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.

Respiratory activity inhibition by toxic compounds in bacteria and yeast has been used to detect toxic compounds in the environment. Often the age of culture contributes towards the sensitivity of detection. In the present work, the effect of growth period on the sensitivity of an inhibitive assay for heavy metals using bacterial respiratory assay system based on the reduction of the water soluble tetrazolium dye MU is reported. A silver-sensitive isolate was discovered to exhibit different sensitivities towards silver at different growth periods. An exponential decay model adequately described the inhibition due to silver. Analysis using ANOVA with post-hoc Tukey's test showed that the IC50 obtained by strain DRYS8 grown at the 12 hr-period in nutrient broth at 28 degrees C gave the lowest value compared to other growth periods. This study highlights the importance of taking into accounts growth conditions and age of culture in developing cellular-based bioassays.

In a previous study, we isolated Leifsonia sp. strain SIU, a new bacterium from agricultured soil. The bacterium was tested for its ability to degrade caffeine. The isolate was encapsulated in gellan gum and its ability to degrade caffeine was compared with the free cells. The optimal caffeine degradation was attained at a gellan gum concentration of 0.75% (w/v), a bead size of 4 mm diameter, and 250 beads per 100 mL of medium. At a caffeine concentration of 0.1 g/L, immobilised cells of the strain SIU degraded caffeine within 9 h, which is faster when compared to the case of free cells, in which it took 12 h to degrade. The immobilised cells degraded caffeine completely within 39 and 78 h at 0.5 and 1.0 g/L, while the free cells took 72 and 148 h at 0.5 and 1.0 g/L, respectively. At higher caffeine concentrations, immobilised cells exhibited a higher caffeine degradation rate. At concentrations of 1.5 and 2.0 g/L, caffeine-degrading activities of both immobilised and free cells were inhibited. The immobilised cells showed no loss in caffeine-degrading activity after being used repeatedly for nine 24-h cycles. The effect of heavy metals on immobilised cells was also tested. This study showed an increase in caffeine degradation efficiency when the cells were encapsulated in gellan gum.

A locally isolated Acinetobacter sp. Strain AQ5NOL 1 was encapsulated in gellan gum and its ability to degrade phenol was compared with the free cells. Optimal phenol degradation was achieved at gellan gum concentration of 0.75% (w/v), bead size of 3 mm diameter (estimated surface area of 28.26 mm(2)) and bead number of 300 per 100 ml medium. At phenol concentration of 100 mg l(-1), both free and immobilized bacteria exhibited similar rates of phenol degradation but at higher phenol concentrations, the immobilized bacteria exhibited a higher rate of degradation of phenol. The immobilized cells completely degrade phenol within 108, 216 and 240 h at 1,100, 1,500 and 1,900 mg l(-1) phenol, respectively, whereas free cells took 240 h to completely degrade phenol at 1,100 mg l(-1). However, the free cells were unable to completely degrade phenol at higher concentrations. Overall, the rates of phenol degradation by both immobilized and free bacteria decreased gradually as the phenol concentration was increased. The immobilized cells showed no loss in phenol degrading activity after being used repeatedly for 45 cycles of 18 h cycle. However, phenol degrading activity of the immobilized bacteria experienced 10 and 38% losses after the 46 and 47th cycles, respectively. The study has shown an increased efficiency of phenol degradation when the cells are encapsulated in gellan gum.

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.

A diesel-degrading bacterium was isolated from a diesel-contaminated site in Selangor, Malaysia. The isolate was tentatively identified as Acinetobacter sp. strain DRY12 based on partial 16S rDNA molecular phylogeny and Biolog (R) GN microplate panels and Microlog (R) database. Optimum growth occurred from 3 to 5% diesel and the strain was able to tolerate as high as 8% diesel. The optimal pH that supported growth of the bacterium was between pH 7.5 to 8.0. The isolate exhibited optimal growth in between 30 and 35 degrees C. The best nitrogen source was potassium nitrate (between 0.6 and 0.9% (w/v)) followed by ammonium chloride, sodium nitrite and ammonium sulphate in descending order. An almost complete removal of diesel components was seen from the reduction in hydrocarbon peaks observed using Solid Phase Microextraction Gas Chromatography analysis after 10 days of incubation. The best growth kinetic model to fit experimental data was the Haldane model of substrate inhibiting growth with a correlation coefficient value of 0.97. The maximum growth rate- p(max) was 0.039 hr(-1) while the saturation constant or half velocity constant Ks and inhibition constant Ki, were 0.387% and 4.46%, respectively. MATH assays showed that 75% of the bacterium was found in the hexadecane phase indicating that the bacterium was hydrophobic. The characteristics of this bacterium make it useful for bioremediation works in the Tropics.

Due to the latest industrial development, many dangerous chemicals have been released directly or indirectly which resulted in the polluted water bodies. Water rehabilitation is an alternative way to restore the quality of water, followed by the environmental management to control the waste discharge to ensure the balance of the degradation rates or detoxifying by environmental factors. However, this process consumed a lot of time and cost. Besides, most of the metal ions, especially copper which is capable to bioaccumulate in aquatic organism and at the elevated level may cause physiological and biochemical alteration which leads to mortality. Environmental monitoring is the initial step presupposed evaluating the potential toxicity of effluent gushing at its purpose to discharge, avoiding the determining effects of contaminant in water bodies. Due to the high sensitivity of the aquatic life towards dissolving toxicant, the fish has been utilized as the biological measurement (Biomarker) to indicate the existence of toxicant exposure and/or the impact towards the evaluation of molecular, cellular to physiological level. Thus, this paper gives an overview of the manipulation of fish as a biomarker of heavy metals through behavior response, hepatocyte alteration, enzymatic reaction and proteomic studies which have proven to be very useful in the environmental pollution monitoring. (c) All Rights Reserved

Biodegradation of agricultural wastes, generated annually from poultry farms and slaughterhouses, can solve the pollution problem and at the same time yield valuable degradation products. But these wastes also constitute environmental nuisance, especially in Malaysia where their illegal disposal on heavy metal contaminated soils poses a serious biodegradation issue as feather tends to accumulate heavy metals from the surrounding environment. Further, continuous use of feather wastes as cheap biosorbent material for the removal of heavy metals from effluents has contributed to the rising amount of polluted feathers, which has necessitated the search for heavy metal-tolerant feather degrading strains. Isolation, characterization and application of a novel heavy metal-tolerant feather-degrading bacterium, identified by 16S RNA sequencing as Alcaligenes sp. AQ05-001 in degradation of heavy metal polluted recalcitrant agricultural wastes, have been reported. Physico-cultural conditions influencing its activities were studied using one-factor-at-a-time and a statistical optimisation approach. Complete degradation of 5 g/L feather was achieved with pH 8, 2% inoculum at 27 degrees C and incubation period of 36 h. The medium optimisation after the response surface methodology (RSM) resulted in a 10-fold increase in keratinase production (88.4 U/mL) over the initial 8.85 U/mL when supplemented with 0.5% (w/v) sucrose, 0.15% (w/v) ammonium bicarbonate, 03% (w/v) skim milk, and 0.01% (w/v) urea. Under optimum conditions, the bacterium was able to degrade heavy metal polluted feathers completely and produced valuable keratinase and protein-rich hydrolysates. About 83% of the feathers polluted with a mixture of highly toxic metals were degraded with high keratinase activities. The heavy metal tolerance ability of this bacterium can be harnessed not only in keratinase production but also in the bioremediation of heavy metal polluted feather wastes. (C) 2016 Published by Elsevier Ltd.

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.

A molybdenum-reducing bacterium from Antarctica has been isolated. The bacterium converts sodium molybdate or Mo6+ to molybdenum blue (Mo-blue). Electron donors such as glucose, sucrose, fructose, and lactose supported molybdate reduction. Ammonium sulphate was the best nitrogen source for molybdate reduction. Optimal conditions for molybdate reduction were between 30 and 50 mM molybdate, between 15 and 20 degrees C, and initial pH between 6.5 and 7.5. The Mo-blue produced had a unique absorption spectrum with a peak maximum at 865 nm and a shoulder at 710 nm. Respiratory inhibitors such as antimycin A, sodium azide, potassium cyanide, and rotenone failed to inhibit the reducing activity. The Mo- reducing enzyme was partially purified using ion exchange and gel filtration chromatography. The partially purified enzyme showed optimal pH and temperature for activity at 6.0 and 20 degrees C, respectively. Metal ions such as cadmium, chromium, copper, silver, lead, and mercury caused more than 95% inhibition of the molybdenum- reducing activity at 0.1 mM. The isolate was tentatively identified as Pseudomonas sp. strain DRY1 based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system. The characteristics of this strain would make it very useful in bioremediation works in the polar and temperate countries.

The first purification of the Mo-reducing enzyme from Serratia sp. strain DRY5 that is responsible for molybdenum reduction to molybdenum blue in the bacterium is reported. The monomeric enzyme has an apparent molecular weight of 105 kDalton. The isoelectric point of this enzyme was 7.55. The enzyme has an optimum pH of 6.0 and maximum activity between 25 and 35 degrees C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50 degrees C (between 54 and 70 degrees C). A plot of initial rates against substrate concentrations at 15mM 12-MP registered a V-max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent k(m) for NADH was 0.79mM. At 5mM NADH, the apparent V-max and apparent k(m) values for 12-MP of 12.05 nmole/min/mg protein and 3.87mM, respectively, were obtained. The catalytic efficiency (k(cat)/k(m)) of the Mo-reducing enzyme was 5.47M(-1) s(-1). The purification of this enzyme could probably help to solve the phenomenon of molybdenum reduction to molybdenum blue first reported in 1896 and would be useful for the understanding of the underlying mechanism in molybdenum bioremediation involving bioreduction.

Gold mining companies are known to use cyanide to extract gold from minerals. The indiscriminate use of cyanide presents a major environmental issue. Serratia marcescens strain AQ07 was found to have cyanide-degrading ability. Optimisation of biodegradation condition was carried out utilising one factor at a time and response surface methodology. Cyanide degradation corresponded with growth rate with a maximum growth rate of 16.14 log cfu/mL on day 3 of incubation. Glucose and yeast extract are suitable carbon and nitrogen sources. Six parameters including carbon and nitrogen sources, pH, temperature, inoculum size and cyanide concentration were optimised. In line with the central composite design of response surface methodology, cyanide degradation was optimum at glucose concentration 5.5 g/L, yeast extract 0.55 g/L, pH 6, temperature 32.5 A degrees C, inoculum size 20 % and cyanide concentration 200 mg/L. It was able to stand cyanide toxicity of up to 700 mg/L, which makes it an important candidate for bioremediation of cyanide. The bacterium was observed to degrade 95.6 % of 200 mg/L KCN under the optimised condition. Bacteria are reported to degrade cyanide into ammonia, formamide or formate and carbon dioxide, which are less toxic by-products. These bacteria illustrate good cyanide degradation potential that can be harnessed in cyanide remediation.

The purpose of this study was to investigate the ability of Acinetobacter sp. strain AQ5NOL 1 immobilised in gellan gum beads to degrade phenol in the presence of heavy metals. Sewn different heavy metals, namely, As5+, Cu2+ Cd2+, Ni2+, Cr6+, Ph2+, and He at 1 ppm were tested. Results of the study showed that degradation of phenol by free cells was inhibited by Hg2+, Cu6+ and Cr6+ after 48 hours of incubation by 97.91 %, 77.58 % and 75.26 %, respectively. Only Hg2+ and Cr6+ inhibited phenol degradation by immobilised Acinetobacter cells in 18 hours by 67.55 % and 53.19 %. Phenol degradation by immobilised cells was affected when Cr and Hg2+ concentrations exceeded 0.5 and 0.1 ppm, respectively. However, inhibitory effects of heavy metals can be overcome by prolonging the incubation time for immobilised Acinetobacter sp. strain AQ5NOL 1 from 18 hours to 24 and 30 hours for Cr6+ (46.80 %) and Hg2+ (21.40 %), respectively.

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.