Forgot your password?
New user?
New Window Opens on the Secret Life of Microbes: Scientists Develop First Microbial Profiles of Ecosystems
CAS announces the release of SciFinder Scholar for Mac OS X
Press Releases
Press Releases
| Check out our New Publishers' Select for Free Articles |
Applied Biochemistry and Microbiology (v.45, #1)
Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography by D. V. Kurek; S. A. Lopatin; V. P. Varlamov (pp. 1-8).
Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label.
An ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions by E. I. Karaseva; E. I. Tarun; D. I. Metelitsa (pp. 9-16).
The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37–59°C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constantsk in, k*in andk in(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO·—DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.
Catalytic properties of Rhodotorula aurantiaca KM-1 phenylalanine ammonia-lyase by I. L. Bazukyan; A. E. Vardanyan; A. A. Hambardzumyan; P. V. Tozalakyan; Yu. G. Popov (pp. 17-21).
L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K M 1.75 ± 0.44 mM and V max 3.01 ± 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K i 3.38 ± 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57°C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.
A new serine protease gene from Trichoderma harzianum is expressed in Saccharomyces cerevisiae by Yan Liu; Qian Yang; Jinzhu Song (pp. 22-26).
Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to biocontrol processes. Difficulties in heterologous expression in a bacterial or yeast host have hampered engineering of these proteases for industrial application. We report here a successful expression of the serine protease SL41 from a biocontrol fungus Trichoderma harzianum in Saccharomyces cerevisiae. A new serine proteases gene SL41 has been cloned from T. harzianum. The full-length cDNA was isolated by 5′ and 3′ rapid amplification of cDNA ends. The isolated cDNA of SL41 was then sequenced. The results showed that the open reading frame of SL41 was 1.617 bp long, encoding 538 amino acids. The cloning vector pMD18-T and an E. coli DH5α host were used to yield clones as E. coli DH5α/SL41. The SL41 gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2/SL41. Serine protease expressed by pYES2/SL41 was induced by galactose (maximal activity 16.5 units ml−1 at 40°C, pH 8.0) and was produced in fermentation liquid cultured for 60 h. Northern blot analysis indicated that SL41 transcripts are differentially accumulated at different culture time.
Synthesis of electroconductive polyaniline using immobilized laccase by I. S. Vasil’eva; O. V. Morozova; G. P. Shumakovich; A. I. Yaropolov (pp. 27-30).
A new method for synthesis of the conductive complex between polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward’s reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4°C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm.
Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) gene and studying the enzymatic properties of its expression product by A. S. Rozanov; S. N. Zagrebelny; A. B. Beklemishev (pp. 31-37).
The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21(DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45°C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15–20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45°C and was completely inactivated after incubation at 65°C for 1 h.
Evaluation of thermal resistance of Azotobacter chroococcum 66 using atomic force microscopy by L. N. Olyunina; Yu. A. Matskova; T. A. Goncharova; Yu. Yu. Gushina (pp. 38-42).
A possibility to use atomic force microscopy (AFM) for comparative analysis of thermal resistance of Azotobacter chroococcum 66 cells has been studied. The sizes of bacteria cells and the structuredness of the cytoderm have been shown to vary depending on the dose of hyperthermic action and on the composition of the media for heating and subsequent incubation. A thermally induced increase of a standard roughness parameter (R a) and of cell sizes has been revealed to reflect an increased level of their resistance to hyperthermia.
Phenol degradation by Rhodococcus opacus strain 1G by E. S. Shumkova; I. P. Solyanikova; E. G. Plotnikova; L. A. Golovleva (pp. 43-49).
During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two catechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to catechol 1,2-dioxygenases of the classical ortho-pathway. Chlorocatechols and chlorophenols served as competitive inhibitors of catechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.
The effect of cultivation conditions on the physicochemical properties of the exopolysaccharide ethapolan by T. P. Pirog; Yu. V. Korzh; T. A. Shevchuk (pp. 50-55).
The physicochemical properties of the complex exopolysaccharide ethapolan (EPS) produced by Acinetobacter sp. 12S during growth on media with various C/N ratios and different concentrations of mineral components and phosphate buffer were studied. Irrespective of the cultivation conditions, the concentrations of carbohydrates (38–44%) and pyruvic acid (3.2–3.7%) in the total EPS, as well as in the acylated (AP) and nonacylated (NAP) polysaccharides obtained from them, were practically the same. The EPS, AP, and NAP were also identical in their monosaccharide composition: the molar ratio of glucose, mannose, galactose, and rhamnose was 3: 2: 1: 1. The polysaccharides contained different concentrations of mineral salts (6–28%), uronic acid (3.7–22.0%), and fatty acids (5.8–15.4%); they also differed in the ratio of acetylated and nonacetylated polysaccharides. Due to the differences in the chemical composition and molecular mass (500 kDa–1.5 MDa), the viscosities of the EPS solutions (in the presence of 0.1 M KCl, in the H+-form, and in Cu2+-glycine system) were different as well. The mechanisms responsible for changes in the physicochemical properties of the total EPS, AP, and NAP synthesized on various media are discussed.
A two-stage technology for bacterial and chemical leaching of copper-zinc raw materials by Fe3+ ions with their subsequent regeneration by chemolithotrophic bacteria by N. V. Fomchenko; V. V. Biryukov (pp. 56-60).
A two-stage technology for bacterial and chemical leaching of nonferrous metals in a specifically designed laboratory unit has been proposed. At the first stage of leaching, ferric iron formed during the second stage of oxidation of Fe2+ ions by mesophilic chemolithotrophic microorganisms was used. The optimal parameters of the first stage of the process (flow rate, temperature, and the process duration) were 2 l/h, 75°C, and 24 h, respectively. The results of testing of the two-stage technology for leaching copper-zinc raw materials indicated that the depth of zinc and copper leaching can be increased from 70 to 93% and from 40 to 58.8%, respectively, and the process duration can be reduced from 120 to 24 h as compared to the commonly used one-stage technology.
Anaerobic solid-phase fermentation of plant substrates by Bacillus subtilis by N. A. Ushakova; E. S. Brodskii; A. A. Kozlova; A. V. Nifatov (pp. 61-67).
Solid-phase growth of Bacillus subtilis 8130 on cellulose-rich plant substrates (presscakes or pulp) under hypoxic conditions was accompanied by cellulose depolymerization, protein hydrolysis, and degradation of other plant components, including some processes of mixed-type carbohydrate fermentation. The bacterial fermentation yielded propionic, butyric, hexanoic acids and butyric acid derivatives. The bacterial metabolism and fermentation degree can be characterized by the proportions of fatty acids in the reaction mixture. The product of sea buckthorn cake fermentation has a good sorption quality.
Decolorization of anaerobically digested molasses spent wash by Pseudomonas putida by M. Ghosh; A. Ganguli; A. K. Tripathi (pp. 68-73).
The distillery wastewater (spent wash) contains dark brown colored recalcitrant organic compounds that are not amenable to conventional biological treatment. The characteristic recalcitrance to decolorization is due to the presence of brown melanoidin polymers. In the present study, feasibility of using Pseudomonas putida putida strain U for decolorization of spent wash was demonstrated. Batch cultures of P. putida decolourized spent wash by 24%, twofold higher decolorization was achieved following immobilization in calcium alginate beads. Glucose concentration was critical for decolourization, and improved color removal efficiency was obtained by periodic replenishment of glucose. Decolourization was also observed with lactose or whey as alternative carbon sources. The results of our study suggest that P.putida could be used for biological decolorization of molasses spent washes and that supplementation with whey (a by-product from cheese industry) can offer economical viability to the process.
The effects of natural and hybrid lectins on the legume-rhizobium interactions by Al. Kh. Baimiev; I. I. Gubaidullin; An. Kh. Baimiev; A. V. Chemeris (pp. 74-80).
The effects of hybrid lectins—full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)—on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.
A new endophytic taxane production fungus from Taxus chinensis by Z. Miao; Y. Wang; X. Yu; B. Guo; K. Tang (pp. 81-86).
More than 50 kinds of endophytic fungi associated with Taxus chinensis were isolated and examined as a potential source of the imposing anticancer drug taxol. Of these, four isolates show ability to produce taxane when measured with the competitive inhibition enzyme immunoassay method. The most promising clone, DA10, identified as Mucor rouxianus sp., is the first rouxianus reported as taxol production fungus. The presence of taxol and its important precursors, such as 10-diacetyl baccatinIII (10-DAB) and baccatinIII, in theculture of this fungus was confirmed by reactivity with a taxane-specific monoclonal antibody, comparative chromatographic and mass spectrometric behavior, cytotoxity to liver carcinoma 7402, and molecular cloning of kernel fragment of taxadiene synthase gene.
Effect of synthetic cyclopentane β,β′-triketones on amino acid metabolism in roots of buckwheat (Fagopyrum esculentum Moench.) seedlings by E. A. Demina; L. Ya. Tishchenko; O. P. Shestak; V. L. Novikov; M. M. Anisimov (pp. 87-92).
Germination of buckwheat seeds in solutions of synthetic mono- and tricyclic cyclopentane β,β′-triketones of various concentrations was accompanied by inhibition of seedling root growth and changes in the contents of glutamate, γ-aminobutyric acid, proline, glutamine, and alanine. The monocyclic triketone also affected the amount of isoleucine. It is likely that the increase in proline content is a nonspecific response significant for enhancing stress tolerance in seedlings.
The effect of ethylene biosynthesis regulators on metabolic processes in the banana fruits in various physiological states by E. A. Bulantseva; Nguyen Tien Thang; A. O. Ruzhitsky; M. A. Protsenko; N. P. Korableva (pp. 93-96).
The effects of ethylene-evolving preparations—2-chloroethylphosphonic acid (2-CEPA), the new generation binary preparation ethacide, and the specific inhibitor of ethylene biosynthesis aminooxyacetic acid (AOA)—on the ethylene evolution by banana (Musa sp.) fruits at various ripening stages and the content of protein inhibitor of polygalacturonase (PIPG), associated with prevention of fruit tissue softening, were studied. It was demonstrated that the ripening stage was of significant importance for the results of treatment with the mentioned preparations. Their effects were most pronounced in the fruits of medium ripeness. 2-CEPA and ethacide increased the ethylene evolution in banana fruits on the average by 25–30%. AOA treatment decreased the ethylene evolution in these fruits by 30%. The PIPG content in fruit pulp was insignificant; 2-CEPA almost did not change its content in banana skin, while ethacide and AOA somewhat decreased it. Consequently, the regulators of ethylene biosynthesis have a potential for optimizing the state of banana fruits during storage and sale.
The effect of precipitation of the complexes of streptavidin with biotinylated proteins in agar gel by M. J. Navakouski; I. I. Vashkevich; O. V. Sviridov (pp. 97-103).
The effect of precipitation of complexes of streptavidin with biotinylated proteins under conditions of simple (according to Mancini) and double (according to Ouchterlony) radial diffusion in agar gel was studied. The position and form of precipitation lines depended primarily on the initial concentration of components and the degree of protein biotinylation. Free biotin, 1% SDS, and 6 M urea contained in the gel, as well as thermal denaturation of streptavidin inhibited the precipitate formation. Mannose, glucose, fucose, galactose, sucrose, and NaCl at high concentrations had no effect on biospecific precipitation. A model of interaction of streptavidin with biotinylated macromolecules is suggested, which accounts for the observed effect, and the prospects of practical application of the precipitation effect are discussed.
Investigation of the photochemical properties of biopterin and its reduced forms by T. A. Lyudnikova; O. A. Dashina; T. A. Telegina; M. S. Kritsky (pp. 104-109).
UV irradiation (270–390 nm, 20 min, I = 3.2 W m−2) of deaerated biopterin solution containing electron donor (Na2-EDTA) led to the formation of 7,8-dihydrobiopterin (H 2 BPT), which, when excited, underwent reduction to form 5,6,7,8-tetrahydrobiopterin (H 4 BPT). Protonated molecules of H4BPT were resistant to oxidation by O2 both in the “dark” incubated and UV-irradiated solutions at pH below 3.0. The rate of H4BPT oxidation dramatically increased at pH above 3.0, and, then, up to pH 10.0, it did not change, showing no dependence on UV irradiation. At the initial stage (5 min) of H4BPT oxidation in neutral solution, UV irradiation stimulated the accumulation of quinonoid 6,7-dihydrobiopterin (q-H 2 BPT) in addition to H2BPT. UV irradiation of H2BPT induced its oxidation to biopterin and unidentified products.
Recent developments in early identification and prevention of risks in food safety
by E. Sokolova; A. Zherdev (pp. 110-111).