Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.     

Thermal limits of wild and laboratory strains of two African malaria vector species, <Emphasis Type="Italic">Anopheles arabiensis</Emphasis> and <Emphasis Type="Italic">Anopheles funestus</Emphasis>

ABSTRACT: Malaria affects large parts of the developing world and is responsible for almost 800,000 deaths annually. As climates change, concerns have arisen as to how this vector-borne disease will be impacted by changing rainfall patterns and warming temperatures. Despite the importance and controversy surrounding the impact of climate change on the potential spread of this disease, little information exists on the tolerances of several of the vector species themselves. METHODS: Using a ramping protocol (to assess critical thermal limits - CT) and plunge protocol (to assess lethal temperature limits - LT) information on the thermal tolerance of two of Africa's important malaria vectors, Anopheles arabiensis and Anopheles funestus was collected. The effects of age, thermal acclimation treatment, sex and strain (laboratory versus wild adults) were investigated for CT determinations for each species. The effects of age and sex for adults and life stage (larvae, pupae, adults) were investigated for LT determinations. RESULTS: In both species, females are more tolerant to low and high temperatures than males; larvae and pupae have higher upper lethal limits than do adults. Thermal acclimation of adults has large effects in some instances but small effects in others. Younger adults tend to be more tolerant of temperature changes than older age groups. Long-standing laboratory colonies are sufficiently similar in thermal tolerance to field-collected animals to provide reasonable surrogates when making inferences about wild population responses. Differences between these two vectors in their thermal tolerances, especially in larvae and pupae, are plausibly a consequence of different habitat utilization. CONCLUSIONS: Limited plasticity is characteristic of the adults of these vector species relative to others examined to date, suggesting limited scope for within-generation change in thermal tolerance. These findings and the greater tolerance of females to thermal extremes may have significant implications for future malaria transmission, especially in areas of current seasonal transmission and in areas on the boundaries of current vector distribution.     

Effect of low temperatures on the activity of oxygen-scavenging enzymes in two populations of the C4 grass Echinochloa crus-galli

To discriminate among possible mechanisms responsible for the differential response to cold temperatures among ecotypes of the C₄ grass weed species Echinochloa crus-galli (L.) Beauv., the specific activities of five oxygen-scavenging enzymes responsible for the elimination or reduction of free radicals and hydrogen peroxide during cold-induced photoinhibition were determined in 5-week-old plants of two populations of the species collected from sites of contrasting climates, Québec (QUE) and Mississippi (MISS). Enzyme activities were measured at temperatures ranging from 5 to 30°C. The specific activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase were significantly higher in cold-adapted QUE plants at low assay temperatures than in warm-adapted MISS plants at the same temperature. The specific activities of superoxide dismutase assayed at 5 and 25°C were similar among plants of the two E. crus-galli populations. Ascorbate concentrations were not different among plants of the two populations, suggesting that the observed differences in the specific activities of ascorbate peroxidase assayed at 5°C, truly reflect a better capacity of the QUE enzyme to reduce H₂O₂ to water at temperature conditions associated with the photoinhibitory process. The enhanced specific activity of four of the five oxygen-scavenging enzymes measured in the cold-adapted QUE population at low assay temperatures correlates with the syndrome of cold-adapted features reported for plants of this population in earlier studies.     

Temperature stress induces notochord abnormalities and heat shock proteins expression in larval green sturgeon (Acipenser medirostris Ayres 1854)

