a two-step resuscitation protocol using non-selective and selective media, did not improve recovery. Although human specific bifidobacteria hold promise as indicators of diffuse faecal contamination, methodological constraints now limit its application to situations of gross contamination, or sampling potential sources during environmental conditions conducive to bifid persistence.