The Role of Electrostatic Interactions in Complex Formation between Bacterial Luciferase and NADPH:FMN-oxidoreductase

A possible mechanism of complex formation between bacterial luciferase and NADPH:FMNoxidoreductase from Vibrio harveyi sustained by electrostatic forces is studied. The complex between the enzymes is important for a direct FMNH2 transfer without a contact with solvent, which could cause a rapid autooxidation and the formation of reactive oxygen species. In the current work the diversity of possible relative positions of NADPH:FMN-oxidoreductase and luciferase was obtained with Monte-Carlo sampling governed by oxidoreductase internal charged groups and electrostatic field caused by luciferase. Among the structures with the minimal energies, the one was found that has a proper active sites orientation for a direct FMNH2 transfer. Possible role of hydrogen bonding between Arg291 and Gln197 of luciferase and oxidoreductase, respectively, in stabilization of this complex is propose

Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination

Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5’-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FM

Bioluminescent enzyme inhibition based assay of metal nanoparticles

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Is Body Mass Index a potential biomarker for anemia in obese adolescents?

The two paradoxical major health problems namely obesity and anemia are confirmed to affect millions around the world. Hepcidin, a protein synthesized in liver is a negative iron binding regulator. There is an affirmative relation between hepcidin and leptin levels and an inverse co-relation between hepcidin and iron status due to inflammation mediated by obesity in adolescents. So this implicates an alliance between anemia and obesity wherein weight reduction can be a powerful medium to improve iron absorption in obese adolescents. Also the Body Mass Index can serve as a preliminary non-invasive screening tool to identify potential adolescents prone to anemia. Keywords: Anemia, Obesity, Hepcidin, Leptin, Body Mass Inde

Functional divergence between evolutionary related LuxG and Fre oxidoreductases of luminous bacteria

In luminous bacteria NAD(P)H:flavin-oxidoreductases LuxG and Fre there are homologous enzymes that could provide a luciferase with reduced flavin. While Fre functions as a housekeeping enzyme, LuxG appears to be a source of reduced flavin for bioluminescence as it is transcribed together with luciferase. This study is aimed at providing the basic conception of Fre and LuxG evolution and revealing the peculiarities of the active site structure resulted from a functional variation within the oxidoreductase family. A phylogenetic analysis has demonstrated that Fre and LuxG oxidoreductases have evolved separately after the gene duplication event, and consequently, they have acquired changes in the conservation of functionally related sites. Namely, different evolutionary rates have been observed at the site responsible for specificity to flavin substrate (Arg 46). Also Tyr 72 forming a part of a mobile loop involved into FAD binding has been found to be conserved among Fre in contrast to LuxG oxidoreductases. The conservation of different amino acid types in NAD(P)H binding site has been defined for Fre (arginine) and LuxG (proline) oxidoreductases

Development of a university’s intellectual capital within “ontologisation”

This article introduces the concept of Networked Self-Developing Education System (NSDES). Such a system could be considered a modern university with a triple network structure where network team interactions between representatives of science, education and production are developed. A new model to explain NSDES intellectual capital development management is described. The model includes four interrelated parts (intellectual property, human, organisation and consumer types of capital). An important NSDES function is to provide conditions for lifelong self-development of scholars and students’ mastery of the scientific method. This is especially important for interdisciplinary natural sciences (biophysics, etc.). The NSDES has its integrative resource potential to meet this challenge. Such potential is accumulated in the open knowledge base and can be transferred to the education process provided knowledge is standardised in an ontology format. To build a course ontology to study the bioluminescent enzyme technology, the experience of developing ontology-based education solutions for railway universities which are evolving into “knowledge factories” is useful. Thus, “ontologisation” is proved to be a trend in the digital transformation of NSDES. The model’s implementation through ontology-based education solutions will create a foundation for the development of students’ research competences to solve urgent problems in sciences and industries

Биолюминесцентный ферментативный ингибиторный анализ наночастиц на основе металлов

The bioluminescent enzymatic bioassays for assessment of nanomaterial biotoxicity using the soluble or immobilized coupled enzyme system of luminous bacteria NAD(P)Н:FMN-oxidoreductase + luciferase (Red + Luc) as a test system were employed in this study. This method specifically detects the toxic properties of substances based on their effect on the parameters of the bioluminescent enzyme reactions. The commercially available metal nanoparticles (MNPs), including silver nanoparticles (Ag), nanoparticles of silicon dioxide (SiO2), and titanium dioxide (TiO2), of different sizes were tested in the study. The inhibitory effects of MNPs on the bioluminescent Red + Luc enzyme system were measured. Results indicated that the soluble Red + Luc coupled enzyme system was more sensitive to the inhibition effect of MNPs than its immobilized form. The inhibitory activity of MNPs decreased in the following order: Ag > TiO2 > SiO2. That correlated well with results of other biological methods. Due to substantial advantages such as technical simplicity, short response time and high sensitivity to analysis, this bioluminescent enzymatic bioassay has the potential to be developed as a general bioassay for safety assessment of a wide variety of nanomaterialsПредложен метод оценки биотоксичности наноматериалов, основанный на использовании в качестве объекта воздействия растворимой и иммобилизованной биолюминесцентной биферментной системы: НАД(Ф)·Н:ФМН-оксидоредуктаза и люцифераза. Принцип метода состоит в обнаружении токсических свойств тестируемых веществ по их влиянию на параметры биолюминесценции используемой биферментной системы. Проведено тестирование коммерчески доступных наночастиц на основе металлов (МНЧ), в том числе наночастиц серебра (Ag), и различающихся по размеру наночастиц диоксидов кремния (SiO2) и титана (TiO2). Эти МНЧ оказывают ингибирующий эффект на активность биферментной системы, причем растворимые ферменты в большей степени подвержены ингибирующему воздействию МНЧ по сравнению с иммобилизованными. Степень ингибирующего воздействия уменьшается в ряду Ag > TiO2 > SiO2, что согласуется с результатами других биологических методов. Биолюминесцентный ферментативный метод анализа занимает 2-3 мин, отличается высокой чувствительностью, технической простотой и может использоваться для оценки безопасности различных классов наноматериало

APPLICATION OF BIOLUMINESCENT ENZYMATIC TESTS IN ECOTOXICOLOGY

This paper examines the general principles of bioluminescent enzymatic toxicity bioassays and describes the applications of these methods and the implementation in commercial biosensors. Bioluminescent Enzyme System Technology (BEST) has been proposed in the bacterial coupled enzyme system, wherein NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST was introduced to facilitate and accelerate the development of cost-competitive enzymatic systems for use in biosensors for medical, environmental, and industrial applications. For wide-spread use of BEST, the multi-component reagent ‘Enzymolum’ has been developed, which contains the bacterial luciferase, NADH:FMN-oxidoreductase and their substrates, co-immobilized in starch or gelatin gel. ‘Enzymolum’ can be integrated as a biological module into the portable biodetector-biosensor originally constructed for personal use. Based on the example of ‘Enzymolum’ and the algorithm of creating new enzyme biotests with tailored characteristics, a new approach was demonstrated in biotechnological design and construction. The examples of biotechnological design of various bioluminescent methods for ecological monitoring were provided. The advantages of enzymatic assays are their rapidity (the period of time required does not exceed 3-5 minutes), high sensitivity, simplicity and safety of procedure, possibility of automation of ecological monitoring; required luminometer is easily available

Immobilization of Firefly Bioluminescent System: Development and Application of Reagents

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL−1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination