The main strategy for lowering blood cholesterol levels is through the inhibition of the NADPH-dependent HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase). The enzyme catalyses the reduction of HMG-CoA to mevalonate and this process is inhibited by statins that form the bulk of the therapeutic agents to treat high cholesterol since the 1970s. Newer drugs that are safer than statins are constantly being developed. The inhibition of candidate drugs to HMG-CoA reductase remains the mainstay of drug development research. The determination of the enzyme activity is important for the correct assessment of potency of the enzyme as well as determining the inhibition of potential therapeutic agents from the plant and microbial extracts. Also, this chapter covers the use of the popular four-parameter logistics model that can yield accurate estimation of the IC50 values of therapeutic agents and their 95% confidence intervals.
Arsenic is a toxic metalloid which is widely distributed in nature. It is normally present as arsenate under oxic conditions while arsenite is predominant under reducing condition. The major discharges of arsenic in the environment are mainly due to natural sources such as aquifers and anthropogenic sources. It is known that arsenite salts are more toxic than arsenate as it binds with vicinal thiols in pyruvate dehydrogenase while arsenate inhibits the oxidative phosphorylation process. The common mechanisms for arsenic detoxification are uptaken by phosphate transporters, aquaglyceroporins, and active extrusion system and reduced by arsenate reductases via dissimilatory reduction mechanism. Some species of autotrophic and heterotrophic microorganisms use arsenic oxyanions for their regeneration of energy. Certain species of microorganisms are able to use arsenate as their nutrient in respiratory process. Detoxification operons are a common form of arsenic resistance in microorganisms. Hence, the use of bioremediation could be an effective and economic way to reduce this pollutant from the environment.
A novel molybdate-reducing bacterium, tentatively identified as Klebsiella sp. strain hkeem and based on partial 16s rDNA gene sequencing and phylogenetic analysis, has been isolated. Strain hkeem produced 3 times more molybdenum blue than Serratia sp. strain Dr.Y8; the most potent Mo-reducing bacterium isolated to date. Molybdate was optimally reduced to molybdenum blue using 4.5 mM phosphate, 80 mM molybdate and using 1% (w/v) fructose as a carbon source. Molybdate reduction was optimum at 30 °C and at pH 7.3. The molybdenum blue produced from cellular reduction exhibited absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide, and potassium cyanide did not inhibit the molybdenum-reducing enzyme. Mercury, silver, and copper at 1 ppm inhibited molybdenum blue formation in whole cells of strain hkeem.
Heavy metals pollution has become a great threat to the world. Since instrumental methods are expensive and need skilled technician, a simple and fast method is needed to determine the presence of heavy metals in the environment. In this study, an inhibitive enzyme assay for heavy metals has been developed using crude proteases from Coriandrum sativum. In this assay, casein was used as a substrate and Coomassie dye was used to denote the completion of casein hydrolysis. In the absence of inhibitors, casein was hydrolysed and the solution became brown, while in the presence of metal ions such as Hg²⁺ and Zn²⁺, the hydrolysis of casein was inhibited and the solution remained blue. Both Hg²⁺ and Zn²⁺ exhibited one-phase binding curve with IC₅₀ values of 3.217 mg/L and 0.727 mg/L, respectively. The limits of detection (LOD) and limits of quantitation (LOQ) for Hg were 0.241 and 0.802 mg/L, respectively, while the LOD and LOQ for Zn were 0.228 and 0.761 mg/L, respectively. The enzyme exhibited broad pH ranges for activity. The crude proteases extracted from Coriandrum sativum showed good potential for the development of a rapid, sensitive, and economic inhibitive assay for the biomonitoring of Hg²⁺ and Zn²⁺ in the aquatic environments.
Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae) originates from Papua Island, Indonesia and grows in tropical areas. The different parts of the fruit of P. macrocarpa were evaluated for antioxidant, anti-inflammatory, and cytotoxic activities.
Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae) is commonly known as 'Crown of God', 'Mahkota Dewa', and 'Pau'. It originates from Papua Island, Indonesia and it grows in tropical areas. Empirically, it is potent in treating the hypertensive, diabetic, cancer and diuretic patients. It has a long history of ethnopharmacological usage, and the lack of information about its biological activities led us to investigate the possible biological activities by characterisation of flavonoids and antimicrobial activity of various part of P. macrocarpa against pathogenic bacteria and fungi. The results showed that kaempferol, myricetin, naringin, and rutin were the major flavonoids present in the pericarp while naringin and quercetin were found in the mesocarp and seed. Furthermore, the antibacterial activity of different parts of P. macrocarpa fruit showed a weak ability to moderate antibacterial activity against pathogenic tested bacteria (inhibition range: 0.93-2.17 cm) at concentration of 0.3 mg/disc. The anti fungi activity was only found in seed extract against Aspergillus niger (1.87 cm) at concentration of 0.3 mg/well. From the results obtained, P. macrocarpa fruit could be considered as a natural antimicrobial source due to the presence of flavonoid compounds.
An inhibitive assay of insecticides using Acetylcholinesterase (AChE) from the local fish Clarias batrachus is reported. AChE was assayed according to the modified method of Ellman. Screening of insecticide and heavy metals showed that carbofuran and carbaryl strongly inhibited C. batrachus AChE. The inhibition concentration (IC) IC50 values (and the 95% confidence interval) for both carbofuran and carbaryl inhibition on C. batrachus AChE at 6.66 (5.97-7.52) and 130.00 (119.3-142.5) microg l(-1), respectively was within the IC50 range of Electrophorus electricus at 6.20 (6.03-6.39) and 133.01 (122.40-145.50) microg l(-1), respectively and were much lower than bovine AChE at 20.94 (19.53-22.58) and 418.80 (390.60-451.60) microg l(-1), respectively. The results showed that C. batrachus have the potential to be used as a cheaper and more readily available source of AChE than other more commercially available sources.
A stab-culture method was adapted to screen for azo dyes-decolorizing bacteria from soil and water samples. Decolorized azo dye in the lower portion of the solid media indicates the presence of anaerobic azo dyes-decolorizing bacteria, while aerobic decolorizing bacteria decolorizes the surface portion of the solid media. Of twenty soil samples tested, one soil sample shows positive results for the decolourisation of two azo dyes; Biebrich scarlet (BS) and Direct blue 71 (DB) under anaerobic conditions. A gram negative and oxidase negative bacterial isolate was found to be the principal azo dyes degrader The isolate was identified by using the Biolog identification system as Serratia marcescens.
In this work, a subtractive inhibition assay (SIA) based on surface plasmon resonance (SPR) for the rapid detection of Campylobacter jejuni was developed. For this, rabbit polyclonal antibody with specificity to C. jejuni was first mixed with C. jejuni cells and unbound antibody was subsequently separated using a sequential process of centrifugation and then detected using an immobilized goat anti-rabbit IgG polyclonal antibody on the SPR sensor chip. This SIA-SPR method showed excellent sensitivity for C. jejuni with a limit of detection (LOD) of 131 ± 4 CFU mL-1 and a 95% confidence interval from 122 to 140 CFU mL-1. The method has also high specificity. The developed method showed low cross-reactivity to bacterial pathogens such as Salmonella enterica serovar Typhimurium (7.8%), Listeria monocytogenes (3.88%) and Escherichia coli (1.56%). The SIA-SPR method together with the culturing (plating) method was able to detect C. jejuni in the real chicken sample at less than 500 CFU mL-1, the minimum infectious dose for C. jejuni while a commercial ELISA kit was unable to detect the bacterium. Since the currently available detection tools rely on culturing methods, which take more than 48 hours to detect the bacterium, the developed method in this work has the potential to be a rapid and sensitive detection method for C. jejuni.
