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Acetylcholinesterase (AChE) is a well-known enzyme sensitive to pesticide exposure, but heavy metal sensitivity is rarely reported. Here, AChE extracted from the brain tissue of Diodon hystrix was exploited and used in a fast and economical way to sensitively detect the existence of heavy metals in a water sample. An inhibitive enzyme assay was conducted, and the activity of AChE was found to be sensitive (> 20% inhibition) to sub-million levels of arsenic, cadmium, nickel, and zinc and less sensitive (< 20% inhibition) to copper and lead. While exposure to argentum, cobalt, and chromium shows no significant inhibition, AChE is not sensitive to those metal ions (p < 0.05). Field test work has proved that the assay is suitable for preliminary detecting heavy metal contamination in the river, especially near industrial and mining sites. Secondary validation was performed using ICP-OES to identify and measure the number of elements in the sample and compare them to the inhibition level of AChE activity.

Pollutant treatment methods are available for membrane isolation, exchange of ions, precipitation, transformation and biosorption. Biosorption has many positive aspects of all this technology, including low running costs, very effective toxicant detoxification at low concentrations, low levels of disposal materials. In many applications, adsorption plays an important role in concentrating useful compounds or eliminating contaminants. The thermodynamic parameters of the adsorption are often obtained from the relation between the adsorption constant (KC in Lmol-1 unit) and the Gibbs free adsorption energy. When the van’t Hoff plot for the temperature range 298.2 K to 328.2 K was plotted, a nonlinear curve was obtained with poor R2 value (R=0.491). In order to calculate the ΔH° and ΔS° more accurately, the thermodynamics parameters were analysed at two processes: one between 298.2 and 301.2 K and another at between 301.2 and 328.2 K which gave a better R2 values of 1.00 and 0.995, respectively. The ∆G°, ∆H° (kJ/mol) and ∆S° (kJ/mol×K) values for temperatures between 298.2 and 301.2 K and between 301.2 and 328.2 K were –29.4524, 14.65, 0.145 and –30.4787, –14.88 and 0.049, respectively. This paper depicts with the proposed workflow in the analysis of liquid-phase adsorption data from the data acquisition to data analysis and thermodynamics appropriate calculation of the dimensionless KC parameter.

Cholinesterase-based biosensor well known as a sensitive method to detect the existence of harmful dissolved compounds in any type of water source, especially the river. This alternative biosensor can be used to determine the level of pollution of the water in a short period of time as well as to evaluate the low cost and simple service. The aim of this study was to exceed the effectiveness of acetylcholinesterase source extracted from the brain tissue of Asian swamp eel; Monopterus albus as a potential environmental biosensor. Purified acetylcholinesterase exposed to a different type of metal ions and mercury showed the highest percentage of inhibition at 62.9% followed by chromium at 59.22% while silver, arsenic, cadmium, cobalt, copper, nickel, zinc and lead at not more than 50% (approximately 37-50%). Metal ions such as mercury, zinc, chromium and copper showed exponential decay type inhibition curves with calculated half maximal inhibitory concentration; IC50 in the ascending sensitivity order 0.005, 0.595, 0.687 and 1.329 mgL-1, respectively. Field trial works exhibited that the acetylcholinesterase was applicable in sensing heavy metals pollution from the river which closed to the industrial and agricultural sites at near real-time and verified using ICP-OES. This study proves the potential use of acetylcholinesterase sourced from M. albus as a biomonitoring tool to assess the contamination level of the river.

Copper is extensively released into the environment, affecting the natural habitat of surrounding organisms especially the aquatic life. The aim of this study is to explore the different protein expressions on different concentrations of CuSO4. Puntius javanicus liver was used as a tool to investigate the biomarkers and mechanisms of copper toxicity to P. javanicus through proteomic assessment after being exposed with different concentrations of CuSO4 (0, 0.1, 0.3, 0.5, 1.0 and 5.0 mg/L). P. javanicus liver was extracted (TCA-acetone extraction) and electrophoresed (IEF and 2D PAGE). Ten protein spots were found to be significantly different in their expression and subsequently identified via matrix assisted laser desorption/ionization tandem time-of-flight (MALDITOF). Three protein spots were further identified as new alternative biomarkers for copper toxicity namely, zinc finger C4H2 domain-containing protein, insulin gene enhancer protein isl-2a and synaptic vesicle membrane protein VAT-1 homolog-like. The present study could provide an additional understanding on the hepatotoxic mechanisms of copper as well as enhance the development of biomarkers for an efficient monitoring of the contamination level in the environment based on proteomic assessment.

Diodon hystrix, commonly known as spot-fin porcupine fish is a salt-water fish belonging to the Diodontidae family. It is widely distributed in Sabah wet market due to its commercial value. This study exploits the effectiveness of Cholinesterase (ChE) obtained from the brain tissue of D. hystrix in detecting carbamates inhibitory activities. Carbamate pesticides known to inhibit ChE and toxic towards living organisms can contaminate the water bodies. By using diethylaminoethanol (DEAE) Sepharose ion exchange chromatography, a total of 40% recovery yield of ChE was obtained with a 165.77 purification fold. Furthermore, the ChE showed a high affinity towards acetylthiocholine iodide (ATC) with an optimum activity at pH 7.45 and temperature ranging from 20 to 40℃. Among five different types of carbamates, methomyl was found to have the highest percentage of inhibition analyzed using ChE inhibitory assay, followed by carbofuran, bendiocarb, carbaryl and propoxur with >85% inhibition rate. The results concluded that ChEs extracted from the brain tissue of D. hystrix are applicable to be used as a bioindicator in detecting the presence of carbamates.

The partially purified enzyme of indigenously isolated Serratia marcescensstrain AQ07 was utilised to develop the best form of cyanide detoxification method that is eco-friendly and cost effective. The present study evaluates the feasibility of the enzyme to degrade high cyanide concentrations and the possible metabolic pathways involved, for which the protein concentration and cyanide detoxification activity were quantified. Bacterial cells grown in cyanide incorporated medium were disrupted by sonication and the resultant cell free extract were tested for metabolic pathway. The cell free extract was precipitated by ammonium sulphate precipitation and partially purified by ion exchange chromatography using DEAE cellulose. The maximum enzyme activity achieved was 2125 µM/min. The partially purified enzyme was found to be able to detoxify 82% of 2 mM KCN in 10 min of incubation and cyanide degradation (or depletion) rate showing a linear increase with increasing enzyme concentration. The effective accruing of ammonia as metabolite illustrated that the detoxification was ensued via the function of cyanide dihydratase. Additional confirmation through SDS-Page showed that the molecular weight of enzyme was assessed to be ~38 kDa, which is tandem with the reported cyanide dihydratases. Hence, the use of enzyme as a substitute to live bacterial cells in detoxification of cyanide illustrates various advantages such as the capacity to withstand and detoxify higher cyanide concentration and total reduction in the total cost of process since nutrient provision is immaterial.

The discharge of industrial effluents into nearby water bodies affects the inhabitants including living organisms. The presence of foreign materials such as heavy metals can be a threat to the ecosystem as they are enormously carcinogenic even though in minute concentration. Hence, an economical and time-efficient preliminary screening test is crucial to be developed for the detection of heavy metals, prior to employment of high technology instruments. In this study, cholinesterase (ChE) from Sabah porcupine fish, Diodon hystrix was purified to test for its potential as an alternative biosensor in detecting metal ions. Few enzymatic parameters including specificity of substrate, temperature and pH were applied to determine its optimal enzymatic activity. ChE enzyme was found to be more sensitive towards the presence of substrate, butyrylthiocholine iodide (BTC), in contrast to acetylthiocholine iodide (ATC) and propionylthiocholine iodide (PTC) with the effective coefficient at 7193, 3680.15 and 2965.26 Vmax/Km, respectively. Moreover, the extracted ChE enzyme showed the optimum activity at pH 9 of 0.1 M Tris-HCl and at 25°C to 30°C range of temperature. When subjected to heavy metals, ChE enzyme was significantly inhibited as the enzyme activity was reduced in the sequence of Hg > Ag > Cr > Cu > Cd > Pb ≥ Zn > As. As a conclusion, the partially purified ChE enzyme proved its sensitivity towards metal ion exposure and can be used as an alternative method in screening the level of contamination in the environment.

Aim : Cholinesterase (ChE) inhibition and histopathological features of brain tissues of Clarias gariepinus were investigated with a purpose to develop biomarkers for monitoring heavy metals. Methodology : The effects of cadmium toxicity on were assessed by ChE inhibition and characterisation of ChE by affinity chromatography and histological variations in brain tissues were studied by H&E staining under light microscope. Results : The purified enzyme gave 10.58 fold purification with the recovery of 12% with specific activity of 159.729 U-1 mg . The Michaelis-Menten constant (K ) m and V value obtained was 1.985 mM max -1 -1 and 0.4479 µmol min mg , respectively. The enzyme showed the ability to hydrolyse acetylthiocholine iodide (ATC) at a faster rate compared to other two synthetic substrates, propionylcholine iodide (PTC) and butyrylthiocholine iodide (BTC). ChE showed highest activity at 35°C in Tris-HCl pH 7.5. The metal inhibition study also found the maximal inhibitory concentration (IC ) 50 -1 value for cadmium to be 0.6517 mg l . Histological study revealed decrease in the number of cells, degeneration of Purkinje cells, increased aggregation area and cells necrosis. Interpretation : Inhibition of cholinesterase activity and degeneration of Purkinje cells observed in the present study can be used as a tool for further developing a biomarker for detecting heavy metals in the aquatic ecosystems. C. gariepinus

Aim : The cholinesterase (ChE) based inhibition and histopathological studies from fish were investigated and represented in this study to develop as one of the great potential biomarkers for heavy metals monitoring. Methodology : In this study, the histopathological study of gills were observed a under microscope. The capability of ChE extracted from the gills of Clarias gariepinus was assessed for declining Cd. ChE was purified through affinity chromatography and continued with the optimisation and inhibition study (IC50) of cholinesterase. Results : Histopathological study of gills was carried out and several changes such as aneurysm, necrosis and lamella fusion were noted. Purification fold obtained from purified enzyme was 1.15 with 30% a yield specific activity 20.726. The optimum temperature for purified AChE was 35°C along with acetylthiocholine iodide (ATC) as a preferable substrate that had the highest Vmax value of 0.5452 U mg'1 and the lowest Km value of 0.0311 mM. The optimum pH was observed to be 10 of Tris-HCl as a medium. Meanwhile, the IC50 of cadmium was 6.808 mg ľ with R2 value of 0.9532. Interpretation : The result of the study can be used as a tool for further developing a biomarker for the detection of heavy metals in aquatic ecosystems. In addition, the baseline data provided can also be used for designing a kit, which would give rapid and accurate result.

The gill is the site of acid-base balance, ionic regulation, gas exchange, and nitrogenous waste excretion by fish. The physiological, histopathological and enzymatic alterations from Clarias gariepinus gills were examined to assess the effects induced in gill tissues within 96 h exposure to waterborne Al 2 (SO 4 ) 3 with different concentrations (25 to 300 mg/L). The exposure of C. gariepinus to aluminium showed deleterious effects to normal fish behaviours and varying degrees of gill damage when visualised under inverted light microscopy (stained with haematoxylin-eosin), scanning electron microscopy and transmission electron microscopy. The mortality rate was observed increasing with increased Al concentration showing noticeably disrupted lamellae, irregular nucleus shape and increasing area of vacuolation in treated fish gills associated with Al 2 (SO 4 ) 3 exposure concentration. Furthermore, electron microscopic X-ray microanalysis of fish gills exposed to sublethal Al revealed Al accumulated on the surface of the gill lamella. Al toxicity also gave inhibitory effects on cholinesterase (ChE) extracted from the fish gills with 90.0% inhibition at 300 mg/L Al highest concentration exposure (in vivo), whereas the Al metal ion inhibited 50% of ChE activity at 4.12 mg/L (IC 50 ).