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Comparative investigation of steroid transforming activity and ultrastructural changes of bis(2-ethylhexyl)phthalate (BEHP, phthalate) treated Mycobacterium sp. NRRL B-3805 cells was carried out. Transformation of β-sitosterol into androstenedione (AD) and androstadienedione (ADD) was performed in phthalate medium by resting cells preincubated in the organic solvent for a period from 3 to 24 h. It was observed that a preincubation greater than 12 h leads to the development of dense formations on the cells surface, reduction in the cell turgor, disruption in the cell walls, and formation of zones with reduced electron density. The preincubation for 24 h causes deeper changes in the cell ultrastructure but the treated cells retain their steroid transforming activity, allowing up to 80% of the substrate to be converted into AD and ADD. A preincubation of the resting Mycobacterium cells in BEHP for 6 h might be recommended as it leads to an achievement of stoichiometrical transformation of the substrate into AD and ADD and slightly higher initial rate of the reaction performed. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

In recent years, there has been a rapid development in the wearable industry. The growing number of wearables has led to the demand for new lightweight, flexible wearable antennas. In order to be applicable in IoT wearable devices, the antennas must meet certain electrical, mechanical, manufacturing, and safety requirements (e.g., specific absorption rate (SAR) below worldwide limits). However, the assessment of SAR does not provide information on the mechanisms of interaction between low-intensity electromagnetic fields emitted by wearable antennas and the human body. In this paper, we presented a detailed investigation of the SAR induced in erythrocyte suspensions from a fully textile wearable antenna at realistic (net input power 6.3 mW) and conservative (net input power 450 mW) conditions at 2.41 GHz, as well as results from in vitro experiments on the stability of human erythrocyte membranes at both exposure conditions. The detailed investigation showed that the 1 g average SARs were 0.5758 W/kg and 41.13 W/kg, respectively. Results from the in vitro experiments demonstrated that the short-term (20 min) irradiation of erythrocyte membranes in the reactive near-field of the wearable antenna at 6.3 mW input power had a stabilizing effect. Long-term exposure (120 min) had a destabilizing effect on the erythrocyte membrane.

Yeasts are rich source of proteins, antioxidants, vitamins, and other bioactive compounds. The main drawback in their utilization as valuable ingredients in functional foods and dietary supplements production is the thick, indigestible cell wall, as well as the high nucleic acid content. In this study, we evaluated the feasibility of pulsed electric field (PEF) treatment as an alternative method for extraction of proteins and other bioactive intracellular compounds from yeasts. Baker's yeast water suspensions with different concentration (12.5-85 g dry cell weight per liter) were treated with monopolar rectangular pulses using a continuous flow system. The PEF energy required to achieve irreversible electropermeabilization was significantly reduced with the increase of the biomass concentration. Upon incubation of the permeabilized cells in water, only relatively small intracellular compounds were released. Release of 90% of the free amino acids and low molecular UV absorbing compounds, 80% of the glutathione, and ∼40% of the total phenol content was achieved about 2 h after pulsation and incubation of the suspensions at room temperature. At these conditions, the macromolecules (proteins and nucleic acids) were retained largely inside. Efficient protein release (∼90% from the total soluble protein) occurred only after dilution and incubation of the permeabilized cells in buffer with pH 8-9. Protein concentrates obtained by ultrafiltration (10 kDa cut off) had lower nucleic acid content (protein/nucleic acid ratio ∼100/4.5) in comparison with cell lysates obtained by mechanical disintegration. The obtained results allowed to conclude that PEF treatment can be used as an efficient alternative approach for production of yeast extracts with different composition, suitable for application in food, cosmetics and pharmaceutical industries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Ganeva, Angelova, Galutzov, Goltsev and Zhiponova.)

А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H. polymorpha cells to the lytic enzyme. Consequently, the subsequent incubation of the permeabilised cells with lyticase led to the recovery of approximately 90% of the recombinant protein, with a purification factor of 1.8. A similar efficiency was obtained by using the industrial lytic enzyme Glucanex. The released FTH1 appears in the form of an oligomer with a molecular mass of approximately 480 kDa, which is able to bind iron. The possibility for scaling the proposed protocol is discussed.

Yeasts are one of the most commonly used systems for recombinant protein production. When the protein is intracelullarly expressed the first step comprises a cell lysis, achieved usually by a mechanical disintegration. This leads to non-selective liberation of the cytoplasmic content, which complicates the following downstream process. Here, we present a new approach suitable for more selective and efficient recovery of large intracellular proteins from yeast, based on the combination of electropermeabilisation and subsequent treatment with lytic enzyme. The experiments were performed with Saccharomyces cerevisiae strains expressing LYTAG-β-galactosidase from Escherichia coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25ms duration and field intensity of 4.3-5.4kV/cm. In the presence of a reducing agent the cells released approximately 80% of the total protein 4h after electrical treatment. At the same conditions the release of the recombinant protein was very slow, reaching 45% from total activity 20h after pulse application. The great difference in the release kinetics enabled to remove a part of the total protein, without significant loss of β-galactosidase activity, only by substituting the incubation buffer. The subsequent addition of lyticase (1-2U/ml) led to recovery of approximately 70% from the recombinant enzyme, with a factor of purification 2.6, without provoking a significant cell lysis. The applicability of similar protocol for liberation of large recombinant and native proteins from yeast is discussed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Studied was the effect of temperature in the range 12-46 degrees C on the rate of bacterial decolorization of the mono-azo dye Acid Orange 7 by Alcaligenes faecalis 6132 and Rhodococcus erythropolis 24. With both strains the raise of temperature led to a corresponding raise of decolorization rate better manifested by R. erythropolis. The analysis of the Arrhenius plot revealed a break near the middle of the temperature range. The regression analysis showed practically complete identity of the observed break point temperatures (T (BP)): 20.7 degrees C for Alc. faecalis and 20.8 degrees C for R. erythropolis. The values of the activation energy of the decolorization reaction (E (a)) were found to depend on both the organism and the temperature range. In the range below T (BP) the estimated values of E (a) were 138 +/- 7 kJ mol(-1) for Alc. faecalis and 160 +/- 8 kJ mol(-1) for R. erythropolis. In the range above T (BP) they were 54.2 +/- 1.8 kJ mol(-1) for Alc. faecalis and 37.6 +/- 4.1 kJ mol(-1) for R. erythropolis. Discussed are the possible reasons for the observed abrupt change of the activation energy.

