Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH? group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.     

Utilizing pigment-producing fungi to add commercial value to American beech (<Emphasis Type="Italic">Fagus grandifolia</Emphasis>)

American beech (Fagus grandifolia) is an abundant, underutilized tree in certain areas of North America, and methods to increase its market value are of considerable interest. This research utilized pigment-producing fungi to induce color in American beech to potentially establish its use as a decorative wood. Wood samples were inoculated with Trametes versicolor, Xylaria polymorpha, Inonotus hispidus, and Arthrographis cuboidea to induce fungal pigmentation. Black pigmentation (T. versicolor, X. polymorpha, I. hispidus) was sporadic, occurred primarily on the surfaces of the heartwood, but not internally. Pink pigmentation (A. cuboidea) occurred throughout all of the tested beech samples, but was difficult to see in the heartwood due to the darker color of the wood. To increase the visibility of the pink stain, beech blocks were pretreated with T. versicolor for 4 weeks before being inoculated with A. cuboidea. This method significantly increased the saturation of the pink stain on both beech heartwood and sapwood, creating coloration similar to that found on sugar maple. This value-adding process should be particularly effective for small-scale wood pigmentation, and should help establish a market for this currently underutilized wood species.     

Flooding affects uptake and distribution of carbon and nitrogen in citrus seedlings

Soil flooding has been widely reported to affect large areas of the world. In this work, we investigated the effect of waterlogging on citrus carbon and nitrogen pools and partitioning. Influence on their uptake and translocation was also studied through 1?N and 13C labeling to provide insight into the physiological mechanisms underlying the responses. The data indicated that flooding severely reduced photosynthetic activity and affected growth and biomass partitioning. Total nitrogen content and concentration in the plant also progressively decreased throughout the course of the experiment. After 36 days of treatment, nitrogen content of flooded plants had decreased more than 2.3-fold compared to control seedlings, and reductions in nitrogen concentration ranged from 21 to 55% (in roots and leaves, respectively). Specific absorption rate and transport were also affected, leading to important changes in the distribution of this element inside the plant. Additionally, experiments involving labeled nitrogen revealed that 1?N uptake rate and accumulation were drastically decreased at the end of the experiment (93% and 54%, respectively). 13CO? assimilation into the plant was strongly reduced by flooding, with δ13C reductions ranging from 22 to 37% in leaves and roots, respectively. After 36 days, the relative distribution of absorbed 13C was also altered. Thus, 13C recovery in flooded leaves increased compared to controls, whereas roots exhibited the opposite pattern. Interestingly, when carbohydrate partitioning was examined, the data revealed that sucrose concentration was augmented significantly in roots (37-56%), whereas starch was reduced. In leaves, a marked increase in sucrose was detected from the first sampling onwards (36-66%), and the same patter was observed for starch. Taken together, these results indicate that flooding altered carbon and nitrogen pools and partitioning in citrus. On one hand, reduced nitrogen concentration appears to be a consequence of impaired uptake and transport. On the other hand, the observed changes in carbohydrate distribution suggest that translocation from leaves to roots was reduced, leading to significant starch accumulation in leaves and further decreases in roots.     

S-layer homology domain proteins Csac_0678 and Csac_2722 are implicated in plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus

The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.     

Arbuscular mycorrhiza confers Pb tolerance in <Emphasis Type="Italic">Calopogonium mucunoides</Emphasis>

Heavy metals (HMs) are environmental pollutants of great concern to humans because of their high potential toxicity. Lead is a HM that is present in the soil in very small amounts, but anthropogenic activities have increased its content in some locations, which can make these areas unproductive or inappropriate for crop production. However, there are some plants that can grow in contaminated soils and, thus, can be useful for the removal or stabilisation of such contaminants. In addition, plants that are not able to tolerate high concentrations of HMs in the soil can become tolerant or increase their performance when associated with arbuscular mycorrhizal (AM) fungi. Accordingly, this study was carried out to verify whether the inoculation of Glomus etunicatum, an AM fungus species, in Calopogonium mucunoides would influence plant tolerance to increasing concentrations of Pb in the soil. The experimental design was completely randomised, in a 2 × 4 factorial design, and the treatments consisted of inoculation (or not) with the AM fungus, G. etunicatum, and the addition of four Pb concentrations (0, 250, 500 or 1,000 mg kg⁻¹) to the soil. The results showed that the association of C. mucunoides with G. etunicatum promoted biomass production, and nutrient uptake (P, S and Fe) was also positively influenced by mycorrhization. The malondialdehyde content was higher in non-mycorrhizal leaves, suggesting a reduction in the damage to membranes by lipid peroxidation in plants associated with mycorrhizae. However, the Pb concentration in the shoots did not differ between the mycorrhizal and non-mycorrhizal plants. The results of our study suggest that the AM symbiosis can be considered very effective in contributing to the tolerance of C. mucunoides to Pb.     

Convective heat transfer from a nude body under calm conditions: assessment of the effects of walking with a thermal manikin

The present experimental work is dedicated to the analysis of the effect of walking on the thermal insulation of the air layer (I _a ) and on the convective heat transfer coefficients (h _conv ) of the human body. Beyond the standing static posture, three step rates were considered: 20, 30 and 45 steps/min. This corresponds to walking speeds of approximately 0.23, 0.34 and 0.51 m/s, respectively. The experiments took place in a climate chamber with an articulated thermal manikin with 16 independent parts. The indoor environment was controlled through the inner wall temperatures since the objective of the tests was restricted to the influence of the walking movements under calm conditions. Five set points were selected: 10, 15, 20, 25 and 30°C, and the operative temperature within the test chamber varied between 11.9 and 29.6°C. The highest and lowest I _a values obtained were equal to 0.87 and 0.71 clo, respectively, and the reduction in insulation due to walking ranged between 9.8 and 11.5%. The convective coefficients (h _conv ) for the whole body and for the different body segments were also determined for each step rate. In the case of the whole body, for the standing static reference posture, the mean value of h _conv was equal to 3.3 W/m²°C and a correlation [Nu = Nu(Gr)] for natural convection is also presented in good agreement with previous results. For the other postures, the values of h _conv were equal to 3.7, 3.9 and 4.2 W/m²°C, respectively for 20, 30 and 45 steps/min.     

Climate change and future temperature-related mortality in 15 Canadian cities

The environmental changes caused by climate change represent a significant challenge to human societies. One part of this challenge will be greater heat-related mortality. Populations in the northern hemisphere will experience temperature increases exceeding the global average, but whether this will increase or decrease total temperature-related mortality burdens is debated. Here, we use distributed lag modeling to characterize temperature-mortality relationships in 15 Canadian cities. Further, we examine historical trends in temperature variation across Canada. We then develop city-specific general linear models to estimate change in high- and low-temperature-related mortality using dynamically downscaled climate projections for four future periods centred on 2040, 2060 and 2080. We find that the minimum mortality temperature is frequently located at approximately the 75th percentile of the city’s temperature distribution, and that Canadians currently experience greater and longer lasting risk from cold-related than heat-related mortality. Additionally, we find no evidence that temperature variation is increasing in Canada. However, the projected increased temperatures are sufficient to change the relative levels of heat- and cold-related mortality in some cities. While most temperature-related mortality will continue to be cold-related, our models predict that higher temperatures will increase the burden of annual temperature-related mortality in Hamilton, London, Montreal and Regina, but result in slight to moderate decreases in the burden of mortality in the other 11 cities investigated.