Catalytic properties of aminoacylase of strain <Emphasis Type="Italic">Rhodococcus armeniensis</Emphasis> AM6.1

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.     

Sucrose dependent mineral phosphate solubilization in <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> PSI3 by heterologous overexpression of periplasmic invertases

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.     

Genetic variability and population structure of grey wolf (<Emphasis Type="Italic">Canis lupus</Emphasis>) in Serbia

Results of previous morphometric and genetic analyses of grey wolf (Canis lupus L.) population from Serbia indicated different patterns of population subdivision. In order to explore population structure, level of genetic variability, genetic drift, inbreeding and signals of bottleneck for grey wolves from Serbia, we applied highly polymorphic genetic markers (microsatellites). Obtained data are valuable in determination of conservation units and creation of appropriate management plans. We have amplified 18 highly polymorphic microsatellites, in a total sample of 75 grey wolves, from different localities across Serbia and multilocus genotypes were analyzed using appropriate software. Observed values of the basic genetic parameters (H_O = 0.69; H_E = 0.75) indicated moderate level of genetic variability, similar to genetic variability in other populations belonging to the Dinaric-Balkan population of grey wolf. In STRUCTURE analysis, although ΔK was estimated to be at first peak K = 2, and second peak K = 4, CLUMPAK analyses showed that there’s no structuring for any of assumed K, and therefore the population of grey wolf from Serbia may be considered as one continuous population and treated as one conservation unit in future management plans. Signals of bottleneck haven’t been observed (Wilcoxon test two phase mutation model p = 0.247; and stepwise mutation model p = 0.815).     

Evaluation of role of the peptide transport system in absorption of dipeptides in the rat small intestine in chronic experiments <Emphasis Type="Italic">in vivo</Emphasis>

Hydrolysis and absorption of glycylglycine and glycyl-L-leucine as well as absorption of glycine and leucine were studied in chronic experiments on rats with their isolated small intestine loop. Values of the “true” kinetic constants (with taking into account effect of the preepithelial layer) were determined to be as follows: (1) K _t = 46.7 ± 4.0 and 2.15 ± 0.59 mM, J _max = 0.74 ± 0.15 and 0.16 ± 0.03 μmol min^?1 cm^?1 (for transport of free glycine and leucine, respectively); (2) K _t = 4.4 ± 0.6 and 4.8 ± 0.9 mM, J _max = 0.24 ± 0.02 and 0.23 ± 0.02 μmol min^?1 cm^?1 (for transport of glycylglycine and glycyl-L-leucine, respectively); (3) K _M = 5.4 ± 1.0 and 38.2 ± 4.4 mM, V _max = 0.09 ± 0.02 and 0.24 ± 0.07 μmol min^?1 cm^?1 (for membrane hydrolysis of these dipeptides, respectively). According to our calculations, in the wide range of the initial glycylglycine concentrations (2.5–40 mM) a part of the peptide component in its total absorption accounts for 0.77–0.80. In the case of glycyl-L-leucine a part of the peptide component in the total glycine absorption decreases from 0.89 to 0.84, while in the total leucine absorption—from 0.86 to 0.71, the initial dipeptide concentration rising from 5 to 40 mM. The obtained results show that the peptide component prevails in absorption of the studied dipeptides in the rat small intestine, but its role is much lesser than what many authors believe. In the case of glycyl-L-leucine, the peptide component can achieve saturation in the range of high substrate concentrations, its part decreasing essentially to become compared with absorption of free amino acids formed as a result of the dipeptide membrane hydrolysis.     

Screening and identification of an <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> strain expressing an active <Emphasis Type="Italic">β</Emphasis>-xylosidase

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29?±?0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant K_M values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The k_cat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s^?1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s^?1?×?mM^?1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.     

Stress influenced the aerotolerance of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> hsryfm 1301

Objective

To investigate the aerotolerance of Lactobacillus rhamnosus hsryfm 1301 and its influencing factors.