The effects of the non-ionic surfactant Triton X-100, the cationic surfactant cetyltri-methylammonium bromide (CTAB) and the anionic surfactant sodium N-lauroyl sarcosinate (SLS) on the decolorization of the reaction medium containing the monoazo dye Acid Orange 7 (AO7) by Alcaligenes faecalis and Rhodococcus erythropolis were studied. It was found that the surfactants influenced in different ways the rate of decolorization. At all concentrations tested the non-ionic surfactant Triton X-100 decreased the decolorization rate of R. erythropolis. At concentrations above the critical micelle concentration (CMC) Triton X-100 upset the usually observed exponential decay of the dye with A. faecalis due probably to the existence of an outer membrane in this organism. In concentrations above the CMC the anionic surfactant SLS inhibited the decolorization and, at prolonged incubation, caused partial release of the bound dye. The cationic surfactant CTAB in concentrations above and below the CMC accelerated drastically the binding of AO7 to the cells causing a rapid staining of the biomass and complete decolorization of the reaction medium. An attempt was made for explanation of the observed differences by the negative electrostatic charge of the living bacterial cell.

We describe a method for continuous cultivation of microorganisms utilizing liquid or gaseous water insoluble substrates as a single source of carbon and energy. The water insoluble substrate, which is also the growth-limiting factor, enters the cultivation space as a manually adjustable single-compound material flow. All nonlimiting nutrients (with the exception of oxygen) enter the cultivation space as ingredients of titrant solution which feed rate is reliably coupled to the rate of substrate addition by means of the system titrator. The method provides mild starting conditions appropriate for primary isolation of microorganisms utilizing substrates with growth-inhibiting properties such as BTEX (benzene, toluene, ethylbenzene, and xylene). The sound control over the microbial specific growth rate makes it suitable for precise kinetic studies as well. We provide a detailed description of both the principles of the method and the equipment used. The dependence of the systems operative range on the concentration of titrant solution is illustrated in the case of continuous cultivation of a mixed bacterial culture on toluene., (Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 160-169, 2003.)

Lipophilic compounds are of significant importance in modern biotechnology. Centerly of interest are the biodegradation as well as the biotransformation of such lipophilic and often water-immiscible substances. Both whole cells and/or enzymes are used for these processes. It is obvious that a wide range of problems arise in an application of such complex systems consisting of biocatalysts substrate(s), product(s), water, (in some cases water-immiscible organic solvents): (i) interactions between lipophilic compounds and the membranes resulting in the change of some physiological characteristics of the living system; (ii) problems in the transport of these compounds (substrates and/or products) within the complex structured reaction systems; (iii) the problem of increasing the solubility of the lipophilic and mostly water-immiscible compounds with a minimum of inhibition effects on the processes; (iv) the presence of lipophilic components may also cause changes of the transport processes within the system (e.g. immobilized cells) resulting in changed yield or activity of the biological system. These problems are critically discussed in this review in relation to the known modes of interaction of lipophilic compounds with membranes, the bioavailability of the substrates, and the cases of steroid biotransformations. An outlook of future aspects in research, development and application of such processes is given.

The production of extracellular inhibitors by Streptomyces sp. 1-72, specific either to trypsin (TI-1), or to chymotrypsin (CI-1), was investigated in relation to the cell growth and composition of the cultivation media. The biosynthesis of the inhibitors was found to be of a growth-associated type which reached a maximum level at late exponential phase. All of the investigated carbon sources, including starch, disaccharides and monosaccharides, equally supported the production of both inhibitors, while the effect of the different nitrogen sources considerably varied, some of them being totally ineffective. The sources which mostly supported the biosynthesis of the inhibitors were tryptone (3%) for TI-1, and peptone (3%) for CI-1 respectively.

In the course of a screening for extracellular inhibitors of 8 different proteinases 140 actinomycete and 150 mould strains were examined. 130 fungal and 50 actinomycete strains were similarly tested for the production of extracellular inhibitors of dihydrofolate reductase (DHFR) and alkaline phosphatase (APh). Most of the inhibitors found in the culture media of the actinomycete and mould strains were specific against serine proteinases. Inhibitors of DHFR were discovered in the culture media of different actinomycetes and moulds. Inhibiting activities towards APh were found mainly in the fermentation filtrates of the mould strains. The approximate molecular sizes of the active substances were determined by using membrane filtration.