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卷 54, 编号 9 (2018)
- 年: 2018
- 文章: 10
- URL: https://journals.rcsi.science/0003-6838/issue/view/9134
Problems and Prospects
Wheat Gluten and Its Hydrolysates. Possible Fields of Practical Use
摘要
Wheat gluten is a group of water-insoluble proteins of wheat endosperm. As a co-product of the starch and bioethanol production, gluten is available in fairly large quantities. Therefore, the search for optimal ways of its industrial utilization is an important economic task. The food industry is a traditional area for gluten application. However, the study of its structural and functional properties allowed expanding the area of the gluten use due to its various modifications, in particular, enzymatic hydrolysis. As compared to native gluten, the gluten hydrolysates have better foaming and emulsifying abilities, as well as higher water solubility. Biologically active properties of hydrolysates (antioxidant, hepatoprotective and angiotensin-converting enzyme inhibiting) were also shown. The search for and selection of proteolytic enzymes or their combinations, and optimization of the technological process will permit to obtain the hydrolysis products with the required characteristics, expand their use in industry and reduce the cost of their production.
825-833
Producing Organisms, Biology, Selection, and Genetic Engineering
Laccase Isoenzymes of Trametes hirsuta LE-BIN072: Degradation of Industrial Dyes and Secretion under the Different Induction Conditions
摘要
It is well-known that various oxidative enzymes of wood-degrading fungi are widely demanded in various fields of biotechnology. Laccases attract particular attention, since they have different substrate specificities and can be used not only in pulp and paper industry, but also for the degradation of various xenobiotics, including decolorization of dyes. In this work, a comparative analysis of the native major (LacA) and the recombinant minor (rLacC, rLacD, rLacF) Trametes hirsuta 072 laccase isoenzymes applicability for bleaching of four widely used recalcitrant dyes was conducted. It was shown that all isoenzymes decolorized dyes to different extent, and the minor isoenzymes rLacD and rLacF are the most promising for decolorization of congo red and phenol red dyes. Moreover, we provide detailed exoproteome analysis of T. hirsuta 072 growing on the media supplemented with compounds that were previously shown to increase transcription levels of the minor laccase isoenzymes.
834-841
Modification of a System Based on the Use of Selection and Sorting Markers for the Screening of Stable Transfectants
摘要
The р-hook technique, which is based on protein domain expression on the cell surface, has been modified and used for the selection and highly sensitive and efficient sorting of stably transfected CHO-S cells. The model protein EYFP served as both the target protein produced by the cell and the expression marker in this study. Chimeric proteins TiBP-PDGFR and CBD-PDGFR, which enabled the binding of protein-producing cells to titanium carriers and cellulose supports, respectively, have been constructed. Combined selection with the use of the chimeric CBD-PDGFR protein exposed on the cell membrane resulted in the production of a clonal line series characterized by a high and stable level of model protein synthesis. The level of model protein expression (inferred from fluorescence) in three clones from the selected series was more than 30 times higher than the average level for the original cell pool. The expression stability in EYFP-positive clones was preserved at late (20th and 35th) culture passages. The use of the chimeric CBD-PDGFR selection and sorting marker developed in this study enables the rapid selection of highly productive stable transfectants and thus reduces the costs associated with the creation of a productive cell line collection.
842-848
Inactivation of Malic Enzymes Improves the Anaerobic Production of Four-Carbon Dicarboxylic Acids by Recombinant Escherichia coli Strains Expressing Pyruvate Carboxylase
摘要
The genes maeA and maeB, encoding NADH- and NADPH-dependent malic enzymes, have been deleted in a recombinant Escherichia coli strain with inactivated mixed-acid fermentation pathways and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. During anaerobic glucose utilization, the parental strain synthesized malic, fumaric, and succinic acids as the main fermentation end products, while pyruvic acid was accumulated as the main by-product resulting from the functioning of the pyruvate–oxaloacetate–malate–pyruvate futile cycle. Upon individual deletions of the maeA and maeB genes, the mutant strains converted glucose into four-carbon dicarboxylic acids with increased efficiency still secreting notable amounts of pyruvic acid. The combined inactivation of both malic enzymes in the constructed strain significantly elevated the portion of malic, fumaric, and succinic acids among the fermentation end products with a concomitant decrease in the secretion of pyruvic acid and other by-products due to the abolishment of the action of the futile cycle competing with the target biosynthetic processes.
849-854
Biological Preparation Technology
Immunological Efficacy and Safety of Synthesized CpG Oligodeoxynucleotides
摘要
The technology for synthesis is described, and the adjuvant properties of CpG oligodeoxynucleotides (CpG-ODNs) are assessed. CpG-ODN sequences were generated according to the available sequences on an automatic synthesizer. The adjuvant activity was evaluated with CpG-ODNs in combination with a recombinant protective antigen and EA1, an S-layer protein of the anthrax agent. It was established that the use of the synthesized adjuvant CpG 2006, along with immunogenic antigens, leads to the development of long-term, high-level immunity in test animals. The synthetic CpG 2006 antigenic product was shown to have an advantage over alhydrogel in terms of adjuvant activity. Experiments on biomodels provided data confirming the absence of toxic and damaging effects of CpG-ODNs on cells and tissues of the macroorganism. Comparison of the cell-mediated immunity (content of CD4+ and CD8+) after immunization by the B. anthracis STI-1 strain or a recombinant anthrax vaccine prototype with CpG 2006 or alhydrogel as an adjuvant is evidence of the activation of the cellular component of the immune system in all of the compared groups.
855-862
Obtainment of Succinic Acid and Higher Alcohols (C8–С10) Diesters by Biphasic Esterification
摘要
Biphasic esterification of model aqueous solutions of crystalline succinic acid or culture liquid obtained as a result of the growth of a succinic acid producer Yarowia lypolitica was studied. The main part of the study was performed with isooctyl (2-ethylhexyl) alcohol. Laurylsulfonic acid (another name dodecylsulfonic acid) was introduced as a catalyst. It was shown that the diester yield increases with an increase in the ratio alcohol/succinic acid solution, and it was higher than 90% when the ratio was 2 : 1. At a constant ratio of the phases described above, the product yield increased with a decrease in the concentration of succinic acid in the aqueous solution. More than 10% of the DSA catalyst passes into the alcohol phase; the compound can be extracted by Na2CO3 solution. The application of real culture liquid resulted in a significant decrease in the product yield as compared to the model succinic acid solutions. The process may become industrially promising with the selection of other efficient catalysts and improved microbial fermentation in order to decrease the content of related organic acid in the culture liquid.
863-868
Biologicals Use
Immunochemical and Protective Properties of Conjugated Capsular Polysaccharide of Streptococcus pneumoniae Serotype 9N
摘要
Conjugates of Streptococcus pneumoniae type 9N capsular polysaccharide with tetanus toxoid have been prepared. Their interactions with specific antibodies to tetanus toxoid and polysaccharide 9N were assessed, and the conjugate with optimal ratio between polysaccharide and carrier protein was chosen for further investigations (3 : 4, wt/wt). Adsorption characteristics of tetanus toxoid and polysaccharide on aluminum hydroxide were determined using the quantitative ELISA for further in vivo experiments; the optimum ratio of the conjugate and adjuvant was proved to be ≤1 : 1 (wt/wt). Repetitive immunization with selected conjugate adsorbed on aluminum hydroxide evoke the anti-PS antibody with titers equal to 70 400 (p < 0.001) which were 140-fold higher comparing to those for the unconjugated form of the antigen. The investigation of the formation of protective immunity from Streptococcus pneumoniae serotype 9N was performed by intranasal challenge of wild-type mice and revealed that the titers of bacterial contamination in the lungs of immunized mice were 10-fold lower comparing with non-immunized mice 72 h after the challenge (p > 0.05).
869-875
A Strain Rhodococcus wratislaviensis КТ112-7 as a Basis for Bioregeneration of PCB-Contaminated Metal/Carbon Catalyst
摘要
A possibility of the bioregeneration of polychlorinated biphenyl-contaminated metal/carbon catalysts using aerobic bacteria as biological agents has been shown. Using the 5%-Pd/Sibunit catalyst contaminated by the products of the hydrodechlorinaton of the PCB commercial mixture of the Sovol brand and a strain of Rhodococcus wratislaviensis КТ112-7 it was proved that the application of the bacterial culture permits to rapidly clean the catalyst with the minimal technical and economic costs. It was established that the degree of the catalyst decontamination for 24 h with the help of the intact bacteria cells was equal to 99% (0.77 mg/mL). According to the gas-liquid chromatography, the R. wratislaviensis КТ112-7 strain decomposed all the components occurring on 5%-Pd/Sibunit after the reaction of PCB hydrodechlorination. The efficiency of the bacterial destruction made up 97% (0.76 mg/mL). The residual portion of contaminating compounds was adsorbed by the bacterial cells. Therefore, the R. wratislaviensis КТ112-7 strain can be used as a biological component in the systems for PCB-contaminated metal/carbon catalyst cleaning.
876-885
Metrology, Standardization, Control
A Method for the Simultaneous Detection of Vibrio cholerae Strains and Drug-Resistant Genes in their Genome by Real-Time PCR
摘要
A method was developed for the simultaneous detection of V. cholerae strains and the presence of drug-resistant genes in their genomes by real-time PCR. Resistance to four antibiotics used for cholera treatment (tetracycline, trimethoprim, chloramphenicol and ciprofloxacin) was analyzed. The sensitivity of the panel was 1 × 103 CFU/mL for pure cultures, simulated clinical material, and environmental samples. PCR efficiency was confirmed by the analysis of 60 natural V. cholerae strains isolated from patients and the environment in different years. It was established that all of the studied V. cholerae strains of the O1 serogroup El Tor biovar isolated before 1993 did not contain the tested drug-resistant genes. At the same time, 18 toxigenic clinical strains (90%) imported within 1993–2010 were characterized by multiple drug resistance; their genomes contained trimethoprim resistance genes (dfrA1) and chloramphenicol resistance genes (floR), and 11 strains additionally contained a tetracycline-resistant gene tetR. In addition, nontoxigenic clinical and aqueous V. cholerae strains carrying a ciprofloxacin-resistant gene, the qnrVC (qnrVC1) gene, have been detected in the last few years (Kalmykia, 2011–2013).
886-893
Comparative Analysis of the Expression Efficiency of the Bacterial Phytase Genes in Pichia pastoris Yeast by the Plate Test
摘要
Comparative analysis of the efficiency of the expression of the Escherichia coli, Obessumbacterium proteus, and Citrobacter freundii phytase genes in Pichia pastoris methylotrophic yeast has been carried out. A simple model with a genetic construct based on the pPICά vector and the plate test with a modified diagnostic medium containing methanol (instead of glucose) and calcium phytate was proposed for the first time in order to assess the expression level. The optimal pH value for the phytase activity was provided by the addition of lactic acid. The highest expression in the studied heterologous genes was shown for the C. freundii phytase gene.
894-898