`Reverse Chemical Evolution': A New Method to Search for Thermally Stable Biopolymers

The primitive sea on Earth may have had high-temperature and high-pressure conditions similar to those in present-day hydrothermal environments. If life originated in the hot sea, thermal stability of the constituent molecules would have been necessary. Thus far, however, it has been reported that biopolymers hydrolyze too rapidly to support life at temperatures of more than 200 °C. We herein propose a novel approach, called reverse chemical evolution, to search for biopolymers notably more stable against thermal decomposition than previously reported. The essence of the approach is that hydrolysis of a protein or functional RNA (m-, t-, r-RNA) at high temperature and high pressure simulating the ancient sea environment may yield thermally stable peptides or RNAs at higher concentrations than other peptides or RNAs. An experimental test hydrolyzing bovine ribonuclease A in aqueous solution at 205 °C and 25 MPa yielded three prominently stable molecules weighing 859, 1030 and 695 Da. They are thermally some tens or hundreds times more stable than a polyglycine of comparable mass. Sequence analyses of the 859- and 1030-Da molecules revealed that they are a heptapeptide and its homologue, respectively, elongated by two amino acids at the N-terminal region, originally embedded as residues 112–120 in the protein. They consist mainly of hydrophobic amino acids.     

Water relations and mucopolysaccharide increases for a winter hardy cactus during acclimation to subzero temperatures

Summary Thickness, relative water content (RWC), osmotic pressure, water potential isotherms, and mucopolysaccharide content were measured for the photosynthetic chlorenchyma and the water-storage parenchyma of the winter hardy cactus, Opuntia humifusa, after shifting from day/night air temperatures of 25° C/15° C to 5° C/–5° C. After 14 d at 5° C/–5° C, the average fraction of water contained in the symplast decreased from 0.92 to 0.78, the water potential of saturated (fully hydrated) tissue was essentially unchanged, but the osmotic pressure of saturated tissue decreased (by 0.15 MPa for the chlorenchyma and 0.12 MPa for the water-storage parenchyma). After 7 weeks at 5° C/–5° C, tissue thickness was reduced by 61% for the chlorenchyma and 65% for the water-storage parenchyma, and the RWC decreased by 42% and 68%, respectively; these changes contributed to an osmotic pressure increase of 0.55 MPa for the chlorenchyma and 0.34 MPa for the water-storage parenchyma. During the 7 week acclimation to low temperature, mucopolysaccharide increased by 114% for the chlorenchyma and by 89% for the water-storage parenchyma. The water potential of the extracted mucopolysaccharide was relatively constant for an RWC between 1.00 and 0.30, decreasing abruptly below 0.30. Changes in water relations parameters and in mucopolysaccharide content during low-temperature acclimation may reduce water efflux from the cells, and thus reduce damage due to rapid dehydration during extracellular freezing.     

Effect of temperature,hydrogen ion concentration and osmotic potential on oospore germination of fivePythium spp. isolated from pond water

Oospore germination occurred over a temperature ranging of 15–35°C forPythium coloratum, 10–35°C forP. diclinum, 15–30°C forP. dissotocum, 7–30°C forP. monospermum, and 10–30°C forP. pleroticum. Optimum temperature was 25°C for all species tested. In case of pH, oospore germination occurred over a range of 4.76–8.55 with an optimum of 6.40–7.40. The least germination occurred at pH 4.76 forP. coloratum, P. diclinum, P. monospermum andP. pleroticum, whileP. dissotocum germinated from pH 5.02. Oospores of the all tested pythia were able to germinate at –0.13 to –1.65 MPa and could not germinate at –3.40 MPa, with the highest germination rate at –0.27 to –0.47 MPa. The effect of temperature, pH and osmotic potential on oospore germination was discussed in relation to pollution of pond water.     

Localization and primary characterization of bile salt hydrolase from Lactobacillus reuteri

The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH 4.5–5.5 and 37–45 °C. The enzyme (80 kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.     

