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Applied Biochemistry and Microbiology (v.44, #3)

Proteinase inhibitors in plant biotechnology: A review by V. V. Mosolov; T. A. Valueva (pp. 233-240).

Possible utilities for natural inhibitors of proteolytic enzymes in plant biotechnology have been reviewed. Among the potential areas of use of the inhibitors are (1) construction of transgenic plants with increased resistance to insects and other pests and (2) development of procedures for biosynthesis of recombinant proteins. In the latter case, the inhibitors will serve to prevent the protein degradation by proteinases.

Preparation and catalytic properties of trypsin immobilized on cryogels of polyvinyl alcohol by E. N. Lysogorskaya; T. V. Roslyakova; A. V. Belyaeva; A. V. Bacheva; V. I. Lozinskii; I. Yu. Filippova (pp. 241-246).

Commercial preparations of trypsin, varying in activity, were immobilized on a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In organic media, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-arginine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.

Isolation and purification of glutathione peroxidase by K. K. Shulgin; T. N. Popova; T. I. Rakhmanova (pp. 247-250).

Electrophoretically homogeneous glutathione peroxidase (EC 1.11.1.9) preparation from rat liver with a specific activity of 1.46 U/mg of protein and a yield of 7.2% was obtained using the purification procedure developed. The K _M values for reduced glutathione and hydrogen peroxide were 0.033 and 0.208 mM, respectively. The enzymatic reaction had the following characteristics: the temperature optimum, 32°C; the pH optimum, 7.4; and the activation energy, 29.1 kJ/mol. The molecular weight of the enzyme was 88 kDa.

Glycolytic activity of enzyme preparation from the red king crab (Paralithodes camtschaticus) hepatopancreas by K. S. Rysakova; V. Yu. Novikov; V. A. Mukhin; E. M. Serafimchik (pp. 251-255).

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei by E. N. Sautkina; N. A. Potapenko; T. I. Bulycheva; N. M. Vladimirova (pp. 256-263).

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35–40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C-and N-terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.

Micellar laccase-catalyzed synthesis of electroconductive polyaniline by A. V. Streltsov; G. P. Shumakovich; O. V. Morozova; M. A. Gorbacheva; A. I. Yaropolov (pp. 264-270).

A method of enzymatic synthesis of electroconductive polyaniline on the micelles of dodecylbenzenesulfonic acid sodium salt (DBSNa) is proposed. The high potential laccase from the basidiomycete Trametes hirsuta was used as a biocatalyst. The conditions for polyaniline synthesis were optimized (pH 4.0; reagent concentrations, 10–20 mM; and aniline/DBSNa ratio, 2 : 1). The resulting product was electrochemically active in the range of potentials from −200 to 600 mV, electroconductive, and capable of reversible dedoping with a change in pH of solution.

Methods for increasing nitrile biotransformation into amides using Mesorhizobium sp. by Y. S. Feng; C. M. Lee; C. C. Wang (pp. 271-275).

Nitriles are potential soil pollutants from industrial wastewater. There has been increased demand for an efficient process for the nitrile degradation process. Nitrile hydratase (NHase) has been extensively used in the production of acrylamide and treatment of organocyanide-contaminated industrial effluents. The NHase of Mesorhizobium sp., isolated from polyacrylonitrile (PAN) activated sludge from fiber manufacturing wastewater treatment systems was studied in the whole bacterial cells. Different chemicals were added to observe the variation in the percentage of acrylonitrile converted into acrylamide. The result indicated that cobalt ions were the NHase cofactor and could increase the NHase activity. The addition of propionaldehyde, or butyraldehyde, could enhance the acrylonitrile conversion rate. Therefore, acrylamide could be accumulated effectively and the percentage of acrylonitrile converted into acrylamide increased. Propionaldehyde was the most effective NHase activator. The percentage of acrylonitrile converted into acrylamide was nearly 100% at 3.8 h when propionaldehyde was added at about 207.4 mg/l. The addition of benzaldehyde was unable to increase the percentage of acrylonitrile converted into acrylamide. EDTA and acrylamide showed no effect on NHase activity. However, 0.1 mg/l of Ag₂SO₄ would slightly inhibit NHase activity, producing an acrylonitrile conversion rate of 492.9 mg/l with 54.9% converted at 29.1 h. The ability of the acrylonitrile biotransformation was completely inhibited if the Ag₂SO₄ concentration was above 0.5 mg/l.

Phenol biodegradation by a Pseudomonas sp. strain tagged with the gfp gene by A. T. Adylova; T. N. Chernikova; A. A. Abdukarimov (pp. 276-281).

