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Applied Biochemistry and Microbiology (v.48, #5)
Biogas production from cellulose-containing substrates: A review by E. A. Tsavkelova; A. I. Netrusov (pp. 421-433).
Anaerobic microbial conversion of organic substrates to various biofuels is one of the alternative energy sources attracting the greatest attention of scientists. The advantages of biogas production over other technologies are the ability of methanogenic communities to degrade a broad range of substrates and concomitant benefits: neutralization of organic waste, reduction of greenhouse gas emission, and fertilizer production. Cellulose-containing materials are good substrate, but their full-scale utilization encounters a number of problems, including improvement of the quality and amount of biogas produced and maintenance of the stability and high efficiency of microbial communities. We review data on microorganisms that form methanogenic cellulolytic communities, enzyme complexes of anaerobes essential for cellulose fiber degradation, and feedstock pretreatment, as biodegradation is hindered in the presence of lignin. Methods for improving biogas production by optimization of microbial growth conditions are considered on the examples of biogas formation from various types of plant and paper materials: office paper and cardboard.
Catalytic properties of a nitrile hydratase immobilized on activated chitosan by Yu. G. Maksimova; T. A. Rogozhnikova; G. V. Ovechkina; A. Yu. Maksimov; V. A. Demakov (pp. 434-439).
The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V max), whereas the Michaelis constant (K M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.
Production of cellulase by immobilized whole cells of Haloarcula by A. Ogan; O. Danis; A. Gozuacik; E. Cakmar; M. Birbir (pp. 440-443).
Halophilic Archaea are adapted to a life in the extreme conditions and some of them are capable of growth on cellulosic waste as carbon and energy source by producing cellulase enzyme. The production of cellulase using free and immobilized cells of halophilic archaeal strain Haloarcula 2TK2 isolated from Tuzkoy Salt Mine and capable of producing cellulose was studied. The cells were cultured in a liquid medium containing 2.5 M NaCl to obtain the maximum cellulase activity and immobilized on agarose or polyacrylamide or alginate. Optimal salt dependence of free and immobilized cells of Haloarcula 2TK2 was established and the effects of pH and temperature were investigated. Immobilization to Na-alginate enhanced the enzymatic activity of the Haloarchaeal cells when compared to free cells and other polymeric supports. From the results obtained it is reasonable to infer that decomposition of plant polymers into simpler end products does occur at high salinities and cellulase producing haloarchael cells may be potentially utilized for the treatment of hypersaline waste water to remove cellulose.
Fructose 6-phosphate phosphoketolase activity in wild-type strains of Lactobacillus, isolated from the intestinal tract of pigs by E. Bolado-Martínez; E. Acedo-Félix; A. B. Peregrino-Uriarte; G. Yepiz-Plascencia (pp. 444-451).
Phosphoketolases are key enzymes of the phosphoketolase pathway of heterofermentative lactic acid bacteria, which include lactobacilli. In heterofermentative lactobacilli xylulose 5-phosphate phosphoketolase (X5PPK) is the main enzyme of the phosphoketolase pathway. However, activity of fructose 6-phosphate phosphoketolase (F6PPK) has always been considered absent in lactic acid bacteria. In this study, the F6PPK activity was detected in 24 porcine wild-type strains of Lactobacillus reuteri and Lactobacillus mucosae, but not in the Lactobacillus salivarius or in L. reuteri ATCC strains. The activity of F6PPK increased after treatment of the culture at low-pH and diminished after porcine bile-salts stress conditions in wild-type strains of L. reuteri. Colorimetric quantification at 505 nm allowed to differentiate between microbial strains with low activity and without the activity of F6PPK. Additionally, activity of F6PPK and the X5PPK gene expression levels were evaluated by real time PCR, under stress and nonstress conditions, in 3 L. reuteri strains. Although an exact correlation, between enzyme activity and gene expression was not obtained, it remains possible that the xpk gene codes for a phosphoketolase with dual substrate, at least in the analyzed strains of L. reuteri.
Effect of cultivation conditions on the adhesive activity of Rhodococcus cells towards n-Hexadecane by E. V. Rubtsova; M. S. Kuyukina; I. B. Ivshina (pp. 452-459).
The effect of cultivation conditions (the composition, acidity, and salinity of the cultivation medium; temperature; and the hydrodynamic conditions of cultivation) on the adhesion of actinobacteria of the genus Rhodococcus to n-hexadecane has been investigated. A study performed showed that the adhesive activity of rhodococci depends on the composition of the cultivation medium and on the cultivation temperature. The possible mechanisms underlying the effect of growth conditions on the adhesion of rhodococci to liquid hydrocarbons and involving changes in the cell lipid content or the zeta potential of cells are addressed. Rhodococcal strains displaying high adhesive activity (80–90%) at a low temperature (18°C), high salinity (5.0% NaCl), and acidity (pH 6.0) of the cultivation medium have been selected as a result of the present work; these strains have a considerable potential for use in bioremediation of soil and water contaminated by hydrocarbons.
Degradation of nicosulfuron by Bacillus subtilis YB1 and Aspergillus niger YF1 by X. H. Lu; Z. H. Kang; B. Tao; Y. N. Wang; J. G. Dong; J. L. Zhang (pp. 460-466).
The optimal degrading conditions for the nicosulfuron degradation by Bacillus subtilis YB1 and Aspergillus niger YF1, and site of their action on nicosulfuron were studied. The results showed that the degradation efficiency of free cells of B. subtilis YB1 and A. niger YF1 was respectively 87.9 and 98.8% in basic medium III containing 2 mg/l of nicosulfuron after inoculation with 1 ml of culture containing 2.3 × 107 CFU ml−1 and incubation for 5 days at 35°C. Moreover, the degradation rate of nicosulfuron by the mixture of microorganisms was much higher than for every of them taken separately in the same conditions. The mass spectrometric analysis of the products degraded by B. subtilis YB1 revealed that the sulfonylurea bridge in nicosulfuron molecule had been broken. Extracellular (EXF) and endocellular (ENF) fractions obtained from bacterium and fungus were tested for the ability to degrade nicosulfuron. The degradation efficiency of fractions extracted from B. subtilis YB1 was 66.8% by EXF and 15.8% by ENF, but neither EXF nor ENF extracted from A. niger YF1 had the activity of degrading nicosulfuron.
Functional analysis of the genome fragment involved in aerobic dichloromethane degradation by Methylobacterium dichloromethanicum DM4 by Yu. E. Firsova; D. N. Fedorov; Yu. A. Trotsenko (pp. 467-472).
The hypothetical genes of Methylobacterium dichloromethanicum DM4, METDI 2671 (bioD 2), and METDI 2680 located within the chromosomal fragment (126 kb) associated with dichloromethane (DCM) degradation have been studied. The reverse transcription polymerase chain reaction method (RT-PCR) showed the presence of transcripts of both genes in cells grown on DCM and methanol. The mobilized suicidal vector pK18mob was used to obtain knockout mutants in these genes. The BIO mutant (with an insertion in the bioD 2 gene) after cultivation on methanol was characterized by a lower growth rate on DCM compared to the wild-type DM4 strain, while the MT mutant (with an insertion in the METDI 2680 gene) did not differ from the initial strain in respect of these characteristics. The results demonstrate the involvement of the bioD 2 gene in biotin biosynthesis coupled with DCM degradation.
Cloning and bioinformatic analysis of an acidophilic β-mannanase gene, Anman5A, from Aspergillus niger LW-1 by S. G. Zhao; M. C. Wu; C. D. Tang; S. J. Gao; H. M. Zhang; J. F. Li (pp. 473-481).
Using 3′ and 5′ rapid amplification of cDNA ends (RACE) techniques, the full-length cDNA sequence of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated to AnMan5A), was identified from the total RNA. The cDNA sequence was 1417 bp in length, harboring 5′- and 3′-untranslated regions, as well as an open reading frame (ORF) which encodes a 21-aa signal peptide, a 17-aa propeptide and a 345-aa mature peptide. Based on the topology of the phylogenetic tree of β-mannanases from glycoside hydrolase (GH) family 5, the AnMan5A belongs to the subfamily 7 of the GH family 5. Its 3-D structure was modeled by the bitemplate-based method using both MODELLER 9.9 and SALIGN programs, based on the known β-mannanase crystal structures of Trichoderma reesei (1QNO) and Lycopersicon esculentum (1RH9) from the GH family 5. In addition, the complete DNA sequence of the Anman5A was amplified from the genomic DNA using the pUCm-T vector-mediated PCR and conventional PCR methods. The DNA sequence was 1825 bp in length, containing a 5′-flanking regulatory region, 2 introns and 3 exons when compared with the full-length cDNA.
Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25 by A. Nadhman; F. Hasan; Z. Shah; A. Hameed; A. A. Shah (pp. 482-487).
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.
Aspergillus ochraceus micromycetes—producers of extracellular proteinases—protein C activators of blood plasma by A. A. Osmolovskiy; V. G. Kreier; A. V. Kurakov; N. . Baranova; N. S. Egorov (pp. 488-492).
Natural isolates of Aspergillus ochraceus micromycetes from soil and plant remains from various regions have been screened. The isolated strains were characterized by similar cultural and morphological features and an identical nucleotide sequence in the ITS1-5,8S-ITS2 region of rDNA. The ability of the extracellular proteinases of A. ochraceus micromycetes to activate protein C of blood plasma has been established. Differences are revealed in the accumulation of proteinases activating protein C and proteinases with thrombinand plasmin-like activities in the growth dynamics of producers.
Development of complex enzymatic preparations of pactinases and celulases for sugar beet marc digestion by E. V. Bushina; A. M. Rozhkova; I. N. Zorov; A. D. Satrutdinov; A. O. Bekkarevich; A. V. Koshelev; O. N. Okunev; A. P. Sinitsyn (pp. 493-499).
Complex enzymatic preparations demonstrating activities homologous to pectinlyase A and heterologous to endo-1,4-β-glucanase from Penicilliumverruculosum and β-glycosidase from Aspergillusniger have been obtained on the basis of recombinant strains of the fungus Penicilliumcanescens. Two approaches were utilized: development of an enzymatic preparation on the basis of a new strain, which produced all three enzymes, and development of an enzymatic preparation via combined cultivation of three strains, each of which produced one of the enzymes.
Induction of heat resistance of wheat coleoptiles by salicylic and succinic acids: Connection of the effect with the generation and neutralization of reactive oxygen species by Yu. E. Kolupaev; T. O. Yastreb; N. V. Shvidenko; Yu. V. Karpets (pp. 500-505).
The influence of salicylic (SaA) and succinic (SuA) acids on the generation of reactive oxygen species (ROS) and the heat resistance of wheat (Triticum aestivum L.) coleoptiles has been studied. The treatment of coleoptiles with 10 μM SaA or SuA results in the accumulation of hydrogen peroxide and enhanced formation of a superoxide anion radical. This effect was partially suppressed by both α-naphthol (the NADPH oxidase inhibitor) and salicylhydroxamic acid (peroxidase inhibitor). SaA and SuA cause an increase in the activity of antioxidant enzymes, such as superoxide dismutase, catalase, and soluble peroxidase, and improve the heat resistance of coleoptiles. Antioxidant ionol and inhibitors of the NADPH oxidase and peroxidase significantly reduce the positive influence of SaA and SuA on the heat resistance of wheat coleoptiles. ROS are considered to be intermediates for heat resistance induction in coleoptiles, treated with SaA and SuA; enhanced ROS generation can be caused by an increased activity of the NADPH oxidase and peroxidase.
Effect of conditions of monoclonal antibody adsorption on antigen-binding activity by Yu. N. Tarakanova; D. A. Dmitriev; Yu. S. Massino; M. B. Smirnova; O. L. Segal; O. V. Fartushnaya; D. A. Yakovleva; G. I. Kolyaskina; V. F. Lavrov; A. D. Dmitriev (pp. 506-512).
The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013–0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.
Comparison of the antiradical activity of ionol, components of fresh ginger, and its extracts by E. S. Alinkina; T. A. Misharina; L. D. Fatkullina; E. B. Burlakova (pp. 513-518).
The antiradical properties of three samples of ginger (Zingiber officinale R.)—juice from fresh rhizome, essential oil, and extracts (oleoresin)—were studied and compared with the properties of synthetic antioxidant ionol (butylatedhydroxy-toluene, BHT). Reaction antioxidants with stable free 2,2-diphenyl-1-picrylhydrozyl radicals were used as model systems. DPPH equivalents per gram of ginger sample, EC50, and antiradical efficiency (AE) were determined. The EC50 and AE values for ginger oleoresin and BHT were similar. They were the same as those of highly active natural antioxidants, and the values for essential oil and ginger juice were lower by two orders of magnitude. On the base of kinetic parameters, the ginger samples may belong to antiradical compounds with prolonged action.
Receptor elements for biosensors in two ways of methylotrophic yeast immobilization by M. G. Zaitsev; V. A. Arlyapov; V. A. Alferov; A. N. Reshetilov (pp. 519-524).
Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.