Use of hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 expressed thermophilic alcohol dehydrogenases as whole-cell catalyst in solvent-free organic media

The hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 was employed as a whole-cell biocatalyst to examine its potential for bioconversion in solvent-free organic media. The genes encoding two different thermostable alcohol dehydrogenases (ADH_Tt1 and ADH_Tt2) of Thermus thermophilus HB27 were expressed in R. rhodochrous cells. To inactivate indigenous mesophilic enzymes in R. rhodochrous, transformant cells were heated at 70 °C for 10 min. Heat-treated hydrophobic wet cells were used for the bioconversion of 2,2,2-trifluoroacetophenone (TFAP) to α-(trifluoromethyl) benzyl alcohol (TFMBA) as a model reaction with ADH_Tt1. NADH, which was supplied in aqueous solution, was regenerated by converting cyclohexanol to cyclohexanone by ADH_Tt2. All reactions were performed by suspending heat-treated cells in solvent-free organic media consisting of 3.7 M TFAP and 4.8 M cyclohexanol (1:1, v/v ratio) at 60 °C. When 800 mg heat-treated R. rhodochrous cells were dispersed in 2 mL of solvent-free organic media (400 mg cells/mL), the product concentration reached about 3.6 M TFMBA by 48 h with a total NADH turnover number of approximately 900. The overall productivity was 190 mol TFMBA/kg cells/h.     

Development response of Spodoptera exigua to eight constant temperatures: Linear and nonlinear modeling

Temperature-dependent development of Spodoptera exigua (Hübner) were evaluated at eight constant temperatures of 12, 15, 20, 25, 30, 33, 34 and 36 °C with a variation of 0.5 °C on sugar beet leaves. No development occurred at 12 °C and 36 °C. Total developmental time varied from 120.50 days at 15 °C to 14.50 days at 33 °C. As temperature increased from 15 °C to 33 °C, developmental rate (1/developmental time) of S. exigua increased but declined at 34 °C. The lower temperature threshold (T_min) was estimated to be 12.98 °C and 12.45 °C, and the thermal constant (K) was 294.99 DD and 311.76 DD, using the traditional and Ikemoto–Takai linear models, respectively. The slopes of the Ikemoto–Takai linear model for different immature stages were different, violating the assumption of rate isomorphy. Data were fitted to three nonlinear models to predict the developmental rate and estimate the critical temperatures. The T_min values estimated by Lactin-2 (12.90 °C) and SSI (13.35 °C) were higher than the value estimated by Briere-2 (8.67 °C). The estimated fastest development temperatures (T_fast) by the Briere-2, Lactin-2 and SSI models for overall immature stages development of S. exigua were 33.4 °C, 33.9 °C and 32.4 °C, respectively. The intrinsic optimum temperature (T_Φ) estimated from the SSI model was 28.5 °C, in which the probability of enzyme being in its native state is maximal. The upper temperature threshold (T_max) values estimated by these three nonlinear models varied from 34.00 °C to 34.69 °C. These findings on thermal requirements can be used to predict the occurrence, number of generations and population dynamics of S. exigua.     

A reduced core to skin temperature gradient,not a critical core temperature,affects aerobic capacity in the heat

The purpose of this study was to determine the impact of the core to skin temperature gradient during incremental running to volitional fatigue across varying environmental conditions. A secondary aim was to determine if a “critical” core temperature would dictate volitional fatigue during running in the heat. 60 participants (n=49 male, n=11 female; 24±5 yrs, 177±11 cm, 75±13 kg) completed the study. Participants were uniformly stratified into a specific exercise temperature group (18 °C, 26 °C, 34 °C, or 42 °C) based on a 3-mile run performance. Participants were equipped with core and chest skin temperature sensors and a heart rate monitor, entered an environmental chamber (18 °C, 26 °C, 34 °C, or 42 °C), and rested in the seated position for 10 min before performing a walk/run to volitional exhaustion. Initial treadmill speed was 3.2 km h⁻¹ with a 0% grade. Every 3 min, starting with speed, speed and grade increased in an alternating pattern (speed increased by 0.805 km h⁻¹, grade increased by 0.5%). Time to volitional fatigue was longer for the 18 °C and 26 °C group compared to the 42 °C group, (58.1±9.3 and 62.6±6.5 min vs. 51.3±8.3 min, respectively, p<0.05). At the half-way point and finish, the core to skin gradient for the 18 °C and 26 °C groups was larger compared to 42 °C group (halfway: 2.6±0.7 and 2.0±0.6 vs. 1.3±0.5 for the 18 °C, 26 °C and 42 °C groups, respectively; finish: 3.3±0.7 and 3.5±1.1 vs. 2.1±0.9 for the 26 °C, 34 °C, and 42 °C groups, respectively, p<0.05). Sweat rate was lower in the 18 °C group compared to the 26 °C, 34 °C, and 42 °C groups, 3.6±1.3 vs. 7.2±3.0, 7.1±2.0, and 7.6±1.7 g m⁻² min⁻¹, respectively, p<0.05. There were no group differences in core temperature and heart rate response during the exercise trials. The current data demonstrate a 13% and 22% longer run time to exhaustion for the 18 °C and 26 °C group, respectively, compared to the 42 °C group despite no differences in beginning and ending core temperatures or baseline 3-mile run time. This capacity difference appears to result from a magnified core to skin gradient via an environmental temperature advantageous to convective heat loss, and in part from an increased sweat rate.     

