Heat resistance of Salmonella senftenberg 775W at various sucrose concentrations in distilled water

Summary The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l^–1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l^–1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k₅=ln k₀-_T [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l^–1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l^–1.     

Thermal stability of <Emphasis Type="Italic">α</Emphasis>-amylase in aqueous cosolvent systems

The activity and thermal stability of α-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 ± 2°C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (T _m )_app and activation energy (E _a ) of α-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (T _m )_app were 20°C, 14°C, 13°C and 9°C, respectively. The E _a of thermal denaturation of α-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.     

Heat resistance of Klebsiella pneumoniae in media with various sucrose concentrations

Summary The heat resistance of Klebsiella pneumoniae, an organism of widespread occurrence in nature has been determined in media containing various amounts of sucrose at temperatures between 47° and 59°C.In the presence of sucrose and at all temperatures the inactivation curves show a fast initial drop (logarithmic phase) in the number of survivors followed by a less rapid one (tail phase). The influence of the sucrose concentration can be described withln k _s = ln k _O – _T [sucrose] for media with more than 0.52 mol/l sucrose for the logarithmic as well as for the tail phase of inactivation.The heat-injured cells were recovered on various media to investigate the influence of the presence of small metabolites and nutrients on the shape of the inactivation curves and on the death rate. For cells heated in media without sucrose, the recovery on a rich medium was much better than on a poor one; for cells heated in media with more than 0.26 mol/l sucrose, no difference was observed between the various recovery media.The activation energies as determined on the various media are always nearly the same, which strongly suggests that the critical sites in the heat inactivation were not enzymes playing a key role in the synthesis of small molecules such as amino acids or nucleotides.     

Heat resistance ofCitrobacter freundii in media with various water activities

Summary The heat resistance ofCitrobacter freundii NCTC 9750 between 45–65°C in media with various water activities has been determined.At a water activity of nearly 1.00, the Arrhenius plot of the death rate shows a sharp breakpoint at 56.5°C, suggesting the existence of at least two different thermal inactivation processes causing lethality of the bacterial cell. The activation energy below 56.5°C is 0.4186 MJ/mol (100 000 cal/mol), above 56.5°C it is 0.1863 MJ/mol (44 500 cal/mol). After addition of sucrose (1.8 mol/l) or NaCl (0.77 mol/l) to the heating medium, such a breakpoint is not observed. The activation energy for these processes are, for sucrose; 0.2097 MJ/mol, for NaCl; 0.3641 MJ/mol. However, at an NaCl concentration of 1.54 mol/l there is a breakpoint at 53.3°C.The influence of the sucrose concentration on the heat resistance can be described by the formula: ln k_S=ln k_O–a [sucrose]. Such a simple correlation does not exist for the influence of NaCl or glycerol.The heat inactivation of whole cells ofC. freundii was also measured with a differential scanning calorimeter. The first irreversible conformation change took place at 323 K, the main conformation change at 343 K.     

Kinetics of Thermal Inactivation of Lactate Dehydrogenase from Rabbit Muscle

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.     

Effect of Sugars on Rabbit Serum Albumin Stability and Induction of Secondary Structure

The effect of sugars (sucrose, maltose, and glucose) on the thermal and chemical denaturation of rabbit serum albumin (RSA) has been examined by viscosity and far UV circular dichroism measurements. The viscosity measurements indicate a change in the reduced viscosity from 4.18 to 16.23 ml/g in the temperature range from 20 to 90°C. The T _m value for RSA obtained by viscosity measurements in the absence of sugar was found to be 63.2°C, but this value increased to 68.4, 70.3, and 73.2°C in the presence of 0.5 M sucrose, 0.5 M glucose, and 0.5 M maltose, respectively. Further, the stability of RSA in the presence of 0.5 M sugars was also investigated by measuring the mean residue ellipticity at 222 nm (MRE₂₂₂) using chemical (0-6 M guanidine hydrochloride) and thermal (20-90°C) transition processes. At the midpoint of the chemical denaturation, the increase in the MRE values at 222 nm in the presence of 0.5 M sugars were of the same order as the increase in the T _m values, i.e., maltose > glucose > sucrose. Interestingly, a mixture of 0.25 M glucose and 0.25 M fructose showed a cumulative effect on the thermal as well as chemical stability as compared to 0.5 M sucrose alone. In the case of both thermal and chemical denaturation, there was an increase in the MRE₂₂₂ values upon addition of various sugars, this indicating induction of secondary structure in the protein.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Effects of pH and deuterium oxide on the heat-inactivation temperature of chloroplasts

The inactivation temperature for Hill activity and for the long-lived delayed fluorescence of isolated Pisum sativum L. chloroplasts was found to depend on pH, the maximal value being in the pH region 5–7. Salts increase the inactivation temperature by 4–7°C. Effects of D₂O and some other substances that modify the thermostability of chloroplasts are dependent on pH. It is concluded that thermal denaturation of proteins is the most probable mechanism for heat inactivation of chloroplasts.Abbreviations Hepes 4-(2-hydroxymethyl)-1-piperazineethane sulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Comparative Study of the Conformational Lock,Dissociative Thermal Inactivation and Stability of <Emphasis Type="Italic">Euphorbia</Emphasis> Latex and Lentil Seedling Amine Oxidases

The thermal stability of copper/quinone containing amine oxidases from Euphorbia characias latex (ELAO) and lentil seedlings (LSAO) was measured in 100 mM potassium phosphate buffer (pH 7.0) following changes in absorbance at 292 nm. ELAO was shown to be about 10°C more stable than LSAO. The dissociative thermal inactivation of ELAO was studied using putrescine as substrate at different temperatures in the range 47–70°C, and a “conformational lock” was developed using the theory pertaining to oligomeric enzyme. Moreover ELAO was shown to be more stable towards denaturants than LSAO, as confirmed by dodecyl trimethylammonium bromide denaturation curves. A comparison of the numbers of contact sites in inter-subunits of ELAO relative to LSAO led us to conclude that the higher stability of ELAO to temperature and towards denaturants was due to the presence of larger number of contact sites in the conformational lock of the enzyme. This study also gives a putative common mechanism for thermal inactivation of amine oxidases and explains the importance of C-terminal conserved amino acids residues in this class of enzymes.     

Growth regulation in Neurospora crassa effects of nutrients and of temperature

Summary Mycelia of Neurospora crassa growing with quite different rates at a given temperature (30°C) are obtained by changing the composition of the culture medium: for instance a duplication time of 220 min is found when cultures are growing in Vogel's minimal medium supplemented with glycerol and one of 80 min when cultures are growing in Vogel's minimal medium supplemented with sucrose and casein hydrolysate. The kinetics of shifth-down and shift-up transitions of growth between culture conditions supporting different rates of growth are described. Temperatures above 40°C and below 20°C severely restrict growth in Vogel's minimal medium supplemented with sucrose. The Arrhenius plot of the constant of the rate of growth suggests that an inactivation process occurs at low temperatures: the molecular basis for such a process is discussed.Abbreviations used A₄₅₀ absorbance at 450 mm     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

Mechanism of dehydration inactivation of Lactobacillus plantarum

The mechanism of dehydration inactivation of Lactobacillus plantarum cells after vacuum-drying above saturated salt solutions was studied. The method used is based on the hypothesis that DNase diffuses into cells with damaged cell membranes/walls and hydrolyses the intracellular DNA. Intact, undamaged cells and cells inactivated by either dehydration or heat treatent were incubated in the presence of DNase. The release of DNA hydrolysis products into the incubation medium was measured. It was shown that dehydration inactivation of L. plantarum, but not thermal inactivation, was associated with clear evidence of membrane damage. The residual glucose-fermenting activity of the dehydrated cells related to the release of hydrolysed DNA in the medium, but there was no such relationship with heat-treated cells. Addition of sorbitol to cells before dehydration increased the residual glucose-fermenting activity after drying and this was associated with a reduced rate of DNA hydrolysis. It is concluded that cell wall and/or cell membrane damage is an important mechanism of dehydration inactivation, but that thermal inactivation (up to 60°C) occurs by a different mechanism.Correspondence to: K. van't Riet     

N-substituted maleimide inactivation of the response to taste cell stimulation

N-Ethylmaleimide (NEM) irreversibly inactivates the response of gustatory cells to stimulation by NaCl, sucrose and hydrogen ions. The rate of inactivation can be measured by monitoring the decay of NaCl-stimulated summated electrophysiological activity at the chorda tympani nerve in the presence of NEM. The observed pseudo first-order rate constants are linear with NEM concentration, and the second-order rate constant is 0.38 M^?1 sec^?1. Other N-substituted maleimides, such as N-methylmaleimide and N-butylmaleimide, which have ether:water partition coefficients similar to NEM, inactivate the NaCl-stimulated response at rates comparable to NEM. However, the hydrophobic derivative, 4-(N-maleimido)phenyltrimethylammonium has a significantly lower ether:water partition coefficient and is essentially ineffective as an inactivator of the NaCl response. These results, together with the observation that the inactivation rate is independent of pH between 4.5 and 7.0, indicate the inactivation site is either intracellular or buried within the cell membrane at a locus inaccessible to most extracellular fluids. The rate of inactivation of the sucrose and HCl responses were measured indirectly and found to be comparable to the NaCl-stimulated inactivation rate, indicating the inhibited event is common to the transduction of the response for all of the stimuli examined. Possible sites of inactivation by N-Substituted maleimides are considered in the context of the results. This kinetic approach should have application in searching for and characterizing receptor-specific as well as other classes of taste cell inhibitors.     

