C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Improvement of laccase production and its properties by low-energy ion implantation

Low-energy ion implantation was employed to breed laccase producing strain Paecilomyces sp. WSH-L07 and a mutant S152 that exhibited an activity of more than three times over the wild strain was obtained. The optimum substrate of both the wild and mutant laccases was 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), and followed by guaiacol with optimal pH at 3.4 and 5.0, respectively, while the mutant laccase exhibited a broader active pH range. The mutant laccase had a higher optimal catalytic temperature (60–65 °C) than the wild one (55 °C), and the wild laccase deactivated rapidly when temperature increased above 55 °C. Furthermore, the mutant laccase was more stable under neutral and alkaline conditions. A thermostability experiment revealed that the mutant laccase was superior to the wild laccase. Both laccases were stable in the presence of metal ions, mildly inhibited by SDS (0.5 mM), EDTA (1 mM) and 1,4-dithiothreitol (0.5 mM), and almost completely inhibited by 0.1 mM NaN₃.     

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2 ng/bee) on the thorax temperatures of foragers exposed to low (22 °C) and high (33 °C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1–2 h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60–90 min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5 min at 4 °C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.     

Effect of Physicochemical Modifications on Antioxidant Activity of Water-soluble Chitosan

Water-soluble chitosan was processed using ultrasonication, microfluidisation and homogenisation to modify its physicochemical properties. The effect of using sonicated chitosan on formation of chitosan-glucose conjugates was investigated. Untreated and sonicated chitosan conjugates were prepared under varying reaction conditions (such as ratios of reactants, pH and temperature). The conjugates formed were evaluated for colour development and antioxidant activity. For both untreated and sonicated chitosans, the reactivity was found to be higher at pH 6.0/121 °C than at pH 4.9/121 °C. The reactivity was found to be lower at 105 °C at both pH conditions than at 121 °C. This clearly demonstrated the greater reactivity at higher temperature irrespective of the reaction pH. Antioxidant activity studies indicated that the conjugates formed at 121 °C had higher activity. Although sonication of water-soluble chitosan led to slightly enhanced viscosity indicating higher reactivity, it did not improve the antioxidant activity.     

Biochemical characterization of a novel thermostable xyloglucanase from an alkalothermophilic Thermomonospora sp.

Xyloglucanase from an extracellular culture filtrate of alkalothermophilic Thermomonospora sp. was purified to homogeneity with a molecular weight of 144 kDa as determined by SDS-PAGE and exhibited specificity towards xyloglucan with apparent K _m of 1.67 mg/ml. The enzyme was active at a broad range of pH (5–8) and temperatures (40–80°C). The optimum pH and temperature were 7 and 70°C, respectively. The enzyme retained 100% activity at 50°C for 60 h with half-lives of 14 h, 6 h and 7 min at 60, 70 and 80°C, respectively. The kinetics of thermal denaturation revealed that the inactivation at 80°C is due to unfolding of the enzyme as evidenced by the distinct red shift in the wavelength maximum of the fluorescence profile. Xyloglucanase activity was positively modulated in the presence of Zn²⁺, K⁺, cysteine, β-mercaptoethanol and polyols. Thermostability was enhanced in the presence of additives (polyols and glycine) at 80°C. A hydrolysis of 55% for galactoxyloglucan (GXG) from tamarind kernel powder (TKP) was obtained in 12 h at 60°C and 6 h at 70°C using thermostable xyloglucanases, favouring a reduction in process time and enzyme dosage. The enzyme was stable in the presence of commercial detergents (Ariel), indicating its potential as an additive to laundry detergents.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

Temperature‐dependent developmental model of Neoseiulus californicus (McGregor) (Acari,Phytoseiidae)