In this work, we report on the isolation of a phenol-degrading Rhodococcus sp. with a high tolerance towards phenol. The isolate was identified as Rhodococcus sp. strain AQ5NOL 2, based on 16S rDNA analysis. The strain degraded phenol using the meta pathway, a trait shared by many phenol-degraders. In addition to phenol biodegradation, the strain was also capable of degrading diesel. Strain AQ5NOL 2 exhibited a broad optimum temperature for growth on phenol at between 20 °C and 35 °C. The best nitrogen sources were ammonium sulphate, glycine or phenylalanine, followed by proline, nitrate, leucine, and alanine (in decreasing efficiency). Strain AQ5NOL 2 showed a high tolerance and degradation capacity of phenol, for it was able to register growth in the presence of 2000 mg l(-1) phenol. The growth of this strain on phenol as sole carbon and energy source were modeled using Haldane kinetics with a maximal specific growth rate (μ(max)) of 0.1102 hr(-1), a half-saturation constant (K(s) ) of 99.03 mg l(-1) or 1.05 mmol l(-1), and a substrate inhibition constant (K(i)) of 354 mg l(-1) or 3.76 mmol l(-1). Aside from phenol, the strain could utilize diesel, 2,4-dinitrophenol and ρ-cresol as carbon sources for growth. Strain AQ5NOL 2 exhibited inhibition of phenol degradation by Zn(2+), Cu(2+), Cr(6+), Ag(+) and Hg(2+) at 1 mg l(-1).
As well as for chemical and environmental reasons, acrylamide is widely used in many industrial applications. Due to its carcinogenicity and toxicity, its discharge into the environment causes adverse effects on humans and ecology alike. In this study, a novel acrylamide-degrading yeast has been isolated. The isolate was identified as Rhodotorula sp. strain MBH23 using ITS rRNA analysis. The results showed that the best carbon source for growth was glucose at 1.0% (w/v). The optimum acrylamide concentration, being a nitrogen source for cellular growth, was at 500 mg l(-1). The highest tolerable concentration of acrylamide was 1500 mg l(-1) whereas growth was completely inhibited at 2000 mg l(-1). At 500 mg l(-1), the strain MBH completely degraded acrylamide on day 5. Acrylic acid as a metabolite was detected in the media. Strain MBH23 grew well between pH 6.0 and 8.0 and between 27 and 30 °C. Amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, and propionamide supported growth. Toxic heavy metals such as mercury, chromium, and cadmium inhibited growth on acrylamide.
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.
The development of glyphosate-resistant genetically modified organisms (GMO) has increased the use of herbicide glyphosate by several magnitudes in recent years. It is now the most commonly used pesticide globally that affects aquatic habitats, especially fish. This study aims to add new knowledge on the effect of technical grade glyphosate on several toxicity parameters and to identify the most effective parameter in predicting technical grade glyphosate chronic toxicity (seven weeks) to fish, especially Malaysia's heavily farmed red tilapia. The results show that a relatively high concentration of technical grade glyphosate is needed to induce significant changes in all tested parameters. However, the results also indicate that the bodyweight index is the most sensitive toxicity parameter in that a reduction in body weight was observed at 25 mg/L of glyphosate. Negative correlations between the glyphosate concentration and toxicity parameters such as specific growth rate (SGR), hepato-somatic index (HIS), and gonado-somatic index (GSI) were observed. The fish condition factor and feed conversion ratio were found not to be affected at the highest glyphosate concentration tested (150 mg/L). To conclude, crossbred red tilapia (O. niloticus × O. mossambicus) is one potential species for evaluating the toxic effects of technical grade glyphosate on fish.
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.
The enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase is the key enzyme of the mevalonate pathway that produces cholesterol. Inhibition of HMG-CoA reductase reduces cholesterol biosynthesis in the liver. Synthetic drugs, statins, are commonly used for the treatment of hypercholesterolemia. Due to the side effects of statins, natural HMG-CoA reductase inhibitors of plant origin are needed. In this study, 25 medicinal plant methanol extracts were screened for anti-HMG-CoA reductase activity. Basella alba leaf extract showed the highest inhibitory effect at about 74%. Thus, B. alba was examined in order to investigate its phytochemical components. Gas chromatography with tandem mass spectrometry and reversed phase high-performance liquid chromatography analysis revealed the presence of phenol 2,6-bis(1,1-dimethylethyl), 1-heptatriacotanol, oleic acid, eicosyl ester, naringin, apigenin, luteolin, ascorbic acid, and α-tocopherol, which have been reported to possess antihypercholesterolemic effects. Further investigation of in vivo models should be performed in order to confirm its potential as an alternative treatment for hypercholesterolemia and related cardiovascular diseases.