Diversity and enzyme properties of protease-producing bacteria isolated from sub-Antarctic sediments of Isla de Los Estados, Argentina

Protease-producing bacteria isolated from sub-Antarctic marine sediments of Isla de Los Estados (Argentina) were characterized, and the thermal inactivation kinetics of their extracellular proteases compared. Isolates were affiliated with the genera Pseudoalteromonas, Shewanella, Colwellia, Planococcus, and a strain to the family Flavobacteriaceae. Colwellia strains were moderate psychrophiles (optimal growth at about 15°C, maximum growth temperature at around 25°C). 16S rRNA phylogenetic analysis revealed that these strains and Colwellia aestuarii form a distinct lineage within the genus. The remaining isolates were psychrotolerant and grew optimally between 20 and 25°C; two of them represent potentially novel species or genus (16S rRNA < 97% sequence similarity). The thermostability of the extracellular proteases produced by the isolates was analysed, and the inactivation rate constant (k _in), the activation energy (Ea_in) and the activation Gibbs free energy of thermal inactivation (ΔG ^* _in) determined. ΔG ^* _in, calculated at 30°C, varied between 97 and 124 kJ/mol. Colwellia enzyme extracts presented the highest thermosensitivity, while the most thermostable protease activity was shown by Shewanella spp. These results demonstrated that the stability to temperature of these enzymes varies considerably among the isolates, suggesting important variations in the thermal properties of the proteases that can coexist in this environment.     

Stabilization of firefly luciferase against thermal stress by osmolytes

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.     

Sucrose transport by the alkaliphilic, thermophilic Bacillus sp. strain TA2.A1 is dependent on a sodium gradient

An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70°C. Sodium chloride (>5 mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ΔpNa⁺) across the cell membrane. Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin. The K_t for sucrose tran-sport was 33 μM and the Eadie–Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose. The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium. Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously. Received: March 15, 2000 / Accepted: July 17, 2000     

Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae

In 12-h-starved larvae of the tobacco hornworm, Manduca sexta, fat body glycogen phosphorylase was quickly inactivated when insects were refed with normal diet and agar which contained 3% sucrose. Only the first 2 min of refeeding were necessary to induce enzyme inactivation. During this short period, larvae did not ingest enough sucrose to increase the hemolymph glucose concentration. This may indicate that the gut released a hormone(s) which directly or indirectly led to the inactivation of fat body glycogen phosphorylase. Inactivation of the enzyme could also be induced by injection of glucose (30 mg) into the hemolymph of starving M. sexta larvae suggesting that there may be separate control from a neuroendocrine site such as the brain or the corpora cardiaca. Trehalose was less effective. Bovine insulin (2 and 4 μg/starved larva) did not induce phosphorylase inactivation over 20 min or decrease hemolymph carbohydrate or lipid concentrations within 60 min. It is, therefore, necessary to screen insect tissues for substances which could bring about inactivation of fat body glycogen phosphorylase. © 1992 Wiley-Liss, Inc.     

Efficacy of agar media for enumerating two Saccharomyces species in sucrose syrups

Experiments were carried out to determine the suitability of acidified and antibiotic-supplemented agars for supporting colony formation by cells ofSaccharomyces cerevisiae andSaccharomyces rouxii. Yeasts had been suspended in sucrose syrups buffered at pH 3.0, 5.0 and 7.0, and stored at 4 and 21°C for periods of time ranging to 10 weeks. TheSaccharomyces species were recovered in equal numbers, regardless of the type of enumeration agar. Sucrose, at concentrations up to 60%, protected cells against inactivation during storage, and survival was greater at 21° than at 4°C.     

UV-sensitivity and repair of UV-damages in Salmonella of wild type.

Summary The UV-sensitivity of wild type Salmonella strains has been compared to that of wild type E. coli and its UV-sensitive mutants. Many wild type Salmonella strains are 4–5 times more sensitive than wild type E. coli and their inactivation curve is similar to that for E. coli with a mutation in the polA gene. Alkaline sucrose gradient centrifugation has shown a deficiency of these strains in normal excision repair of UV-damaged DNA. This deficiency is not a Salmonella genus feature because one strain as resistant as wild type E. coli was found. This resistant strain showed normal excision repair in alkaline sucrose gradient centrifugation experiments. The possible influence of plasmids and mutations in repair genes on the ability of Salmonella to repair UV-damaged DNA is discussed.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.