The environmental toxicants such as copper are known to affect vital organ especially liver. This study examined the effects of copper sulfate (CuSO4) on the liver morphological structure of P. javanicus. The untreated control, 0.1 and 0.3 mg/L CuSO4 treated groups displayed normal polygonal structure of the hepatocyte. However, at the concentrations of 0.5, 1.0 and 5.0 mg/L CuSO4, the hepatostructure was significantly affected, as shown by the increasing number of dilation and congestion of sinusoids, vacuolation, macrophage activities and peliosis. The damage level and HSI value were increased while the number of hepatic nuclei per mm(2) was decreased with the increasing of copper concentration. In conclusion, this study shows that the degree of liver damage in P. javanicus is dependent to the dose exposure. (c) All Rights Reserved

Climbing Perch or its scientific name, Anabas testudineus is one of the freshwater fish belonging to the family of Anabantidae. It is widely distributed in ponds, swamps and estuaries in Asia. In this study, cholinesterase (ChE) was partially purified from the liver of A. testudineus through ion exchange chromatography. This purification method provided a recovery yield of 5.36% with a purification fold of 6.6. The optimum conditions for ChE assay were identified to be 2.5 mM of butyrylthiocholine iodide (BTC) with pH 8.0 in Tris-HCl buffer at 40 degrees C. Substrate specificity profile also indicated that ChE favours BTC as substrate because it records the highest catalytic efficiency (V-max/K-m). Protein analysis through Native-PAGE showed that ion exchange chromatography is an effective method to partially purify ChE. Metal ion inhibition tests were conducted and mercury (Hg) was found to show the highest inhibition effect (87.30%) whereas lead (Pb) shows the lowest inhibition effect (28.01%). All these findings showed that partially purified ChE from the liver of A. testudineus is suitable to be used as a bioindicator to detect the presence of metal ions. (c) All Rights Reserved

Aim : The cholinesterase (ChE) based inhibition studies from fish were investigated and presented here emerged to be one of the great potential biomarkers for heavy metals monitoring. Methodology : In this study, the capability of ChE extracted from the kidney of Lates calcarifer was assessed for of metal. ChE was purified through ammonium sulphate precipitation and ion exchange chromatography. Results : The purified enzyme gave 12 fold purification with the recovery of 12.17% with specific activity of 2.889 U mg(-1). The Michaelis-Menten constant (K-m) and V-max value obtained was 0.1426 mM and 0.0217 mu mol min(-1)mg(-1), respectively. The enzyme has the ability to hydrolyse acetylthiocholine iodide (ATC) at a faster rate compared to other two synthetic substrates, propionylthiocholine iodide (PTC) and butyrylthiocholine iodide (BTC). ChE gave highest activity at 20-30 degrees C in Tris-HCI buffer pH 8.0. The results showed that cholinesterase from L. calcarifer kidney was very sensitive to sensitive to copper and lead after being tested argentum, arsenic, cadmium, chromium, copper, cobalt, mercury, nickel, lead and zinc. Interpretation : The effect of heavy metals studied on the activity of ChE differed from each other. 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 information can also be used for designing a kit, that would give a rapid and accurate result.

Acetylcholinesterase (AChE) from the brain tissue of local freshwater fish, Tor tambroides was isolated through affinity purification. Acetylthiocholine iodide (ATCi) was preferable synthetic substrate to purified AChE with highest maximal velocity (V-max) and lowest biomolecular constant (K-m) at 113.60 Umg(-1) and 0.0689 mM, respectively, with highest catalytic efficiency ratio (V-max/K-m) of 1648.77. The optimum p11 was 7.5 with sodium phosphate butler as medium, while optimal temperature was in the range of 25 to 35 degrees C. Bendiocarp, carbofuran, carbaryl, methomyl and propoxur significantly lowered the AChE activity greater than 50%, and the IC50 value kvas estimated at inhibitor concentration of 0.0758, 0.0643, 0.0555, 0.0817 and 0.0538 ppm, respectively.

Another alternative source of cholinesterase (ChE) that is sensitive towards metal ion has been revealed. ChE from muscle and blood of Anabas testudineus were extracted and purified through ammonium sulphate precipitation followed by an ion exchange chromatography with a total recovery of 47.66% and 7.92%, respectively. Kinetic study measured that BTC was the most preferable synthetic substrate to blood ChE while muscle ChE preferred PTC with the biomolecular constant of 1.07 and 0.53 mM, respectively. Optimum pH for blood and muscle ChE were determined at 8 and 9. Both ChE shared an optimum temperature of 30 degrees C. Inhibition study showed that muscle ChE has inhibited more than 50% of metal ions namely arsenic, chromium, copper, mercury and zinc compared to blood ChE with only copper and mercury. Studies on half inhibitory effect (IC50) of blood and muscle ChE were tested with series concentration of mercury calculated at 1.003 and 1.048 mg/L. This result will be used as a reference for future development of biosensor.

The aim of this study is to determine the inhibitory effect of copper towards butyrylcholinesterase (BChE) activity. Using the Lineweaver-Burk plot, Puntius javanicus BChE activity was found to be noncompetitively inhibited by copper. The maximal velocities of untreated (control) BChE, 0.5 and 10 mg/L copper-treated BChE are 53.70, 31.81 and 14.30 Umg(-1), respectively, while the biomolecular constant (K-m) values of both tests shows no significant difference (p>0.05). The in vitro IC50 of copper ion to the BChE was found to be 0.0948 (0.0658 to 0.1691) mg/L. In vivo tests showed that in the presence of 0.1 mg/L copper, the BChE activity was slightly higher compared to the untreated control. Copper sulfate at 0.3 mg/L concentration showed no significant inhibition compared to control. However, the activity decreased with increasing copper concentrations of 0.5, 1.0 and 5.0 mg/L, with the remaining activity at 87.60, 84.60 and 73.00 %, respectively. This study suggests that BChE isolated from P. javanicus liver tissue is a potentially new source of biomarker for copper contamination.

The characterization of bacterial enzymatic pathways of phenol metabolism is important to better understand phenol biodegradation. Phenol hydroxylase is the first enzyme involved in the oxidative metabolism of phenol, followed by further degradation via either meta-or ortho-pathways. In this study, the first known instance of phenol degradation via the meta-pathway by a member of the genus Acinetobacter (Acinetobacter sp. strain AQ5NOL 1) is reported. Phenol hydroxylase converts phenol to catechol, which is then converted via the meta-pathway to 2-hydroxymuconic semialdehyde by the catechol 2,3-dioxygenase enzyme. Phenol hydroxylase extracted from strain AQ5NOL 1 was fully purified using DEAE-Sepharose((R)), DEAE-Sephadex((R)), Q-Sepharose((R)) and Zorbax((R)) Bioseries GF-250 gel filtration and was demonstrated by SDS-PAGE to have a molecular weight of 50 kDa. The phenol hydroxylase was purified to about 210.51 fold. The optimum pH and temperature for enzyme activities are 20 degrees C and 7- 7.5, respectively. The apparent K-m and V-max values of phenol hydroxylase with phenol as the substrate were 13.4 mu M and 2.5 mu mol min(-1) mg(-1), respectively. The enzyme was stable at -20 degrees C for 36 days.

The purification of a soluble cholinesterase (ChE) from Puntius javanicus liver using affinity chromatography was studied. Affinity matrix was synthesised through the cooling system of ligands procainamide to epoxy-activated Sephacryl 6B and purification process was performed using calibrated flow rate at 0.2 mL/min. Non-denaturing electrophoresis condition was employed and the single band native form of ChE was detected at 66.267 kDa after being stained with commasie brilliant blue. ChE detection was performed using gel filtration; ZORBAX column attached to the HPLC with the flow rate of 1 mL/min. Only a single peak was detected at the retention time of 3.720. From the assay evaluation, the final purified ChE procedure displayed the highest sensitivity of detecting the anticholinesterase namely mercury, copper, malaoxon and carbofuran compared to the impure ChE and the results were further discussed in detail to the potential application of ChE from P. javanicus as a biomarker for those toxicants. (C) 2015 Friends Science Publishers

Fish are ubiquitous organisms that have many features that designate their potential as a biomarker of heavy metals pollution. Thus, an investigation was done to detect the effect of heavy metals on cholinesterase (ChE) activity from Lates calcarifer organs which were gill and muscle. Ammonium sulphate precipitation was performed along with ion exchange chromatography to purify the enzyme. In the substrate specificity study, ChE from L. calcarifer gills was capable of breaking down acetylthiocholine iodide (ATC) at a faster rate compared to the other two synthetic substrates, which are butyrylthiocholine iodide (BTC) and propionylthiocholine iodide (PTC). In contrast, the muscle ChE has a higher affinity towards PTC. The maximum activity of ChE observed at the temperature ranging from 20 to 30 A degrees C in Tris-HCl buffer pH 8. ChE from the two organs of L. calcarifer showed an inhibitive reaction towards heavy metals, but with different effects. ATC from gills showed 50 % inhibition by Cu, Hg and Pb, while PTC from muscle showed 50 % inhibition by Pb. The variation of inhibitory effect that was shown by ChE from L. calcarifer organs can be further studied in designing a biosensor kit that is sensitive towards heavy metal.