Effects of different temperatures on the life history of Evania appendigaster L. (Hymenoptera: Evaniidae), a solitary oothecal parasitoid of Periplaneta americana L. (Dictyoptera: Blattidae)

The influence of temperatures on the life parameters of the solitary oothecal parasitoid Evania appendigaster, was investigated in the laboratory. Parasitized oothecae of Periplaneta americana were left to develop under seven constant temperatures: 15, 17, 20, 25, 30, 35, and 40 °C. At the end, we found that: (i) E. appendigaster was able to complete development within the temperature range of 17–34 °C; (ii) mean adult longevity decreased as temperature increased, with the temperature of 40 °C being fatal in a matter of hours; (iii) males lived longer than females between 15 and 30 °C; (iv) adult emergence rate was the highest at 25 °C, and (v) no wasps emerged at 15 or 40 °C. Non-emerged oothecae contained either unhatched eggs or dead larvae. We determined the theoretical lower developmental threshold and thermal constant for the complete development as 12.9 °C and 584.8 day-degrees for males, and 13.1 °C and 588.2 day-degrees for females, respectively. A good balance between faster development, maximum adult longevity and good egg viability was obtained between 25–30 °C, and that would be the best temperature range for rearing E. appendigaster.     

Development of robust in vitro RNA-dependent RNA polymerase assay as a possible platform for antiviral drug testing against dengue

NS5 is the largest and most conserved protein among the four dengue virus (DENV) serotypes. It has been the target of interest for antiviral drug development due to its major role in replication. NS5 consists of two domains, the N-terminal methyltransferase domain and C-terminal catalytic RNA-dependent RNA polymerase (RdRp) domain. It is an unstable protein and is prone to inactivation upon prolonged incubation at room temperature, thus affecting the inhibitor screening assays. In the current study, we expressed and purified DENV RdRp alone in Esherichia coli (E. coli) cells. The N-terminally His-tagged construct of DENV RdRp was transformed into E. coli expression strain BL-21 (DE3) pLysS cells. Protein expression was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM. The induced cultures were then grown for 20 h at 18 °C and cells were harvested by centrifugation at 6000 x g for 15 min at 4 °C. The recombinant protein was purified using HisTrap affinity column (Ni-NTA) and then the sample was subjected to size exclusion chromatography, which successfully removed the degradation product obtained during the previous purification step. The in vitro polymerase activity of RdRp was successfully demonstrated using homopolymeric polycytidylic acid (poly(rC)) RNA template. This study describes the high level production of enzymatically active DENV RdRp protein which can be used to develop assays for testing large number of compounds in a high-throughput manner. RdRp has the de novo initiation activity and the in vitro polymerase assays for the protein provide a platform for highly robust and efficient antiviral compound screening systems.     

Can acclimation of thermal tolerance,in adults and across generations,act as a buffer against climate change in tropical marine ectotherms?

Thermal acclimation capacity was investigated in adults of three tropical marine invertebrates, the subtidal barnacle Striatobalanus amaryllis, the intertidal gastropod Volegalea cochlidium and the intertidal barnacle Amphibalanus amphitrite. To test the relative importance of transgenerational acclimation, the developmental acclimation capacity of A. amphitrite was investigated in F₁ and F₂ generations reared at a subset of the same incubation temperatures. The increase in CT_max (measured through loss of key behavioural metrics) of F₀ adults across the incubation temperature range 25.4–33.4 °C was low: 0.00 °C (V. cochlidium), 0.05 °C (S. amaryllis) and 0.06 °C (A. amphitrite) per 1 °C increase in incubation temperature (the acclimation response ratio; ARR). Although the effect of generation was not significant, across the incubation temperature range of 29.4–33.4 °C, the increase in CT_max in the F₁ (0.30 °C) and F₂ (0.15 °C) generations of A. amphitrite was greater than in the F₀ (0.10 °C). These correspond to ARR's of 0.03 °C (F₀), 0.08 °C (F₁) and 0.04 °C (F₂), respectively. The variability in CT_max between individuals in each treatment was maintained across generations, despite the high mortality of progeny. Further research is required to investigate the potential for transgenerational acclimation to provide an extra buffer for tropical marine species facing climate warming.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Thermal tolerance,growth and oxygen consumption of Labeo rohita fry (Hamilton, 1822) acclimated to four temperatures

