Determination of Bactericidal Activity Spectrum of Recombinant Endolysins of ECD7, Am24, Ap22, Si3, and St11 Bacteriophages.

The bactericidal activity of recombinant endolysins LysECD7, LysAm24, LysAp22, LysSi3 and LysSt11 was assayed in multidrug resistant strains (n=120) of Salmonella enterica, E. coli, Acinetobacter baumannii, Enterobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, and Campylobacter jejuni. The assay showed that the recombinant endolysins had a wide spectrum of bactericidal activity compared to endolysins of their progenitor phages. Among examined endolysins, we selected the active pharmaceutical substances with broad spectrum of bactericidal activity. Most strains were sensitive to LysECD7 (70.7%), LysAm24 (65%), and LysAp22 (58.6%), which seems to be promising causative agents for the development of finished dosage form.

Biotransformation of Progesterone by the Ascomycete Aspergillus niger N402.

The ability of the ascomycete Aspergillus niger N402 to transform exogenous progesterone was investigated. We found that this strain has steroid-hydroxylating activity and can introduce a hydroxyl group into the progesterone molecule mainly at positions C11(α) and C21 with predominant formation of 21-hydroxyprogesterone (deoxycortone). In addition, formation of 6ß,11α-dihydroxyprogesterone was also observed. Studying the effects of the growth medium composition and temperature on progesterone conversion by A. niger N402 showed that the most intense accumulation of 21-hydroxyprogesterone occurred in minimal synthetic medium at 28°C. Increasing the cultivation temperature to 37°C resulted in almost complete inhibition of the hydroxylase activity in the minimal medium. In the complete medium, a similar increase in temperature inhibited 11α-hydroxylase activity and completely suppressed 6ß-hydroxylase activity, but it produced no effect on 21-hydroxylating activity.

[Preparation of protoplasts of the fungus Trametes hirsuta 072 and study of the effect of antioxidants on their formation and regeneration].

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 5­7%. Studies of the effect of antioxidants with different antioxidant capacities expressed in mmol equivalents of Trolox (ascorbate, 0.99; α-tocopherol, 1.0; ß-carotene, 2.14; quercetin, 3.98) indicated that the yield of protoplasts was increased in the presence of ß-carotene and quercetin by 18­24%. 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 ß-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 ß-carotene and quercetin.

Comparative proteomic study of the basidiomycete Trametes hirsuta grown on different substrates.

Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO4 were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO4 as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (ß-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.

[Range of Gene candidates involved in biosynthesis of laccase from basidiomycete Trametes hirsuta].

A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.