Discovery of a novel series of benzimidazole derivatives as diacylglycerol acyltransferase inhibitors

A novel series of benzimidazole derivatives was prepared and evaluated for their diacylglycerol acyltransferase (DGAT) inhibitory activity using microsome from rat liver. Among the newly synthesized compounds, furfurylamine containing benzimidazole carboxamide 10j showed the most potent DGAT inhibitory effect (IC₅₀ = 4.4 μM) and inhibited triglyceride formation in HepG2 cells. Furthermore, compound 10j reduced body weight gain of Institute of Cancer Research mice on a high-fat diet and decreased levels of total triglyceride, total cholesterol, and LDL-cholesterol in the blood accompanied with a significant increase in HDL-cholesterol level.     

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.     

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.     

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.     

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.     

Isolation and properties of β-xylosidase from Aspergillus niger GS1 using corn pericarp upon solid state fermentation

There is growing interest in developing high-yield and low-cost production of xylanolytic enzymes for industrial applications using agroindustrial byproducts. A native strain of Aspergillus niger GS1 was used to produce β-xylosidase (EC 3.2.1.37) on solid state fermentation using corn pericarp (CP) with innovative alkaline electrolyzed water (AEW) pretreatment at room temperature. β-xylosidase was purified by ammonium sulfate fractionation followed by anion exchange and hydrophobic interaction chromatographies. β-Xylosidase showed a molecular weight of 111 kDa, isoelectric point of 5.35 and specific activity of 386.7 U (mg protein)^?1, using p-nitrophenyl-β-d-xylopyranoside as substrate, at pH 5 and 60 °C, and optimal activity at pH 4.5. Optimal temperature was 65 °C, showing full activity after 1 h at 60 °C. Activity was reduced by 1 mM β-mercaptoethanol (55.6 ± 0.1%), and enhanced by 1 mM SDS (11.0 ± 0.03%). K_m and V_max were 6.1 ± 0.9 mM and 1364 ± 105 U (mg protein)^?1, respectively, whereas k_cat was 5.1 s^?1. A predominant α-helix (41%) was determined from circular dichroism on β-xylosidase, while thermal transition profiles produced a T_m of 54.1 ± 5.8 °C, enthalpy change for unfolding of 67.4 ± 6.7 kJ/mol, and onset temperature of 37 °C. Pre-treatment of CP using AEW is an ecologically friendly alternative to chemical and heat treatments for the production of relatively high levels of β-xylosidase.     

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.     

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.     

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.     

Purification of a laccase from fungus combs in the nest of Odontotermes formosanus

A new enzyme was isolated from the fungus combs in the nest of Odontotermes formosanus and identified as a laccase. The single laccase was purified with a purification factor of 16.83 by ammonium sulphate precipitation and anion exchange chromatography, to a specific activity of 211.11 U mg⁻¹. Its molecular mass was 65 kDa. The optimum pH value and temperature were 4.0 °C and 10 °C with ABTS as the substrate, respectively. The enzyme activity stabilized at temperatures between 10 °C and 30 °C and decreased rapidly when the temperature was above 30 °C. The V_max and K_m values were 3.62 μmol min⁻¹ mg⁻¹ and 119.52 μM, respectively. Ethanol concentration affected laccase activity, inhibiting 60% of enzyme activity at a concentration of 70%. Metal ions of Mg²⁺, Ba²⁺ and Fe²⁺ showed inhibition on enzyme activity of 17.2%, 5.3% and 9.4%, respectively, with the increase of metal ions concentration from 1 mM to 5 mM. Especially Fe²⁺ strongly inhibited enzyme activity up to 89% inhibition at a concentration of 1 mM.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

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.     

Growth and development of cotton (Gossypium hirsutum L.) in response to CO2 enrichment under two different temperature regimes

An increase in atmospheric CO₂ concentration ([CO₂]) together with other climate change factors could greatly affect agricultural productivity. Understanding the impact of the change in atmospheric [CO₂] in conjunction with the ongoing global change is crucial to prepare for mitigation and any adaptation for future agricultural production. The main goal of this project was to study the time-course pattern of cotton plant growth in response to [CO₂] and temperature to investigate the hypothesis that whether response to elevated [CO₂] would change at different temperatures. An experiment was conducted in the controlled-environment chambers of the Georgia Envirotron with two different day/night temperatures levels, e.g., 25/15 °C and 35/25 °C, and three CO₂ concentrations, e.g., 400, 600 and 800 μmol l^?1. The experimental design was completely randomized with four replicates (plastic containers) per treatment. Growth analysis was conducted at bi-weekly intervals during the growing season. In addition, leaf area, leaf dry mass, root dry mass, square dry mass, boll dry mass and total above dry mass per plant were also measured at each sampling. Plant traits, including plant height, number of leaves, number of squares and number of bolls were recorded weekly. The number of days to emergence, squaring, flowering and maturity were also observed. The results showed that by increasing [CO₂] to 600 μmol l^?1 total biomass increased at both temperature levels, but a further increase of [CO₂] up to 800 μmol l^?1 increased total biomass only at the temperature of 35/25 °C. Throughout the growing season, there was no significant effect of [CO₂] levels on LAI. Increasing temperature from 25/15 °C to 35/25 °C had a positive impact on LAI across all CO₂ levels (P < 0.05). Increasing CO₂ from 400 to 600 μmol l^?1 significantly increased the number of squares by 31.4%, but a further increase to 800 μmol l^?1 caused a 6.6% decrease (non-significant) in the number of squares. The interactive effects of [CO₂] and temperature indicated that at a higher temperature, CO₂ would be more beneficial as we proceed towards the end of the growing season. However, further studies are needed to really understand the interaction between higher [CO₂] and temperature levels and cultivar characteristics.     

