Title: Isolation and Characterization Of An Acrylamide-Degrading Bacillus cereus
Author: yunus
Last saved by: Kimia
Company: upm

Vol. 2, No. 1, September 2008		J. Ris. Kim.

J. Ris. Kim.		Vol. 2, No. 1, September 2008

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

Neni Gusmanizar^1, Yunus Shukor^2, Johari Ramli^2 and Mohd. Arif Syed^2
1)	Dept. of Feed and Animal Nutrition, Faculty of Animal Husbandry,
Andalas University, Padang, Indonesia
^2) Dept. of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
email : gusmanizar@gmail.com

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, Bacterium

INTRODUCTION

Acrylamide is the building block for the polymer, polyacrylamide. It is known that commercial polyacrylamide preparations may be contaminated with its toxic monomer, acrylamide. Thus, regulations have been set on the amount of acrylamide that is present in polyacrylamide. For example, a limit of 500 ppm in polyacrylamide preparations is used in agriculture or water treatment. Other uses of acrylamide include as sewage-flocculating agent^[1], stabilising tunnels and dams, and in the industry as adhesives^[2]. Thus it is not surprising that one of the known cases of acrylamide toxicity is due to the application of acrylamide for the stabilizing of tunnel in Sweden. Acrylamide pumped to surrounding soil had resulted in acrylamide polluting the Hallandas village in Sweden causing death to cows and fish^[3,4]. Other source of high level of pollution originates from acrylonitrile-acrylamide producing industry reaching a level as high as 1 g L^-1 ^[5]. In Malaysia, large amount of polyacrylamide are used yearly for drinking water treatment. For example, in the Sarawak state alone, almost 800 kgs of polyacrylamide additive (Superfloc and Praestol 2530) are used yearly by the Kuching Water Board^[6]. A direct introduction of acrylamide into agriculture soil comes from glyphosate application in agriculture areas, where 20-30% polyacrylamide is added as dispersing agent in the glyphosate formulation^[7]. It is not known whether vegetables absorbs acrylamide from soil despite the scare of reports of high levels of acrylamide in food although it is known that occurrence of acrylamide in food is due to Maillard’s reaction^[8]. It is known that all polyacrylamide from any sources will degrade to acrylamide under environmental conditions with the half life of acrylamide ranging from weeks to months in rivers^[9]. 

Since acrylamide is a well known neurotoxicant, carcinogen and teratogen^[10] its presence in the environment poses a hazard and its bioremediation must be sought. Many investigators have isolated bacteria from the environment capable of utilizing acrylamide as the sole carbon and/or nitrogen source.  Bacteria such as Rhodococcus sp., Arthrobacter^[11], Pseudomonas stutzeri^[12] have been isolated for the biodegradation of acrylamide. Currently, there is no study being carried out on microbial acrylamide degradation in Malaysia. Here we report on the isolation and characterization of a locally-isolated acrylamide-degrading bacterium.

MATERIALS AND METHODS

Isolation of Acrylamide-Degrading Bacterium and Phenotype Identification 

Soil samples were collected from several agricultural locations in Malaysia with a history of polyacrylamide application in the form of herbicide glyphosate commercial formulation ROUNDUP^® which is known to contain between 25 to 30% polyacrylamide solutions to reduce spray drift and to act as a surfactant^[7]. Soil samples were collected 5-15 cm from the surface and were placed in sterilized plastic bags and stored on ice during   transfer from site to the laboratory. Bacteria were isolated from soil samples by enrichment culture using a basal medium containing 100 mg L^-1 acrylamide as the sole nitrogen source.  Bacteria were isolated from soil samples by enrichment culture using basal medium containing 100 mg L^-1 acrylamide as the sole nitrogen source.  The growth and maintenance of the isolates were carried out according to the modified medium of Ciskanik^[13]. Briefly the medium contained (per liter) 8.6 g of KH_2PO_4, 0.01 g of yeast extract, 10 g of glucose, 0.5 g of MgSO_4.7H_2O, 0.05 g of FeSO_4.H_2O, 100 mg L^-1 acrylamide and 1 mL of the following trace elements; ZnSO_4·7H_2O, 0.34 μM; MnCl_2·4H_2O, 0.15 μM; H_3BO_3, 4.85 μM; CoCl_2·6H_2O, 0.84 μM; CuCl_2·2H_2O, 0.05 μM; NiCl_2·6H_2O, 0.08 μM; Na_2MoO_4·2H_2O, 0.123 μM. Subcultures were prepared by incubating the bacterium on a rotary shaker (200 rpm for 48 h at 25°C in 50-mL conical flasks containing the medium described. Subcultures were prepared by incubating organisms for four days at 10°C in universal bottle containing 10 mL of medium described above. Isolates were maintained and grown in basal medium containing 100 mg L^-1 acrylamide. Subcultures were inoculated into 150 mL volumes of the same medium and incubated with rotary shaking (150 rpm) for four days at room temperature. During optimization studies, cellular numbers measured as CFU/mL on day 2 was subtracted from cellular numbers at the start of incubation. Identification at species level was performed by using Biolog GN MicroPlate (Biolog, Hayward, CA, USA) according to the manufacturer's instructions and molecular phylogenetics studies.

Monitoring of Acrylamide Degradation Using HPLC Method

Acrylamide degradation was monitored on an HPLC system (Agilent, 1100 series) consisted of a pump, a manual-injection system, an online degasser and a diode array detector.  A pre-filtered (0.45µm polytetrafluoroethylene (PTFE) 20 µL sample was injected into Rheodyne^TM sample injector and separation was performed on a column (Microsorb MV100-5 C_18, 4.6 x 250 mm, particle size 5 µm, Alltech Associates, Deerfield, Illinois, USA). Sample was eluted with filtered ultra pure water at a flow rate of 1 mL/min. Detection was performed by monitoring the absorbance at λ = 196 nm using a reference wavelength of 360 nm^[14].  

Bacteria capable of utilizing acrylamide as a sole source of nitrogen (best degrader) were isolated by enrichment in basal salts medium containing 100 mg L^-1 acrylamide as the nitrogen source.  Acrylamide degradation was quantified by observing the decrease of the acrylamide amount during the experimental period. Degradation was determined by comparing the amount of acrylamide recovered from bacterial growth. Periodically, several millilitre aliquots from the acrylamide-degrading culture media was removed and divided into two portions. A portion is filtered using 0.45µm polytetrafluoroethylene (PTFE) syringe filter to separate cells from the supernatant. The separated supernatant samples were analyzed to determine residual acrylamide level.  At the same time, suitable serial dilutions of the other portion were carried out for bacterial plate count. 

16s rDNA Gene Sequencing 

Genomic DNA was extracted from bacterial colonies by alkaline lysis. PCR amplification was performed using a thermal cycler (Biometra, Gottingen, Germany). The PCR mixture contained 0.5 pM of each primer, 200 µM of each deoxynucleotide triphosphate,10x reaction buffer, 2.5 U of Taq DNA polymerase (Promega) to achieve a final volume of 50 µL. The 16s rDNA gene from the genomic DNA was amplified by PCR using the following primers; 5’-AGAGTTTGATCCTGGCTCAG-3’ and 5’-AAGGAGGTGATCCAGCCGCA-3’corresponding to the forward and reverse primers of 16s rDNA respectively^[15]. PCR was performed under the following conditions: initial denaturation at 94°C for 3 min; 25 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min; and a final extension at 72°C for 10 min. Cycle sequencing was subsequently performed with the Big Dye terminator kit (Perkin-Elmer Applied Biosystems) as recommended by the manufacturer. Sequence data were initially recorded and edited using CHROMAS Version 1.45. The resultant 711 bases were compared with the Gen Bank database using the Blast server at NCBI. This analysis showed this sequence to be closely related to rrs from Bacillaceae. The 16s rRNA ribosomal gene sequence for this isolate have been deposited in Gen Bank under the following accession number DQ 851857.

Phylogenetic Analysis

A multiple alignment of 20 16S rRNA gene sequences closely matches Strain Dr.Y27 were retrieved from Gen Bank and were aligned using clustal_W^[16] with the PHYLIP output option. The alignment was checked by eye for any obvious mis-alignments. Alignment positions with gaps were excluded from the calculations. A phylogenetic tree was constructed by using PHYLIP, version 3.573 (J. Q. Felsenstein, PHYLIP—phylogeny inference package, version 3.573, Department of Genetics, University of Washington, Seattle, W. A. with Lactobacillus casei as the  out group in the cladogram. Evolutionary distance matrices for the neighbour-joining/UPGMA method were computed using the DNADIST algorithm program. The program reads in nucleotide sequences and writes an output file containing the distance matrix. The model of nucleotide substitution is from Jukes and Cantor^[17]. Phylogenetic tree was inferred by using the neighbour-joining method of Saitou and Nei^[18]. With each algorithm, confidence levels for individual branches within the tree were checked by repeating the PHYLIP analysis with 1000 bootstraps^[19] by the SEQBOOT program in the PHYLIP package. Majority rule (50%) consensus trees were constructed for the topologies found using a family of consensus tree methods called the Ml methods^[20] using the CONSENSE program and the tree was viewed using TreeView^[21]. 

RESULTS AND DISCUSSION 

Isolation of Acrylamide-Degrading Bacteria

Five isolates were obtained based on their ability to grow on acrylamide as the sole nitrogen source with CFU/mL as the measurement goal. They were isolate 5, 12, 20, 27, 88, 97 and 135. All isolates show optimum growth on day 4 of aerobic incubation with isolate 27 giving the highest growth (Figure 1). On this basis, isolate 27 was selected for further study.

