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Biomedical subjects

A Mulchandani

Publications and source records attributed to A Mulchandani.

33 records · Page 2Linked to original sources
PubMed1998

Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 1. Potentiometric microbial electrode.

A potentiometric microbial biosensor for the direct measurement of organophosphate (OP) nerve agents was developed by modifying a pH electrode with an immobilized layer of Escherichia coli cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosporus pesticides to release protons, the concentration of which is proportional to the amount of hydrolyzed substrate. The sensor signal and response time were optimized with respect to the buffer pH, ionic concentration of buffer, temperature, and weight of cells immobilized using paraoxon as substrate. The best sensitivity and response time were obtained using a sensor constructed with 2.5 mg of cells and operating in pH 8.5, 1 mM HEPES buffer. Using these conditions, the biosensor was used to measure as low as 2 microM of paraoxon, methyl parathion, and diazinon. The biosensor had very good storage and multiple use stability. The use of cells with the metabolic enzyme expressed on cell surface as a biological transducer provides advantages of no resistances to mass transport of the analyte and product across the cell membrane and low cost due to elimination of enzyme purification, over the conventional microbial biosensors based on cells expressing enzyme intracellularly and enzyme-based sensors, respectively.

Biosensing Techniques↗
PubMed1998

The use of live biocatalysts for pesticide detoxification.

During the past decade, numerous microorganisms capable of degrading pesticides have been isolated, and detoxification processes based on these live biocatalysts have been developed. Recently, novel detoxification strategies using genetically engineered microorganisms with extended degradative capabilities have been investigated and, in some cases, shown to be more effective. One promising approach for the detoxification of organophosphate pesticides uses genetically engineered Escherichia coli with surface-expressed organophosphorus hydrolase. Continuous efforts in this direction are required, in conjunction with a search for microorganisms capable of degrading pesticides rapidly, to establish efficient and cost-effective large-scale processes for pesticide detoxification.

Bacteria↗
PubMed1997

A receptor protein-based bioassay for quantitative determination of paclitaxel.

A novel receptor-based bioassay for the quantitative measurement of Taxol was developed. The assay was based on the well-investigated and established finding that Taxol, its active analogs, and active metabolites bind reversibly to the receptor protein tubulin, a process similar to antibody and antigen interaction. The assay was performed in a competitive format by allowing a mixture of horseradish peroxidase-labeled Taxol and Taxol in the analyte sample to compete for the Taxol binding site of a polystyrene microtiter plate wall coated with purified tubulin and subsequently measuring the tubulin-Taxol complex by determining the activity of the horseradish peroxidase label. Using this method, Taxol was measured very sensitively, linear range of 0.0001-1 nM, and selectively, without interference from non-tumor-active compounds such as baccatin III, cephalomaninne, and 10-deacetyl taxol. The method was applied for the determination of picomolar concentrations of Taxol in human plasma.

Animals↗
PubMed1997

Innovative bioreactors.

Recent papers have described new bioreactor designs. Most innovations addressed either oxygen transfer, shear induced by stirring, control of water activity in organic phase systems or waste biotreatment. Innovations made during the past year were reported in mainly three areas: bioreactor designs for increases in oxygen transfer and decreases in shear stress; bioreactors for two-phase reactions with water activity control; and environmental bioreactors.

Journal Article↗
PubMed1997

Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane fractions was verified by immunoblotting with OmpA antisera. Inclusion of the organophosphorus hydrolase signal sequence was necessary for achieving enzymatic activity on the surface. More than 80% of the OPH activity was located on the cell surface as determined by protease accessibility experiments. Whole cells expressing OPH on the cell surface degraded parathion and paraoxon very effectively without any diffusional limitation, resulting in sevenfold higher rates of parathion degradation compared with whole cells with similar levels of intracellular OPH. Immobilization of these live biocatalysts onto solid supports could provide an attractive means for pesticide detoxification in place of immobilized enzymes, affording a reduced diffusional barrier.

Bacterial Outer Membrane Proteins↗
PubMed1996

Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

Animals↗
PubMed1995

Amperometric determination of lipid hydroperoxides.

A new amperometric analytical technique for measuring lipid hydroperoxides is described. The technique is based on the measurement of cathodic current due to the reduction of ferricinium ion formed as result of the oxidation of ferrocene by lipid hydroperoxides. The effects of pH and applied potential were investigated to determine the optimum pH and working potential for the determination of linoleic acid and linolenic acid hydroperoxides. The analysis, performed in pH 5.5, 0.1 M phosphate buffer and at -100 mV (vs Ag/AgCl) applied potential, responded linearly to linoleic acid hydroperoxide and linolenic acid hydroperoxide up to 1.5 and 1.2 microM, respectively. The lower detection limits were 20 nM for linoleic acid hydroperoxide and 25 nM for linolenic acid hydroperoxide. Reductants such as ascorbate and urate present in the biological samples, as well as other peroxides, did not interfere in the amperometric analyses of lipid hydroperoxides.

Ascorbic Acid↗
PubMed1995

Principles and applications of biosensors for bioprocess monitoring and control.

Biosensors are useful analytical devices that can be integrated with on-line process monitoring schemes. In this article, the principles and applications of these devices for bioprocess monitoring are considered. Several different types of biosensors are described, and the applications and limitations of flow injection analysis (FIA) for these applications are discussed. It is hoped that the background provided here can be useful to researchers in this area.

Animals↗
PubMed1991

A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells.

A biosensor system based on the difference in the oxygen uptake response of two microbial electrodes was developed to monitor trimethylamine (TMA). The first electrode, constructed using Pseudomonas aminovorans grown on TMA, was sensitive to TMA, trimethylamine N-oxide (TMAO), dimethylamine (DMA) and monomethylamine (MMA). The second electrode responding to TMAO, DMA and MMA was prepared using Ps. aminovorans grown on TMAO. The difference in oxygen uptake was linearly related to the TMA concentration in the range of 5-26 microM. The minimum detectable level was 2.6 microM and the relative standard deviation was determined to be 14% for 16 repeated analyses. When operated and stored at 30 degrees C, the response of the system was stable for only 2 days. However, when the biosensor system was operated at 30 degrees C but stored overnight at 4 degrees C, the system was stable up to 20 days. The biosensor system was applicable for the determination of TMA in fish tissue extracts and the results compared well with those determined by HPLC.

Animals↗
PubMedDate not supplied

Cell surface display of organophosphorus hydrolase using ice nucleation protein.

A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability.

Aryldialkylphosphatase↗
PubMedDate not supplied

Whole-cell immobilization using cell surface-exposed cellulose-binding domain.

Specific adhesion of Eshcherichia coli with surface-exposed cellulose-binding domain (CBD) to cellulosic materials was investigated. Whole-cell immobilization was very specific, forming essentially a monolayer of cells onto the different supports. Cells with surface-exposed CBD bound specifically and tightly to cellulose supports at a wide range of pH. In contrast to CBD, which shows the highest binding to cellulose at 4 degrees C, highest cell loading was observed at 37 degrees C. The extent of immobilization was dependent on the amount of surface-exposed CBD. Cells binding increased with increasing amount of CBD until binding was saturated. Even induction of very low level of CBD (0.05 mM IPTG) was sufficient to provide specific and tight binding to cellulose support. Because optimal binding can be obtained under physiological conditions such as pH 7 and 37 degrees C, the results demonstrate the general utility of surface-exposed CBD as an efficient means of whole-cell immobilization.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Factors influencing parathion degradation by recombinant Escherichia coli with surface-expressed organophosphorus hydrolase.

A unique approach for organophosphorus pesticides detoxification was developed previously by anchoring organophosphorus hydrolase (OPH) onto the surface of Escherichia coli with a tightly regulated tac promoter. The resulting recombinant cells degraded parathion very effectively without the diffusional limitation observed in cells expressing OPH intracellularly. However, the precise conditions for surface targeting or pesticide degradation were not fully understood. In this paper, several factors influencing parathion degradation were investigated. Production of active OPH onto the cell surface was highly host-specific; a high rate of parathion degradation was observed from strains JM105 and XL1-Blue, which regulated production of the OPH fusion very tightly. However, in the absence of ampicillin selection, plasmids were only favorably maintained in strain XL1-Blue. OPH activity was highly dependent on growth conditions. Optimal OPH activity was observed when cells were grown in Luria-Bertani (LB)-buffered medium at 37 degrees C. OPH activity was further improved by supplementing the growth medium with cobalt chloride, which favors the formation of the metal active center. The timing of cobalt addition also influenced parathion degradation. Maximum OPH activity was obtained by adding cobalt to induced cultures during the late stationary phase. The resulting cultures grown under the optimized conditions had an eight-fold increase in parathion degradation.

Aryldialkylphosphatase↗
PubMedDate not supplied

Fiber-optic enzyme biosensor for direct determination of organophosphate nerve agents.

A fiber-optic enzyme biosensor for the direct measurement of organophosphate nerve agents was developed. The basic element of this biosensor is organophosphorus hydrolase immobilized on a nylon membrane and attached to the common end of a bifurcated optical fiber bundle. The enzyme catalyzes the hydrolysis of organophosphate compounds to form stoichiometric amounts of chromophoric products that absorb light at specific wavelengths. The back-scattered radiation of the specific incident radiation was measured using a photomultiplier detector and correlated to the organophosphate concentration. The effects of buffer pH, temperature, and the units of enzyme immobilized on the steady-state and kinetic response of the biosensor were investigated to optimize the operating conditions for the fiber-optic enzyme biosensor. These conditions were then used to measure parathion, paraoxon, and coumaphos selectively without interference from carbamates and triazines. Concentrations as low as 2 microM can be measured in less than 2 min using the kinetic response. When stored in buffer at 4 degreesC the biosensor shows long-term stability.

Biosensing Techniques↗