Zazali Alias

Beta class glutathione S-transferase (GST) activity is known to be associated with antibiotic resistance, one of the most serious threats to global health. In this research, the study of antibiotic resistance developed by beta class GST was conducted using KKSG6, one of the GST isozymes found in Acidovorax sp. KKS102. The KKSG6 gene has been successfully expressed in Escherichia coli BL21 Star™ (DE3) using pET101/D-TOPO®-KKSG6 as an expression vector, resulting in the presence of a protein band of approximatly 20 kDa after purification using GSTrap™ HP column. Over-expression of KKSG6 made Escherichia coli BL21 Star™ (DE3) to be less susceptible towards kanamycin, streptomycin, gentamycin, tetracycline and chloramphenicol, suggesting the antibiotics binding with KKSG6. Our study has revealed the KKSG6 conjugation activity towards chloramphenicol, but not with kanamycin and tetracycline. The inhibition of protein conjugation activity towards CDNB by the presence of chloramphenicol has also been demonstrated. An in-silico study using protein-ligand docking predicted that antibiotics binding could take place at the protein dimer interface and H-site depending on their properties.

Glutathione S-transferases (GSTs) are enzymes involved in phase II of detoxification metabolism and could be used as biomarkers for water pollution. This study aims to determine heavy metal concentrations in the soft tissue of the mangrove clam Geloina expansa, as well as the expression of GSTs in the species. The acid digestion method was used to digest the samples, followed by a standard USEPA 6010B procedure using inductively coupled plasma optical emission spectrometry (ICP-OES) to measure the heavy metal contents in the samples. GST enzyme activity was measured using 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. One-way ANOVA was performed to compare the mean values of heavy metal concentration, protein concentration, enzyme activity, and specific activity. There was a significant difference (p<0.05) for Zn, total protein, and specific activity in G. expansa, but no significant difference in Pb, Cu and enzyme activity. GST enzyme activities were estimated at 0.16 ± 0.01 µmol/min, with a protein content of 1.24 ± 0.04 mg. The specific activity for GST was 0.13 ± 0.01 µmol/min/mg, calculated as the ratio of enzyme activity to the total protein. GST-specific activity positively correlates with Pb concentration in the soft tissue of G. expansa. Detailed studies on the effects of pollution on the expression of GST need to be further investigated for the future use of this species as an efficient biomarker model.

Glutathione transferase Pi (GST-P) reportedly overexpressed markedly in cancer cell lines. It was correlated to the resistance towards drugs used in chemotherapy treatment. The study demonstrated the isolation of peptide as potential inhibitor to the enzyme. Glutathione transferase Pi (GST-P) was purified from human colon adenocarcinoma HT-29 cell line using glutathione (GSH)-affinity chromatography. Active components presence in ethanolic extract (50% ethanol) of leaves Leptospermum flavescens Sm. was shown possessing inhibitory property (IC₅₀ of 0.088 mg/mL) towards GST-P in vitro. Further fractionation using polyamide the 50% methanol in 2% acetic acid eluate possessed an inhibitory property at IC₅₀ of 0.191 mg/mL. In the study the IC₅₀ values of doxorubicin are 0.788 and 0.816 μg/mL on HT-29 and MRC-5 cell lines respectively while IC₅₀ values of cisplatin were at 9.49 and 4.07 μg/mL on HT-29 and MRC-5 cell lines respectively. The 50% methanol eluate has significantly non-toxic to both of the cell lines with 100% cell viability at more than 100 μg/mL sample applied. In combination with doxorubicin, 50% methanol eluate enhanced cytotoxicity of the drug towards HT-29 by reduction of IC₅₀ value significantly to 66%. The eluate however only reduced the IC₅₀ to 11% when combined with cisplatin. The study indicates that the eluate can potentiate cytotoxicity of doxorubicin on HT-29 cell line and this effect is correlated to the ability of the eluate to inhibit GST-P in vitro. The purified active molecule was a peptide with molecular weight of 3.5 kDa.

Glutathione S-transferase (GST) is an ancient enzyme that widely spread in aerobic organisms, including bacteria. GST catalyzes the conjugation reaction between the various electrophilic compound and glutathione (GSH) through nucleophilic substitution mechanism. Unlike higher eukaryote GSTs which are commonly play a role in detoxification, bacterial GSTs are mainly involve within primary metabolism to support microorganism survival in the low-nutrient environment. Beta is a cytosolic GST class which only found in bacteria. Several unique GST activities such as binding with antibiotics, oxidoreductase activity and polycyclic aromatic hydrocarbons (PAHs) have only been observed in beta class GST. The special catalytic activity of bacterial GST has been extensively proposed to address environment contaminations. Unlike other approaches, utilizing bacteria in bioremediation has some benefits: it is simple, inexpensive, less harmful, and provide the long-term protection. Some genetically modified organisms exploiting bacterial GST have been developed to detect and solve PAHs contamination.

In this study, the induction of glutathione S-transferase (GST) enzymatic activities in Aedes albopictus under 24 h of xenobiotic challenges was investigated. From LCMS analysis, 23 GST isoforms were identified under Delta, Epsilon, Sigma, Zeta, Omega, and Iota classes, together with one GSTX1-1 isoform, in both treated and untreated samples. Using STRING 11.5, the functional enrichment network of Gene Ontology (GO) analysis, the identified peptides were found to be involved in the glutathione metabolic biological process (GO:0006749, p-value: 1.93 × 10⁻²⁹), and the molecular functions involved are due to glutathione transferase (GO:0016848, p-value: 2.92 × 10⁻⁸) aside from carbon-halide lyase activity (GO:004364, p-value: 1.21 × 10⁻³¹). The Protein-Protein Interaction (PPI) network (STRING 11.5) showed significant interactions within the GST superfamily and some of the GST classes interacted with other proteins among the input domain of the identified peptides (p-value < 1.0 × 10⁻¹⁶). In TMT labeling for the quantification of peptide abundance, isoforms from Delta (GSTD1-2, GSTD1-3, GSTD1-4) and Epsilon (GSTE3-1, GSTE4-2) were found to be overexpressed (between 1.5-fold and 2-fold changes). In the PPI analysis, 12 common enriched pathways of Kyoto Encyclopedia of Genes and Genomes (KEGG) were found to be intercorrelated with the identified GSTs at PPI enrichment p-value < 1.0 × 10⁻¹⁶. Overall, this study indicates that distinct GST enzymes, which were identified up to their specific protein isoforms, are involved in the metabolic mechanisms underlying xenobiotic stress.

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.

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.

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 K_i 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.