Strains of halophilic bacteria from samples of Barsakelmes saline soil were screened for ability to synthesize carotinoid pigments. An active strain that accumulated β-carotene as the main pigment was selected. The β-carotene was shown to be identical to the standard pigment.Anaerobic photosynthetic bacteria, cyanobacteria, yeast, algae, higher plants, fungi, and extremely halophilic bacteria are known microbial producers of carotinoids [1]. Cells of halophiles contain many carotinoid pigments that color the colonies from pink to red. This is very important for halophilic microorganisms because it protects them from the intense radiation that is typical of their habitat [2]. Several halophilic microorganisms have been previously isolated and characterized from samples of saline soils of the Republic [3]. However, their pigment-forming activity has not be examined. Our goal was to identify carotene-forming halophiles and study the array of carotinoids synthesized by them.We previously isolated 37 bacterial isolates growing in media with salt concentrations 15-25% from saline soils in various regions of the Republic of Uzbekistan. A characteristic signature of most isolated strains was the ability to accumulate various red shaded pigments [4]. Based on the literature [5], we assumed that the characteristic color of the isolated bacteria was due to the presence of carotinoid pigments. Fourteen cultures colored various shades of red and pink were selected for the investigation of their carotene-forming properties. We found that the selected isolates were able to grow in a medium containing 25% NaCl. This salt concentration was optimal for some of them. All selected cultures to one degree or another were able to accumulate carotinoid pigments. Figure 1 shows that the total carotinoid content in most cultures varied in the range 0.157-1.5 mg/L of culture liquid. Moreover, the carotinoid level in three cultures, designated K38, K91, and K91r, was significantly higher. Thus, isolate K91r accumulated up to 5 mg/L; K91, 4.6; K38, 4.3 mg/L. According to the literature, the accumulation level of carotinoid pigments in microorganism producers reaches 8 mg/L of culture liquid [6,7]. For example, the single-celled alga Dunaliella, the most famous producer of β-carotene, could synthesize up to 30 mg of carotinoids per liter under the optimal growth conditions. Of these, 60% were the cis-isomers of β-carotene, which are much more active than chemically synthesized trans-isomers of the pigment [8].β-Carotene is commonly regarded as the principal biotechnologically valuable carotinoid pigment. Therefore, we performed HPLC analysis of extracts from cultures K38, K91, and K91r using ethylacetate as the solvent and mobile phase in order to determine the β-carotene content in the total carotinoid preparations.Isolate K91 contained a fraction corresponding with standard β-carotene according to the chromatographic separation.

The activities of nuclear enzymes involved in NAD+ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD+ producer were investigated. The presence of extracellular nicotinamide increased the total NAD+ pool in the cells and increased [3H]nicotinic acid incorporation; however, NAD+ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD+ correlated with increases in ADP-ribosyl transferase, NAD+-pyrophosphorylase, and NAD+ase activities.

It has been shown that the yeast S. cerevisiae has the ability to synthesize nicotinic acid and NAD from 3-methylpyridine. Fractionation of intracellular yeast proteins established that the fraction of molecular weight 65-90 kDa had the highest activity for transformation of pyridine derivatives into nicotinate. HPLC showed that, in addition to nicotinic acid, nicotinamide was also present in the intermediates during transformation of 3-methylpyridine. The results were consistent with the presence in yeast cells of an enzymatic system that transforms 3-methylpyridine into vitamin PP.Nicotinic acid (NA) is known to be produced chemically from the pyridine derivatives β-picoline (3-methylpyridine, 3-MP) or 3-acetylpyridine (3-AP) [1]. The question of NA production in microbiological conversions was first raised in the 1970s by researchers [2-4] who isolated more than 100 cultures of Mycobacterium, Nocardia, Corynebacterium, and Arthrobacter and others that not only decomposed the pyridine ring but also oxidized alkyl substituents on it without destroying it. Also, the ability to oxidize the methyl on 3-MP was first demonstrated at that time. However, the enzymatic aspects of the microbiological transformation of pyridine derivatives have been poorly studied despite the other successes.We observed previously that local strains of the yeast Saccharomyces cerevisiae 913a-1, which were selected as producers of nicotinamideadeninedinucleotide (NAD), accumulate high concentrations of the coenzyme in the presence of 3-MP and 3-AP [5]. This suggests that enzymatic transformation of these compounds into nicotinate and its subsequent participation in NAD biosynthesis is possible. Therefore, our goal was to find intracellular proteins that are involved in the bioconversion of 3-MP into NA by S. cerevisiae 913a-1.Fermentation of S. cerevisiae 913a-1 biomass in the presence of 3-MP is accompanied by more than a doubling of the intracellular NAD concentration (from 35.6 to 68.2 µg/mL) whereas the free NA content did not change (8.6 µg/mL).However, it should be considered that intracellular NA is an intermediate in many exchange transformations of pyridinenucleotides whereas the intracellular content of free NA is low compared with that of nicotinamide or NAD. Thus, the ability of yeast cells to accumulate high concentrations of NAD indicates that biosynthesis can supply the required amount of NA. The increased intracellular pool of NAD observed in yeast in the presence of 3-MP may be due to its preliminary transformation into nicotinate due to its enzymatic transformation in cytoplasm. According to the literature, various microorganism groups can not only completely degrade pyridine bases but also partially transform them. Thus, the ability of the bacteria Nocardia and Arthrobacter to oxidize under co-oxidation conditions alkylpyridines to the corresponding acids, 3-and 2-MP to nicotinic and picolinic acids, has been demonstrated. Transformation of pyridine into nicotinamide also occurs through NA or other intermediates such as ...

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The light-dependent protein bacteriorhodopsin deserves special attention among physiologically active compounds from halophilic Archeans. It is associated with the development of a new type of information-storage system, the creation of an artificial eye, and the use as biologically active additives with clearly pronounced antioxidant and radioprotector properties [1].We isolated previously two strains of extreme halophilic bacteria from Barsakelmes salt marsh (South Sub-Aral) that were capable of synthesizing carotinoid pigments [2]. The goal of the present study was to identify and determine quantitatively bacteriorhodopsin in cells of the previously isolated strain Halobacterium sodomense K91r and to study the protein accumulation as a function of culture age.The capability of cells of an extreme halophile to synthesize bacteriorhodopsin was studied for the first time in Uzbekistan. Cell-free extract of H. sodomense K91r was obtained by the standard method using CFE buffer [3]. The composition of the resulting protein extract was analyzed by electrophoresis under denaturing conditions according to Laemmli [4]. The electrophoregram of the studied extract showed 16 bands corresponding to proteins of molecular weights in the range 110-7 kDa. Among them, a protein band of molecular weight of the order of 26 kDa was observed. This corresponded to bacteriorhodopsin standard (Fluka). The identification of this protein in the extract from the halophilic strain was promising for further work to isolate and purify bacteriorhodopsin.The quantitative content of the synthesized bacteriorhodopsin was determined as a function of the cultivation time of the strain and the composition of the growth medium for No. 44 and Braun liquid media using spectrophotometry based on the light-sensitivity of the protein [5]. For this, cells were collected from growth medium (2 mL) by centrifugation and disintegrated by distilled water containing DNAase 1 (0.01 mg). The lysate was stirred until homogeneous and treated with NaOH (4 M) and NH 4 OH (4 M) in a 9:0.5:0.5 (lysate:NaOH:NH 4 OH) ratio in the dark. Absorption at 568 nm (A 568 ) was first measured in the dark (A 0 568 ). The mixture was exposed to light for 24 h. The absorption was measured again (A 24 568 ). Considering that the molecular weight of bacteriorhodopsin is 26 kDa and the molar extinction coefficient is 63,000 M -1 ·cm -1 , the bacteriorhodopsin concentration was determined from the equation BR (g/L) = 26,000 (A 0 568 -A 24 568 )/63,000. Bacteriorhodopsin began to accumulate heavily starting on the 5-6 th day after the start of cultivation. This was due to an increase of the NaCl concentration in the medium and; therefore, a decrease of the aeration of the growth medium and a switch to the conversion of light energy into chemical mediated by bacteriorhodopsin. Despite the fact that H. sodomense K91r biomass grew more vigorously in Braun medium, this did not affect the accumulation of bacteriorhodopsin. A study of bacteriorhodopsin accumulation during growth of H. sodomen...