Whole-genome sequencing of Xanthomonadaceae strain Alg18-2.2, isolated from the Saline Lake Gudzhirganskoe in the Republic of Buryatia, Russia

A draft genome sequence of the bacterial isolate Alg18-2.2, recovered from the highly saline and alkaline lake Gudzhirganskoe (Buryatia, Russia), was determined. The results of bacterial identification using 16S rRNA gene sequence and whole-genome analyses suggest that the bacterium belongs to a novel genus. Some genomic features are discussed here

Сравнительный анализ способов асептического введения в культуру in vitro растений Melittis sarmatica Klok

Разработаны способы стерилизации и введения в культуру in vitro редкого пряноароматического растения Белорусской флоры Melittis sarmatica Klok. Материалом для введения служили семена и черенки с пазушными почками. В качестве основного стерилизующего агента для семян использовали 0,1%-ный раствор нитрата серебра, для черенков – 0,1%-ный раствор диацида. Изучено влияние 6-бензиламинопурина (6-БАП), 24-эпибрассинолида (ЭБ), их сочетаний и концентраций на всхожесть in vitro семян растений Melittis sarmatica. Наиболее высокие показатели по стерильности и всхожести семян наблюдались при сочетании 6-БАП и ЭБ в концентрациях по 0,5 мг/л (всхожесть 3–5%), при добавлении в среду только 0,5–1,0 мг/л 6-БАП (2–6%), или только 0,1–1,0 мг/л ЭБ (1–2%). Наиболее высокие результаты по стерилизации (72,6%), регенерации (62,7%) и укореняемости (28,7%) стерильных черенков были получены при семиминутной обработке 0,1% раствором диацида. Ключевые слова: Melittis sarmatica, in vitro, стерилизация, эксплант, нитрат серебра, диацид, бензиламинопурин, эпибрассинолид. There were developed the methods of sterilization and introduction in vitro of a rare aromatic plants of the Belarusian flora of Melittis sarmatica Klok. The seeds and cuttings with axillary buds were used as a material for introduction in vitro culture. As the main sterilizing agents there were used the 0.1% solution of silver nitrate and the 0.1% solution of the diacid. Influence of a 6-benzilaminopurin (6-BAP), 24-epibrassinolid (EB), their combinations and concentration on in vitro viability and germination of plant seeds Melittis sarmatica in vitro is studied. The highest rates on sterility and viability of seeds were ob-served at a combination 6-BAP and EB in concentration on 0.5 mg/l (viability of 3-5%), at addition on medium only of 0.5-1.0 mg/l 6-BAP (viability of 2–6%), or only of 0.1–1.0 mg/l of EB (viability of 1–2%). The most high influence on sterilization (72.6%), regeneration (62.7%) and rooting (28.7%) of sterile cuttings have been received at seven-minute processing of 0.1% solution of the diacid. Key words: Melittis sarmatica, in vitro, sterilization, explant, silver nitrate, the diacid, benzylaminopurine, epibrassinolide

Reversible self-association of ovalbumin at air-water interfaces and the consequences for the exerted surface pressure

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ovalbumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces

Reversible self-association of ovalbumin at air-water interfaces and the consequences for the exerted pressure

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ovalbumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces

Solubilization and refolding of inclusion body proteins in reverse micelles

Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Hare, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water-sodium bis-2-ethylhexyl sulfosuccinate-isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with Fusarium galactose oxidase and Stigmatella aurantiaca putative galactose oxidase. (C) 2003 Elsevier Science (USA). All rights reserved

Galactonolactone oxidoreductase from Trypanosoma cruzi employs a FAD cofactor for the synthesis of vitamin C.

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are interesting targets for drug therapy. The terminal step in ascorbate biosynthesis is catalyzed by flavin-dependent aldonolactone oxidoreductases belonging to the vanillyl-alcohol oxidase (VAO) protein family. Here we studied the properties of recombinant T. cruzi galactonolactone oxidoreductase (TcGAL), refolded from inclusion bodies using a reverse micelles system. The refolded enzyme shows native-like secondary structure and is active with both l-galactono-1,4-lactone and d-arabinono-1,4-lactone. At odd with an earlier claim, TcGAL employs a non-covalently bound FAD as redox-active cofactor. Moreover, it is shown for the first time that TcGAL can use molecular oxygen as electron acceptor. This is in line with the absence of a recently identified gatekeeper residue that prevents aldonolactone oxidoreductases from plants to act as oxidases

Chemical modification causes similar change in dependence on water activity of chymotrypsin hydration and catalysis in hexane

Amino groups in alpha-chymotrypsin were reacted with pyromellitic anhydride, introducing 17 to 32 additional carboxyl groups. This modification causes a major change in the water adsorption isotherm of the lyophilized protein powder. Little water is bound by the modified enzyme at water activity (a(w)) below 0.35, but it shows increased water binding at a(w) over 0.5. This correlates with a similar change in the a(w) dependence of the catalytic activity of the enzyme powder suspended in hexane, with a much steeper increase in activity of the modified chymotrypsin

Molecular details of ovalbumin-pectin complexes at the air/water interface: a spectroscopic study.

To stabilize air-water interfaces, as in foams, the adsorption of surface-active components is a prerequisite. An approach to controlling the surface activity of proteins is noncovalent complex formation with a polyelectrolyte in the bulk phase. The molecular properties of egg white ovalbumin in a complex with pectin in the bulk solution and at air/water interfaces were studied using drop tensiometry (ADT) and time-resolved fluorescence anisotropy techniques. The complex formation of ovalbumin with pectin in the bulk resulted in the formation of a compact structure with a different spatial arrangement depending on the protein/pectin ratio. Complex formation did not provide an altered protein structure, whereas the conformational stability was slightly increased in the complex. In excess pectin, an overall condensed complex structure is formed, whereas at limited pectin concentrations the structure of the complex is more "segmental". The characteristics of these structures did not depend on pH in the 7.0 to 4.5 regime. Interaction with pectin in the bulk solution resulted in a significantly slower adsorption of the protein to the air/water interface. The limited mobility of the protein at the interface was found for both ovalbumin and ovalbumin-pectin complexes. From both the rotational dynamics and total fluorescence properties of the protein in the absence and presence of pectin, it was suggested that the complex does not dissociate at the interface. Ovalbumin in the complex retains its initial "aqueous" microenvironment at the interface, whereas in the absence of pectin the microenvironment of the protein changed to a more nonpolar one. This work illustrates a more general property of polyelectrolytes, namely, the ability to retain a protein in its microenvironment. Insight into this property provides a new tool for better control of the surface activity of complex biopolymer systems

Structure and dynamics of egg white ovalbumin adsorbed at the air/water interface

The molecular properties of egg white ovalbumin adsorbed at the air/water interface were studied using infrared reflection absorption spectroscopy (IRRAS) and time-resolved fluorescence anisotropy (TRFA) techniques. Ovalbumin adsorbed at the air/water interface adopts a characteristic partially unfolded conformation in which the content of the beta-sheet is 10% lower compared to that of the protein in bulk solution. Adsorption to the interface leads to considerable changes in the rotational dynamics of ovalbumin. The results indicate that the end-over-end mobility of the ellipsoidal protein becomes substantially restricted. This is likely to reflect a preferential orientation of the protein at the interface. Continuous compression of surface layers of ovalbumin causes local aggregation of the protein, resulting in protein-network formation at the interface. The altered protein-protein interactions contribute to the strong increase in surface pressure observed