Study of the Effect of Squalene Epoxidase Activity on Squalene Biosynthesis by Yeast Saccharomyces Cerevisiae VGSh-2

The researchers of this study investigated the biosynthesis of squalene by the yeast S. cerevisiae VGSH-2 through the activity of squalene epoxidase, which is a key enzyme in the conversion of squalene to ergosterol. It has been established that under aerobic conditions the antimycotic drug terbinafine promotes the switching of ergosterol formation to squalene synthesis. This switch occurs through specific inhibition of the squalene epoxidase of the yeast S. cerevisiae VGSH-2, thus increasing the biosynthetic ability of the yeast towards squalene. According to the results of this study, the optimal concentration of terbinofine in the nutrient medium was 0.3 μmol / cm3 . This concentration led to a 5-fold decrease in squalene epoxidase activity and a 7-8 times increase in squalene synthesis. The results obtained can be used to develop a competitive technology for the industrial production of squalene by microbial synthesis. Keywords: squalene, yeast, biosynthesis, inhibition of activity, terbinafine, squalene epoxidase, Saccharomices cerevisiae VGSH-2

The effects of different compositions of growth media on the development of microplants of the Solnechny potato variety

The results of studying the effect of nutrient media of various compositions on the growth of improved micro-plants of potatoes of the Solnechny variety grown under laboratory conditions in vitro are presented. Six compositions of the nutrient medium were studied: standard Murashige-Skuga medium modified for micropropagation (considered as a control), modified Murashige-Skuga medium with a reduced content of mineral components (up to 1/2 and up to 1/3), modified Murashige-Skuga medium with an increased content of agar-agar (10 g/l), modified Murashige-Skuga medium with a reduced content of agar-agar (4 g/l), Murashige-Skuga medium modified with the addition of 3 mg/L giberrellinic acid and 1 mg/L indoliacetic acid. The following parameters of cultivated plants were taken into account: plant length, root presence, number of internodes, total plant mass, leaf mass, root mass, leaf plate surface area. The use of modified nutrient media with a reduced content of mineral components led to an increase in plant length (by 28-30%), stem mass (by 25%) due to leaf mass (by 18%) and stem mass (by 31%) and the total surface area of leaf plates (by 12%). In the variant using a medium with 1/3 mineral components an increase in the mass of the root system was observed (by 20%). When growing plants on a modified nutrient medium with a high content of agar-agar, a decrease in the length of plants (by 6%), a decrease in the mass of the scion (by 12%) due to a decrease in the mass of the stem (by 15%) was observed. Plants grown on a modified nutrient medium with a reduced content of agar-agar were distinguished by a larger mass of the root system (by 10%), scion (by 17%) (due to an increase in leaf mass (by 27%), as well as the total surface area of leaf plates (by 22%). When growth regulators (giberrellin and indoliacetic acid) were added to the modified nutrient medium, a significant increase in plant height (by 70%), a decrease in the mass of the root system (by 50%) and leaves (by 46%), and an increase in the mass of the stem (by 23%) were observed. The total leaf surface area was 28% lower than the control values. For accelerated micropropagation of improved potato plants of the Solnechny variety and preparation of plants for transplanting to aerohydroponic systems in order to produce mini-tubers, the following modified nutrient media are optimal options: with a reduced number of mineral components (1/2 and 1/3) and with a reduced content of agar-agar.

Influence of biotechnological parameters on the yield of macrostructures from unfertilized seed germs of diploid sugar beet

Purpose. To determine the influence of biotechnological parameters on the yield of macrostructures from unfertilized seed germs of diploid sugar beet. Methods. Biotechnological, laboratory, analytical, statistical. Results. It was found that the use of 35% sodium hypochlorite solution at an exposure of 40 min allows to obtain from 73.13 to 75.83% of sterile seed germs. Exposure of 50 min allows to obtain the sterility of the source material from 83.58 to 85.39%. Sterilization of explants for 60 min allows to obtain sterility of the source material from 86.88 to 92.80%. The share of infected seed germs with increasing exposure decreased from 20.09–22.14 to 6.52–12.61%. The yield of macrostructures has been experimentally confirmed to significantly depend on breeding genotype and type of medium. The largest number of calluses (10–80%) was formed with the use of the Hamburg and Eveleg’s medium. With the use of he Murasige and Skoog’s medium, their share was 10–35%. Noteworthy, in breeding genotypes 07–181, 80% of genotypes formed buds and 35% formed calluses in the Hamburg and Eveleg’s medium. Of breeding genotypes 07–178, 55% of genotypes formed a callus and 80% buds. Conclusions. As a result of the conducted researches the influence of biotechnological parameters (exposure to 35% solution of sodium hypochlorite, type of nutrient medium) on the yield of macrostructures from unfertilized seed germs of diploid sugar beet was determined. It is optimal to carry out treatment with 35% sodium hypochlorite solution for 50–60 minutes, regardless of the selection number of sugar beet. To obtain macrostructures from unfertilized seed germs of diploid sugar beet, it is necessary to use the Hamburg and Eveleg’s medium for breeding genotypes 07–188, 07–178 and 07–181.

