The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.
BACKGROUND: Millions of tons of feather waste from the poultry industry are disposed of annually despite containing a high level of keratin. The aim of this study was to compare the hydrolysis of non-treated feather waste using three different approaches (whole cell microbial digestion, enzymatic and chemical cleavage) and to test the use of hydrolysates as peptone substitutes in a culture medium. RESULTS: Among bacterial isolates, Pseudomonas sp. P5 exhibited the highest keratinolytic activity and efficiency to hydrolyse raw feather material. The hydrolysates contained up to 301 mg L −1 of free amino acids and 6.2 g L −1 of peptides. Hydrolysates obtained by digestion using semi-purified keratinase from Pseudomonas sp. P5 were richer in amino acids (1191 mg L −1 , 56% essential ones) but peptides were present in lower amounts (up to 3.3 g L −1 ). The third approach was feather treatment under mild alkaline conditions. This provided the highest amount of peptides (17.2 g L −1 ) but a significantly lower level of amino acids, especially the essential ones. CONCLUSIONS: All approaches tested could convert raw feather waste into products of commercial value with proven use in a cultivation medium. The level of peptides, their molecular size and amino acid composition was dependent on the method used.
Electrospun polyamide (PA) nanofibers have great potential for
medical applications (in dermatology as antimicrobial compound carriers
or surgical sutures). However, little is known about microbial colonization
on these materials. Suitable methods need to be chosen and optimized
for the analysis of biofilms formed on nanofibers and the influence
of their morphology on biofilm formation. We analyzed 11 PA nanomaterials,
both nonfunctionalized and functionalized with AgNO3, and
tested the formation of a biofilm by clinically relevant bacteria
(Escherichia coli CCM 4517, Staphylococcus aureus CCM 3953, and Staphylococcus epidermidis CCM 4418). By four different
methods, it was confirmed that all of these bacteria attached to the
PAs and formed biofilms; however, it was found that the selected method
can influence the outcomes. For studying biofilms formed by the selected
bacteria, scanning electron microscopy, resazurin staining, and colony-forming
unit enumeration provided appropriate and comparable results. The
values obtained by crystal violet (CV) staining were misleading due
to the binding of the CV dye to the PA structure. In addition, the
effect of nanofiber morphology parameters (fiber diameter and air
permeability) and AgNO3 functionalization significantly
influenced biofilm maturation. Furthermore, the correlations between
air permeability and surface density and fiber diameter were revealed.
Based on the statistical analysis, fiber diameter was confirmed as
a crucial factor influencing biofilm formation (p ≤ 0.01). The functionalization of PAs with AgNO3 (from 0.1 wt %) effectively suppressed biofilm formation. The PA
functionalized with a concentration of 0.1 wt % AgNO3 influenced
the biofilm equally as nonfunctionalized PA 8% 2 g/m2.
Therefore, biofilm formation could be affected by the above-mentioned
morphology parameters, and ultimately, the risk of infections from
contaminated medical devices could be reduced.
Stabilized sewage sludge is applied to agricultural fields and farmland due to its high organic matter content. The aim of this study was to investigate the effects of two types of sludge stabilization, mesophilic anaerobic digestion (MAD) and thermophilic anaerobic digestion (TAD), on bacterial communities in sludge, including the presence of pathogenic microorganisms. Bacterial community structure and phylogenetic diversity were analyzed in four sewage sludge samples from the Czech Republic. Analysis of 16S ribosomal RNA (rRNA) genes showed that investigated sludge samples harbor diverse bacterial populations with only a few taxa present across all samples. Bacterial diversity was higher in sludge samples after MAD versus TAD treatment, and communities in MAD-treated sludge shared the highest genetic similarities. In all samples, the bacterial community was dominated by reads affiliated with Proteobacteria. The sludge after TAD treatment had considerably higher number of reads of thermotolerant/thermophilic taxa, such as the phyla Deinococcus-Thermus and Thermotogae or the genus Coprothermobacter. Only one operational taxonomic unit (OTU), which clustered with Rhodanobacter, was detected in all communities at a relative abundance >1 %. All of the communities were screened for the presence of 16S rRNA gene sequences of pathogenic bacteria using a database of 122 pathogenic species and ≥98 % identity threshold. The abundance of such sequences ranged between 0.23 and 1.57 % of the total community, with lower numbers present after the TAD treatment, indicating its higher hygienization efficiency. Sequences clustering with nontuberculous mycobacteria were present in all samples. Other detected sequences of pathogenic bacteria included Streptomyces somaliensis, Acinetobacter calcoaceticus, Alcaligenes faecalis, Gordonia spp., Legionella anisa, Bordetella bronchiseptica, Enterobacter aerogenes, Brucella melitensis, and Staphylococcus aureus.
Although nanomaterials are used in many fields, little is known about the fundamental interactions between nanomaterials and microorganisms. To test antimicrobial properties and retention ability, 13 electrospun polyamide (PA) nanomaterials with different morphology and functionalization with various concentrations of AgNO3 and chlorhexidine (CHX) were analyzed. Staphylococcus aureus CCM 4516 was used to verify the designed nanomaterials’ inhibition and permeability assays. All functionalized PAs suppressed bacterial growth, and the most effective antimicrobial nanomaterial was evaluated to be PA 12% with 4.0 wt% CHX (inhibition zones: 2.9 ± 0.2 mm; log10 suppression: 8.9 ± 0.0; inhibitory rate: 100.0%). Furthermore, the long-term stability of all functionalized PAs was tested. These nanomaterials can be stored at least nine months after their preparation without losing their antibacterial effect. A filtration apparatus was constructed for testing the retention of PAs. All of the PAs effectively retained the filtered bacteria with log10 removal of 3.3–6.8 and a retention rate of 96.7–100.0%. Surface density significantly influenced the retention efficiency of PAs (p ≤ 0.01), while the effect of fiber diameter was not confirmed (p ≥ 0.05). Due to their stability, retention, and antimicrobial properties, they can serve as a model for medical or filtration applications.
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