Crude extract of ChE from the liver of Puntius javanicus was purified using procainamide-sepharyl 6B. S-Butyrylthiocholine iodide (BTC) was selected as the specific synthetic substrate for this assay with the highest maximal velocity and lowest biomolecular constant at 53.49 µmole/min/mg and 0.23 mM, respectively, with catalytic efficiency ratio of 0.23. The optimum parameter was obtained at pH 7.5 and optimal temperature in the range of 25 to 30°C. The effect of different storage condition was assessed where ChE activity was significantly decreased after 9 days of storage at room temperature. However, ChE activity showed no significant difference when stored at 4.0, 0, and -25°C for 15 days. Screening of heavy metals shows that chromium, copper, and mercury strongly inhibited P. javanicus ChE by lowering the activity below 50%, while several pairwise combination of metal ions exhibited synergistic inhibiting effects on the enzyme which is greater than single exposure especially chromium, copper, and mercury. The results showed that P. javanicus ChE has the potential to be used as a biosensor for the detection of metal ions.
Atherosclerosis is the process of hardening and narrowing the arteries. Atherosclerosis is generally associated with cardiovascular diseases such as strokes, heart attacks, and peripheral vascular diseases. Since the usage of the synthetic drug, statins, leads to various side effects, the plants flavonoids with antiartherosclerotic activity gained much attention and were proven to reduce the risk of atherosclerosis in vitro and in vivo based on different animal models. The flavonoids compounds also exhibit lipid lowering effects and anti-inflammatory and antiatherogenic properties. The future development of flavonoids-based drugs is believed to provide significant effects on atherosclerosis and its related diseases. This paper discusses the antiatherosclerotic effects of selected plant flavonoids such as quercetin, kaempferol, myricetin, rutin, naringenin, catechin, fisetin, and gossypetin.
Luminescence-based assays for toxicants such as Microtox, ToxAlert, and Biotox have been used extensively worldwide. However, the use of these assays in near real time conditions is limited due to nonoptimal assay temperature for the tropical climate. An isolate that exhibits a high luminescence activity in a broad range of temperatures was successfully isolated from the mackerel, Rastrelliger kanagurta. This isolate was tentatively identified as Photobacterium sp. strain MIE, based on partial 16S rDNA molecular phylogeny. Optimum conditions that support high bioluminescence activity occurred between 24 and 30°C, with pH 5.5 to 7.5, 10 to 20 g/L of sodium chloride, 30 to 50 g/L of tryptone, and 4 g/L of glycerol as the carbon source. Assessment of near real time capability of this bacterial system, Xenoassay light to monitor heavy metals from a contaminated river running through the Juru River Basin shows near real time capability with assaying time of less than 30 minutes per samples. Samples returned to the lab were tested with a standard Microtox assay using Vibrio fishceri. Similar results were obtained to Xenoassay light that show temporal variation of copper concentration. Thus, this strain is suitable for near real time river monitoring of toxicants especially in the tropics.
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°C. The Mo-reducing enzyme was extremely sensitive to temperatures above 50°C (between 54 and 70°C). A plot of initial rates against substrate concentrations at 15 mM 12-MP registered a V max for NADH at 12.0 nmole Mo blue/min/mg protein. The apparent K m for NADH was 0.79 mM. At 5 mM NADH, the apparent V max and apparent K m values for 12-MP of 12.05 nmole/min/mg protein and 3.87 mM, respectively, were obtained. The catalytic efficiency (k cat/K m ) of the Mo-reducing enzyme was 5.47 M(-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.
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 GN microplate panels and Microlog 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- micromax 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.