Spontaneously reduced body temperature and gasping ability as a mechanism of extreme tolerance to asphyxia in neonatal rats

The aim of the investigation was to verify our hypothesis that extreme tolerance of newborn rodents to anoxia is determined by their ability to maintain reduced body temperature and to keep on gasping.Newborn Wistar rats were used. In separate experiments we checked (1) effect of extreme thermal conditions on rectal temperature (T_re) of the newborns in their nests; (2) effect of ambient temperature (T_a) on oxygen consumption; (3) effects of controlled changes in T_re on thermoregulatory and respiratory responses to anoxia and on anoxia tolerance.In their nests rat pups controlled T_re at 32–36 °C while the T_re–T_a difference changed within a range of 1–20 °C. The lowest oxygen consumption of ∼24 ml O₂ kg⁻¹ min⁻¹ was recorded at T_a of 32 °C. Pups, exposed to anoxia at their normal T_re of 33 °C, were able to decrease T_re by another 1.7 °C and they kept on extremely slow and quiescent gasping for scheduled 25 min. In contrast, rats at T_re of 37 °C and 39 °C reached a critical phase of accelerated and shallow gasping after 14.95±0.40 min and 9.25±0.30 min, respectively.In conclusion, reduced T_re and unique gasping ability make newborn rats extremely tolerant to asphyxia.     

Metabolic,hygric and ventilatory physiology of the red-tailed phascogale (Phascogale calura; Marsupialia,Dasyuridae): Adaptations to aridity or arboreality?

The red-tailed phascogale is a small arboreal dasyurid marsupial that inhabits semi-arid to arid regions of Western Australia's wheat belt. Its body mass (34.7 g) is only ～15% of that predicted based on its phylogenetic position among other dasyuromorphs; we interpret this as an adaptation to its scansorial and semi-arid/arid lifestyle. The standard physiology of this species at a thermoneutral ambient temperature of 30 °C conforms to that of other dasyurid marsupials; body temperature (34.7 ± 0.37 °C), basal metabolic rate (0.83 ± 0.076 mL O₂ g^?1 h^?1), evaporative water loss (1.68 ± 0.218 mg H₂O g^?1 h^?1) and wet thermal conductance (3.8 ± 0.26 J g^?1 h^?1 °C^?1) all fall within the 95% predication limits for the respective allometric relationships for other dasyurid species. Thermolability confers an energy savings at low T_a and water savings at high T_a. Torpor, observed at low T_a, was found to be more beneficial for energy savings than for water economy. The red-tailed phascogale therefore has a physiology suitable for the challenges of arid environments without any obvious requirement for adaptations to its scansorial lifestyle, other than its considerably lower-than-expected body mass.     

Cell-associated acid β-xylosidase production by Penicillium sclerotiorum

In recent decades, β-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 °C, under stationary condition for six days. The optimum activity temperature was 60 °C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 °C, with half-life of 240 and 232 min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The β-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V_max and K_m values was 0.48 μmol PNXP min^?1 mg^?1 protein and 0.75 mM, respectively. The enzyme was xylose tolerant with a K_i of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.     

Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate (K_m = 11 μM, k_cat = 0.79 s^?1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP₅, K_m = 0.07 μM, k_cat = 0.024 s^?1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg²⁺ (10–20 mM) or Mn²⁺ (1 mM) were preferred for nucleotide hydrolysis and Mg²⁺ (0.5–1 mM) or Co²⁺ (1–100 μM) for PP-InsP₅ hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H₂O₂ and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP₅ to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP₅ found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP₅ during development.     

The effect of sub-optimal temperature on specific sulfidogenic activity of mesophilic SRB in an H2-fed membrane bioreactor

The sulfidogenic activity of two mesophilic sulfate reducing enrichment cultures was studied in H₂-fed membrane bioreactors. The two enrichment cultures had different origins; one of them was a mesophilic and the other a psychrotolerant mesophilic culture. The operational temperatures of the reactors were gradually changed: for one the temperature was increased from 9 to 30 °C and for the other it was decreased from 35 to 9 °C. The specific sulfidogenic activities were 21–31, 52–53 and 57–92 mmol SO₄²⁻ g VSS⁻¹ d⁻¹ at 9, 15 and 30–35 °C, respectively. The sulfate reduction rate of the SRB stabilized to a lower level after the temperature was decreased. The percent electron flow to sulfate reduction was on average 24–32, 50 and 47–69% at 9, 15 and 30–35 °C, respectively. The capability of mesophilic SRB to oxidize electron donor decreased as the temperature was decreased. The results indicate that starting of the reactor operation at 9 °C resulted in higher sulfidogenic activity at sub-optimal temperatures and selective enrichment of the psychrotolerant species improved. The start-up of the reactor at 35 °C resulted in decreased sulfidogenic activity as the temperature was decreased. This indicates that the operational temperature of bioreactors with mesophilic SRB can be decreased to 15–20 °C and the sulfidogenic activity will decrease by 10–40%. Moreover, an operational temperature of 9 °C seems to be close to the lower limit of active sulfate reduction for the mesophilic enrichment cultures used in this study.     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.