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Applied Biochemistry and Microbiology (v.43, #4)

Induced plant resistance and salicylic acid: A review by N. I. Vasyukova; O. L. Ozeretskovskaya (pp. 367-373).

Current understanding of the involvement of salicylic acid (SA) in the formation of plant resistance has been reviewed. SA acts as a signal molecule in the SA-dependent pathway. The so-called salicylate burst observed in tissues of plants after stress increases their resistance. The mechanism whereby SA induces plant resistance depends on the ability of this compound to inhibit the enzymes of the antioxidant system of plants, which results in the accumulation of active oxygen species and the expression of defense genes.

Catalytic properties of glucoamylase immobilized on synthetic carbon material Sibunit by G. A. Kovalenko; L. V. Perminova; T. G. Terent’eva; G. V. Plaksin (pp. 374-378).

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousand-fold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10⁵. The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60°C exceeded 30 days.

Bifunctional inhibitor of α-amylase/trypsin from wheat grain by R. A. Islamov; O. V. Fursov (pp. 379-382).

A trypsin inhibitor, isolated from whole-wheat grain (Triticum aestivum L.) by the method of biospecific chromatography on trypsin-Sepharose, was potent in inhibiting human salivary α-amylase. The bifunctional α-amylase/trypsin inhibitor was characterized by a narrow specificity for other α-amylases and proteinases. The high thermostability of the inhibitor was lost in the presence of SH group-reducing agents. The inhibitor-trypsin complex retained its activity against α-amylase. The inhibitor—α-amylase complex was active against trypsin. Studies of the enzyme kinetics demonstrated that the inhibition of α-amylase and trypsin was noncompetitive. Our results suggest the existence of two independent active sites responsible for the interaction with the enzymes.

Production of fructooligosaccharide syrup from sucrose in combination with palatinose and trehalose by A. A. Markosyan; L. A. Abelyan; M. O. Adamyan; Z. D. Ekazhev; Zh. I. Akopyan; V. A. Abelyan (pp. 383-389).

The fructofuranosidases (EC 3.2.1.26) of Aspergillus niger St-0018 and A. foetidus St-0194 were used to produce fructooligosaccharides (FOS) under periodic and continuous conditions. The incorporation of cells into calcium alginate gel gave the most efficient immobilized biocatalysts. The feasibility of transforming residual sucrose into palatinose and trehalulose using isomaltulose synthase (EC 5.4.99.11) was demonstrated.

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases by I. A. Goptar’; G. N. Balandina; E. N. Lysogorskaya; I. Yu. Filippova (pp. 390-393).

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.

Detection of C-P-lyase activity in a cell-free extract of Escherichia coli by S. V. Kononova; S. M. Trutko; K. S. Laurinavichus (pp. 394-398).

A system has been developed for in vitro testing of E. coli C-P-lyase (the enzyme cleaving C-P bonds in phosphonates). NADH, ATP, and the system of ATP regeneration were necessary, but not sufficient, for expression of the C-P-lyase activity in cell-free extracts of E. coli. Experimental evidence suggests that glucose 6-phosphate and (or) glucose activate C-P-lyase, serving as precursors in the formation of (alkylphosphono)ribose, an intermediate in the reaction. Guanine is the most likely acceptor of the phosphate group.

Biological fixation of nitrogen and growth of bacteria of the genus Azotobacter in liquid media in the presence of perfluorocarbons by M. K. Bakulin; A. S. Grudtsyna; A. Yu. Pletneva (pp. 399-402).

The addition of perfluorocarbons (perfluorodecalin, carbogal, and perfluoromethyldecalin) to nitrogen-free liquid media during the submerged cultivation of bacteria of the genus Azotobacter was followed by (1) increases in biomass accumulation and nitrogenase activity and (2) fixation of molecular nitrogen. Addition of perfluorodecalin (5 vol %) to the culture medium of A. chroococcum ACB 121 contributed to increases in biomass accumulation, cell concentration (of more than by five times), nitrogenase activity (of 3.4 times), and total nitrogen content in the medium (of 4.5 times).

