Immobilization and characterization of recombinant esterase enzyme on chitosan nanoparticles

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Patterns of functional enzyme activity in fungus farming ambrosia beetles

Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

Campylobacter monitoring in German broiler flocks: an explorative time series analysis

Campylobacter, a major zoonotic pathogen, displays seasonality in poultry and in humans. In order to identify temporal patterns in the prevalence of thermophilic Campylobacter spp. in a voluntary monitoring programme in broiler flocks in Germany and in the reported human incidence, time series methods were used. The data originated between May 2004 and June 2007. By the use of seasonal decomposition, autocorrelation and cross-correlation functions, it could be shown that an annual seasonality is present. However, the peak month differs between sample submission, prevalence in broilers and human incidence. Strikingly, the peak in human campylobacterioses preceded the peak in broiler prevalence in Lower Saxony rather than occurring after it. Significant cross-correlations between monthly temperature and prevalence in broilers as well as between human incidence, monthly temperature, rainfall and wind-force were identified. The results highlight the necessity to quantify the transmission of Campylobacter from broiler to humans and to include climatic factors in order to gain further insight into the epidemiology of this zoonotic disease.

Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST-21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis.

New copper(II) complexes with isoconazole: Synthesis, structures and biological properties

There is an increasing demand for novel metal-based complexes with biologically relevant molecules in technology and medicine. Three new Cu(II) coordination compounds with antifungal agent isoconazole (L), namely mononuclear complexes CuCl2(L)(2) (1), and Cu(O2CMe)(2)(L)(2)center dot 2H(2)O (2) and coordination polymer Cu(pht)(L)(2)(n) (3) (where H(2)pht - o-phthalic acid) were synthesized and characterized by IR spectroscopy, thermogravimetric analysis and X-ray crystallography. X-ray analysis showed that in all complexes, the isoconazole is coordinated to Cu(II) centres by a N atom of the imidazole fragment. In complex I, the square-planar environment of Cu(II) atoms is completed by two N atoms of isoconazole and two chloride ligands, whereas the Cu(II) atoms are coordinated by two N atoms from two isoconazole ligands and two O atoms from the different carboxylate residues: acetate in 2 and phthalate in 3. The formation of an infinite chain through the bridging phthalate ligand is observed in 3. The biosynthetic ability of micromycetes Aspergillus niger CNMN FD 10 in the presence of the prepared complexes 1-3 as well as the antifungal drug isoconazole were studied. Complexes 2 and 3 accelerate the biosynthesis of enzymes (beta-glucosidase, xylanase and endoglucanase) by this fungus. Moreover, a simplified and improved method for the preparation of isoconazole nitrate was developed.

Response of chironomid assemblages to environmental change during the early Late-glacial at Gerzensee, Switzerland

Prior to ca. 14,660 yr BP, during the early Late-glacial (Oldest Dryas), larval assemblages of Chironomidae (Insecta: Diptera) in Gerzensee, Switzerland, were dominated by cold stenothermic taxa as well as by taxa typical of subalpine lakes today. This was the coldest period of the entire sequence. After ca. 14,660 yr BP, in the Late Glacial Interstadial (Bølling–Allerød), a temperature increase is recorded by a sharp rise in the oxygen-isotope ratio in lake marl and by an increase in the organic-matter content of the sediments. Changes in the chironomid fauna then are consistent with rising temperatures. This warming trend is interrupted between 14,070 and 13,940 yr BP, coinciding with the GI-1d cold oscillation, but the change in the chironomid assemblage is more consistent with a response to increasing lake depth and density of aquatic macrophytes than falling temperature. A rise in cold-adapted chironomid taxa between 13,840 and 13,710 yr BP suggests that summer air temperatures may have declined. Changes in the chironomid assemblage after 13,710 yr BP suggest a decline in submerged macrophytes coupled with a rise in lake productivity and summer temperature, although the latter is not reflected in the oxygen-isotope record. This suggests that there may have been increasing seasonality during this period when summer temperatures were rising, driven by rising summer insolation, and winters becoming cooler, which is largely reflected in the oxygen-isotope record. A decline in thermophilic chironomids and a rise in cold-adapted taxa after 13,180 yr BP suggest a response to cooling at the beginning of the Gerzensee Oscillation.

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight {HSPs} families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the {HSP60} family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.