The effects of thermal stress on survival, development and heat shock protein (hsp) expression of green sturgeon (GS) yolk‐sac larvae, from hatching through yolk depletion were investigated to provide insight into effects of highly altered natural river hydrographs. Hatched GS larvae were reared at constant water temperatures 18°C (control) through 28°C at 2°C increments. Larval survival significantly decreased at 26–28°C, with 28°C being lethal. Significant proportions of deformed larvae were found at sub‐lethal (20–26°C) and lethal 28°C rearing temperatures, with kyphosis (i.e. backward flexion of notochord) accounting for >99% of morphological deformities. Histological analysis of larvae preparations indicate that elevated water temperature affects notochord cell function and physiology. At rearing temperatures 20–28°C, thermal stress elicited a quick (24 h) and long lasting (yolk‐sac absorption) significant over‐expression of measured heat shock proteins (hsps), all of which are known components of intracellular protein repair and stabilization mechanism. Thermal sensitivity, as indicated by the incidence of abnormalities and expression of different hsps, varied significantly between crosses. Thermally tolerant progeny exhibited a short but rapid hsp72 (size in kDa) over‐expression, and more pronounced hsp60 and hsp90 over‐expression, than less tolerant progeny which exhibited a prolonged hsp72 and hsp78 over‐expression. At environmentally relevant water temperatures bent larvae exhibited spiral swimming, which in the wild would compromise the ability of emerging larvae to forage, avoid predators, and migrate downstream, ultimately compromising survival and recruitment. Before larvae hsp content can be used as a thermal‐stress biomarker for GS, field validation studies are needed.     

ECOLOGICAL NOVELTY BY HYBRIDIZATION: EXPERIMENTAL EVIDENCE FOR INCREASED THERMAL TOLERANCE BY TRANSGRESSIVE SEGREGATION IN TIGRIOPUS CALIFORNICUS

Early generations of hybrids can express both genetic incompatibilities and phenotypic novelty. Insights into whether these conflicting interactions between intrinsic and extrinsic selection persist after a few generations of recombination require experimental studies. To address this question, we use interpopulation crosses and recombinant inbred lines (RILs) of the copepod Tigriopus californicus, and focus on two traits that are relevant for the diversification of this species: survivorship during development and tolerance to thermal stress. Experimental crosses between two population pairs show that most RILs between two heat‐tolerant populations show enhanced tolerance to temperatures that are lethal to the respective parentals, whereas RILs between a heat‐tolerant and a heat‐sensitive population are intermediate. Although interpopulation crosses are affected by intrinsic selection at early generational hybrids, most of the sampled F₉ RILs have recovered fitness to the level of their parentals. Together, these results suggest that a few generations of recombination allows for an independent segregation of the genes underlying thermal tolerance and cytonuclear incompatibilities, permitting certain recombinant lineages to survive in niches previously unused by parental taxa (i.e., warmer thermal environments) without incurring intrinsic selection.     

Can Winter-Active Bumblebees Survive the Cold? Assessing the Cold Tolerance of Bombus terrestris audax and the Effects of Pollen Feeding

There is now considerable evidence that climate change is disrupting the phenology of key pollinator species. The recently reported UK winter activity of the bumblebee Bombus terrestris brings a novel set of thermal challenges to bumblebee workers that would typically only be exposed to summer conditions. Here we assess the ability of workers to survive acute and chronic cold stress (via lower lethal temperatures and lower lethal times at 0°C), the capacity for rapid cold hardening (RCH) and the influence of diet (pollen versus nectar consumption) on supercooling points (SCP). Comparisons are made with chronic cold stress indices and SCPs in queen bumblebees. Results showed worker bees were able to survive acute temperatures likely to be experienced in a mild winter, with queens significantly more tolerant to chronic cold temperature stress. The first evidence of RCH in any Hymenoptera is shown. In addition, dietary manipulation indicated the consumption of pollen significantly increased SCP temperature. These results are discussed in the light of winter active bumblebees and climate change.     

Cold adaptation of enzyme reaction rates

A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.     

Cold adaptation of enzymes: structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the Arctic psychrophile Colwellia psychrerythraea and of human leukotriene A(4) hydrolase

The relationships between structure, activity, stability and flexibility of a cold-adapted aminopeptidase produced by a psychrophilic marine bacterium have been investigated in comparison with a mesophilic structural and functional human homolog. Differential scanning calorimetry, fluorescence monitoring of thermal- and guanidine hydrochloride-induced unfolding and fluorescence quenching were used to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a low structural stability versus thermal and chemical denaturants and a greater structural permeability to a quenching agent relative to the mesophilic homolog. These findings support the hypothesis that cold-adapted enzymes maintain their activity at low temperatures as a result of increased global or local structural flexibility, which results in low stability. Analysis of the thermodynamic parameters of irreversible thermal unfolding suggests that entropy-driven factors are responsible for the fast unfolding rate of the cold-adapted aminopeptidase. A reduced number of proline residues, a lower degree of hydrophobic residue burial and a decreased surface accessibility of charged residues may be responsible for this effect. On the other hand, the reduction in enthalpy-driven interactions is the primary determinant of the weak conformational stability.     