Ochrobactrum intermedium ANKI, a nitrogen-fixing bacterium able to decolorize azobenzene by N. D. Wackerow-Kouzova (pp. 403-406).

Morphological and biochemical properties of the nitrogen-fixing strain Ochrobactrum intermedium ANKI, intensely growing on media with azo compounds, and its resistance to various common xenobiotics were investigated. The kinetics of azobenzene transformation by O. intermedium ANKI was studied. Under cometabolism conditions, up to 40 mg of azobenzene per liter of medium were decolorized within one week. It was shown that the strain possessed molybdenum-dependent nitrogenase activity, and its nitrogenase system was sensitive to oxygen and fixed nitrogen in the medium.

Microbioluminescent study of the general toxicity and mutagenicity of pollutants by I. L. Maslennikova; N. V. Golyasnaya (pp. 407-413).

Bacterial bioluminescence was applied to detection of general toxicity (MIT test) and genotoxicity (SOS-lux test) of some chemicals, seawater, and fresh water. The SOS-induced luminescence of E. coli WP2s (cda::luxCDABE) cells was higher than in E. coli C 600 (cda::luxCDABE) at 37°C and pH 6.5. The mutagenic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide determined from the induction of E. coli WP2s cell luminescence was detected at lower concentrations than in the assessment of reversion frequencies. General toxicity was demonstrated by using luminescence inhibition for hydrogen peroxide, Zn²⁺, and Cd²⁺ at low concentrations. Regions of the Krasnodar Krai where sea and fresh waters exerted toxic action on luminescence were determined by the microbioluminescent method.

Cloning and expression of variants of the glutaryl-7-aminocephalosporic acid acylase of the bacterium Brevundimonas diminuta in Escherichia coli cells by S. A. Khatuntseva; M. A. El’darov; S. A. Lopatin; O. A. Zeinalov; K. G. Skryabin (pp. 414-421).

The gene coding for glutaryl-7-aminocephalosporic acid acylase (Gl7ACA acylase) of the bacterium Brevundimonas diminuta (BrdGl7ACA), a commercial enzyme widely used in modern biocatalytic technologies for manufacture of β-lactam antibiotics, was cloned. Efficient expression systems for producing a “native” recombinant BrdGl7ACA and its analogs modified by attaching affinity groups—the chitin-binding domain of chitinases A1 and hexahistidine sequence—were designed. It was demonstrated that both the recombinant hybrid proteins and the native Gl7ACA acylase produced in E. coli cells underwent a correct autoproteolytic processing with generation of functionally active enzymes and could be isolated with a high yield using one-step affinity chromatography.

Determination of antibiotics using luminescent Escherichia coli and blood serum by I. I. Vlasova; T. V. Asrieli; E. M. Gavrilova; V. S. Danilov (pp. 422-428).

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 ± 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 ± 0.5; for tetracycline, 45 ± 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.

The effect of the phenolic compounds exuded by pea roots in darkness on the reproduction of Rhizobium by L. E. Makarova; S. E. Latysheva; T. E. Putilina (pp. 429-434).

The exudation, composition, and biological activity of the phenolic compounds (PC) of pea (Pisum sativum L.) roots in the light and darkness were studied. The roots of leguminous plants grown for 5 days in darkness exuded a smaller amount of PC that displayed a weaker stimulation of Rhizobium reproduction. Moreover, the root exudates contained antimicrobial compounds, stilbenes. It is assumed that a lower PC exudation by roots and the specific features of PC composition influencing the biological activity are among the reasons causing a delayed nodulation of legumes grown in darkness.

Synthesis of α-cyclopiazonic acid by fungi of the genus Aspergillus by N. G. Vinokurova; N. E. Ivanushkina; I. I. Khmel’nitskaya; M. U. Arinbasarov (pp. 435-438).

The presence of α-cyclopiazonic acid has been studied among metabolites of Aspergillus fungi. The study was performed with 138 cultures of 13 species obtained from the All-Russia Collection of Microorganisms and the collection of our institute. α-Cyclopiazonic acid was most frequently encountered among the metabolites of the section Flavi (the ability to synthesize α-cyclopiazonic acid was expressed in 61% of the strains of A. flavus, 83% of the strains of A. oryzae, and all strains of A. tamarii). This expression index for A. versicolor was less than 5%. We showed for the first time that α-cyclopiazonic acid is produced by A. fumigatus and A. phoenicis (expression in 30% of the strains of either species).

Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei by X. Zhou; Z. Wang; K. Jiang; Y. Wei; J. Lin; X. Sun; K. Tang (pp. 439-443).

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography, and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compound-producing endophytic fungi, which can improve prominently screening efficiency.

Antioxidant activities of cultured Armillariella mellea by L. T. Ng; S. J. Wu; J. Y. Tsai; M. N. Lai (pp. 444-448).

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate.

Study of chitin-glucan complexes from the soil micromycete Cephaliophora tropica D3 by K. M. Zlotnikov; A. V. Kazakov; N. G. Vinokurova; A. K. Zlotnikov (pp. 449-452).

We studied chitin-glucan complexes from the soil micromycete Cephaliophora tropica D3. The yield and purity of chitin-glucan complexes depended on the medium used for micromycete culturing and the method of mycelium disintegration. Disintegration with liquid nitrogen allowed us to obtain preparations of chitin-glucan complexes with a low content of proteins and a high yield of chitosan (per dry fungal biomass unit weight).

Enzyme-linked immune sorbent assay for PR-toxin in taxonomical assessment of fungi belonging to the genus Penicillium link by A. A. Burkin; G. P. Kononenko; G. A. Kochkina; S. M. Ozerskaya (pp. 453-458).

The use of an indirect competitive enzyme-linked immune sorbent assay (ELISA) involving polyclonal rabbit antibodies against BSA-conjugated PR-toxin (sensitivity, 1 ng/ml) established the ability to synthesize PR-toxin in 18 out of 35 morphologically identified strains of Penicillium roqueforti and P. chrysogenum. The results indicate that ELISA for PR-toxin may be used in assessing the taxonomical position of terverticillate penicillia in the presence of other micotoxins.

The action of Laminaria japonica extractives on 1,3-β-D-glucanase, a digestive enzyme of the Strongylocentrotus intermedius sea urchin by V. V. Agarkova; T. N. Krupnova; S. P. Ermakova; N. M. Shevchenko; T. N. Zvyagintseva (pp. 459-464).

Nutritional attractiveness of the brown alga Laminaria japonica for the sea urchin Strongylocentrotus intermedius was studied. The composition of L. japonica was analyzed after one and two years of its life under natural conditions, in its seedlings, and in the alga partially degraded by natural factors. Substances extracted with various solvents were tested for the presence of inhibitors and activators of 1,3-β-D-glucanase, a digestive enzyme of the sea urchin. Ethanolic extract of freshly harvested L. japonica was found to suppress the enzyme activity. Substances present in ethanolic extracts of the alga after one or two years of its life cycle and in the alga, partly degraded by natural factors, activated the sea urchin enzyme. This fact is in agreement with earlier natural observations concerning the nutritional attractiveness of such L. japonica samples for Strongylocentrotus intermedius.

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris by E. A. Günter; Yu. S. Ovodov (pp. 465-472).

Ultraviolet radiation (wavelength, 280–315 nm; power, 0.2–13.0 W/m²; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m²) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1: (3.4–8.3).

Chronicle of the eighth international conference “Modern perspectives in chitin and chitosan studies” by E. P. Feofilova (pp. 473-473).

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Applied Biochemistry and Microbiology (v.43, #4)