The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

Polysulphone and poly(phenylene sulphide) blends: 1. Thermal characterization and phase morphology

The phase behaviour and morphology of injection moulded specimens of polysulphone (PSF) and poly(phenylene sulphide) (PPS) blends were studied by differential scanning calorimetry (d.s.c.), dynamical mechanical thermal analysis (d.m.t.a.) and transmission electron microscopy (TEM). The blends are phase separated regardless of the blend composition as revealed by d.s.c., d.m.t.a. and TEM. Upon annealing at 160°C for 2 h, d.m.t.a. results indicate that the PPS phase remains in the amorphous state at compositions <10%. At compositions between 20 and 35%, the PPS appears to be dispersed in a mixed mode of amorphous and crystalline domains. Above 35% the PPS phase appears to become fully crystallized upon annealing of the blends. At 10% PPS, TEM results showed 35–200 nm size dispersion both in the as-moulded and in the annealed specimens. At 20% the PPS phase varied widely in size, from 35 nm to tens of micrometres but remained as an included phase. TEM also revealed a compound morphology of the included phase at a composition of 50 wt% of each component.     

Crop residue,manure and fertilizer in dryland maize under reduced tillage in northern China: I grain yields and nutrient use efficiencies

The rapidly increasing population and associated quest for food and feed in China has led to increased soil cultivation and nitrogen (N) fertilizer use, and as a consequence to increased wind erosion and unbalanced crop nutrition. In the study presented here, we explored the long-term effects of various combinations of maize stover, cattle manure and nitrogen (N) and phosphorus (P) fertilizer applications on maize (Zea mays L.) yield and nutrient and water use efficiencies under reduced tillage practices. In a companion paper, we present the effects on nutrient balances and soil fertility characteristics. The ongoing factorial field trial was conducted at Shouyang Dryland Farming Experimental Station in northern China from 1993 onwards. The incomplete, determinant-optimal design comprised 12 treatments, including a control treatment, in duplicate. Grain yields and N, P, and potassium (K) uptakes and N, P and K use efficiencies were greatly influenced by the amount of rain during the growing season (GSR), and by soil water at sowing (SWS). There were highly significant interactions between GSR and added stover and manure, expressed in complex annual variations in grain yield and N, P and K use efficiencies. Annual mean grain yields ranged from 3,000 kg ha⁻¹ to 10,000 kg ha⁻¹ and treatment mean yields from 4,500 kg ha⁻¹ to 7,000 kg ha⁻¹. Balanced combination of stover (3,000–6,000 kg), manure (1,500–6,000 kg) and N fertilizer (105 kg) gave the highest yield. Stover and manure were important for supplying K, but the effects differed greatly between years. Overall mean N recovery efficiency (NRE) ranged from 28% to 54%, depending on N source. NRE in wet years ranged from 50% to 90%. In conclusion, balanced combinations of stover, manure and NP fertilizer gave the highest yield and NRE. Reduced tillage with adding stover and manure in autumn prior to ploughing is effective in minimizing labor requirement and wind erosion. The potentials of split applications of N fertilizer, targeted to the need of the growing crop (response farming), should be explored to further increase the N use efficiency.     

The influence of temperature on the impact behavior of unidirectional glass fiber-epoxy composite

The influence of temperature on the impact behavior of unidirectional glass fiber-epoxy composite was studied in the range of −20 and 150°C using a 3-point drop weight test at a constant impact velocity of 2.2 m/s (5 mph). The impact energy per unit deformed volume was found to be very sensitive to the ratio length/thickness of the specimens. When the ratio was 4, the impacted specimens exhibited extensive delamination (shear failure) and the energy absorbed by the composite was about 14 MJ/m³ between −20 and 100°C, and 18 MJ/m³ at 150°C. At length/thickness above 16 the failure was mostly in tension, and the impact energy approached a constant value of about 3.5 MJ/m³, independent of temperature.     

The Rheology of Wetting By Polymer Melts

Theoretically, the rate of capillary penetration of a polymer melt into a slit, a model for a surface irregularity, has been shown to depend on γcosθ/η) where γ refers to the surface tension of the liquid, η its viscosity and θ a time-dependent contact angle. Analytical expressions relating the depth of penetration with time have been experimentally verified by observations of the penetration of molten polyethylene and poly-(ethylene-vinyl acetate) into aluminum channels. Values of η, calculated from the observed data, agree closely with independent determinations of this material parameter. A theoretical treatment has also been developed which describes the velocity of spreading of a liquid drop over a flat surface. Flow equations for the flow of free films were adapted for this purpose. The spreading velocity is predicted to depend on the product of three factors (1) a scaling factor, (γ/η1R_o), where R_o is the initial radius of curvature, (2) cosθ_∞. (l-cosθ/cosθ_∞) where θ_∞ refers to the equilibrium value of θ, and (3) geometric terms. After demonstrating that a drop of molten polymer may be treated as a spherical cap, the predicted dependence of spreading rate on drop size, cosθ_∞ (nature of the substrate) and the scaling factor was experimentally verified. Some discrepancies noted at long times and high temperatures are discussed.     

