Purification And Characterization Of Acrylamide-Degrading Enzyme From Burkholderia Sp. Dr.Y27

Acrylamide is a toxic and carcinogenic compound. There are many sources of acrylamide pollution in soil. Three major documented sources are polyacrylamide used liberally as a flocculating agent in water treatment, acrylamide waste from acrylic industries, and the other is from the formulation in the herbicide glyphosate. It has been documented that approximately 0.1% of polyacrylamide is degraded yearly to the carcinogenic acrylamide in soil by soil bacteria. Some of the acrylamide is used as carbon and nitrogen sources by soil bacteria whilst it is suggested that the remaining becomes a source of contamination in vegetables and potatoes. Understanding acrylamide degradation in soil is vital not only to the microbiological point of view, but the prospect of lowering acrylamide concentrations via bioremediation would lower the potential of acrylamide as a pollutant and contaminant. Several local bacteria have been isolated from glyphosate-contaminated soils at various locations throughout Malaysia. Out of these isolates we have singled out a potent acrylamide-degrading bacterium, which could be potentially used in the bioremediation of acrylamide. Quantitative degradation of acrylamide was performed using High Performance Liquid Chromatography (HPLC), whilst bacterial growth was carried out by plate counting. Isolate 2.7 could degrade 99.84% of 100 mg/L acrylamide as the sole nitrogen source after 48 hours of incubation. Isolate 2.7 was identified as Burkholderia sp. Strain DR.Y27 using 16S rRNA and BiologTM microbial identification system. Burkholderia sp. Strain DR.Y27 showed an optimum temperature for growth at 30°C, and optimum initial pH medium for bacterial growth at pH 7.5. Burkholderia sp. strain DR.Y27 showed maximum growth in medium containing 1 % glucose and when 500 mg/L acrylamide was provided. The acrylamide-degrading enzyme, amidase, from this bacterium was stable at pH 8 when stored at 4 and -20 °C. Amidase activity was not affected by 1 mM of all metal ions tested, such as WO42-, L12+, Fe2+, As4+, Ni2+, Se2+, Zn2+, Cs2+, Cr2+, Al3+, Mn2+, Co2+, Mg2+, Cu2+, Pb2+, Cd2+, Ag2+, Hg2+, the enzyme activity also was not affected by EDTA, β-Merchaptoethanol and DTT. The maximum velocity in the order of decreasing rates using various substrates were 1.99 ± 0.11 Units/mg protein, 1.50 ± 0.09 Units/mg protein, 1.5 ± 0.02 Units/mg protein, 0.6 ± 0.04 Units/mg protein, 0.48± 0.01 Units/mg protein and 0.34 ± 0.02 Units/mg protein for propionamide, acrylamide, urea, acetamide and 2-cloroacetamide, respectively. The apparent Km for these substrates in the order of decreasing affinity are 0.27 ± 0.19 mM, 1.21 ± 0.13 mM, 1.88 ± 0.28 mM, 2.39 ± 1.84 mM and 4.29 ± 0.87 mM for acetamide, 2-cloroacetamide, urea, acrylamide and propionamide, respectively. The amidase from Burkholderia sp. strain DR.Y27 could not use metachrylamide and nicotinamide as substrate. The amidase exhibited maximal activity at 40°C and at pH 8.0 of phosphate buffer. The apparent Km and Vmax values for amidase were 2.39 ± 1.84 mM mM acrylamide and, 1.50 ± 0.09 μmol min-1 mg-1 protein, respectively using acrylamide as a substrate. The amidase was purified to homogeneity by a combination of anion exchange and gel filtration chromatography. The purification strategy achieved 11.15 of purification fold and a yield of 1.55%. Pure amidase showed a homogenous protein band with approximate MW of 186 kDa using gel filtration ZorbaxR GF-250 column chromatography. The purified enzyme migrated as a single band in SDS-PAGE in the presence of β-mercaptoethanol with a molecular mass of 47 kDa. It indicates that the native enzyme was a homotetramer

ISOLATION AND CHARACTERIZATION OF AN ACRYLAMIDE-DEGRADING Burkholderia sp. STRAIN DR.Y27

 ABSTRACT Several local bacteria have been isolated from glyphosate-contaminated soils at various locations throughout Malaysia. Quantitative monitoring of acrylamide degradation was performed using High Performance Liquid Chromatography (HPLC) whilst bacterial growth was carried out by plate counting. The isolate was tentatively identified as Burkholderia sp. strain DR.Y27 based on carbon utilization profiles using Biolog GN plates and partial 16s rDNA molecular phylogeny. Highest growth was obtained at acrylamide concentrations of between 100 to 2000 mg L-1.  Complete degradation of 850 mg L-1 of acrylamide occurs after ten days of incubation with concomitant cell growth. The isolate grew optimally in between pH 6.0 and 8.0. The effect of incubation temperature on the growth of this isolate shows an optimum growth at 30°C. Glucose, lactose, maltose, fructose, mannitol, citric acid and sucrose at an initial concentration of 1.0% (w/v) supported growth with glucose being the best carbon source. Aliphatic amides such as 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, propionamide and urea supported growth with increasing assimilative capability from 2-chloroacetamide to urea. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamide.  Keywords:  isolation, characterization, acrylamide-degrading, Bacteriu