Conjugal transfer of the pAG408 suicide vector from E. coli S17-1 to Pseudomonas sp. cells able to consume phenol yielded transconjugates brightly luminescing under UV illumination. It was shown that tagging of the Pseudomonas sp. cells with the gfp gene did not affect their ability to consume phenol. The change of the population density of the tagged bacteria after their introduction to soil was studied. The potential of the resulting bacterial strain in remediation of phenol-polluted soils is discussed.

Change of oil-degrading activity in microorganisms stored under laboratory conditions by N. I. Belousova; L. M. Baryshnikova; A. N. Shkidchenko (pp. 282-286).

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50–60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.

Study on the Lake Baikal microbial community in the areas of the natural oil seeps by O. N. Pavlova; T. I. Zemskaya; A. G. Gorshkov; V. V. Parfenova; M. Yu. Suslova; O. M. Khlystov (pp. 287-291).

We studied the composition of a natural microbial community, the distribution of different groups of microorganisms (including those able to degrade oil hydrocarbons) within the areas of natural oil seeps in the Lake Baikal. It was revealed that, in the bottom sediments, the oil-degrading microorganisms dominating the community have included the bacteria of g. Bacillus, while in the water column, dominating microbes are presented by species of genera Rhodococcus, Pseudomonas, and Micrococcus. Under the conditions of the model experiment, the potential activity of Baikal microbes towards utilization of n-alcanes has been assessed. Under such conditions it was shown that the concentration of n-alcanes decreases to 60% during 20 days of the experiment (the initial oil concentration was 0.5 mg/l, i.e., ten maximal permissible concentrations, MPC).

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors by D. G. Deryabin; E. S. Aleshina (pp. 292-296).

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl⁻, SO ₄ ²⁻ , HCO ₃ ⁻ , and CO ₃ ²⁻ on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.

A new source of methane in boreal forests by V. A. Mukhin; P. Yu. Voronin (pp. 297-299).

Methane was found among the gases evolved during natural wood decay caused by bracket fungi in boreal forests. Methane was detected both in decaying wood and fungal fruiting bodies. A scheme of symbiotic association of wood-degrading fungi and anaerobic microorganisms providing the methanogenesis in the wood was proposed. The scale of mycogenic methane emission has to be consistent with the huge volume of decaying wood in boreal forest ecosystems.

In vitro inhibitory effect of cranberry (Vaccinium macrocarpum Ait.) juice on pathogenic microorganisms by H. L. E. Magariños; C. Sahr; S. D. C. Selaive; M. E. Costa; F. E. Figuerola; O. A. Pizarro (pp. 300-304).

The purpose of this study was to determine the inhibitory effects of cranberry juice on pathogenic microorganisms. The microorganisms analyzed were Escherichia coli from patients with urinary infections, Salmonella spp., Listeria monocytogenes, Pseudomona aeruginosa, and Staphylococcus aureus. The disc method was used to determine the sensitivity of bacteria to cranberry juice (CJ, both concentrated and diluted). A lawn of 10⁶ cfu/ml was grown on agar surfaces in Petri dishes and on Whatman discs that had been previously saturated with CJ and CJ : water. 1 : 1 to 1 : 50 juice solutions had been placed on the discs, which were cultured and incubated. The results indicated that S. aureus was more susceptible to cranberry juice inhibition than the other microorganisms. L. monocytogenes was the most resistant to the inhibitory action of cranberry juice, showing a significant difference from the inhibition of P. aeruginosa, uropathogenic E. coli, Salmonella spp., and S. aureus. This study also demonstrated that the inhibitory activity of cranberry juice for E. coli took place up to a dilution of 1 : 20.

Comparative chemical composition of the Barents Sea brown algae by E. D. Obluchinskaya (pp. 305-309).

Comparative study of phytochemical compositions of the most widespread brown algae species (one laminarian and four fucoid algae) from Barents Sea has been performed. A modified technique for mannitol determination in brown algae is proposed. It was revealed that fucus algae (fam. Fucaceae) contain 3% (of total dry weight) less mannitol than laminaria (Laminaria saccharina). The contents of alginic acid and laminaran in the Barents Sea fucoids are more than 10% less compared to laminaria. The alga L. saccharina contains almost two times more iodine than the species of fam. Fucaceae. The amounts of fucoidan and sum lipids in the Barents Sea fucoid algae is higher than in Laminaria saccharina (4–7% and 1–3%, respectively). In terms of contents of main biologically active compounds, fucus and laminarian algae from Barents Sea are inferior to none of the Far-Eastern species. The Barents Sea algae may become an important source of biologically active compounds.