Differential responses of thermal tolerance to acclimation in the sub-Antarctic rove beetle Halmaeusa atriceps

Abstract. Investigations of the responses to acclimation of upper and lower lethal limits and limits to activity in insects have focused primarily on Drosophila. In the present study, Halmaeusa atriceps (Staphylinidae) is examined for thermal tolerance responses to acclimation, and seasonal acclimatization. In summer and winter, lower lethal temperatures of adults and larvae are approximately −7.6 ± 0.03 and −11.1 ± 0.06 °C, respectively. Supercooling points (SCPs) are more variable, with winter SCPs of −5.4 ± 0.4 °C in larvae and −6.3 ± 0.8 °C in adults. The species appears to be chill susceptible in summer and moderately freeze tolerant in winter, thus showing seasonal acclimatization. Similar changes cannot be induced solely by acclimation to low temperatures in the laboratory. Upper lethal temperatures show a weaker response to acclimation. There are also significant responses to acclimation of critical thermal limits. Critical thermal minima vary between −3.6 ± 0.2 and −0.6 ± 0.2 °C in larvae, and from −4.1 ± 0.1 to −0.8 ± 0.2 °C in adults. By contrast, critical thermal maxima vary much less within adults and larvae. These findings are in keeping with the general pattern found in insects, although this species differs in several respects from others found on Marion Island.     

Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats

The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n = 123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 °C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.     

Thermal behavior of the Pacific sardine (Sardinops sagax) acclimated to different thermal cycles

The study of thermal behavior of fishes provides useful data to enable predictions of the effect of climatic change on populations so as to ensure good management of fisheries. The Pacific sardine has a complex population dynamics; three subpopulation have been proposed (cold, temperate and warm).We exposed Sardinops sagax caeruleus (temperate sub-population) to two different thermal cycles, which were chosen to be consistent with the temperatures reported in two distant places Cedros Island (CIc: 18–23 °C) and San Pedro (SPc: 13–18 °C).The thermal behavior of the SPc and CIc sardines was affected by acclimation treatment: the interval of thermal preference was 17.1–19.9 and 16.0–18.8 °C, while the lethal temperatures interval (LT50) was 7.7–25.6 and 6.9–24.3 °C, and the critical limits CTMax and CTMin were 7.1–32.2 and 5.5–30.4 °C, respectively.The results of thermal behavior showed that sardines of the temperate subpopulation are more tolerant to cold; this might suggest that they would be more able to survive in California and Oregon than in Baja California Sur and the Gulf of California.     

Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase (IPMDH) from a random mutant library was a double mutant containing the mutations I11V and S92F that were found in cold-adapted mutant IPMDHs previously isolated. To elucidate the effect of each mutation on enzymatic activity, I11V and six multiple mutant IPMDHs were constructed and analyzed. All of the multiple mutant IPMDHs were found to be improved in catalytic activity at moderate temperatures by increasing the k(cat) with a simultaneous increase of K(m) for the coenzyme NAD(+). k(cat) was improved by a decrease in the activation enthalpy, DeltaH( not equal). The multiple mutants did not show large reduction in thermal stability, and one of them showed enhanced thermal stability. Mutation from I11 to V was revealed to have a stabilizing effect. Mutants showed increased thermal stability when the mutation I11V was combined. This indicates that it is possible to construct mutants with enhanced thermal stability by combining stabilizing mutation. No additivity was observed for the thermodynamic properties of catalytic reaction in the multiple mutant IPMDHs, implying that the structural changes induced by the mutations were interacting with each other. This indicates that careful and detailed tuning is required for enhancing activity in contrast to thermal stability.     

Effects of temperature and exposure time on mortality of stored-product insects exposed to low pressure.

