The need to isolate efficient heavy metal reducers for cost effective bioremediation strategy have resulted in the isolation of a potent molybdenum-reducing bacterium. The isolate was tentatively identified as Serratia sp. strain DRY5 based on the Biolog GN carbon utilization profiles and partial 16S rDNA molecular phylogeny. Strain DRY5 produced 2.3 times the amount of Mo-blue than S. marcescens strain Dr.Y6, 23 times more than E. coli K12 and 7 times more than E. cloacae strain 48. Strain DRY5 required 37 degrees C and pH 7.0 for optimum molybdenum reduction. Carbon sources such as sucrose, maltose, glucose and glycerol, supported cellular growth and molybdate reduction after 24 hr of static incubation. The most optimum carbon source that supported reduction was sucrose at 1.0% (w/v). Ammonium sulphate, ammonium chloride, glutamic acid, cysteine, and valine supported growth and molybdate reduction with ammonium sulphate as the optimum nitrogen source at 0. 2% (w/v). Molybdate reduction was optimally supported by 30 mM molybdate. The optimum concentration of phosphate for molybdate reduction was 5 mM when molybdate concentration was fixed at 30 mM and molybdate reduction was totally inhibited at 100 mM phosphate. Mo-blue produced by this strain shows a unique characteristic absorption profile with a maximum peak at 865 nm and a shoulder at 700 nm, Dialysis tubing experiment showed that 95.42% of Mo-blue was found in the dialysis tubing suggesting that the molybdate reduction seen in this bacterium was catalyzed by enzyme(s). The characteristics of isolate DRY5 suggest that it would be useful in the bioremediation ofmolybdenum-containing waste.
In this work the development of an inhibitive assay for copper using the molybdenum-reducing enzyme assay is presented. The enzyme is assayed using 12-molybdophosphoric acid at pH 5.0 as an electron acceptor substrate and NADH as the electron donor substrate. The enzyme converts the yellowish solution into a deep blue solution. The assay is based on the ability of copper to inhibit the molybdenum-reducing enzyme from the molybdate-reducing Serratia sp. Strain DRY5. Other heavy metals tested did not inhibit the enzyme at 10 mg l(-1). The best model with high regression coefficient to measure copper inhibition is one-phase binding. The calculated IC50 (concentration causing 50% inhibition) is 0.099 mg l(-1) and the regression coefficient is 0.98. The comparative LC50, EC50 and IC50 data for copper in different toxicity tests show that the IC50 value for copper in this study is lower than those for immobilized urease, bromelain, Rainbow trout, R. meliloti, Baker's Yeast dehydrogenase activity Spirillum volutans, P. fluorescens, Aeromonas hydrophilia and synthetic activated sludge assays. However the IC50 value is higher than those for Ulva pertusa and papain assays, but within the reported range for Daphnia magna and Microtox assays.
A new inhibitive heavy metals determination method using trypsin has been developed. The enzyme was assayed using the casein-Coomassie-dye-binding method. In the absence of inhibitors, casein was hydrolysed to completion and the Coomassie-dye was unable to stain the protein and the solution became brown. In the presence of metals, the hydrolysis of casein was inhibited and the solution remained blue. The bioassay was able to detect zinc and mercury with IC50 (concentration causing 50% inhibition) values of 5.78 and 16.38 mg l(-1) respectively. The limits of detection (LOD), for zinc and mercury were 0.06 mg l(-1) (0.05-0.07, 95% confidence interval) and 1.06 mg l(-1) (1.017-1.102, 95% confidence interval), respectively. The limits of quantitation (LOQ) for zinc and mercury were 0.61 mg l(-1) (0.51-0.74 at a 95% confidence interval) and 1.35 mg l(-1) (1.29-1.40 at a 95% confidence interval), respectively. The IC50 value for zinc was much higher than the IC50 values for papain and Rainbow trout, but was within the range of Daphnia magna and Microtox. The IC50 value for zinc was only lower than those for immobilized urease. Other toxic heavy metals, such as lead, silver arsenic, copper and cadmium, did not inhibit the enzyme at 20 mg l(-1). Using this assay we managed to detect elevated zinc concentrations in several environmental samples. Pesticides, such as carbaryl, flucythrinate, metolachlor glyphosate, diuron, diazinon, endosulfan sulphate, atrazine, coumaphos, imidacloprid, dicamba and paraquat, showed no effect on the activity of trypsin relative to control (One-way ANOVA, F(12,26)= 0.3527, p> 0.05). Of the 17 xenobiotics tested, only (sodium dodecyl sulphate) SDS gave positive interference with 150% activity higher than that of the control at 0.25% (v/v).
A diesel-degrading bacterium from Antarctica has been isolated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ3 based on partial 16S rDNA molecular phylogeny and Biolog GN microplate panels and Microlog database. Growth on diesel was supported optimally by ammonium sulphate, nitrate and nitrite. The bacterium grew optimally in between 10 and 15 degrees C, pH 7.0 and 3.5% (v/v) diesel. The biodegradation of diesel oil by the strain increased in efficiency from the second to the sixth day of incubation from 1.4 to 18.8% before levelling off on the eighth day n-alkane oxidizing and aldehyde reductase activities were detected in the crude enzyme preparation suggesting the existence of terminal n-alkane oxidizing activity in this bacterium.
