Assessment of recombinant glutathione-S-transferase (HaGST-8) silica nano-conjugates for effective removal of pesticides.

Diverse glutathione-S-transferases (GSTs) are produced by insect pests including Helicoverpa armigera (HaGSTs) for detoxification of insecticides or xenobiotic compounds that they encounter. In an earlier study, the HaGST-8 gene was isolated from H. armigera larvae exposed to pesticide mixtures and the recombinant protein was expressed in the yeast Pichia pastoris. In this investigation, HaGST-8 was successfully immobilized on glutaraldehyde-activated APTES functionalized silica nanoparticles to obtain SiAPT-HaGST-8 nano-conjugates. Although enzyme activity associated with these conjugates was comparable to that of free HaGST-8, the specific activity of the former was found to be 1.25 times higher than the latter. In comparison with the free enzyme (that demonstrated a pH optimum of 9.0), for the nano-conjugates, the pH range was extended between pH 8.0 to 9.0. The optimum temperature for activity of both forms of the enzyme was found to be 30 °C. Stability of the enzyme was improved from 20 d for free HaGST-8 to 30 d for SiAPT-HaGST-8 nano-conjugates. Some loss in GST activity was detected after every reuse cycle of nano-conjugates and in all, 63% reduction was observed after three cycles. When 3 kinds of pesticides (namely, chlorpyrifos, dichlorvos and cypermethrin) were reacted with SiAPT-HaGST-8, more than 80% reduction in levels were observed. On the basis of the results obtained, the use of such silica nanoparticle-based systems for stable enzyme conjugation followed by effective removal of pesticides from aqueous media is envisaged.

Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: Noctuidae) HaGST-8 in detoxification of pesticides.

The present study deals with glutathione-S-transferase (GST) based detoxification of pesticides in Helicoverpa armigera and its potential application in eliminating pesticides from the environment. Dietary exposure of a pesticide mixture (organophosphates - chlorpyrifos and dichlorvos, pyrethroid - cypermethrin; 2-15ppm each) to H. armigera larvae resulted in a dose dependant up-regulation of GST activity and gene expression. A variant GST from H. armigera (HaGST-8) was isolated from larvae fed with 10ppm pesticide mixture and it was recombinantly expressed in yeast (Pichia pastoris HaGST-8). HaGST-8 had a molecular mass of 29kDa and was most active at pH 9 at 30°C. GC-MS and LC-HRMS analysis validated that HaGST-8 was effective in eliminating organophosphate type of pesticides and partially reduced the cypermethrin content (53%) from aqueous solutions. Unlike the untransformed yeast, P. pastoris HaGST-8 grew efficiently in media supplemented with pesticide mixtures (200 and 400ppm each pesticide) signifying the detoxification ability of HaGST-8. The amino acid sequence of HaGST-8 and the already reported sequence of HaGST-7 had just 2 mismatches. The studies on molecular interaction strengths revealed that HaGST-8 had stronger binding affinities with organophosphate, pyrethroid, organochloride, carbamate and neonicotinoid type of pesticides. The abilities of recombinant HaGST-8 to eliminate pesticides and P. pastoris HaGST-8 to grow profusely in the presence of high level of pesticide content can be applied for removal of such residues from food, water resources and bioremediation.

Silica nanoparticle based techniques for extraction, detection, and degradation of pesticides.

Silica nanoparticles (SiNPs) find applications in the fields of drug delivery, catalysis, immobilization and sensing. Their synthesis can be mediated in a facile manner and they display broad range compatibility and stability. Their existence in the form of spheres, wires and sheets renders them suitable for varied purposes. This review summarizes the use of silica nanostructures in developing techniques for extraction, detection and degradation of pesticides. Silica nanostructures on account of their sorbent properties, porous nature and increased surface area allow effective extraction of pesticides. They can be modified (with ionic liquids, silanes or amines), coated with molecularly imprinted polymers or magnetized to improve the extraction of pesticides. Moreover, they can be altered to increase their sensitivity and stability. In addition to the analysis of pesticides by sophisticated techniques such as High Performance Liquid Chromatography or Gas chromatography, silica nanoparticles related simple detection methods are also proving to be effective. Electrochemical and optical detection based on enzymes (acetylcholinesterase and organophosphate hydrolase) or antibodies have been developed. Pesticide sensors dependent on fluorescence, chemiluminescence or Surface Enhanced Raman Spectroscopic responses are also SiNP based. Moreover, degradative enzymes (organophosphate hydrolases, carboxyesterases and laccases) and bacterial cells that produce recombinant enzymes have been immobilized on SiNPs for mediating pesticide degradation. After immobilization, these systems show increased stability and improved degradation. SiNP are significant in developing systems for effective extraction, detection and degradation of pesticides. SiNPs on account of their chemically inert nature and amenability to surface modifications makes them popular tools for fabricating devices for 'on-site' applications.