Results

The growth rate of L. rhamnosus hsryfm 1301 weakened noticeably when the concentration of supplemented H₂O₂ reached 1 mM, and only 2% of all L. rhamnosus hsryfm 1301 cells survived in MRS broth supplemented with 2 mM H₂O₂ for 1 h. After pretreatment with 0.5 mM H₂O₂, the surviving cells of L. rhamnosus hsryfm 1301 in the presence of 5 mM H₂O₂ for 1 h increased from 3.7 to 7.8 log CFU. Acid stress, osmotic stress, and heat stress at 46 °C also enhanced its aerotolerance, while heat stress at 50 °C reduced the tolerance of L. rhamnosus hsryfm 1301 to oxidative stress. Moreover, treatment with 0.5 mM H₂O₂ increased the heat stress tolerance of L. rhamnosus hsryfm 1301 by approximately 150-fold.

Conclusions

Lactobacillus rhamnosus hsryfm 1301 possesses a stress-inducible defense system against oxidative stress, and the cross-adaptation to different stresses is a promising target to increase the stress tolerance of L. rhamnosus hsryfm 1301 during probiotic food and starter culture production.

     

Genetic diversity of the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> and geographic distribution of its subspecies-specific RAPD markers on the territory of Russia

Genetic diversity and geographic distribution of taxon-specific RAPD markers was examined in ten local populations of the house mouse Mus musculus (n = 42). The house mice were generally characterized by moderate genetic variation: polymorphism P ₉₉ = 60%, P ₉₅ = 32.57%; heterozygosity H = 0.12; the observed allele number n _a = 1.6; the effective allele number n _e = 1.18; the within-population differentiation ?_s = 0.388; and Shannon index I = 0.19. The degree of genetic isolation of individual local populations was greatly variable. The genetic subdivision index G _st varied from 0.162 to 0.770 at the gene flow of Nm = 2.58?0.149, while the among-population distances D _N varied from 0.026 to 0.178. The largest part of the genetic diversity was found among the populations (H _T = 0.125), while the within-population diversity was twice lower (H _S = 0.06). The samples examined were well discriminated relative to the sets of RAPD markers. The character distribution pattern provided conditional subdivision of the mice into the “western” and the “eastern” groups with the putative boarder along the Baikal Lake. The first group was characterized by the prevalence of the markers typical of M. m. musculus and M. m. domesticus. The second group was characterized by the prevalence of the markers typical of M. m. musculus, M. m. gansuensis, M. m. castaneus, M. m. domesticus, and M. m. wagneri. The genotype of the nominative subspecies M. m. musculus was background for all populations. In the populations examined some of earlier described subspecies-specific molecular markers were found at different frequencies, pointing to the involvement of several subspecies of M. musculus in the process of hybridization.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

Feruloyl esterase from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (K_M) and catalytic efficiencies (k_cat/K_M) were determined for typical substrates of feruloyl esterase, and the lowest K_M of 50.6 μM (i.e., the highest affinity) and the highest k_cat/K_M (3.1 × 10⁵ s^—1 M^–1) were observed for methyl ^p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and ^p-coumaric acids.     

Purification and characterization of two glutathione peroxidases from embryo of the camel tick <Emphasis Type="Italic">Hyalomma dromedarii</Emphasis>

Two glutathione peroxidase isoenzymes were purified from 24-day old embryos of the camel tick Hyalomma dromedarii and designated tick embryo glutathione peroxidase 1 and 2 (TEGPx1 and TEGPx2). The purification procedure involved ammonium sulfate precipitation, as well as ion exchange and gel filtration column chromatography. Glutathione peroxidase isoenzymes subunit molecular mass was determined by SDS-PAGE to be 36 ± 2 kDa and 59 ± 1.5 kDa for TEGPx1 and TEGPx2, respectively. TEGPx1 isoenzyme exhibited a dimeric structure with native molecular mass of 72 kDa while TEGPx2 was a monomeric protein. TEGPx1 and TEGPx2 displayed their pH optima at 7.6 and 8.2. Both isoenzymes cleaved preferentially H₂O₂ with K _m values of 24 and 49 μM. Iodoacetamide competitively inhibited TEGPx1 with K _i value of 0.45 mM and 1.10; phenanthroline competitively inhibited TEGPx2 with K _i value of 0.12 mM. These results indicate the presence of two different forms of glutathione peroxidase in the developing camel tick embryos. This finding enhances our knowledge and understanding of the physiology of these ectoparasites and will encourage the development of new and untraditional control methods.     

Purification,characterization and gene analysis of a new α-glucosidase from <Emphasis Type="Italic">shiraia</Emphasis> sp. SUPER-H168

A new α-glucosidase from Shiraia sp. SUPER-H168 under solid-state fermentation was purified by alcohol precipitation and anion-exchange and by gel filtration chromatography. The optimum pH and temperature of the purified α-glucosidase were 4.5 and 60 °C, respectively, using p-nitrophenyl-α-glucopyranoside (α-pNPG) as a substrate. Ten millimoles of sodium dodecyl sulfate, Fe²⁺, Cu²⁺, and Ag⁺ reduced the enzyme activity to 0.7, 7.6, 26.0, and 6.2 %, respectively, of that of the untreated enzyme. The K _m, V _max, and k _cat/K _m of the α-glucosidase were 0.52 mM, 3.76 U mg^?1, and 1.3?×?10⁴ L s^?1 mol^?1, respectively. K _m with maltose was 0.62 mM. Transglycosylation activities were observed with maltose and sucrose as substrates, while there was no transglycosylation with trehalose. DNA and its corresponding full-length cDNA were cloned and analyzed. The α-glucosidase coding region consisted of a 2997-bp open reading frame encoding a 998-amino acid protein with a 22-amino acid signal peptide; one 48-bp intron was located. The α-glucosidase was a monomeric protein with a predicted molecular mass of 108.2 kDa and a predicted isoelectric point of 5.08. A neighbor-joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 α-glucosidase is an ascomycetes α-glucosidase. This is the first report of α-glucosidase from a filamentous fungus that had good glycoside hydrolysis with maltose and α-pNPG, transglycosylation and conversion activity of maltose into trehalose.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Inheritance of Traits Controlled by Odd Chromosomes Using Data on Transmission of Monosomic Addition S<Superscript>t</Superscript> Chromosome of the <Emphasis Type="Italic">Elymus Sibiricus</Emphasis> Genome

On the basis of the winter bread wheat cultivar Obryi, two independent disomic addition lines BC₁₂F_∞ with the chromosome of the E. sibiricus S^t genome are created. A practical algorithm for determining the probabilities of transmission of the odd chromosome separately through male and female gametes in selfpollination of hemizygous hybrids from the equation p²–(1 + f₁–f₄) × p + f₁ = 0 is proposed, where p is the probability of the formation of viable gametes with the considered chromosome and f₁ and f₄ are the empirical frequencies of the corresponding homozygotes with and without the trait. The probability of transmission of an alien univalent chromosome through pollen (p_♂) is associated with the frequency of its transmission through the egg cell (p_♀) in backcrosses and in self-pollination (1–f₄) by the equation p_♂ = 1–f₄/(1–p_♀). The calculated empirically dependent estimates of the probabilities of transmission of the added chromosome through the egg cell p_♀ = 18.7% and through pollen p_♂ = 4.3% correspond to the empirical frequencies obtained for backcrosses. The coefficients of the gamete selection V_♀ = 0.748 and V_♂ = 0.172 are calculated, and the expected segregation for the alien trait controlled by a dominant gene located in the added chromosome is determined—with the trait: without the trait is 0.222: 0.778 in F₂; 0.187: 0.813 in equational and 0.043: 0.957 in certational backcrosses.     

Reassessment of hydrogen tolerance in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Caldicellulosiruptor saccharolyticus has the ability to produce hydrogen (H₂) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H₂ (P_H2) is a critical aspect to eliminate the need for continuous stripping of the produced H₂ from the bioreactor.

Results

Herein, we demonstrate that, under given conditions, growth and H₂ production in C. saccharolyticus can be sustained at P_H2 up to 67 kPa in a chemostat. At this P_H2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (K i of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of C. saccharolyticus was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of C. saccharolyticus to grow and produce H₂ under relatively high P_H2.

Conclusion

C. saccharolyticus is able to grow and produce hydrogen at high P_H2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H₂ yield.