A 30 day feeding trial was conducted using a freshwater fish, Labeo rohita (rohu), to determine their thermal tolerance, oxygen consumption and optimum temperature for growth. Four hundred and sixteen L. rohita fry (10 days old, 0.385±0.003 g) were equally distributed between four treatments (26, 31, 33 and 36 °C) each with four replicates for 30 days. Highest body weight gain and lowest feed conversion ratio (FCR) was recorded between 31 and 33 °C. The highest specific growth rate was recorded at 31 °C followed by 33 and 26 °C and the lowest was at 36 °C. Thermal tolerance and oxygen consumption studies were carried out after completion of growth study to determine tolerance level and metabolic activity at four different acclimation temperatures. Oxygen consumption rate increased significantly with increasing acclimation temperature. Preferred temperature decided from relationship between acclimation temperature and Q₁₀ values were between 33 and 36 °C, which gives a better understanding of optimum temperature for growth of L. rohita. Critical thermal maxima (CTMax) and critical thermal minima (CTMin) were 42.33±0.07, 44.81±0.07, 45.35±0.06, 45.60±0.03 and 12.00±0.08, 12.46±0.04, 13.80±0.10, 14.43±0.06, respectively, and increased significantly with increasing acclimation temperatures (26, 31, 33 and 36 °C). Survival (%) was similar in all groups indicating that temperature range of 26–36 °C is not fatal to L. rohita fry. The optimum temperature range for growth was 31–33 °C and for Q₁₀ values was 33–36 °C.     

An organic solvent and thermally stable lipase from Burkholderia ambifaria YCJ01: Purification,characteristics and application for chiral resolution of mandelic acid

A solvent-tolerant bacterium Burkholderia ambifaria YCJ01 was newly isolated by DMSO enrichment of the medium. The lipase from the strain YCJ01 was purified to homogeneity with apparent molecular mass of 34 kDa determined by SDS-PAGE. The purified lipase exhibited maximal activity at a temperature of 60 °C and a pH of 7.5. The lipase was very stable below 55 °C for 7 days (remaining 80.3% initial activity) or at 30 °C for 60 days. PMSF significantly inhibited the lipase activity, while EDTA had no effect on the activity. Strikingly, the lipase showed distinct super-stability to the most tested hydrophilic and hydrophobic solvents (25%, v/v) for 60 days, and different optimal pH in contrast with the alkaline lipase from B. cepacia S31. The lipase demonstrated excellent enantioselective transesterification toward the S-isomer of mandelic acid with a theoretical conversion yield of 50%, ee_p of 99.9% and ee_s of 99.9%, which made it an exploitable biocatalyst for organic synthesis and pharmaceutical industries.     

Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli,purification, characterization and use in continuous cyanide degradation

A cyanide hydratase from Aspergillus niger K10 was expressed in Escherichia coli and purified. Apart from HCN, it transformed some nitriles, preferentially 2-cyanopyridine and fumaronitrile. V_max and K_m for HCN were ca. 6.8 mmol min⁻¹ mg⁻¹ protein and 109 mM, respectively. V_max for fumaronitrile and 2-cyanopyridine was two to three orders of magnitude lower than for HCN (ca. 18.8 and 10.3 μmol min⁻¹ mg⁻¹, respectively) but K_m was also lower (ca. 14.7 and 3.7 mM, respectively). Both cyanide hydratase and nitrilase activities were abolished in truncated enzyme variants missing 18–34 C-terminal aa residues. The enzyme exhibited the highest activity at 45 °C and pH 8–9; it was unstable at over 35 °C and at below pH 5.5. The operational stability of the whole-cell catalyst was examined in continuous stirred membrane reactors with 70-mL working volume. The catalyst exhibited a half-life of 5.6 h at 28 °C. A reactor loaded with an excess of the catalyst was used to degrade 25 mM KCN. A conversion rate of over 80% was maintained for 3 days.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Overexpression and characterization of a glucose-tolerant β-glucosidase from Thermotoga thermarum DSM 5069T with high catalytic efficiency of ginsenoside Rb1 to Rd