Interaction of pressure, time and temperature of pressurization on viability loss of Listeria innocua

In this study, the combined effectiveness of pressures of 137.9–344.7MPa, temperatures of 25–50°C and exposure times of 5–15min on Listeria innocua viability in peptone solution is examined. The results showed that under the study conditions only the combination of 344.7MPa, 50°C and 9.1min can reduce the viability of this species by 7 logs, with a z value of 173.1MPa.     

Prebiotic Oligomerization on or Inside Lipid Vesicles in Hydrothermal Environments

Oligomerization of amino acids proceeded on or inside lipid vesicles as a model of prebiotic cells in a simulated hydrothermal environment. When the suspension of lipid vesiclestaking up monomeric glycine underwent a sudden temperature dropby traversing from a hot (180 °C) to a cold (0 °C) region repeatedly while circulating through a closedreaction circuit, oligopeptides up to heptaglycine were formed even in the absence of condensing agents.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Synthesis of biomolecules from N2, CO,and H2O by electric discharge

A model primitive gas containing a mixture of N₂, CO and water vapor over a water pool (300 mL, 37 °C) was subjected to electric discharges. The discharge vessel (7 L in volume) was equipped with a CO₂ absorber (The CO₂ being formed during the discharge), thus simulating possible absorption of CO₂ in the primitive ocean. The vessel also has a cold trap ( –15 °C), which protects the primary products against the further decomposition in the discharge phase by enabling these products to adhere to the trap. Since the partial pressures of CO and N₂ decreased at rates of 1.5–1.7 cmHg day^–1 and 0.1–0.2 cmHg day^–1, respectively, the gases were added at regular intervals. The solution was analyzed at regular intervals for HCN, HCHO and urea, and maximum concentrations of about 50, 2, and 140 mM were observed. The discharge phase was continued for 6 months. In the solution, glycine (5.6% yield based on the carbon), glycylglycine (0.64%), orotic acid (0.004%) and small amounts of the other amino acids were found.     

Abiotic Formation of Valine Peptides Under Conditions of High Temperature and High Pressure

We investigated the oligomerization of solid valine and the stabilities of valine and valine peptides under conditions of high temperature (150–200 °C) and high pressure (50–150 MPa). Experiments were performed under non-aqueous condition in order to promote dehydration reaction. After prolonged exposure of monomeric valine to elevated temperatures and pressures, the products were analyzed by liquid chromatography mass spectrometry comparing their retention times and masses. We identified linear peptides that ranged in size from dimer to hexamer, as well as a cyclic dimer. Previous studies that attempted abiotic oligomerization of valine in the absence of a catalyst have never reported valine peptides larger than a dimer. Increased reaction temperature increased the dissociative decomposition of valine and valine peptides to products such as glycine, β-alanine, ammonia, and amines by processes such as deamination, decarboxylation, and cracking. The amount of residual valine and peptide yields was greater at higher pressures at a given temperature, pressure, and reaction time. This suggests that dissociative decomposition of valine and valine peptides is reduced by pressure. Our findings are relevant to the investigation of diagenetic processes in prebiotic marine sediments where similar pressures occur under water-poor conditions. These findings also suggest that amino acids, such as valine, could have been polymerized to peptides in deep prebiotic marine sediments within a few hundred million years.     