Isolation and characterisation of a molybdenum-reducing and Metanil Yellow dye-decolourizing Bacillus sp. strain Neni-10 in soils from West Sumatera, Indonesia

A molybdenum reducing bacterium with the novel ability to decolorise the azo dye Metanil Yellow is reported. Optimal conditions for molybdenum reduction were pH 6.3 and at 34°C. Glucose was the best electron donor. Another requirement includes a narrow phosphate concentration between 2.5 and 7.5 mM. A time profile of Mo-blue production shows a lag period of approximately 12 hours, a maximum amount of Mo-blue produced at a molybdate concentration of 20 mM, and a peak production at 52 h of incubation. The heavy metals mercury, silver, copper and chromium inhibited reduction by 91.9, 82.7, 45.5 and 17.4%, respectively. A complete decolourisation of the dye Metanil Yellow at 100 and 150 mg/L occurred at day three and day six of incubations, respectively. Higher concentrations show partial degradation, with an approximately 20% decolourisation observed at 400 mg/L. The bacterium is partially identified based on biochemical analysis as Bacillus sp. strain Neni-10. The absorption spectrum of the Mo-blue suggested the compound is a reduced phosphomolybdate. The isolation of this bacterium, which shows heavy metal reduction and dye-decolorising ability, is sought after, particularly for bioremediation

Isolation and characterization of an acrylamide-degrading Antarctic bacterium

The presence of acrylamide in the environment poses a threat due to its well known neurotoxic, carcinogenic and teratogenic properties. Human activities in various geographical areas are the main anthropogenic source of acrylamide pollution. In this work, an acrylamide-degrading bacterium was isolated from Antarctic soil. The physiological characteristics and optimum growth conditions of the acrylamide-degrading bacteria were investigated. The isolate was tentatively identified as Pseudomonas sp. strain DRYJ7 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. The results showed that the best carbon sources for growth was glucose and sucrose with no significant difference in terms of cellular growth between the two carbon sources (p>0.05). This was followed by fructose and maltose with fructose giving significantly higher cellular growth compared to maltose (p<0.05). Lactose and citric acid did not support growth. The optimum acrylamide concentration as a nitrogen source for cellular growth was at 500 mgl-1. At this concentration, bacterial growth showed a 2-day lag phase before degradation took place concomitant with an increase in cellular growth. The isolate exhibited optimum growth in between pH 7.5 and 8.5. The effect of incubation temperature on the growth of this isolate showed an optimum growth at 15°C. The characteristics of this isolate suggest that it would be useful in the bioremediation of acrylamid

Isolation and characterization of an acrylamide-degrading Bacillus cereus

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°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

Isolation and characterization of a molybdenum-reducing and azo-dye decolorizing Serratia marcescens strain Neni-1 from Indonesian soil

Heavy metals and organic xenobiotics including dyes are important industrial components with their usage amounting to the millions of tonnes yearly. Their presence in the environment is a serious pollution issue globally. Bioremediation of these pollutants using microbes with multiple detoxification capacity is constantly being sought. In this work we screen the ability of a molybdenum-reducing bacterium isolated from contaminated soil to decolorize various azo and triphenyl methane dyes. The bacterium reduces molybdate to molybdenum blue (Mo-blue) optimally at pH 6.0, and temperatures of between 25 and 40oC. Glucose was the best electron donor for supporting molybdate reduction followed by sucrose, trehalose, maltose, d-sorbitol, d-mannitol, d-mannose, myo-inositol, glycerol and salicin in descending order. Other requirements include a phosphate concentration of between 5.0 and 7.5 mM and a molybdate concentration between 10 and 20 mM. The absorption spectrum of the Mo-blue produced was similar to previous Mo-reducing bacterium, and closely resembles a reduced phosphomolybdate. Molybdenum reduction was inhibited bycopper, silver and mercury at 2 ppm by 43.8%, 42.3% and 41.7%, respectively. We screen for the ability of the bacterium to decolorize various dyes. The bacterium was able to decolorize the dye Congo Red. Biochemical analysis resulted in a tentative identification of the bacterium as Serratia marcescens strain Neni-1. The ability of this bacterium to detoxify molybdenum and decolorize azo dye makes this bacteriuman important tool for bioremediation

Isolation and characterization of a molybdenum-reducing and amide-degrading Burkholderia sp. strain NENI-11 in soils from West Sumatra, Indonesia

A molybdenum-reducing bacterium isolated from contaminated soil was able to utilize acrylamide as the electron donor source, and was able utilize acrylamide, acetamide and propionamide for growth. Reduction was optimal at pH between 6.0 to 6.3, at temperatures of between 30 and 37 oC, glucose as the electron donor, phosphate at 5.0 mM, and sodium molybdate at 15 mM. The absorption spectrum of the Mo-blue indicates it is a reduced phosphomolybdate. Molybdenum reduction was inhibited by mercury (ii), silver (i) and chromium (vi) at 2 p.p.m. by 91.9, 82.7 and 17.4 %, respectively. Biochemical analysis resulted in a tentative identification of the bacterium as Burkholderia cepacia strain Neni-11. The growth of this bacterium modelled according to the modified Gompertz model. The growth parameters obtained were maximum specific growth rates of 1.241 d-1, 0.971 d-1, 0.85 d-1 for acrylamide, propionamide and acetamide, respectively, while the lag periods of 1.372 d, 1.562 and 1.639 d were observed for acrylamide, propionamide and acetamide, respectively. The ability of this bacterium to detoxify molybdenum and grown on toxic amides makes this bacterium an important tool for bioremediation

Modelling the effect of copper on the growth rate of Enterobacter sp. strain Neni-13 on SDS

The introduction of tiny amounts of heavy metals into the environment can encourage the growth of a wide variety of microorganisms. The concentration at which enhanced microbial activity is seen, on the other hand, results in a significant decrease in growth rate as well as an increase in lag time (due to the higher lag time). An established link exists between heavy metal toxicity in microorganisms and the process of bioremediation, which has been well-documented. Because heavy metals have an impact on bioremediation, they must be researched, and appropriate countermeasures must be implemented. Copper reduced the growth of the SDS-degrading bacteria Enterobacter sp. strain Neni-13 to a significant extent. Under varying doses of mercury, the SDS-degrading bacteria exhibited a sigmoidal pattern with time periods ranging from 7 to 10 hours. Gompertz's model was used to calculate the growth rates of copper in different concentrations. As the copper concentration rose, the growth of bacteria was suppressed with a concentration of 1.0 g/L, with virtually total stoppage of bacterial development. From the Gompertz model, we got the estimates of growth rates; after which, they were estimated according to the Han-Levenspiel, Shukor, Wang, Liu, Andrews, and Amor models. The modified Han-Levenspiel, Andrews, Liu, and Shukor models could all successfully fit the curve. Results of the statistical analysis showed that the Han-Levenspiel model was the best model based on highest adjusted correlation coefficient (adR2), the lowest values for RMSE and AICc, and values of AF and BF closest to unity. The parameters obtained from the Han-Levenspiel model were Ccrit 0.209 mg/L (95%, C.I., 0.199 to 0.219), μmax 0.209 h-1 (95% C.I., 0.199 to 0.219) and m 0.472 (95% C.I., 0.383 to 0.561. The results obtained in this study indicate the maximum tolerable copper concentration that the conditions for biodegradation should not exceed

Molybdate reduction to molybdenum blue and growth on polyethylene glycol by Bacillus sp. strain Neni-8

The accumulation of heavy metals and xenobiotic compounds in soil and aquatic bodies is caused by inappropriate waste disposal, industrial and mining operations, and excessive use of agricultural pesticides. Bioremediation is a more cost-effective way of removing these pollutants than other approaches. A new molybdenum-reducing bacterium with the ability to grow on a variety of polyethylene glycol (PEG)s has been discovered. Based on biochemical test, the bacterium was partially identified as Bacillus sp. strain Neni-8. Mo-blue production required an optimal pH of between 6.3 and 6.5, and between 30 and 37 oC. The carbon source, D-glucose best supported molybdenum reduction. A narrow requirement for phosphate of between 2.5 and 7.5 mM for molybdenum reduction was seen. Sodium molybdate as a substrate for reduction showed maximal reduction between 20 and 30 mM. The molybdenum blue absorption spectrum indicates that its identity was possibly a reduced phosphomolybdate. Several heavy metals such as silver, mercury, copper and chromium inhibited molybdenum reduction by 67.6, 48.7, 36.8 and 17.4 %, respectively. Bacterial growth modelled using the modified Gompertz model with PEG 600 as the best carbon source predicted a maximum growth rate of 15.4 Ln CFU/ml, a maximum specific growth rate of 0.198 h-1 and a lag period of 10.1 h. The novel characteristics of this bacterium are very useful in future bioremediation works