Expression in Pichia pastoris of Thermostable Endo-1,4-ß-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Biodegradable Interpolycomplexes for Anti-Erosion Stabilization of Soil and Sand.

A linear anionic polysaccharide, sodium alginate, electrostatically interacts with a cationic polysaccharide, quaternized hydroxyethyl cellulose ethoxylate, in aqueous solution, thus giving an interpolyelectrolyte complex. Aqueous solutions of the initial polysaccharides and polycomplexes with an excess of the cationic or anionic polymers were used for the stabilization of soil and sand against water erosion. Physicochemical, mechanical and biological properties of the polymers and coatings were characterized by gravimetric analysis, viscosimetry, mechanical strength assessment, cell viability, and cell-mediated degradation with the following main conclusions. (a) Non-stoichiometric polycomplexes with an excess of cationic or anionic units ("cationic" and "anionic" polycomplexes, respectively) form transparent solutions or stable-in-time dispersions. (b) The complexation results in a decrease in the viscosity of polymer solutions. (c) A complete dissociation of polycomplexes to the initial components is achieved in a 0.2 M NaCl solution. (d) Soil/sand treatment with 1 wt% aqueous solutions of polymers or polycomplexes and further drying lead to the formation of strong composite coatings from polymer(s) and soil/sand particles. (e) Cationic polycomplexes form stronger coatings in comparison with anionic polycomplexes. (f) The polymer-soil coatings are stable towards re-watering, while the polymer-sand coatings show a much lower resistance to water. (g) The individual polysaccharides demonstrate a negligible toxicity to Gram-negative and Gram-positive bacteria and yeast. (h) The addition of Bacillus subtilis culture initiates the degradation of the polysaccharides and polycomplexes. (i) Films from polysaccharides and polycomplexes decompose down to small fragments after being in soil for 6 weeks. The results of the work are of importance for constructing water-resistant, low toxicity and biodegradable protective coatings for soil and sand.

Exobiology of the Venusian Clouds: New Insights into Habitability through Terrestrial Models and Methods of Detection.

The search for life beyond Earth has focused on Mars and the icy moons Europa and Enceladus, all of which are considered a safe haven for life due to evidence of current or past water. The surface of Venus, on the other hand, has extreme conditions that make it a nonhabitable environment to life as we know it. This is in contrast, however, to its cloud layer, which, while still an extreme environment, may prove to be a safe haven for some extreme forms of life similar to extremophiles on Earth. We consider the venusian clouds a habitable environment based on the presence of (1) a solvent for biochemical reactions, (2) appropriate physicochemical conditions, (3) available energy, and (4) biologically relevant elements. The diversity of extreme microbial ecosystems on Earth has allowed us to identify terrestrial chemolithoautotrophic microorganisms that may be analogs to putative venusian organisms. Here, we hypothesize and describe biological processes that may be performed by such organisms in the venusian clouds. To detect putative venusian organisms, we describe potential biosignature detection methods, which include metal-microbial interactions and optical methods. Finally, we describe currently available technology that can potentially be used for modeling and simulation experiments.

Effects of Radiation Intensity, Mineral Matrix, and Pre-Irradiation on the Bacterial Resistance to Gamma Irradiation under Low Temperature Conditions.

Ionizing radiation is one of the main factors limiting the survival of microorganisms in extraterrestrial conditions. The survivability of microorganisms under irradiation depends significantly on the conditions, in which the irradiation occurs. In particular, temperature, pressure, oxygen and water concentrations are of great influence. However, the influence of factors such as the radiation intensity (in low-temperature conditions) and the type of mineral matrix, in which microorganisms are located, has been practically unstudied. It has been shown that the radioresistance of bacteria can increase after their exposure to sublethal doses and subsequent repair of damage under favorable conditions, however, such studies are also few and the influence of other factors of extraterrestrial space (temperature, pressure) was not studied in them. The viability of bacteria Arthrobacter polychromogenes, Kocuria rosea and Xanthomonas sp. after irradiation with gamma radiation at a dose of 1 kGy under conditions of low pressure (1 Torr) and low temperature (-50 °C) at different radiation intensities (4 vs. 0.8 kGy/h) with immobilization of bacteria on various mineral matrices (montmorillonite vs. analogue of lunar dust) has been studied. Native, previously non-irradiated strains, and strains that were previously irradiated with gamma radiation and subjected to 10 passages of cultivation on solid media were irradiated. The number of survived cells was determined by culturing on a solid medium. It has been shown that the radioresistance of bacteria depends significantly on the type of mineral matrix, on which they are immobilized, wherein montmorillonite contributes to an increased survivability in comparison with a silicate matrix. Survivability of the studied bacteria was found to increase with decreasing radiation intensity, despite the impossibility of active reparation processes under experimental conditions. Considering the low intensity of radiation on various space objects in comparison with radiobiological experiments, this suggests a longer preservation of the viable microorganisms outside the Earth than is commonly believed. An increase in bacterial radioresistance was revealed even after one cycle of irradiation of the strains and their subsequent cultivation under favourable conditions. This indicates the possibility of hypothetical microorganisms on Mars increasing their radioresistance.

Soil bacterial communities of Sahara and Gibson deserts: Physiological and taxonomical characteristics.

The purpose of this research was to investigate the structure of soil bacteria communities present in the Gibson (Australia) and the Sahara (Egypt) deserts, as well as to estimate strain survivability under different environmental factors. It should be noticed that the screening of bacterial resistance to wide spectra of principally different stress conditions was performed for the first time. Experiments were conducted with culturable bacterial communities. Strains were identified using 16S rRNA sequencing, and stress-tolerance was estimated by growing strains in various nutrient media. In order to characterize the community the epifluorescent microscopy and multisubstrate testing were also performed. High bacterial abundance in the desert soils was detected, and there was seen a significant proportion of culturable cells. The close numbers of psychotropic and mesophilic bacteria in arid ecosystems were revealed. The representatives of the Actinobacteria phylum were dominant in the microbial communities, and Firmicutes, Proteobacteria, and Bacteroidetes phyla representatives were also identified. Tolerance of the axenic bacterial cultures, isolated from arid desert ecotopes, to temperature, pH, salts (KCl, NaCl, MgSO4, NaHCO3), strong oxidizers (Mg(ClO4)2), and antibiotics (ampicillin, cephalexin, chloramphenicol, tetracycline, doxycycline, kanamycin, rifampicin) was studied. The bacterial isolates were characterized by polyextremotolerance and by the ability to maintain metabolic activity in vitro while influenced by a wide range of physicochemical and biotic factors.