The decomposition of organic compounds in soil

Summary The course of the CO₂ evolution rates of soil samples has been followed continuously in the absence and in the presence of various organic compounds. After an incubation period of 300 hours at 13 and 20°C the CO₂ evolution from pasture soil (containing 1.76% soil organic carbon) amounted to 0.13 and 0.44g CO₂–C.g soil^–1.h^–1, respectively. For arable soil (containing 1.20% soil organic carbon) the rates amounted to 0.04 and 0.09 g CO₂–C.g soil^–1.h^–1, respectively.At 20°C larger amounts of the organic substrates added to the soil supplied with 20 g NH₄NO₃–N.g soil^–1 were lost as CO₂ than at 13°C, indicating a higher efficiency of the growth of microorganisms at lower temperatures. In the absence of NH₄NO₃ the respiration rates were initially higher than in its presence, suggesting that a part of the soil microflora is inhibited by low concentrations of NH₄NO₃. The amounts of carbon lost were low for phenolcarboxylic acids with OH groups in the ortho position. The replacement of one of these groups by a methoxyl group resulted in a larger amount of the C lost as CO₂. The replacement of the COOH group by a C=C–COOH group had a decreasing effect on the decomposition of the phenolic acids tested. The decomposition of vanillic acid,p-hydroxybenzoic acid, and of the benzoic acids with OH groups in the meta position was as complete as that of glucose, amino acids or casein. The decomposition of bacterial cells to CO₂ was considerably less than that of glucose.No evidence could be obtained that the low percentage of substrate converted to CO₂ at the time of maximal respiration rate was due to the decreasing diffusion rate of substrate to the microbial colonies in the soil during the consumption of substrate.     

Catalytic hydrothermal processing of microalgae: decomposition and upgrading of lipids

Hydrothermal processing of high lipid feedstock such as microalgae is an alternative method of oil extraction which has obvious benefits for high moisture containing biomass. A range of microalgae and lipids extracted from terrestrial oil seed have been processed at 350 °C, at pressures of 150-200 bar in water. Hydrothermal liquefaction is shown to convert the triglycerides to fatty acids and alkanes in the presence of certain heterogeneous catalysts. This investigation has compared the composition of lipids and free fatty acids from solvent extraction to those from hydrothermal processing. The initial decomposition products include free fatty acids and glycerol, and the potential for de-oxygenation using heterogeneous catalysts has been investigated. The results indicate that the bio-crude yields from the liquefaction of microalgae were increased slightly with the use of heterogeneous catalysts but the higher heating value (HHV) and the level of de-oxygenation increased, by up to 10%.     

An Evaluation of the Critical Parameters for Abiotic Peptide Synthesis in Submarine Hydrothermal Systems

It has been proposed that oligopeptides may be formed in submarine hydrothermal systems (SHSs). Oligopeptides have been synthesized previously under simulated SHS conditions which are likely geochemically implausible. We have herein investigated the oligomerization of glycine under SHS–like conditions with respect to the limitations imposed by starting amino acid concentration, heating time, and temperature. When 10⁻¹ M glycine solutions were heated at 250°C for < 20 min glycine oligomers up to tetramers and diketopiperazine (DKP) were detectable. At 200°C, less oligomerization was noted. Peptides beyond glycylglycine (gly₂) and DKP were not detected below 150°C. At 10⁻² M initial glycine concentration and below, only gly₂, DKP, and gly₃ were detected, and then only above 200°C at < 20 min reaction time. Gly₃ was undetectable at longer reaction times. The major parameters limiting peptide synthesis in SHSs appear to be concentration, time, and temperature. Given the expected low concentrations of amino acids, the long residence times and range of temperatures in SHSs, it is unlikely that SHS environments were robust sources of even simple peptides. Possible unexplored solutions to the problems presented here are also discussed.     

Different effects of hypothermia on amino acid incorporation and on amino acid uptake in the brainin vivo

The temperature dependence of the incorporation of amino acids into cerebral proteins and that of the transport of amino acids through the blood-brain barrier were studied. We measured the protein synthesis rate in vivo over a wide temperature range (14°C–38°C) in male Sprague-Dawley rats using a flooding dose of labeled valine. There was a linear dependence of the protein synthesis rate on temperature. The temperature quotient expressed as per cent decrease per 1°C was somewhat lower at the lower temperatures, a decrease from 7.8% in the 37.7–32.5°C range to 6.7% in the 25.5–14°C range. The transport of the three amino acids phenylalanine, lysine, and alanine, representing there transport systems, through the blood-brain barrier showed no temperature dependence in vivo. The results show that in hypothermia cerebral metabolic rates are lowered to a great extent, while some aspects of metabolic transport are not affected.     

Pressure Effects on the Abiotic Polymerization of Glycine

Polymerization experiments were performed using dry glycine under various pressures of 5–100 MPa at 150°C for 1–32 days. The series of experiments was carried out under the assumption that the pore space of deep sediments was adequate for dehydration polymerization of pre-biotic molecules. The products show various colors ranging from dark brown to light yellow, depending on the pressure. Visible and infrared spectroscopy reveal that the coloring is the result of formation of melanoidins at lower pressures. High-performance liquid chromatography and mass spectrometry analyses of the products show that: (1) glycine in all the experimental runs oligomerizes from 2-mer to 10-mer; (2) the yields are dependent on pressure up to 25 MPa and decrease slightly thereafter; and (3) polymerization progressed for the first 8 days, while the amounts of oligomers remained constant for longer-duration runs of up to 32 days. These results suggest that pressure inhibits the decomposition of amino acids and encourages polymerization in the absence of a catalyst. Our results further imply that abiotic polymerization could have occurred during diagenesis in deep sediments rather than in oceans.     

Changes in brain temperature and free amino acids in normal and protein deprived suckling rats exposed to room temperature

Previous reports on early-induced protein-calorie malnutrition (PCM) in rats have indicated alterations in the concentration of free amino acids and of protein synthesis in the brain. Recently it was shown that early-induced protein deprivation (PD) retards the development of thermoregulation. This resulted in a failure to maintain a normal rectal temperature after short exposure to room temperature (+22°C) still at the age of 20–25 days corresponding to changes seen in normal rats at an age of 10–15 days. In the present study, 20-day old PD and normal rats where examined with regard to the effect of exposure to room temperature on brain temperature and on brain free amino acids. The results show a similar reduction in brain and rectal temperature of the PD rats occuring within 30 minutes after exposure to room temperature. The reduction was in the range of 5°C. PD rats kept in room temperature for 5 hours and then allowed to recover at 32.5°C showed a slow increase in brain and rectal temperature but normal temperatures were not reached even after 1 hour. The concentration of free amino acids in the brain was examined in rats kept for 1 hour at room temperature or at 32.5°C. In the PD rats kept at 32.5°C, free aspartate and glutamate were reduced whereas taurine, GABA and glycine were increased as compared to their corresponding control rats. As a result of the reduced brain temperature in PD rats exposed to room temperature there was a reduction in free asparagine. The lability of the pool of asparagine may be related to the low levels of aspartate and glutamate in PD rats. On the basis of the present findings it is recommended that temperature-sensitive parameters are examined in PCM rats at a normal body temperature.Special Issue dedicated to Prof. Holger Hydén     

Polyhydric alcohol protective effect on Rhizomucor miehei lipase deactivation enhanced by pressure and temperature treatment

The influence of polyhydric alcohols (sorbitol, xylitol, erythritol, glycerol) on the thermal stability of Rhizomucor miehei lipase has been studied at high hydrostatic pressure (up to 500 MPa). In the absence of additives, a protective effect (PE) (the ratio between the residual activities determined at 480 MPa for the enzyme in the presence or absence of polyhydric alcohols) of low-applied pressures (from 50 MPa to 350 MPa) against thermal deactivations (at 50°C and 55°C) has been noticed. In the presence of additives, a strong correlation between PE and the total hydroxyl group concentration has been obtained, for the first time, under treatments of combining denaturing temperatures and high hydrostatic pressures. This relationship does not seem to be dependent on the nature polyhydric alcohols as the same effect could be observed with 1 M sorbitol and 2 M glycerol. This PE, against thermal and high pressure combined lipase deactivation, increases with polyhydric alcohol concentrations, and when temperature increases from 25°C to 55°C.     

Characteristics of pullulanases from extremely thermophilic archaea isolated from deep-sea hydrothermal vents

Two out of three extremely thermophilic anaerobic archaea, isolated from deep-sea hydrothermal vents, produced pullulanase activity in the presence of maltose in the growth medium. Enzyme activities were mainly extracellular and characterized by optimum temperatures of 95°C and 80–95°C, optimum pH of 5.0–7.0 and a high degree of thermostability. One strain when grown in a fermenter with maltose as inducer produced pullulanase at 35 U/l. © Rapid Science Ltd. 1998     

Evidence for organic synthesis in high temperature aqueous media — Facts and prognosis

Hydrothermal systems are common along the active tectonic areas of the earth. Potential sites being studied for organic matter alteration and possible organic synthesis are spreading ridges, off-axis systems, back-arc activity, hot spots, volcanism, and subduction. Organic matter alteration, primarily reductive and generally from immature organic detritus, occurs in these high temperature and rapid fluid flow hydrothermal regimes. Hot circulating water (temperature range — warm to >400 °C) is responsible for these molecular alterations, expuslion and migration. Compounds that are obviously synthesized are minor components because they are generally masked by the pyrolysis products formed from contemporary natural organic precursors. Heterocyclic sulfur compounds have been identified in high temperature zones and hydrothermal petroleums of the Guaymas Basin vent systems. They can be interpreted as being synthesized from formaldehyde and sulfur or HS _x ^– in the hydrothermal fluids.Other products from potential synthesis reactions have not yet been found in the natural systems but are expected based on known industrial processes and inferences from experimental simulation data. Various industrial processes have been reviewed and are of relevance to hydrothermal synthesis of organic compounds. The reactivity of organic compounds in hot water (200–350 °C) has been studied in autoclaves, and supercritical water as a medium for chemistry has also been evaluated. This high temperature aqueous organic chemistry and the strong reducing conditions of the natural systems suggest this as an important route to produce organic compounds on the primitive earth. Thus a better understanding of the potential syntheses of organic compounds in hydrothermal systems will require investigations of the chemistry of condensation, autocatalysis, catalysis and hydrolysis reactions in aqueous mineral buffered systems over a range of temperatures from warm to >400 °C.Presented in part at the International Society for the Study of the Origin of Life Meeting, Barcelona, Spain, July 1993.     

Responsiveness of Orobanche ramosa L. Seeds to GR 24 as Related to Temperature, Oxygen Availability and Water Potential During Preconditioning and Subsequent Germination

Broomrape (Orobanche ramosa L.) is a root holoparasite responsible for important yield losses in numerous crops, particularly in the Mediterranean area. In this paper, the effects of temperature, oxygen concentration and water potential of the medium on broomrape seed germination were investigated. Seeds became able to germinate in the presence of a strigol analogue (GR 24) only after a preincubation period for at least 3 days at 20 °C. Their responsiveness to GR 24 increased with increasing duration of their preconditioning at 20 °C, and was optimal after 2–3 weeks. The preconditioning treatment was effective at temperatures ranging from 10 to 30 °C. At the optimal temperature (20 °C), it required at least 1% oxygen in the atmosphere and remained effective at a water potential of the medium of –2 MPa. A too prolonged preincubation of seeds at sub- or supraoptimal temperatures (5 and 30 °C) resulted in induction of a secondary dormancy. Seeds preconditioned for 14 days at 20 °C germinated in the presence of 1 mg L^–1 GR 24 at temperatures ranging from 10 to 25 °C, and the thermal optimum was the same (20 °C) than that of preconditioning. At 20 °C, seeds were able to germinate in the presence of GR 24 under atmospheres containing at least 3% oxygen and at a water potential of the medium as low as –3 MPa. The differences observed in the effects of environmental factors on preconditioning efficiency and germination of preconditioned seeds suggest that both processes involve different mechanisms. The results obtained might also help to better understand the regulation of O. ramosa spread in temperate areas.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada