The Role of Glutathione Transferases in the Development of Insecticide Resistance

Glutathione transferases are multifunctional enzymes. Some of the known functions of the enzymes are biotransformation of xenobiotics, countering oxidative stress and participating in cell regulatory functions. As the isoforms present in number of classes the purification of a particular isoform for characterization is a challenging task. In insect, the study of GSTs is focusing on their roles in development of insecticide resistance. There were evident that certain classes of the enzymes are reactive towards conjugating the pesticides. This makes GSTs one of the enzymes of intention in the discipline of pesticide control management

Substrate Specificities and Kinetic Parameters of Recombinant Drosophila melanogaster Glutathione S-Transferases E6 and E7

D. melanogaster glutathione transferases E6 and E7 (DmGSTE6 and DmGSTE7) were successfully cloned, purified, and biochemically characterized. The recombinant proteins were readily purified using the combination of both anionic and BSP/GSH-agarose affinity chromatography. Although both GSTs have significant identity in their amino acid sequence, each enzyme displayed unique biochemical characteristics. Both recombinant proteins were only active toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzyl chloride (p-NBC) with significant difference in catalytic activities. The findings have shown that neither GSTE6 nor GSTE7 was able to counter oxidative stress. Comparatively, GSTE7 was a more efficient enzyme at turning over DCNB and p-NBC, based on its kcat/Km values which were of 0.183 and 2.25 min−1 mM−1, respectively. Thin-layer chromatography analysis showed that both isoforms were not able to conjugate several tested insecticides. The inhibition kinetics of natural products and dyes toward GSTs in vitro revealed that phenol red possessed inhibition effects only on GSTE6 while rose bengal and cardiogreen inhibit significantly on both GSTE6 and GSTE7. In contrast, methylene blue dye and trans-chalcone have been shown to stimulate GSTE7 activity toward CDNB

Purification and partial characterisation of a protease inhibitor from Mimosa diplotricha

Search for inhibitors to insect proteases is one of many strategies to control pests. Previous work has demonstrated successful purification of effective inhibitors from plant origin. Thus, the current study attempted to purify protease inhibitors from locally available medicinal plants. The study demonstrated that the ethanolic extracts of Mimosa diplotricha leaves caused a significant 80% reduction in bovine trypsin activity. The inhibitory property of the proteinaceous nature of the extract was reconfirmed through qualitative analysis using the detection of trypsin inhibitors on the SDS-PAGE technique. The ammonium precipitated trypsin inhibitor was purified using Hi-Trap G25 and resolved into a single band with a molecular weight of approximately 20.8 kDa. By using the Dixon plot the competitive inhibitor has a Ki value of 2.16 × 10-4 mM. The purified protein inhibited the protease extract of Chrysomya megacephala at IC50 of 28 μg/mL. The results highlighted the presence of trypsin inhibitor in Mimosa diplotricha and its potential as a pest control agent

Purification and partial characterisation of a protease inhibitor from Mimosa diplotricha

Search for inhibitors to insect proteases is one of many strategies to control pests. Previous work has demonstrated successful purification of effective inhibitors from plant origin. Thus, the current study attempted to purify protease inhibitors from locally available medicinal plants. The study demonstrated that the ethanolic extracts of Mimosa diplotricha leaves caused a significant 80% reduction in bovine trypsin activity. The inhibitory property of the proteinaceous nature of the extract was reconfirmed through qualitative analysis using the detection of trypsin inhibitors on the SDS-PAGE technique. The ammonium precipitated trypsin inhibitor was purified using Hi-Trap G25 and resolved into a single band with a molecular weight of approximately 20.8 kDa. By using the Dixon plot the competitive inhibitor has a Ki value of 2.16 × 10-4 mM. The purified protein inhibited the protease extract of Chrysomya megacephala at IC50 of 28 μg/mL. The results highlighted the presence of trypsin inhibitor in Mimosa diplotricha and its potential as a pest control agent

Purification of glutathione Transferases (GSTs) from identified Rhizospheric bacteria

The glutathione S-Transferase (GST) enzyme plays an important role in cellular detoxification. This multifunctional enzyme is involved in Phase II detoxification pathways that protect cellular macromolecules from being attacked by harmful compound. The study is an attempt to isolate glutathione transferase-expressing bacteria from the rhizospheric soil of selected herbal plants. Screening showed nine positive isolates out of twelve bacterial samples from a large microbial population in our soil collection. Crude extract from strain E1 which was isolated from Piper sarmentosum (Kadok) showed the highest specific activity against 1-chloro-2, 4-dinitrobenzene substrates (5.78 × 10-06 μmol/min/mg). Based on the carbon utilization of E1 assessed using Biolog system, the strain was identified as Comamonas testosterone E1. Glutathione S-transferase purification using GST trap yielded two distinct subunits with molecular weights of 23 and 24 kDa as visualized on 1D SDS-polyacrylamide gel electrophoresis. The purified GST showed reactivity towards 1-chloro-2, 4-dinitrobenzene, 1, 2-dichloro-4-nitrobenzene and ethacrynic acid with specific activity of 0.264 ± 0.038 nmol/min/mg and 0.056 ± 0.002 nmol/min/mg and 10.500 ± 3.130 nmol/min/mg, respectively. However, no activity was detected against p-Nitrobenzyl chloride, Sulfobromophthalein, trans-4-phenyl-3-butene-2-one, hexa-2, 4- dienal, trans-hepta-2, 4-dienal and trans-oct-2-enal in the study

Defence against Oxidative Stress and Insecticides in <em>Musca domestica</em>

This review is looking at the way Musca domestica defends itself against harmful molecules. One of the most notable enemies is against oxidative stress. Over the years there were reports that indicated the development of resistance on range of pesticides that are used against the flies. Researches have demonstrated that there are several functional protein molecules which contribute directly or indirectly as a response to oxidative stress and resistance against insecticides. As currently, the whole genome sequencing of the organisms has enabled future study to be conducted in evaluating the behaviour of the targeted protein/enzyme in response to oxidative stress and intake of insecticides in the flies

Bioinformatic analysis and purification of glutathione transferase (GST) from Pseudomonas sp. UW4

The study aimed at identifying and purifying cytosolic glutathione transferase isoforms expressed in Pseudomonas sp. UW4. Search at UniProt (https://www.uniprot.org/uniprot/), has indicated that there were 20 genes encoding putative glutathione transferases for the microorganism. The molecular weights of the isoforms ranged from 17.6 to 34.06 kDa. SDS-polyacrylamide gel electrophoresis revealed that the GST purified using Sulfobromophthalein-glutathione (BSP) affinity column, resolved into a single band with a low molecular weight (MW) of 16 kDa with the pI value of 6.0. Purified GST was reactive towards ethacrynic acid, 1-chloro-2,4-dinitrobenzene, cumene hydroxide, and hydrogen peroxide, but no detectable activity with Trans-2-octenal, hepta-2,4-dienal and Trans-4-phenyl-3-butene-2-one. This has proven that putative GST possessed peroxidase activity and proposed to be similar to PputUW4_00801 (putative glutathione S-transferase) of Pseudomonas sp. UW4 according to its estimated molecular weight and the pI values obtained experimentally

Bioinformatic analysis and purification of Glutathione Transferase (GST) from Pseudomonas sp. UW4

The study aimed at identifying and purifying cytosolic glutathione transferase isoforms expressed in Pseudomonas sp. UW4. Search at UniProt (https://www.uniprot.org/uniprot/), has indicated that there were 20 genes encoding putative glutathione transferases for the microorganism. The molecular weights of the isoforms ranged from 17.6 to 34.06 kDa. SDS-polyacrylamide gel electrophoresis revealed that the GST purified using Sulfobromophthalein-glutathione (BSP) affinity column, resolved into a single band with a low molecular weight (MW) of 16 kDa with the pI value of 6.0. Purified GST was reactive towards ethacrynic acid, 1-chloro-2,4-dinitrobenzene, cumene hydroxide, and hydrogen peroxide, but no detectable activity with Trans-2-octenal, hepta-2,4-dienal and Trans-4-phenyl-3-butene-2-one. This has proven that putative GST possessed peroxidase activity and proposed to be similar to PputUW4_00801 (putative glutathione S-transferase) of Pseudomonas sp. UW4 according to its estimated molecular weight and the pI values obtained experimentally

Temperature and pH Profiling of Amylase from Antarctic and Arctic Soil Microfungi

While diversity studies and screening for enzyme activities are important elements of understanding fungal roles in the soil ecosystem, extracting and purifying the target enzyme from the fungal cellular system is also required to characterize the enzyme. This is, in particular, necessary before developing the enzyme for industrial-scale production. In the present study, partially purified α-amylase was obtained from strains of Pseudogymnoascus sp. obtained from Antarctic and Arctic locations. Partially purified α-amylases from these polar fungi exhibited very similar characteristics, including being active at 15 °C, although having a small difference in optimum pH. Both fungal taxa are good candidates for the potential application of cold-active enzymes in biotechnological industries, and further purification and characterization steps are now required. The α-amylases from polar fungi are attractive in terms of industrial development because they are active at lower temperatures and acidic pH, thus potentially creating energy and cost savings. Furthermore, they prevent the production of maltulose, which is an undesirable by-product often formed under alkaline conditions. Psychrophilic amylases from the polar Pseudogymnoascus sp. investigated in the present study could provide a valuable future contribution to biotechnological applications

Fishmeal replacement with Spirulina Platensis and Chlorella vulgaris in African catfish (Clarias gariepinus) diet: Effect on antioxidant enzyme activities and haematological parameters

This study explored fishmeal replacement with two freshwater microalgae: Spirulina Platensis and Chlorella vulgaris in African catfish (Clarias gariepinus) diet. The effect of inclusion of the two microalgae on biomarkers of oxidative stress, haematological parameters, enzyme activities and growth performance were investigated. The juvenile fish were given 3 distinct treatments with isonitrogenous (35.01–36.57%) and isoenergetic (417.24–422.27 Kcal 100 g−1) diets containing 50% S. platensis (50SP), 75% S. platensis (75SP), 50% C. vulgaris (50CL), 75% C. vulgaris (75CL) and 100% fishmeal (100% FM) was used as the control diet. The result shows that all the diets substituted with both S. platensis, and C. vulgaris boosted the growth performance based on specific growth rate (SGR) and body weight gain (BDWG) when compared with the control diet. The feed conversion ratio (FCR) and protein efficiency ratio (PER) was significantly influenced by all the supplementations. The haematological analysis of the fish shows a significant increase in the value of red and white blood cells upon supplementation with 50SP and 50CL but decrease slightly when increased to 75SP and 75CL. Furthermore, the value of haematocrit and haemoglobin also increased upon supplementation with 50SP and 50CL but decrease slightly when increased to 75SP and 75CL. The white blood cell (WBC), red blood cell (RBC) increased, while total cholesterol (TCL), and Plasma glucose levels decreased significantly upon supplementation of algae. This is a clear indication that S. platensis and C. vulgaris are a promising replacement for fishmeal, which is a source protein in the C. gariepinus diet