The developmental time and survival of immature stages of Neoseiulus californicus were studied at nine constant temperatures (12, 16, 24, 24, 28 32, 36, 38 and 40°C), 60–70% RH, and a photoperiod of 16 : 8 (L : D) h. The total mortality of immature N. californicus was lowest at 24°C (4.5%) and highest at 38°C (15.2%). The total developmental time decreased with increasing temperature between 12°C (18.38 days) and 32°C (2.98 days), and increased beyond 32°C. The relationship between the developmental rate and temperature was fitted by five nonlinear developmental rate models (Logan 6, Lactin 1, 2 and Briere 1, 2). The nonlinear shape of temperature development was best described by the Lactin 1 model (r² = 0.98). The developmental variation of each stage was well described by the three‐parameter Weibull distribution model (r² = 0.91–0.93). The temperature‐dependent developmental models of N. californicus developed in this study could be used to determine optimal temperature conditions for its mass rearing, to predict its seasonal population dynamics in fruit tree orchards or greenhouse crops, or to develop a population dynamics model of N. californicus.     

Optimization of aqueous-assisted extraction of polysaccharides from pumpkin (Cucurbita moschata Duch) and their biological activities

The pumpkin pulp contains a greater composition of edible polysaccharides and has reported with excellent biological applications. This research pertains to optimize the extraction of polysaccharides from the fleshy portion of the pumpkin using aqueous assisted extraction (AAE). The result showed that the optimal extraction condition of pumpkin polysaccharide was as follows: extraction temperature at 55 °C, pH 4.5, and enzyme concentration of 4000 µ/g for 80 min. Under the optimal extraction condition, the yield of pumpkin polysaccharide via AAE (15.4) was significantly higher. The biological activities of extracted polysaccharide including α-amylase inhibition (57.41% at 1000 µg/mL) and anti-inflammatory (50.41% at 25 µg/mL) activity increased significantly. Additionally, the antioxidant activities of extracted pumpkin polysaccharides including IC₅₀ values of DPPH and ABTS were 59.87% and 58.74%, respectively. The pumpkin polysaccharide has maximum inhibitory effects against bacterial strains especially for Escherichia coli than that of fungal strains. It is suggested that the aqueous assisted extraction of is a cost-effective promising method to decrease the processing time as well as enhancing extracted polysaccharide yield – times.     

Antioxidant responses of Propylaea japonica (Coleoptera: Coccinellidae) exposed to high temperature stress

Temperature is one of the most important environmental factors, and is responsible for a variety of physiological stress responses in organisms. Induced thermal stress is associated with elevated reactive oxygen species (ROS) generation leading to oxidative damage. The ladybeetle, Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae), is considered a successful natural enemy because of its tolerance to high temperatures in arid and semi-arid areas in China. In this study, we investigated the effect of high temperatures (35, 37, 39, 41 and 43 °C) on the survival and activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST), and total antioxidant capacity (TAC) as well as malondialdehyde (MDA) concentrations in P. japonica adults. The results indicated that P. japonica adults could not survive at 43 °C. CAT, GST and TAC were significantly increased when compared to the control (25 °C), and this played an important role in the process of antioxidant response to thermal stress. SOD and POD activity, as well as MDA, did not differ significantly at 35 and 37 °C compared to the control; however, there were increased levels of SOD, POD and MDA when the temperature was above 37 °C. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play important roles in reducing oxidative damage in P. japonica adults. This study represents the first comprehensive report on the antioxidant defense system in predaceous coccinellids (the third trophic level). The findings provide useful information for predicting population dynamics and understanding the potential for P. japonica as a natural enemy to control pest insects under varied environmental conditions.     

