E. Rainina

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Cell Biosensor for Detection of Phenol in Aqueous Solutions

A microbial sensor for concentration measurement of phenol in aqueous solutions has been developed. Phenol-utilizing cellsPseudomonas putida GFS-8 immobilized in poly(vinyl)alcohol cryogel were used as a biological transducer. Relationships between phenol concentration in the activating medium and endogenic cell respiration have been established. Cell respiration and phenol concentration in the assay solution positively correlated at a phenol concentration range of 0.1–2.0 mg/L and were linearly dependent in the range of 0.1–1.0 mg/L. A Clark membrane electrode was the physiochemical transducer. The assay may be completed within 5 min. The cells oxidize phenol, pyrocatechol, mesityl oxide, aniline, and do not react with a number of xenobiotics, sugars, and alcohol. With the exception of aniline, most components found in waste waters from phenol production affect neither the assay process nor the ability of these cells to use phenol as exogenic respiratory substrate. The immobilized cells retained their ability to utilize phenol as an exogenic respiratory substrate for up to 1 mo.

Production of L-lysine by free and PVA-cryogel immobilizedCorynebacterium glutamicum cells

SummaryFree and immobilized cells ofC. glutamicum were analyzed for production of L-lysine. Non-growing free cells as well as immobilized ones produced higher levels of lysine than growing free cells, when cultured for 24 h in a medium containing 80 g/L glucose as a carbon source. The effect of initial biomass and yeast extract concentrations was investigated in order to improve lysine production.

A biosensor for L-proline determination by use of immobilized microbial cells

A biosensor to quantify L-proline within 10-5–10-3 mole/L concentration is described. ImmobilizedPseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.

Degradation of thiodiglycol, the hydrolysis product of sulfur mustard, with bacteria immobilized within poly(vinyl) alcohol cryogels

Alcaligenes xylosoxidans subsp. xylosoxidans (SH91) capable of biodegradation of thiodiglycol (TDG) were immobilized in poly(vinyl) alcohol (PVA) cryogels. Cryoimmobilized biocatalyst was formed as spherical granules with a diameter of 0.5 mm; the biomass concentration inside the gel matrix was as high as 10% (w/w). The immobilized cells were capable of rapid degradation of TDG in tap water or potassium phosphate buffer (100 mM, pH 8.0) containing only (NH4)2 SO4. The immobilized biocatalyst did not show any substrate inhibition up to 200 mM TDG, and retained 100% activity during three months of continuous use in a repeated-batch bioreactor.

Biosensor to Detect Organophosphorus Insecticides

This chapter presents an overview of a biosensor to detect organophosphorus insecticides. A new potentiometric assay of organophosphorous compounds (OPC) in water and organic media is developed. The assay is based on application of polyvinyl alcohol) cryogel (PVAG)-entrapped E. coli DH-5α bacteria featuring a high-activity organophosphate hydrolase (OPH). OPH catalyzes the hydrolysis of paraoxon and its analogs to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. Measurements are performed in 0.5 mM glycine buffer (pH 9.0). In the assay, the cells immobilized in PVAG-beads (diameter 0.7 mm) or films, are placed into a unit equipped with a combined pH-electrode and a mixing device. When organophosphorous compounds are assayed in organic solvents, the assay is conducted in a biphasic system. The assay was elaborated by using such widely known pesticides, as paroxon, paration, and cumaphos. The linear dependence of pH dynamics through hydrolysis of the pesticides by the immobilized cells was observed in the range 10 −7 –10 −5 M. The assay time is 5 min. The immobilized cells are usable in the assay repeatedly. The activity of the immobilized cells on storage in 50 mM phosphatebuffer (pH 7.2) containing CO 2+ remained unchanged for not less than 2 months.

Bioluminescent Detection of Food-Stuff “Freshness”

This chapter elaborates the bioluminescent detection of food-stuff “freshness.” The research has shown a principal possibility to specify the “freshness” of fish products and to assign the time limits for their storage from the bioluminescent measurement of ATP level in fish samples. The method is based on application of ATP-dependent firefly luciferase, is highly sensitive, and affords detection of up to 1 × 10 −13 mole ATP per 1 g sample. Experimentally, the dynamics of ATP content in fish has been studied at various storage conditions. ATP content in muscles of river carp stored at −70°C for 20 days changed little and was about 2 × 10 −11 mol/mg muscle. At storage temperatures +15 to −20°C, ATP level in muscles fell as early as in 2 days 40, 20 and 4 times at +15, +6, and −20°C, respectively. At longer storage at +6 and −20°C, the decline in ATP content decelerated. ATP content in muscle samples from various body regions differed a few times. This discrepancy held at the storage temperature −70°C.