The prospects of using low pressure that creates a low-oxygen atmosphere to control stored-product insects were investigated in the laboratory. Eggs, larvae, and pupae of Tribolium castaneum (Herbst), Plodia interpunctella (Hübner), and Rhyzopertha dominica (F.) were exposed to 32.5 mmHg in glass chambers at 25, 33, 37, and 40 degrees C for times ranging from 30 min to 144 h. Time-mortality data were subjected to probit analyses and lethal dose ratios were computed to determine differences in lethal time (LT) values among all species-life stage combinations across the four temperatures. Eggs of each species were the life stage most tolerant to low pressure. Pupae of T. castaneum and R. dominica were more tolerant to low pressure than larvae. In all life stages, mortality increased with increasing exposure time to low pressure and also with increasing temperature. Immature stages of R. dominica were more tolerant to low pressure than immature stages of the other two species. The LT99 for R. dominica eggs was 176.32 h at 25 degrees C and that for P. interpunctella eggs was 28.35 h at the same temperature. An increase in temperature to 33 degrees C resulted in a LT99 < of 85.98 h for R. dominica and 6.21 h for P. interpunctella. Higher temperatures resulted in further significant reduction in lethal time values. Low pressure represents a simple, nonchemical alternative to fumigants such as methyl bromide and phosphine for controlling pests of stored-products or other commodities.     

The critical thermal limits for juvenile Arctic charr Salvelinus alpinus

The chief objective was to determine the critical thermal limits for alevins, fry and parr of Arctic charr, Salvelinus alpinus , (L.) from four races living in Windermere (northwest England). The experimental fish were reared in a hatchery but were the progeny of wild parents. As comparisons between tethal temperatures at four acclimation temperatures (5, 10, 15, 20° C) revealed few significant racial differences, the data were pooled to estimate the lethal values for survival over 7 days (incipient lethal temperature) and over only 10 min (ultimate lethal temperature) for each life stage. Upper lethal values increased with acclimation temperatures for alevins but this effect was negligible for fry and parr, Alevins were generally less tolerant than fry and parr at lower, but not higher, acclimation temperatures; e.g. after acclimation at 5° C, mean upper ultimate values were 23·3, 25·1 and 25·7° C and mean upper incipient values were 18·7, 21·5 and 21·5° C for alevins, fry and parr respectively; after acclimation at 20° C, mean upper ultimate and incipient values were 26·2, 26·1 and 26·6° C and 20·8, 20·8 and 21·6° C for alevins, fry and parr respectively. The area of the temperature tolerance polygon (expressed as ° C²) for juvenile Arctic charr is amongst the lowest recorded for salmonids; being 409, 439 and 461° C² for alevins, fry and parr respectively. These low values are due to lower upper tolerance limits, not high lower tolerance limits; the latter being close to 0° C (<1°C for parr and fry, <0·3° C for alevins) at all acclimation temperatures. Arctic charr are therefore amongst the least resistant of salmonids to high temperatures but probably the most resistant to low temperatures.     

Juvenile Ribbontail Stingray, Taeniura lymma (Forssk?l, 1775) (Chondrichthyes, Dasyatidae), demonstrate a unique suite of physiological adaptations to survive hyperthermic nursery conditions

Juvenile ribbontail stingrays, Taeniura lymma (Forssk?l, 1775) of the tropical West Pacific inhabit mangal and seagrass nurseries that often experience rapid and extreme increases in water temperature. We hypothesized that juvenile rays possess a thermal strategy similar to other hyperthermic specialists, in which fish prefer high temperatures, are always prepared for thermal extremes regardless of previous thermal history, and exhibit low metabolic thermal sensitivity. Critical thermal methodology was used to determine the thermal niche, and a thermal gradient used to estimate stingray final preferendum. Temperature quotients (Q ₁₀) were calculated from metabolic rates determined at three temperatures using flow-through respirometry. As predicted, juvenile rays showed a relatively small thermal niche dominated by intrinsic tolerance with limited capacity for acclimation. Thermal preference values were higher than those reported for other elasmobranch species. Interestingly, the temperature quotient for juvenile rays was higher than expected, suggesting that these fish may have the ability to exploit the thermal heterogeneity in their environment. Temperature likely acts as a directing factor in this species, separating warm tolerant juveniles from adults living in deeper, cooler waters.     