Impact energy absorption of unidirectional glass fiber-epoxy composites

The influence of resin and fiber properties on the impact behavior of composites can be assessed in a three-point drop-weight impact test by varying the length-to-thickness ratio of the specimen. The fracture initiation energy per unit deformed volume, w_i, can be described by the expression: where τ₁₁ is the tensile stress, τ₁₂ is shear stress; E₁₁ is tensile modulus; and G₁₂ is shear modulus. A unidirectional glass fiberepoxy composite was tested at impact velocities of 2.2 m/s (5 mph) and 4.5 m/s (10 mph). The energy to initiate fracture, w_i, was in the range of 2 to 3.5 MJ/m³, apparently independent of impact velocity. The total energy absorbed by the impacted composite was also found to be independent of impact rate but very sensitive to the length to thickness ratio: about 13 and 3.5 MJ/m3 at the corresponding ratios of 4.6 and 23. It was generally observed that high fracture energy is associated with extensive specimen delamination, i.e. failure in shear.     

Uncertainties in the fate of nitrogen I: An overview of sources of uncertainty illustrated with a Dutch case study

This study focuses on the uncertainties in the fate of nitrogen (N) in the Netherlands. Nitrogen inputs into the Netherlands in products, by rivers, and by atmospheric deposition, and microbial and industrial fixation of atmospheric N₂ amount to about 4450 Gg N y^–1. About 60% of this N is transported out of the Netherlands in products. The fate of the remaining 40%, however, is less clear. We discuss uncertainties in losses to the atmosphere (as ammonia or through denitrification), by leaching and runoff, and in N accumulation in biomass and soils. These processes may account for the fate of about 40% of the N in the Netherlands, and for the fate of about 60% of the N in Dutch agricultural soils. Reducing uncertainties in the estimates of these fluxes is necessary for reducing the impact of excess N in the environment. In particular, monitoring the environmental effects of ammonia emissions and nitrate leaching to groundwater and aquatic systems requires an increased understanding of the fate of N. Uncertainties arise because (1) some N fluxes cannot be measured directly and are usually quantified indirectly as the balance in N budgets, (2) direct measurements of N fluxes have inevitable inaccuracies, (3) lack of experimental data and other information (e.g. statistics) needed for upscaling, (4) large spatial and temporal variability of fluxes, and (5) poor understanding of the processes involved. These uncertainties can be reduced by additional experimental studies and by further development of process-based models and N budget studies. We prioritize these future research needs according to a range of different criteria.     

Use of the nitrification inhibitor dicyandiamide (DCD) does not mitigate N2O emission from bovine urine patches under Oxisol in Northwest Brazil

Nutrient Cycling in Agroecosystems - Animal production systems are important sources of greenhouse gases (GHGs), especially methane (CH4) and nitrous oxide (N2O). GHG emissions from urine patches...     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

Increased emissions of nitric oxide and nitrous oxide following tillage of a perennial pasture

About 40% of the agricultural land in the European Union (EU) is grassland used for animal production. When grassland is tilled, organically bound carbon and nitrogen are released, providing substrates for nitrifying and denitrifying microorganisms. The aim of this study was to examine the immediate effects of tillage of a perennial grassland carried out on different dates, on the emissions of nitric oxide (NO) and nitrous oxide (N₂O), monitored intensively over a 5-day period, in a humid, dairy farming area of northern Spain. Soil was tilled 12 days and 2 days prior to fertiliser application. Tillage, time of tillage, and N fertiliser application affected NO and N₂O emissions. Tillage 12 days before the start of the flux measurements resulted in higher emissions than tillage one day before, the difference being related to differences in soil mineral N and water-filled pore space (WFPS). Emissions of NO peaked at a WFPS of 50–60%, while N₂O fluxes peaked at 70–90% WFPS. Loss of N was greater as N₂O than as NO. The total loss of N as N₂O plus NO ranged from 0.027 kg N ha^–1 in unfertilised plots to 0.56 kg N ha^–1 in the tilled and N fertilised plot. Thereafter emissions decreased rapidly to low values. The results of this study indicate that tillage of perennial grassland may release large amounts of NO and N₂O, the amounts also depending on moisture conditions and addition of N fertiliser. We suggest that in order to reduce such emissions, application of N fertiliser should not immediately follow tillage of perennial grassland, as there is an extra supply of N from mineralisation of organic matter at this time.