Development of haploids in the winter bread wheat anthers

In the modern world, the use of isolated anther cultivation technology is currently an integral part of the wheat breeding process. The development of haploids in the winter bread wheat anthers will allow obtaining new forms of wheat in the shortest possible time and without large areas. The purpose of the current study was to estimate the F3 winter bread wheat hybrids according to the anthers’ sensitivity to androgenesis and plant regeneration in vitro and to identify the factors affecting the yield of haploid production. There has been studied the ability to androgenesis in vitro in the anthers of four winter bread wheat hybrids of intensive and semi-intensive type of the FSBSI “ARC “Donskoy”. There has been assessed the role of the mineral composition of three induction nutrient media N6, W14 and NPB-99. There has been established a correlation between the main stages of development of haploids and a genotype. The highest regeneration rate of green plants was obtained in the sample F3 623 of intensive type (3.3%). The most suitable medium for androgenesis of the winter bread wheat anthers in vitro is NPB-99. Since the genotype F3 623 of intensive type demonstrated high values of haploid production capacity, it could be successfully used in breeding programs for the rapid production of homozygous wheat anther lines in vitro. Using two-way analysis of variance, there has been identified a correlation between the effects of a genotype, nutrient medium and their interaction with the main parameters of haploid formation in winter wheat. The formation of embryogenic structures is mainly associated with the effect of a genotype (46.52%). The proportion of the nutrient composition of the medium was low (1.82%), and the correlation factor was 2.1%. The genotype had the greatest effect on the indicator of the regenerants’ number. The nutrient medium had little effect. Regarding the regeneration of green plants, which is the main indicator of the haploid production, the share of a genotype effect was the largest (47.32%). The contribution of the medium and the correlation of factors were less important, but statistically significant.

Creation of the bank of breeding material of winter rye in vitro

Аn important issue of the selection process is the preservation of the source and obtained material, especially cross-pollinated crops, which significantly lose their viability by inbreeding. Creating a bank of genetic resources using biotechnological methods will effectively solve this problem. The aim of the work was to determine the conditions for the formation of a genetic bank of valuable winter rye materials with changes in temperature and modeling of the nutrient medium for long-term disposal of cloned plants and the use of active collection of original and created forms in the selection process. To deposit the clones, a nutrient medium, which included macro- and microelements according to the Murashige-Skuga medium was used. The nutrient substrate modified with cytokinins and carbohydrates. The clones in culture rooms at a temperature of 6–12 °С and low light intensity (2 kLk) were stored. In the course of research the conditions of creation of an active collection of plants of winter rye with use of temperature restriction and modification of a nutrient medium are defined. A consistent technological scheme for the conversion of plant material into a state of relative anabiosis has been developed. It is proved that the optimal storage temperature for samples is 6 °С. Survival of plants at the specified temperature regime after 12 months of deposition on average by genotypes at the level of 78,2 % was recorded. Modification of the nutrient medium with agar-agar at a concentration of 12,0 g/l increases the proportion of viable clones to 81,3 %, and the introduction into the substrate of an increased concentration of growth regulators, in particular 6-BAP (2,0 mg/l) and sucrose 40,0 g/l and a gradual decrease in temperature to 10 °С prolongs the period of deposition of cloned plants without changing the substrate and the shelf life of breeding material in isolated crops. Using of biotechnological methods for the preservation and reproduction of valuable material intensifies the selection process of obtaining initial samples of winter rye.