A novel nonionic surfactant- and solvent-stable alkaline serine protease from <Emphasis Type="Italic">Serratia</Emphasis> sp. SYBC H with duckweed as nitrogen source: production,purification, characteristics and application

A novel nonionic surfactant- and hydrophilic solvent-stable alkaline serine protease was purified from the culture supernatant of Serratia sp. SYBC H with duckweed as nitrogen source. The molecular mass of the purified protease is about 59 kDa as assayed via SDS-PAGE. The protease is highly active over the pH range between 5.0 and 11.0, with the maximum activity at pH 8.0. It is also fairly active over the temperature range between 30 and 80°C, with the maximum activity at 40°C. The protease activity was substantially stimulated by Mn²⁺ and Na⁺ (5 mM), up to 837.9 and 134.5% at 40°C, respectively. In addition, Mn²⁺ enhanced the thermostability of the protease significantly at 60°C. Over 90% of its initial activity remained even after incubating for 60 min at 40°C in 50% (v/v) hydrophilic organic solvents such as DMF, DMSO, acetone and MeOH. The protease retained 81.7, 83.6 and 76.2% of its initial activity in the presence of nonionic surfactants 20% (v/v) Tween 80, 25% (v/v) glycerol and Triton X-100, respectively. The protease is strongly inhibited by PMSF, suggesting that it is a serine protease. Washing experiments revealed that the protease has an excellent ability to remove blood stains.     

Effects of heat stress on some reproductive parameters of male cavie (Cavia porcellus) and mitigation strategies using guava (Psidium guajava) leaves essential oil

Climate changes, particularly the increase of temperature are among the main causes behind the decline of fertility in humans as well as animals. In this study, the effects of heat stress on some reproductive parameters of male cavies and mitigation strategies using guava leaves essential oil (GLEO) were studied. For this purpose, 40 male cavies aged 2.5–3 months and weighing between 348 and 446 g were divided into 4 groups of 10 animals each and subjected to the following temperatures: Ambient temperature (20–25 °C) for the control group, 35 °C for group 1, 45 °C for group 2 and 45 °C+100 µl GLEO/kg body weight, administered by gavage to animals for group 3. Exposure time of heat was 7 h per day for 60 days. Results reveal that the relative weights of testes, epididymis, vas deferens and seminal vesicles were hardly affected by the temperature levels considered (P>0.05). The mass and individual sperm motility was significantly lower (P<0.05) in cavies exposed to the temperature of 35 and 45 °C as compared with those which received GLEO and controls. The percentages of abnormal sperm and altered sperm DNA were higher in animals exposed to temperature of 35 and 45 °C as compared with the controls. The activity of superoxide dismutase significantly increased (P<0.05) in animals exposed to temperature of 45 °C and in those of 45 °C and orally treated with GLEO, compared with cavies exposed to temperature of 45 °C without receiving GLEO. The level of malondialdehyde was significantly increased (P<0.05) in animals exposed to temperature of 35 and 45 °C, whereas the level of nitric oxide was significantly lower (P<0.05) in exposed animals as compared with controls. It was concluded that the exposure of male cavies at 35 and 45 °C for 60 days induce heat stress that causes deterioration of sperm characteristics. These effects that can be mitigated by the administration of guava leaves essential oil.     

Biology and thermal requirements of Chrysoperla genanigra (Neuroptera: Chrysopidae) reared on Sitotroga cerealella (Lepidoptera: Gelechiidae) eggs

Chrysoperla genanigra Freitas is a common green lacewing associated with melon pests in the Northeastern Brazil. All life stages of this recently described species were studied under a range of constant temperature conditions (17, 21, 25, 29, 33, 35 and 37 °C), a photoperiod of 12 h:12 h (L:D) and 70 ± 10% relative humidity. Adults of C. genanigra were fed on a diet consisting of a 1:1 (v/v) mixture of brewer’s yeast and honey, while larvae were provided with eggs of Sitotroga cerealella (Olivier) ad libitum. The duration of preimaginal development of the species was inversely proportional to temperature and ranged from approximately 63 days at 17 °C to 15 days at 35 °C. The percentage of adult emergence varied from 6.7% at 17 °C to 76.7% at 25 °C, although no larvae were able to complete development at 37 °C. The lower thermal threshold for total preimaginal development was approximately 10.8 °C and the thermal requirement was 336.7 degree-days. Egg production, along with the longevity of both males and females, were significantly affected by temperature. It is concluded that the best temperature for rearing C. genanigra is 25 °C, with the lowest preimaginal mortality and the highest egg production (992.7 eggs/female).     

A combined pH and temperature precipitation step facilitates the purification of tobacco-derived recombinant proteins that are sensitive to extremes of either parameter

Incubation at pH 4.0 or blanching at ∼65°C facilitates the purification of biopharmaceutical proteins from plants by precipitating most of the host cell proteins (HCPs) before chromatography. However, both methods are compatible only with pH or thermostable target proteins whereas many target proteins may irreversibly denature, e.g., at pH < 4.0. Here, we developed a combined pH/temperature treatment for clarified tobacco extracts and intact leaves. The latter were subjected to a blanching procedure, i.e., the submersion into a hot buffer. Using a design of experiments approach we identified conditions that remove ∼70% of HCPs at ∼55°C, using the thermosensitive antibody 2G12 and the pH-sensitive DsRed as model proteins. We found that pH and temperature exerted a combined effect during the precipitation of HCPs in the pH range 5.0–7.0 at 35°C–60°C. For clarified extracts, the temperature required to achieve a DsRed purity threshold of 20% total soluble protein (TSP) increased from 54°C to 63°C when the pH was increased from 6.4 to 7.3. The pH-stable antibody 2G12 was less responsive to the combined treatment, but the purity of 1% TSP was achieved at 35°C instead of 44°C when the pH was reduced from 6.3 to 5.8. When blanching intact leaves, product losses were not exacerbated at pH 4.0. Indeed, the highest DsRed purity (58% TSP) was achieved at this pH, combined with a temperature of 60°C and an incubation time of 30 min. In contrast, the highest 2G12 purity (0.7% TSP) was achieved at pH 5.1 and 40°C with an incubation time of 20 min. Our data suggest that a combined pH/temperature regime can avoid extreme values of either parameter; therefore, broadening the applicability of these simple purification techniques to other recombinant proteins.     

Production of novel halo-alkali-thermo-stable xylanase by a newly isolated moderately halophilic and alkali-tolerant Gracilibacillus sp. TSCPVG

An aerobic xylanolytic Gracilibacillus sp. TSCPVG growing at moderate to extreme salinity (1–30%) and neutral to alkaline pH (6.5–10.5) was isolated from the salt fields near Sambhar district of Rajasthan, India. β-xylanase (18.44 U/ml) and β-xylosidase (1.01 U/ml) were produced in 60 h in the GSL-2 mineral base medium with additions of (in g/l) Birchwood xylan (7.5), yeast extract (10.0), tryptone (8.0), proline (2.0), thiamine (2.0), Tween-40 (2.0) and NaCl (35) at pH 7.5, 30 °C and 180 rpm. The β-xylanase was active within a broad salinity range (0–30% NaCl), pH (5.0–10.5) and temperature (50–70 °C). It exhibited maximal activity with 3.5% NaCl, pH 7.5 at 60 °C. It was extremely halotolerant retaining more than 80% of activity at 0 and 30% NaCl and alkali-tolerant retaining 76% of activity at pH 10.5. The acetone precipitated xylanase was highly stable (100%) at variable salinities of 0–30% NaCl, pH of 5.0–10.5 and temperatures of 0–60 °C for 48 h. HPLC analysis showed xylose, arabinose and xylooligosaccharides as hydrolysis products of xylan. This is the first report on hemi-cellulose degrading halo-alkali-thermotolerant enzyme from a moderately halophilic Gram-positive Gracilibacillus species.     

An alkali-tolerant xylanase produced by the newly isolated alkaliphilic <Emphasis Type="Italic">Bacillus pumilus</Emphasis> from paper mill effluent

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0–9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.     

Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml⁻¹ at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml⁻¹ at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg⁻¹ protein. The molecular mass of the purified enzyme was 97 KDa. The values of K _m and V _max were 36 mg ml⁻¹ and 222 μmol mg⁻¹ protein min⁻¹, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn²⁺, whereas it was inhibited by Hg²⁺, Cu²⁺, Zn²⁺, Mg²⁺, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.     

Effect of pre-treatment and extraction conditions on the antioxidant properties of persimmon (Diospyros kaki) leaves

Persimmon is a very delicious fruit and the leaves of this tree are used as a traditional drug. This study aimed to investigate the effects of drying method (hot air and freeze-drying), extraction temperature (80, 90 and 100 °C) and extraction time (10, 30, 60 and 120 min) and harvest stage (flowering and fruiting) on the antioxidant contents and antioxidant activity of persimmon leaves. The results showed that the highest antioxidants were obtained in both methods of drying. Also, 100 °C for 120 min of extraction gave the highest antioxidant contents, but with no significant difference compared to 90 °C for 60 min of extraction. Persimmon leaves collected during flowering stage had the maximum amount of antioxidants compared to the fruiting stage. Finally, it can be said that persimmon leaves harvested during flowering stage and treated by hot air drying with these extraction conditions (90 °C for 60 min) are richer in bioactive compounds.     

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

Multicopper oxidases can act on a broad spectrum of phenolic and non‐phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), 2,6‐dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where k_cat and K_m values at 60°C were 8.1 (± 0.8) s^?1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half‐life of over 10 h at 80°C and over 7 h at 90°C. Site‐directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10‐fold increase in activity compared with the wild‐type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta‐position of aromatic substrates.     

Temperature-dependent development of Lista haraldusalis (Walker) (Lepidoptera: Pyralidae) on Platycarya strobilacea

The effect of constant temperatures on development and survival of Lista haraldusalis (Walker) (Lepidoptera: Pyralidae), a newly reported insect species used to produce insect tea in Guizhou province (China), was studied in laboratory conditions at seven temperatures (19 °C, 22 °C, 25 °C, 28 °C, 31 °C, 34 °C, and 37 °C) on Platycarya strobilacea. Increasing the temperature from 19 °C to 31 °C led to a significant decrease in the developmental time from egg to adult emergence, and then the total developmental time increased at 34 °C. Egg incubation was the stage where L. haraldusalis experienced the highest mortality at all temperatures. The survival of L. haraldusalis was significantly higher at 25 °C and 28 °C, whereas none of the eggs hatched at 37 °C. Common and Ikemoto linear models were used to describe the relationship between the temperature and the developmental rate for each immature stage of L. haraldusalis. The estimated values of the lower temperature threshold and thermal constant of the total immature stages using Common and Ikemoto linear models were 11.34 °C and 11.20 °C, and 939.85 and 950.41 degree-days, respectively. Seven nonlinear models were used to fit the experimental data to estimate the developmental rate of L. haraldusalis. Based on the biological significance for model evaluation, Ikemoto linear, Logan-6, and SSI were the best models that fitted each immature stage of L. haraldusalis and they were used to estimate the temperature thresholds. These thermal requirements and temperature thresholds are crucial for facilitating the development of factory-based mass rearing of L. haraldusalis.     

A thermostable recombinant transaldolase with high activity over a broad pH range

Thermophilic enzymes are in high demand for various applications due to their prolonged lifetimes and high reaction rates at elevated temperatures. In this work, an open reading frame TM0295, which encodes a putative transaldolase (TAL) from a hyper-thermophilic microorganism, Thermotoga maritima, was cloned and expressed in Escherichia coli. The enzyme activity of transaldolase at high temperatures (e.g., at 80 °C) was reported here for the first time. The recombinant T. maritima transaldolase was extremely thermostable, with a half-life time of 198 and 13.0 h at 60 °C and 80 °C, respectively. The estimated total turn-over number was 1.5 × 10⁶ mol of product per mol of enzyme at 80 °C. This enzyme also exhibited high activities within a broad pH range of 6.0–9.0. This ultra-thermostable TAL with high activity shows great potential for use in such applications as the production of enzymatic biofuels production and the synthesis of high-value carbohydrates by cell-free synthetic pathway biotransformation.