Effect of latitude and acclimation on the lethal temperatures of the peach‐potato aphid Myzus persicae

• 1 Aphids, similar to all insects, are ectothermic and, consequently, are greatly affected by environmental conditions. The peach potato aphid Myzus persicae (Sulzer) has a global distribution, although it is not known whether populations display regional adaptations to distinct climatic zones along its distribution and vary in their ability to withstand and acclimate to temperature extremes. In the present study, lethal temperatures were measured in nine anholocyclic clones of M. persicae collected along a latitudinal cline of its European distribution from Sweden to Spain. The effects of collection origin and intra‐ and intergenerational acclimation on cold and heat tolerance, as determined by upper and lower lethal temperatures (ULT₅₀ and LLT₅₀, respectively), were investigated.
• 2 Lethal temperatures of M. persicae were shown to be plastic and could be altered after acclimation over just one generation. Lower lethal temperatures were significantly depressed in eight of nine clones after acclimation for one generation at 10°C (range: ?13.3 to ?16.2°C) and raised after acclimation at 25°C (range: ?10.7 to ?11.6°C) compared with constant 20°C (range: ?11.9 to ?12.9°C). Upper lethal temperatures were less plastic, although significantly increased after one generation at 25°C (range: 41.8–42.4°C) and in five of nine clones after acclimation at 10°C. There was no evidence of intergenerational acclimation over three generations.
• 3 Thermal tolerance ranges were expanded after acclimation at 10 and 25°C compared with constant 20°C, resulting in aphids reared at 10°C surviving over a temperature range that was approximately 2–6°C greater than those reared at 25°C.
• 4 There was no clear relationship between lethal temperatures and latitude. Large scale mixing of clones may occur across Europe, thus limiting local adaption in thermal tolerance. Clonal type, as identified by microsatellite analysis, did show a relationship with thermal tolerance, notably with Type O clones being the most thermal tolerant. Clonal types may respond independently to climate change, affecting the relative proportions of clones within populations, with consequent implications for biodiversity and agriculture.

     

Swimming performance and energetics as a function of temperature in killifish Fundulus heteroclitus

Populations of the common killifish Fundulus heteroclitus are found along a latitudinal temperature gradient in habitats with high thermal variability. The objectives of this study were to assess the effects of temperature and population of origin on killifish swimming performance (assessed as critical swimming speed, U(crit)). Acclimated fish from northern and southern killifish populations demonstrated a wide zone (from 7 degrees to 33 degrees C) over which U(crit) showed little change with temperature, with performance declining significantly only at lower temperatures. Although we observed significant differences in swimming performance between a northern and a southern population of killifish in one experiment, with northern fish having an approximately 1.5-fold-greater U(crit) than southern fish across all acclimation temperatures, we were unable to replicate this finding in other populations or collection years, and performance was consistently high across all populations and at both low (7 degrees C) and high (23 degrees C) acclimation temperatures. The poor swimming performance of southern killifish from a single collection year was correlated with low muscle [glycogen] rather than with other indicators of fuel stores or body condition. Killifish acclimated to 18 degrees C and acutely challenged at temperatures of 5 degrees , 18 degrees , 25 degrees , or 34 degrees C showed modest thermal sensitivity of U(crit) between 18 degrees and 34 degrees C, with performance declining substantially at 5 degrees C. Thus, much of the zone of relative thermal insensitivity of swimming performance is intrinsic in this species rather than acquired as a result of acclimation. These data suggest that killifish are broadly tolerant of changing temperatures, whether acute or chronic, and demonstrate little evidence of local adaptation in endurance swimming performance in populations from different thermal habitats.     

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.     

Temperature tolerance polygon of Poecilia sphenops Valenciennes (Pisces: Poeciliidae)

1. The critical thermal maximum (CTMax) and minimum (CTMin), the upper and lower incipient lethal temperature, and the high and low avoidance temperature of Poecilia sphenops were established.2. The area of thermal tolerance of P. sphenops upon considering the lethal limits was 863.9 (°C)², the estimated area using the critical temperatures, as well as the zone of thermal preference were 959 and 323.4 (°C)², respectively.3. P. sphenops is a species highly eurythermical that possesses a high tolerance, resistance and capacity of adaptation to environmental variations.