Development of nutrient medium for riboflavin biosynthesis by Eremothecium ashbyi ascomycetes

The object of the study is the riboflavin producer Eremothecium ashbyi Guilliermond 1935 VKPM F-340, the subject of the study is the regularities of riboflavin biosynthesis by the E. ashbyi F-340 strain under different cultivation conditions. Riboflavin is an important micronutrient that is a precursor of the coenzymes flavin mononucleotide and flavinadine dinucleotide, it is necessary for biochemical reactions in all living cells. Population growth and an increase in human needs for vitamin-fortified food and agricultural products is the reason for an increase in demand for riboflavin preparations. Considering this, it is important and economically beneficial to improve the technology for the production of vitamin B2. An important factor that affects the yield of the product is the nutrient medium. At present, the influence of agricultural waste on the biosynthesis of riboflavin is being actively studied in the world. However, not all of the studied types of raw materials are typical for the agriculture of this or that country. Therefore, in order to determine whether this direction of research is promising, it is important to check the effect on the biosynthetic activity of the riboflavin producer of the most common wastes of the domestic industry. In this work, this is done on the example of Ukraine. In the course of the study, microbiological (surface and deep cultivation of E. ashbyi F-340), physicochemical (determination of the amount of biomass by the gravimetric method, determination of the concentration of riboflavin by the spectrophotometric method) and mathematical methods were used. The proposed media with the addition of agricultural waste, providing a higher yield of riboflavin compared to conventional media. The influence of different types of agricultural waste on the biosynthesis of riboflavin by the producer E. ashbyi F-340 was evaluated. The efficiency of using sunflower cake as a component of the nutrient medium is shown. The optimal sources of carbon for the nutrient medium with oil cake have been determined, which increases the yield of riboflavin. Due to the large amount of sunflower cake obtained in Ukraine, its use for modifying the nutrient medium in order to increase the yield of riboflavin in the future will lead to a decrease in the cost of the target product due to the use of cheap and ecological raw materials.

In vitro regeneration of soybean (a review)

This is an overview of contemporary published works dedicated to the ability of soybean plants to regenerate in vitro and the techniques to achieve high regeneration rates, which is a necessary condition for the inclusion of soybean genotypes in genome editing programs. The main factors that determine the regenerative capacity of explants from various soybean accessions are considered. The greatest effect on the efficiency of regeneration is exerted by the conditions of in vitro culture initiation, type of explant, composition of the nutrient medium, shelf life of seeds, and genotypic characteristics of soybean accessions.

BIOSURFACTANTS SYNTHESIS BY PSEUDOMONAS AERUGINOSA BACTERIA ISOLATED FROM THE SURFACE OF MUSSELS OF THE BLACK SEA

Aim. Establishing of the ability to synthesize surface-active compounds by Pseudomonas aeruginosa bacteria isolated from the surface of Black Sea mussels. Methods. During the research several marine Pseudomonas spp strains isolated from petroleum hydrocarbon contaminated areas of Black Sea wereused: P. aeruginosa M1, P. aeruginosa M4 and P. aeruginosa PA01 as reference strain in suspension and biofilm cultures (LB and Giss media). Cultivation of Pseudomonas aeruginosa strains was performed at 37 °C for 120 and 168 hours. Planktonic culture growth was determined spectrophotometrically on the wave length 600 nm. Biofilm mass was determined spectrophotometrically on the wave length 592 nm by CV-test. The presence of surface-active compounds was determined in a drop test. The quantitative content of rhamnolipids was evaluated by the color reaction of rhamnose with orcin. Results. P. aeruginosa strains M1 and M4 isolated from Black Sea mussel’s surfaces synthesize 25% and 66% more surfactants than the reference strain PA01. All strains in Giss medium synthesized 10–20 times less rhamnolipids than in LB medium. In biofilm cultures the same biosurfactant synthesis dependence on the composition of the nutrient medium is observed as in suspension cultures. According to the intensity of rhamnolipid production in biofilm cultures, the studied strains can be arranged in the following row: P. aeruginosa M4 > P. aeruginosa M1 >> P. aeruginosa PA01.Conclusions. The strains of P. aeruginosa isolated from the Black Sea are more efficient producers of rhamnolipids than the reference strain of P. aeruginosa PA01; the intensity of biosynthesis of surfactants significantly depends on the composition of the nutrient medium and the method of cultivation.

