Prikladnaia biokhimiia i mikrobiologiia最新文献

The consistent application of homogenization and enzymatic treatment is required to obtain protoplasts from the basidiomycete fungus Trametes hirsuta. The maximum yield of protoplasts (∼2.5 × 107/mL) was achieved when mycelium in the exponential growth phase (60 h) was used. The maximum stability was observed in MES+ buffer during 4 h of incubation; in this case the titer reduction was 5–7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; β-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of β-carotene and quercetin by 18–24%. The studied antioxidants did not affect the protoplasts stability. The degree of regeneration of protoplasts correlated with the antioxidant capacity of the studied antioxidants and was maximal (0.4%) in the presence of β-carotene and quercetin; it was 0.1% in the presence of MES+. The rate of protoplast growth was two times higher in the presence of β-carotene and quercetin.

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K m) and maximum reaction velocities (V max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K i = 104.7 ± 21.7 mM, K m = 2.5 ± 0.5 mM, V max = 25.1 ± 1.5 U/mg) was observed.

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H2O2 > OCl– > NO• > RОO• > ONOO–> O 2 •- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO• > H2O2 > ONOO– > RОO• > OCl– > O 2 •- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H2O2 and OCl–, along with O 2 •- and NO•. Among the used reactive oxygen and nitrogen species, H2O2 showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison than the biosensors based on E. coli. .

The effects of two Pseudomonas aeruginosa bacteriophages, vB-Pa 4 and vB-Pa 5, on the formation and development of biofilms of six polyresistant hospital strains of P. aeruginosa have been investigated. Pretreatment of bacteriophages prevented the formation or almost completely prevented the growth of adequate biofilms. The biofilms that had already formed were partially or completely destroyed after phage treatment. The results demonstrate the prospects of using isolated bacteriophages of P. aeruginosa to destroy biofilms and prevent their formation.

The effect of N-phenyl-2-naphthylamine, negative allelochemical isolated from the exudates of roots of pea (Pisum sativum L.), on the growth and activity of the adenylate cyclase signal system and virulence factors of the bacteria Rhizobium leguminosarum bv. viciae and Pseudomonas siringae pv. pisi was studied. It was demonstrated that N-phenyl-2-naphthylamine at a physiological concentration nonspecifically inhibited the growth of these bacteria in both planktonic cultures and biofilms. One of the reasons for this phenomenon is the reduction of intra- and extracellular concentrations of cAMP due to greater activation of phosphodiesterase, which disrupts cAMP, in comparison to soluble adenylyl cyclase, which synthesizes it. At the same time, N-phenyl-2-naphthylamine did not affect activity of either membrane-bound adenylyl cyclase or bacterial virulence factors.

The hydroxylase activities of new strains such as Curvularia lunata, C. geniculata, C. eragrostidis, C. prasadii, Ulocladium botrytis, Alternaria tenuis, and Fusarium oxysporum toward three steroid substrates, namely, androstenedione (AD), cortexolone (S), and dehydroepiandrosterone acetate (DAA), were characterized. The 9α-hydroxylase activity of C. lunata 1011 cells against S to form 9α-hydroxy-S was shown for the first time. It was found that C. geniculata 837 and F. oxysporum 11dn1 strains can hydroxylate substrates to form pharmacologically promising 7α-hydroxysteroids. C. geniculata 837 cells selectively hydroxylate AD, resulting in 7α-hydroxytestosterone, whereas F. oxysporum 11dn1 leads to the transformation of DAA to 7α-hydroxydehydroepiandrosterone.

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5′ untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.

The essential oils from 16 various spice plants were studied as natural antioxidants for the inhibition of autooxidation of polyunsaturated fatty acids methyl esters isolated from linseed oil. The content of methyl oleate, methyl linoleate, and methyl linolenoate after 1, 2, and 4 months of autooxidation were used as criteria to estimate the antioxidant efficiencies of essential oils. In 4 months, 92% of the methyl linolenoate and 79% of the methyl linoleate were oxidized in a control sample of a model system. It was found that the most effective antioxidants were essential oils from clove bud, cinnamon leaves, and oregano. They inhibited autooxidation of methyl linolenoate by 76–85%. The antioxidant properties of these essential oils were due to phenols— eugenol, carvacrol, and thymol. Essential oil from coriander did not contain phenols, but it inhibited methyl linolenoate oxidation by 38%. Essential oils from thyme, savory, mace, lemon, and tea tree inhibited methyl linolenoate oxidation by 17–24%. The other essential oils had no antioxidant properties.

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K M value to be 4.34 mg/mL (for D-xylose) and V max value to be 2028.9 μmoles mg–1 min–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg2+ and Mn2+ were found to be required for the enzyme activity, however, Co2+, Hg2+, Fe2+ and Cu2+ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.

The growth of marine diatoms Phaeodactylum tricornutum was investigated on a medium with artificial sea water under artificial and natural light. The maximum specific growth rate was 0.7 day–1, the productivity was 0.8 g/L day, and the maximum biomass was 3.86 g/L under artificial light in laboratory conditions. In the conditions of Crimea, the maximum productivity of P. tricornutum amounted to 6 g/m2 day under natural light in an outdoor photobioreactor (pool). The results of cultivation of P. tricornutum in a pool with artificial seawater under natural light may serve as a basis for developing technologies for the industrial cultivation of algae.