Vol 52, No 1 (2016)

The review considers recent data that indicate the ability of chitosan and its derivatives to bind reactive oxygen species. The analysis of the results of these studies will promote the selection of a promising natural antioxidant for applications in the cosmetics, food, pharmaceutical, and other industries.

The gene encoding enolase from Fusobacterium nucleatum (FnENO) was cloned and analyzed for the first time. The gene comprises of 1302 nucleotide base pairs and encodes 433 amino acids. The gene sequence alignment demonstrated the presence of several distinct insertions and deletions, compared with the human enzyme. The gene for recombinant FnENO was inserted into the pLATE 31 vector system and expressed in E. coli BL21(DE3) cells as a soluble protein. The protein was purified by affinity chromatography using a Ni-NTA agarose matrix and shown on SDS-PAGE to be a 46 kDa protein. The molecular weight of the octameric form of the purified recombinant protein was determined as being 375 kDa by size exclusion chromatography. Optimal enzyme activity was observed at pH 8.5 and the enzyme remained stable at a range of different temperatures from 30 to 60°C. Using 2-phosphoglyceric acid as substrate for the purified enzyme, K_M, k_cat and k_cat/K_M were determined as 0.48 mM, 20.4 s^–1 and 4.22 × 10⁴ M^–1s^–1, respectively. Potential drug binding sites of FnENO were detected using homology modeling. These data could facilitate the design of new inhibitors of F. nucleatum which has already been shown to be resistant to several known antibiotics.

Spherical gel particles from apple pectin and amidated citrus pectin with a low degree of methyl esterification were obtained by ionotropic gelation in the presence of calcium ions. A commercial preparation of alkaline phosphatase from bovine intestinal mucosa was used for enzyme immobilization by physical adsorption on pectin particles and by incorporation into a hydrogel. Alkaline phosphatase immobilized on pectin gels retained 21–55% of the initial activity. Release of the immobilized enzyme from the gel particles into solutions modeling the physiological conditions in different parts of the gastrointestinal tract was investigated. Alkaline phosphatase desorbed from pectin particles was shown to dephosphorylate bacterial lipopolysaccharide.

We studied the antimicrobial properties of 32 new non-protein compounds synthetized in the Scientific and Production Center “Armbiotechnology” by the method of asymmetric synthesis; new analogs of L-histidine and L-isoleucine were found. Via chemical mutagenesis, we obtained new mutants of the wild strain of Brevibacterium flavum 14067 resistant to (S)-β-[4-phenyl-3-(3’-hydroxypropyl)-5-thioxo-1,2,4-thiazole-1-yl]-α-alanine (an analog of histidine) and to (2S,3R)-oxyleucine (an analog of isoleucine). It was found that the synthesis rate of histidine and isoleucine in some mutants is higher than that of most active strain-producers resistant to one of these amino acids analogs described in literature. Our data prove the efficacy of the use of new analogs as selective agents for the screening of highly active strains producing histidine and isoleucine.

The role of reactive oxygen species in the interaction between brown rust-causing pathogen Puccinia triticina Erikss. and the disease-resistant species Triticum timopheevii Zhuk. was investigated using salicylic acid treatment to amplify the oxidative burst or treatment with verapamil, an inhibitor of Ca²⁺-channels to suppress oxidative the burst. The levels of reactive oxygen species in control T. timopheevii plants increased during the early stages of pathogenesis due to O₂^2← generation by stomatal guard cells contacting the appressoria and by mesophyll cells that developed a hypersensitivity reaction in response to the invasion of haustoria. Hydrogen peroxide was subsequently accumulated in the cytoplasm of cells destroyed by hypersensitivity reaction, as well as on the walls of cells contacted with fungal hyphae. The production of O₂^2←, which results in the death of most of the inoculum on the stomata and rapid accumulation of hydrogen peroxide, were enhanced by salicylic acid pretreatment. Pretreatment of the plants with verapamil inhibited the production of reactive oxygen species and hypersensitivity reaction in disease-resistant T. timopheevii plants and thus facilitated the penetration of the fungus into the stomata and more intensive development of mycelium in plant tissues in the early stages of pathogenesis. Some colonies with single haustoria died regardless of production of reactive oxygen species by the plants.

The antiradical properties of essential oils and extracts from coriander seeds Coriandrum sativum L., cardamom fruits Elettaria cardamomum L., fruits of white and black pepper Piper nigrum L., and pods of red cayenne and green chili pepper Capsicum frutescens L. were studied in model reactions with the stable free 2,2-diphenyl-1-picrylhydrazyl radical. The essential oils consisted of monoand sesquiterpene hydrocarbons, alcohols, oxides and esters as the main components. Spice extracts contained flavonoids, diand triterpenoids, phenolic acids, alkaloids and carotenoids. The values of antiradical efficiency were low and decreased in the following order: black pepper extract > cayenne pepper extract > cardamom essential oil > chili pepper extract > cardamom extract > white pepper extract > coriander extract > black pepper essential oil > white pepper essential oil > coriander essential oil.

R-phycoerithrin, a water soluble photosynthetic pigment of red algae, was used as a matrix and a reducer for the synthesis of Ag⁰ nanoparticles. The diameter of dominating Ag⁰ nanoparticles determined from the electron microscope images was 6.5 ± 0.5 nm. Dynamic light scattering in the regime of heating with a constant rate showed that the hydrodynamic radius of R-phycoerithrin molecules began to increase at a temperature of more than 50°C. The hydrodynamic radius of R-phycoerithrin with Ag⁰ nanoparticles synthesized in the inner cavity remained constant upon heating to 90°C. It was shown by circular dichroism that the temperature at which the portion of the denatured protein reached 50% was 51.7 ± 0.1°C for R-phycoerithrin and 58.0 ± 0.1°C for R-phycoerithrin with synthesized Ag⁰ nanoparticles. This indicated that thermal stability of R-phycoerythrin increased when its cavity was filled with an Ag⁰ nanoparticle. According to the comparative spectral studies of R-phycoerythrin before and after the synthesis of Ag⁰ nanoparticles and the heat treatment, we conclude that an increase in thermal stability of R-phycoerythrin is caused by chemical intramolecular cross-linking of a protein molecule with an Ag⁰ nanoparticle.

An efficient method of highly sensitive, specific determination of botulinic type A neurotoxin (which allows the detection of a toxin in a concentration from 1 pg/mL (10 fg per one PCR reaction) was created based on the technology of immuno-PCR. Solid phase consisting of paramagnetic organo–silicious particles carrying a detection complex of the toxin heavy chain with a pair of polyclonal antibodies to it was the most appropriate in the optimal scheme of the analysis. One of the antibodies bound to DNA (which is a matrix for PCR amplification) by means of biotin–neutravidin interaction. Matrix DNA was a molecular “net” generated by biotinylated DNA molecules with tetravalent neutravidin. The binding signal was detected by real time PCR (by the TaqMan method). An immuno-PCR method for determination of type A botulotoxin was developed.