Selection of Optimal Conditions for Cultivation of Bacillus megaterium UCM B-5710 – Producer of Keratinase

Every year the volume of production of poultry products all over the world is growing steadily. This contributes to a constant increase in the amount of by-products of poultry processing in the form of down and feather waste, which are dangerous for the environment due to the hard-to-degrade keratin protein and a large number of microbial pathogens. Therefore, the use of environmentally friendly methods for the destruction of keratin substrates due to keratinases of microorganisms is an urgent area of research. The aim of this work was to select the optimal cultivation conditions for the Bacillus megaterium strain UCM B-5710 to increase the activity of the keratinase synthesized by it. Methods. The culture was grown at 28°C, 201 rpm for 7 days on a basic nutrient medium containing defatted chicken feathers as the only source of carbon and nitrogen. The selection of optimal cultivation conditions was carried out according to the following parameters: temperature (21°C, 28°C, 42°C), stirring speed (201 rpm, 212 rpm), amount of inoculum (5%, 10%, 15% , 20%, 25%), the initial pH value of the nutrient medium (4.0–11.0), concentration of keratin-containing substrate (0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%), additional carbon source (glucose, galactose, lactose, maltose, sucrose, mannitol, potato and corn starch, soluble starch, soybean meal) and nitrogen (NH4Cl, NH4NO3, (NH4)2SO4, NaNO3, urea, peptone, tryptone, yeast extract and soybean meal) at a concentration of 1%. Keratinase activity was assessed by the UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method. Results. The dynamics of the enzyme synthesis showed that the culture of B. megaterium UCM B-5710 exhibited the highest keratinase activity on the 3rd day, and complete splitting of feathers was observed on the 4–5th days. The selection of the concentration of the keratin-containing substrate showed that 0.5% is the optimal concentration. The study of the influence of the initial pH value of the nutrient medium indicates that the culture grew well at pH 6.0–7.0 and pH 9.0–11.0, but at pH 8.0 its growth was very weak. The culture exhibited the maximum keratinase activity at pH 10.0. In addition, at this pH value, complete splitting of feathers was visually observed. The influence of such a key factor as temperature on the growth and synthesis of the enzyme by B. megaterium UCM B-5710 culture demonstrated complete splitting of feathers already on the 2nd day of cultivation at 42°C, at 21°C the culture split feathers very poorly. The introduction of the inoculum into the composition of the nutrient medium in an amount of 15% of the volume of the medium and the mixing intensity of 212 rpm turned out to be optimal. Besides, it was shown that the introduction of an additional source of carbon or nitrogen had an ambiguous effect on the level of keratinase activity of B. megaterium UCM B-5710. Complete inhibition of enzyme synthesis was observed when ammonium sulfate was added to the nutrient medium, and partial inhibition was observed in the case of glucose, lactose, and maltose. Potato, corn, and soluble starch stimulated keratinase synthesis. The majority of inorganic nitrogen sources (ammonium chloride and nitrate) did not affect the synthesis of B. megaterium UCM B-5710 keratinase, while organic sources (urea, peptone, tryptone, yeast extract) increased the level of keratinase activity by 20–50%. However, the most effective result was obtained using soybean meal, the addition of which to the nutrient medium increased the keratinase activity by 2.5 times. Conclusions. As a result of the studies, the optimal conditions for cultivation of the B. megaterium UCM B-5710 strain were selected: the optimum temperature for the growth and development of the culture is 42°C, the initial pH value is 10.0, the stirring speed is 212 rpm and the amount of inoculum introduced is 15%, an additional source of carbon and nitrogen in the form of soybean meal at a concentration of 0.5%. This made it possible to increase the activity of keratinase by 4 times.

Influence of the composition of nutrient media on the productivity and biological activity of the strain of entomopathogenic fungus Beauveria bassiana

The productivity and biological activity of the entomopathogenic fungus Beauveria bassiana (F-145) strain during the liquid-phase cultivation on various substrates for developing a biopesticide in its native form was analyzed (the research was carried out in 2019). For submerged cultivation, by-products from dairy and beer production (milk whey and brewing spent grain liquor) were used as components of the nutrient medium with addition of diesel fuel (DF) and Tween-80 as inducers of biological activity. It has been established, that the productivity of the strain on industrial by-product substrates was 1.5-2 times higher than on the Czapek medium. A high yield of a mycelial biomass with a titer of 108 -1010 CFU/ml was shown in a 5-day suspension based on a mixture of milk whey and brewing spent grain liquor. The biological activity of the culture suspension of the strain was determined. It was shown that the nematicidal activity of Beauveria bassiana strain with regard to nematodes of the Rhabditis sp. was largely manifested in a suspension obtained on a mixed medium with the addition of inducers. Ninety per cent death at mobile nematode stages was registered within one or two days of test-organism incubation. A complex nutrient medium composition containing by-products and inducers contributed to the preservation of the biological activity of the strain. The strain nematicidal activity was established at the level of 67-80 per cent with a titer of 106 -107 CFU/ml when the suspension was stored for 67 days.