The β-glucosidase gene Tt-bgl from Thermotoga thermarum DSM 5069T was cloned and overexpressed in Escherichia coli. A simple strategy, induction at 37 °C with no IPTG, was explored to reduce the inclusion bodies, by which the activity of Tt-BGL was 13 U/mL in LB medium. Recombinant Tt-BGL was purified by heat treatment followed by Ni–NTA affinity. The optimal activity was at pH 4.8 and 90 °C. The activity of Tt-BGL was significantly enhanced by methanol and Al³⁺. The enzyme was stable over pH range of 4.4–8.0, and had a 2-h half life at 90 °C. The V_max for p-nitrophenyl-β-d-glucopyranoside and ginsenoside Rb1 was 142 U/mg and 107 U/mg, while the K_m was 0.59 mM and 0.15 mM, respectively. The activity of the enzyme was not inhibited by ginsenoside Rb1 (36 g/L). It was activated by glucose at concentrations lower that 400 mM. With glucose further increasing, the activity of Tt-BGL was gradually inhibited, but remained 50% of the original value in even as high as 1500 mM glucose. Under the optimal conditions, Tt-BGL transformed ginsenoside Rb1 (36 g/L) to Rd by 95% in 1 h.     

Effect of heating rate on pDNA production by E. coli

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q_P) and average pDNA yield (Y_p/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y_p/x remained relatively constant, whereas Q_P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.     

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.     

Fructose-1,6-bisphosphatase from a hyper-thermophilic bacterium Thermotoga maritima: Characterization,metabolite stability,and its implications

Fructose-1,6-bisphosphatase gene from a hyperthermophilic bacterium Thermotoga maritima was cloned, and the recombinant protein was produced in E. coli, purified, and characterized. The fructose-1,6-bisphosphatase (FBPase) with a molecular mass of ca. 28 kDa was purified from the fusion protein cellulose-binding module (CBM)-intein-FBPase by affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. The substrate fructose 1,6-bisphosphate was not stable at high temperatures, especially at high pHs. The degradation constants of fructose 1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate were determined at different temperatures (37, 60, and 80 °C) and pH 7.5 or 9.0. The k_cat and K_m values of FBPase were 8.57 s⁻¹ and 0.04 mM at 60 °C, as well as 58.7 s⁻¹ and 0.12 mM at 80 °C. This enzyme was very stable at its suboptimal temperatures, with half-life times of ca. 1330 and 55.6 h at 60 and 80 °C, respectively. At 60 °C, this enzyme had an estimated total turn-over number of 20,500,000 (mol product/mol enzyme) and weight-based total turn-over umber of 192,000 (kg product/kg enzyme), respectively. These results indicated that this enzyme would be a stable building block for cell-free synthetic pathway biotransformation (SyPaB) that can implement complicated biochemical reactions. In order to obtain high-yield desired products, we suggest that over-addition or over-expression of the enzymes responsible for converting easily degraded metabolites should be important to prevent unnecessary metabolite loss for in vitro or in vivo synthetic pathway design.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.     

Nitrate and phosphate uptake kinetics of the harmful diatom Eucampia zodiacus Ehrenberg,a causative organism in the bleaching of aquacultured Porphyra thalli

The diatom Eucampia zodiacus Ehrenberg is a harmful diatom which indirectly causes bleaching of aquacultured Nori (Porphyra thalli) through competitive utilization of nutrients during bloom events. In the present study, we experimentally investigated the nitrate (N) and phosphate (P) uptake kinetics of E. zodiacus, Harima-Nada strain. Maximum uptake rates (ρ_max), which were obtained by short-term experiments, were 0.777 and 0.916 pmol cell^?1 h^?1 for nitrate and 0.244 and 0.550 pmol cell^?1 h^?1 for phosphate at 9 and 20 °C, respectively. The half-saturation constants for uptake (K_s) were 2.59 and 2.92 μM N and 1.83 and 4.85 μM P at 9 and 20 °C, respectively. Although the maximum specific uptake rate (V_max; V_max = ρ_max/Q₀, Q₀; minimum cell quota) and V_max/K_s for nitrate at 9 °C are about 1/2 of those obtained at the optimum temperature (20 °C), they are still higher than those obtained for many other phytoplankton at their optimum temperature conditions for uptake. These results suggest that E. zodiacus utilizes nitrogen efficiently at low water temperature, and it is one of the important factors causing the serious damage to Porphyra thalli by bleaching due of this species. For phosphate, the K_s values of E. zodiacus were higher than those reported for other species; the V_max and V_max/K_s values were much lower than those of other diatoms such as Skeletonema costatum (Greville) Cleve. These results suggest that E. zodiacus is disadvantaged compared to other diatom species during competitive utilization of phosphate.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.