Application of heat shock proteins as stress markers in aquatic organisms using endemic Baikal amphipods as an example by M. A. Timofeev; Zh. M. Shatilina; D. S. Bedulina; M. V. Protopopova; A. V. Kolesnichenko (pp. 310-313).

When heat shock proteins (HSPs) are used as biomarkers in monitoring studies of aquatic ecosystems, it is necessary to take into account the specificity of synthesis of these proteins in various organisms. This especially applies to endemic species and species with narrow ranges of adaptation for specific conditions in certain water bodies. In this study, we assessed the possibility to use HSPs as molecular stress markers in species with a narrow niche breadth using endemic Baikal amphipods (Crustacea, Amphipoda) as an example. The effect of stress induced by toxicants and temperature has been assessed. Proteins of families HSP70 and lowmolecular-weight HSP related to α-crystallins were used as biomarkers. Temperature-and toxicant-induced stresses induced low-molecular-weight HSP synthesis in the endemic amphipod species studied. However, induction of HSP70 synthesis in the same species after temperature stress has not been detected. The specificity of synthesis of HSP70 is discussed. The results obtained in this study suggest that low-molecular-weight HSPs can be used as stress markers in Baikal species and species with a narrow niche breadth.

Biosensors for assay of glycoalkaloids in potato tubers by V. N. Arkhypova; S. V. Dzyadevych; N. Jaffrezic-Renault; C. Martelet; A. P. Soldatkin (pp. 314-318).

The possibility of practical application of biosensors based on pH-sensitive field-effect transistors and butyrylcholinesterase to glycoalkaloid analysis in potato tubers was studied. The main analytical features of the designed biosensors and measurement conditions were optimized. The biosensor was applied to quantitative analysis of glycoalkaloids in tubers of different potato varieties. The results proved to be in good agreement with those obtained by conventional protocols. Experiments on glucose assay were performed. An inverse correlation between the contents of glucose and glycoalkaloids in potato tubers was demonstrated.

Effect of hydrogen peroxide on morphological characteristics and resistance of wheat calluses to T. caries Tul. by N. B. Troshina; L. G. Yarullina; Z. R. Yusupova; O. B. Surina; I. V. Maksimov (pp. 319-322).

We studied the effect of hydrogen peroxide on morphological characteristics and resistance of common wheat calluses ( Triticum aestivum L.) to Tilletia caries Tul. The induction of the defense response and morphogenesis in calluses depended on H₂O₂ concentration. A correlation was revealed between the elevated concentration of hydrogen peroxide in wheat calluses and high activity of oxalate oxidase in the cell wall. Administration of H₂O₂ into the callus culture medium was followed by rhizogenesis, induced the formation of dense regions, and inhibited fungal growth on calluses. Hydrogen peroxide at high concentrations was less potent in inhibiting the growth of fungi. A relationship was found between oxalate oxidase activity, H₂O₂ concentration, and morphogenetic and defense responses of calluses induced by exogenous hydrogen peroxide. These data suggest that the induction of H₂O₂ generation is one of the approaches to increase callus resistance.

Obtaining antibodies to 1,4-Dihydropyridine calcium channel antagonists by A. A. Burkin; M. A. Burkin (pp. 323-327).

Immunization of rabbits with amlodipine conjugated with horseradish peroxidase resulted in raising polyclonal antibodies that allowed group determination of 1,4-dihydropyridine calcium channel antagonists in aqueous solutions by ELISA with a sensitivity of 0.1 to 1.0 ng/ml for amlodipine, felodipine, nifedipine, and isradipine.

Retention of components in a mixture of volatile organic substances by maltodextrins by T. A. Misharina; M. B. Terenina; N. I. Krikunova (pp. 328-331).

The effect of composition and origin of maltodextrins on the retention of 38 components in a mixture of volatile organic substances (odorants) during 6-month storage was studied by means of capillary gas-liquid chromatography. The retention of esters increased with an increase in their molecular weight. The retention of lactones, phenols, linalool, menthone, and damascone was 75–85%. Storage of aldehydes was accompanied by oxidation, and the retention of these substances did not exceed 55%. The retention of odorants increased with a decrease in the molecular weight of maltodextrins. The maximum retention was typical of maltodextrin from amylopectin starch not containing amylose.

L. I. Vorob’eva, The archaea: A textbook for higher education institutions, Moscow: Akademkniga, 2007, 447 pp. by G. I. El-Registan (pp. 332-333).

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Applied Biochemistry and Microbiology (v.44, #3)