Several local acrylamide-degrading bacteria have been isolated. One of the isolate that exhibited the highest growth on acrylamide as a nitrogen source was then further characterized. The isolate was tentatively identified as Bacillus cereus strain DRY135 based on carbon utilization profiles using Biolog GP plates and partial 16S rDNA molecular phylogeny. The isolate grew optimally in between the temperatures of 25 and 30 degrees C and within the pH range of 6.8 to 7.0. Glucose, fructose, lactose, maltose, mannitol, citric acid and sucrose supported growth with glucose being the best carbon source. Different concentrations of acrylamide ranging from 100 to 4000 mg l(-1) incorporated into the growth media shows that the highest growth was obtained at acrylamide concentrations of between 500 to 1500 mg l(-1). At 1000 mg l(-1) of acrylamide, degradation was 90% completed after ten days of incubation with concomitant cell growth. The metabolite acrylic acid was detected in the media during degradation. Other amides such as methacrylamide, nicotinamide, acetamide, propionamide and urea supported growth with the highest growth supported by acetamide, propionamide and urea. Strain DRY135, however was not able to assimilate 2-chloroacetamide. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.
A heavy-metal assay has been developed using bromelain, a protease. The enzyme is assayed using casein as a substrate with Coomassie dye to track completion of hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion, and the solution is brown. In the presence of metal ions such as Hg2+ and Cu2+, the hydrolysis of casein is inhibited, and the solution remains blue. Exclusion of sulfhydryl protective agent and ethylenediaminetetraacetic in the original assay improved sensitivity to heavy metals several fold. The assay is sensitive to Hg2+ and Cu2+, exhibiting a dose-response curve with an IC50 of 0.15 mg 1(-1) for Hg2+ and a one-phase binding curve with an IC50 of 0.23 mg 1(-1) for Cu2+. The IC50 value for Hg2+ is found to be lower to several other assays such as immobilized urease and papain assay, whilst the IC50 value for Cu2+ is lower than immobilized urease, 15-min Microtox, and rainbow trout.
Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, Km for the laboratory-prepared 10:4-phosphomolybdate is 2.56 +/- 0.25 mM (arbitrary concentration), whereas the apparent V(max) is 99.4 +/- 2.85 nmol Mo-blue min(-1) mg(-1) protein. The apparent Michaelis constant or Km for NADH as the electron donor is 1.38 +/- 0.09 mM, whereas the apparent V(max) is 102.6 +/- 1.73 nmol Mo-blue min(-1) mg(-l) protein. The apparent Km and V(max) for another electron donor, NADPH, is 1.43 +/- 0.10 mM and 57.16 +/- 1.01 nmol Mo-blue min(-1) mg(-1) protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V(max) obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.
A molybdate-reducing bacterium has been locally isolated. The bacterium reduces molybdate or Mo(6+) to molybdenum blue (molybdate oxidation states of between 5+ and 6+). Different carbon sources such as acetate, formate, glycerol, citric acid, lactose, fructose, glucose, mannitol, tartarate, maltose, sucrose, and starch were used at an initial concentration of 0.2% (w/v) in low phosphate media to study their effect on the molybdate reduction efficiency of bacterium. All of the carbon sources supported cellular growth, but only sucrose, maltose, glucose, and glycerol (in decreasing order) supported molybdate reduction after 24 h of incubation. Optimum concentration of sucrose for molybdate reduction is 1.0% (w/v) after 24 h of static incubation. Ammonium sulfate, ammonium chloride, valine, OH-proline, glutamic acid, and alanine (in the order of decreasing efficiency) supported molybdate reduction with ammonium sulfate giving the highest amount of molybdenum blue after 24 h of incubation at 0.3% (w/v). The optimum molybdate concentration that supports molybdate reduction is between 15 and 25 mM. Molybdate reduction is optimum at 35 degrees C. Phosphate at concentrations higher than 5 mM strongly inhibits molybdate reduction. The molybdenum blue produced from cellular reduction exhibits a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. The isolate was tentatively identified as Serratia marcescens Strain Dr.Y6 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny.
Extensive use of metals in various industrial applications has caused substantial environmental pollution. Molybdenum-reducing bacteria isolated from soils can be used to remove molybdenum from contaminated environments. In this work we have isolated a local bacterium with the capability to reduce soluble molybdate to the insoluble molybdenum blue. We studied several factors that would optimize molybdate reduction. Electron donor sources such as glucose, sucrose, lactose, maltose and fructose (in decreasing efficiency) supported molybdate reduction after 24 h of incubation with optimum glucose concentration for molybdate reduction at 1.5% (w/v). The optimum pH, phosphate and molybdate concentrations, and temperature for molybdate reduction were pH 6.5, 5.0, 25 to 50 mM and 37 degrees C, respectively. The Mo-blue produced by cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, cadmium, copper, silver and mercury caused approximately 73, 71, 81, 77 and 78% inhibition of the molybdenum-reducing activity, respectively. All of the respiratory inhibitors tested namely rotenone, azide, cyanide and antimycin A did not show any inhibition to the molybdenum-reducing activity suggesting components of the electron transport system are not responsible for the reducing activity. The isolate was tentatively identified as Enterobacter sp. strain Dr.Y13 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny.
A local molybdenum-reducing bacterium was isolated and tentatively identified as Acinetobacter calcoaceticus strain Dr.Y12 based on carbon utilization profiles using Biolog GN plates and 16S rDNA comparative analysis. Molybdate reduction was optimized under conditions of low dissolved oxygen (37 degrees C and pH 6.5). Of the electron donors tested, glucose, fructose, maltose and sucrose supported molybdate reduction after 1 d of incubation, glucose and fructose supporting the highest Mo-blue production. Optimum Mo-blue production was reached at 20 mmol/L molybdate and 5 mmol/L phosphate; increasing the phosphate concentrations inhibited the production. An increase in an overall absorption profiles, especially at peak maximum at 865 nm and the shoulder at 700 nm, was observed in direct correlation with the increased in Mo-blue amounts. Metal ions, such as chromium, cadmium, copper, mercury and lead (2 mmol/L final concentration) caused approximately 88, 53, 80, 100, and 20 % inhibition, respectively. Respiratory inhibitors, such as antimycin A, rotenone, sodium azide and cyanide showed in this bacterium no inhibition of the Mo-blue production, suggesting that the electron transport system is not a site of molybdate reduction.
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°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°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.
1. alpha-Tocopherol (alpha-T) and gamma-tocotrienol (gamma-T) were supplemented continuously for 8 weeks in the diets of normal rats and rats chemically induced with cancer using diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF) and partial hepatectomy. Hepatocarcinogenesis was followed by determining the plasma gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase (ALP) activities as well as placental glutathione S-transferase (PGST) and GGT activities histochemically, at 4-week intervals. 2. Male Rattus norvegicus were supplemented alpha-T and gamma-T at two different doses of 30 and 300 mg/kg diet. The supplementation was started at three different times: simultaneously with DEN administration; 4 weeks; and 8 weeks after DEN administration. 3. Elevation of plasma GGT activities and formation of PGST and GGT positive foci were attenuated significantly (P < 0.05) when alpha-T and gamma-T were supplemented simultaneously with cancer induction. Supplementation begun 4 and 8 weeks after cancer induction did not affect plasma enzyme activities and formation of enzyme-positive foci. 4. alpha-T was more effective than gamma-T, and a lower dose of 30 mg/kg was found to be more effective in reducing the severity of hepatocarcinogenesis.
1. Toxicity evaluations of DDT, lindane, abate and carbaryl were carried out in the larvae of two wild Aedes aegypti strains from Kuala Lumpur and Klang. The Kuala Lumpur strain was more susceptible to the insecticides than the Klang strain. 2. The lethal toxicity time was also determined. The insecticides were found to take a longer time to exert their effect in the Klang strain as compared to the Kuala Lumpur strain. 3. Carboxylesterase activity was determined to be higher in the Kuala Lumpur strain, but glutathione transferase activities were higher in the Klang strain.
1. Glutathione transferases from the liver, lung and kidney tissues of the buffalo (Bubalus bubalis) and the Kedah-Kelantan cattle (Bos indicus) were partially purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration. 2. Liver tissue contains the highest enzyme activity when compared to the lung and kidney tissues. 3. The activity in cattle is higher than that in the buffalo. 4. Isoelectric focusing separates the activities into the acidic, near neutral and basic fractions. 5. The focused patterns are different for each of the tissues and in each of the species investigated.
Ageing is associated with increased oxidative stress accompanied by cognitive decline. The aim of this study was to evaluate oxidative stress biomarkers and their possible relationship with cognitive performances during ageing among the Malay population. Approximately 160 healthy Malay adults aged between 28 and 79 years were recruited around Selangor and Klang Valley. Cognitive function was assessed by Montreal Cognitive Assessment (MoCA), forward digit span (FDS), backward digit span (BDS), digit symbol, Rey Auditory Verbal Learning Test immediate recalled [RAVLT(I)] and delayed recalled [RAVLT(D)], and visual reproduction immediate recalled (VR-I) and delayed recalled (VR-II). DNA damage, plasma protein carbonyl and malondialdehyde (MDA) levels were also determined. Cognitive function test showed significant lower scores of MoCA, BDS, RAVLT(I), RAVLT(D), digit symbol, VR-I, and VR-II in the older age group (60 years old) compared with the 30-, 40-, and 50-year-old group. The extent of DNA damage was sequential with age: 60 > 50 > 40 > 30, whereas protein carbonyl was higher in 40-, 50-, and 60-year-old groups compared with the youngest group (30 years old). However, the MDA level was observed unchanged in all age groups. Approximately 21.88% of the participants had cognitive impairment. Multiple logistic regression analysis revealed that DNA damage and protein carbonyl levels are predictors for cognitive impairment in healthy Malays. In conclusion, cognitive decline occurred in healthy adult Malay population at an early age of 30 years old with corresponding higher DNA damage and protein oxidation.