Enzyme Inhibition-Based Assay to Estimate the Contribution of Formulants to the Effect of Commercial Pesticide Formulations.

Pesticides can affect the health of individual organisms and the function of the entire ecosystem. Therefore, thorough assessment of the risks associated with the use of pesticides is a high-priority task. An enzyme inhibition-based assay is used in this study as a convenient and quick tool to study the effects of pesticides at the molecular level. The contribution of formulants to toxicological properties of the pesticide formulations has been studied by analyzing effects of 7 active ingredients of pesticides (AIas) and 10 commercial formulations based on them (AIfs) on the function of a wide range of enzyme assay systems differing in complexity (single-, coupled, and three-enzyme assay systems). Results have been compared with the effects of AIas and AIfs on bioluminescence of the luminous bacterium Photobacterium phosphoreum. Mostly, AIfs produce a considerably stronger inhibitory effect on the activity of enzyme assay systems and bioluminescence of the luminous bacterium than AIas, which confirms the contribution of formulants to toxicological properties of the pesticide formulation. Results of the current study demonstrate that "inert" ingredients are not ecotoxicologically safe and can considerably augment the inhibitory effect of pesticide formulations; therefore, their use should be controlled more strictly. Circular dichroism and fluorescence spectra of the enzymes used for assays do not show any changes in the protein structure in the presence of commercial pesticide formulations during the assay procedure. This finding suggests that pesticides produce the inhibitory effect on enzymes through other mechanisms.

Toxicity of Different Types of Surfactants via Cellular and Enzymatic Assay Systems.

Surfactants have a widespread occurrence, not only as household detergents, but also in their application in industry and medicine. There are numerous bioassays for assessing surfactant toxicity, but investigations of their impact on biological systems at the molecular level are still needed. In this paper, luminous marine bacteria and their coupled NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc) enzyme system was applied to examine the effects of different types of surfactants, including cationic cetyltrimethylammonium bromide (CTAB), non-ionic polyoxyethylene 20 sorbitan monooleate (Tween 80) and anionic sodium lauryl sulfate (SLS), and to assess whether the Red + Luc enzyme system can be used as a more sensitive indicator of toxicity. It was shown that the greatest inhibitory effect of the surfactants on the activity of luminous bacteria and the Red + Luc enzyme system was in the presence of SLS samples. The calculated IC50 and EC50 values of SLS were 10-5 M and 10-2 M for the enzymatic and cellular assay systems, respectively. The results highlight the benefits of using the enzymatic assay system in ecotoxicology as a tool for revealing surfactant effects on intracellular proteins if the cellular membrane is damaged under a long-term exposure period in the presence of the surfactants. For this purpose, the bioluminescent enzyme-inhibition-based assay could be used as an advanced research tool for the evaluation of surfactant toxicity at the molecular level of living organisms due to its technical simplicity and rapid response time.

Enzymes Immobilized into Starch- and Gelatin-Based Hydrogels: Properties and Application in Inhibition Assay.

The present work is a review of the research on using hydrogels based on natural biodegradable polymers, starch, and gelatin for enzyme immobilization. This review addresses the main properties of starch and gelatin that make them promising materials in biotechnology for producing enzyme preparations stable during use and storage and insensitive to chemical and physical impacts. The authors summarize their achievements in developing the preparations of enzymes immobilized in starch and gelatin gels and assess their activity, stability, and sensitivity for use as biorecognition elements of enzyme inhibition-based biosensors.

Design of bioluminescent biosensors for assessing contamination of complex matrices.

The presence of potentially toxic xenobiotics in complex matrices has become rather the rule than the exception. Therefore, there is a need for highly sensitive inexpensive techniques for analyzing environmental and food matrices for toxicants. Enzymes are selectively sensitive to various toxic compounds, and, thus, they can be used as the basis for detection of contaminants in complex matrices. There are, however, a number of difficulties associated with the analysis of complex matrices using enzyme assays, including the necessity to take into account properties and effects of the natural components of the test media for accurate interpretation of results. The present study describes the six-stage procedure for designing new enzyme sensors intended for assessing the quality of complex matrices. This procedure should be followed both to achieve the highest possible sensitivity of the biosensor to potentially toxic substances and to minimize the effect of the uncontaminated components of complex mixtures on the activity of the biosensor. The proposed strategy has been tested in designing a bioluminescent biosensor for integrated rapid assessment of the safety of fruits and vegetables. The biosensor is based on the coupled enzyme system NAD(P)H:FMN-oxidoreductase and luciferase as the biorecognition element. The study describes methods and techniques for attaining the desired result in each stage. The proposed six-stage procedure for designing bioluminescent enzyme biosensors can be used to design the enzymatic biosensors based on other enzymes.