Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1). Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported.     

The effect of solar radiation on the flavonol content in broccoli inflorescence

The effect of solar radiation on the quercetin and kaempferol contents in the inflorescence of three broccoli cultivars (‘Lord’, ‘Marathon’ and ‘Fiesta’) was investigated from 1999 to 2001. Great differences in the contents of both flavonols, dependent on growing time and cultivar, were found. Quercetin and kaempferol contents varied from 14.3 to 81.0 mg kg⁻¹ f.w. and from 35.9 to 213 mg kg⁻¹ f.w., respectively. Inflorescences of the cultivar ‘Lord’ were characterised by the highest mean content of quercetin and those of cultivar ‘Fiesta’ of kaempferol. The contents of both flavonols were highly positively correlated with total solar radiation in the period from planting to the harvest of broccoli inflorescences.     

The production of communication signals at the air-water and water-substrate boundaries.

The 2 interfaces of the aquatic environment, the boundary between air-water and water-substrate, have distinctive physical characteristics that facilitate the production of communication signals. Recent evidence suggests that animals living on or near these boundaries use the interface to generate signals in 2 ways: (1) by producing a signal that propagates along the interface or (2) by producing a signal at the interface that is transmitted and detected within 1 of the component media. By examining the diversity of behaviors used to produce signals at these boundaries, the authors illustrate how human perception of these environments may cause researchers to incorrectly assume the environmental context of signal-generating behaviors and overlook modalities of communication pertinent to the animal. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Control of morphology and nanostructure of copper and cobalt oxalates: effect of complexing ions, polymeric additives and molecular weight

Precipitated oxalates are often nanostructured and can be used as precursors for nanostructured oxides for different applications. The modification of the particle shape and nanostructures of both copper and cobalt oxalates has been demonstrated using polymeric additives or complexing counter-ions. In the case of cobalt oxalate the characteristic elongated rod particle shape (axial ratio of 10) can be modified by using polymethymethacrylate (PMMA) to produce particles with lower axial ratios of 2, through cubes all the way to platelets (axial ratio 0.2). The PMMA inhibits the growth of the particles along the [101] direction more and more strongly as the concentration of the polymer increases. The crystallite size from XRD line broadening is not modified by the PMMA indicating that the PMMA does not influence the nucleation and growth but modifies the aggregation kinetics. Copper oxalates precipitated in the presence of different cellulose derived polymers with different molecular weights and functional groups (methyl and propyl) showed sensitivity to both molecular weight and functional group. Higher molecular weights did not influence the copper oxalate particle shape, whereas methyl cellulose gave elongated particles and propyl celluloses gave platelet like particles. Copper oxalate precipitated in the presence of acetate counter ions gave platelets with an axial ratio of 0.15 compared to the cushion-like morphology (axial ratio 0.5). The primary crystallites were more elongated along the [001] direction in the presence of acetate, modifying the proportion of the hydrophobic and hydrophilic surfaces and hence influencing the aggregation kinetics and particle shape. The copper and cobalt oxalate particle formation seems to be dominated by the primary particle aggregation with the different additives interacting specifically with different crystallographic faces of the primary particles. By tuning this interaction particles with different shapes and substructures can be formed.     

East African climate: 1880–1980

The paper attempts to scan the East African climate during the period 1880–1980 to find any evidence for climatic change. Four indicators of climatic change were investigated: atmospheric temperature, rainfall, mountain glaciers and lake level fluctuations. Available evidence tends to indicate that there is no conclusive evidence that the East African climate has changed over the last century. What can be observed are short‐term climatic fluctuations without long‐term trends.     

Production and purification of active FGF2 via recombinant fusion protein

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.     

Removal of persistent pharmaceutical micropollutants from sewage by addition of PAC in a sequential membrane bioreactor

The performance of a membrane bioreactor operating in a sequential mode (SMBR) using an external flat-plate membrane was investigated. After 200 days of operation, a single addition of 1 g L⁻¹ Powdered Activated Carbon (PAC) was added directly into the mixed liquor in order to enhance the simultaneous removal of nutrients and pharmaceutical micropollutants from synthetic urban wastewater. Throughout the entire operation (288 days), Chemical Oxygen Demand (COD) removal efficiencies were up to 95%, ammonium nitrogen removal was maintained over 70-80%, whereas phosphorus removal achieved only high values (around 80%) after PAC addition. During the operation of the SMBR without PAC addition, micropollutants which exerted a more recalcitrant behaviour were carbamazepine, diazepam, diclofenac and trimethoprim, with no significant removal. On the other hand, moderate removals (42-64%) were observed for naproxen and erythromycin, whereas ibuprofen, roxithromycin and fluoxetine were removed in the range of 71-97%. The addition of PAC into the aeration tank was a successful tool to improve the removal of the more recalcitrant compounds up to 85%. The highest removal with PAC was observed for carbamazepine, trimethoprim as well as for roxithromycin, erythromycin and fluoxetine. The latter four compounds have amine groups and pKa in the range 6.7-10.1, thus the interaction between PAC and the positively charged amino groups might be the cause of their comparatively better results. Microbial ecology present in the biomass showed a higher abundance of Accumulibacter phosphatis as well as the ammonium oxidizing bacteria belonging to the genus Nitrosomonas after PAC addition.     

Clean production in fish canning industries: recovery and reuse of selected wastes

Fish canning industries generate a large number of different wastewaters that usually are treated together in a complex plant. However, some of these streams can be treated to recover additional subproducts as well as reducing the final volume of wastewaters. As a first example, oil and fat contained in cannery wastewaters are usually removed in conventional treatment plants with flotation devices. As a result of this operation, a stream that contains most of the oil, fat and floating matter is obtained, which can account for a large volume with high costs of disposal and/or treatment. A procedure based on the use of heat and centrifugation has been successfully used to generate three fractions: a solid phase, an aqueous phase and a 40–50% oily phase with similar characteristics to a fuel. A second example is focused on the effluent generated during the cooking of tuna, characterized by a high organic matter (COD 59 g/l) and total protein (20 g/l) content. This stream can be treated individually in an evaporation plant to obtain a concentrate with a high protein content, which could have interest as a complement in animal nutrition. Nutritional experiments were carried out on two groups of rats, showing that the addition of small quantities of concentrate to standard food (under 15% of weight), improve its digestibility and food intake.