A strong bootstrap values (100%) links isolate 27 to Burkholderia sp. strain DR.Y27 indicating strong phylogenetic relationship (Figure 2). Together with the Biolog identification system which gave the closest ID to Burkholderia cepacia isolate 27 was assigned tentatively as Burkholderia sp. strain DR.Y27. The major acrylamide or aliphatic amides-degrading bacteria is the genus Pseudomonas. Strains of Pseudomonas known to degrade acrylamide or aliphatic amide are Pseudomonas aeruginosa^[22], Pseudomonas acidovorans^[23] Pseudomonas putida^[24], Pseudomonas chlororaphis^[13], Pseudomonas azotoformans^[25] and Pseudomonas sp.^[26,27]. Pseudomonas genera remained as one of the important acrylamide-degrading group. Other acrylamide- or aliphatic amide-degrading bacteria reported in the literature include Burkholderia cepacia strain C^[28], Brevibacterium sp. R312^[8], Methylophilus methylotrophus^[8], and Helicobacter pylori^[29] Pseudonocardia thermophilia^[30], Rhodococcus rodochrous^[31], Rhodococcus sp.^[32], Thermococcus hydrothermalis^[33], Brevibacillus borstelensis BCS-1^[34] and Klebsiella pneumoniae NCTR 1^[35].

Effects of Initial pH 

The effect of initial pH on the growth of this strain was studied at room temperature using an overlapping buffer system. The measurement of growth was carried out after 48 hours of incubation.  The result of this study is shown in Figure 3. This strain grows optimally in the range of pH from 6.0 to 8.0 with an optimum between pH 7.5 and pH 8.0. Growth declines dramatically outside of this range. Phosphate is the optimum buffer for buffering medium during growth whilst carbonate and borate gave lower growth at the same concentration.  The result obtained in this work is in agreement with several previous works. Egorova et al.,^[30] reported an optimum pH for the growth of Pseudonocardia thermophilia at 7.2.  Nawaz et al.,^[32] and Komeda et al.,^[27] reported an optimum pH of 7.0 for Rhodococcus sp. and Pseudomonas sp. MCI3434 respectively. A slight departure from neutrality as the requirement of optimum growth was reported by Postec et al.^[33]. In their work they reported that the hyperthermophilic archaeon Thermococcus hydrothermalis requires pH 6. 

Effect of Temperature 

The effect of incubation temperature on the growth of acrylamide-degrading bacteria shows an optimum growth at 30°C (Fig. 4). A dramatic drop in growth is seen at temperatures lower or higher than 30°C. Most of the commonly studied acrylamide/aliphatic amides-degrading bacteria are mesophiles with optimum temperature for growth in the range of 25 to 40°C.  Ciskanik et al.^[13]reported an optimum temperature for the growth of Pseudomonas chlororaphis at 26°C, whereas Kotlova et al.^[31], Nawaz et al.^[36] and Wang and Lee^[12] reported 30°C as the optimum temperature for the growth of Rhodococcus rodochrous, Rhodococcus sp. and Pseudomonas stutzeri respectively. Helicobacter pylori, found in the human gut,  had an optimum temperature of 37°C^[29]. In contrast, thermoactive bacteria need higher temperature for their growth. The thermoactive Pseudonocardia thermophilia showed optimal growth at 50°C^[30] whereas Brevibacillus borstelensis BCS-1 needs 55°C^[34].

Figure 1.  Growth of several acrylamide-degrading strains isolated from soils in media supplemented with 100 mg L^-1 acrylamide as the nitrogen source and 1% glucose as the carbon source. The error bars represent mean and standard deviation for three replicates

Figure 2. Neighbour-joining method cladogram showing phylogenetic relationship between isolate DR.Y27 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.  Species names are followed by the accession numbers of their 16S rDNA sequences.  The numbers at branching points refer to bootstrap values, based on 1000 re-samplings.  The branch lengths in the cladogram are not to scale

Figure 3. Effects of pH (50 mM phosphate) on growth of Strain DR.Y27 in minimal media. The error bars represent the standard deviation for three replicates

Figure 4. Effect of temperature on Strain DR.Y27 growth (Log10 CFU/mL) in minimal media at pH7.5.  The error bars represent the standard deviation for three replicates

The Effects of Carbon Sources on Growth of Bacterium

Different carbon sources such as glucose, fructose, lactose, maltose, mannitol, citric acid and sucrose were used at an initial concentration of 1.0% (w/v) to study their effects on acrylamide degradation with acrylamide as the sole nitrogen source. All of the carbon sources supported cellular growth with glucose as the best carbon source after 48 hours of incubation (Figure 5). The optimum concentration of glucose for maximum growth of Strain DR.Y27 was found to be in the range of 0.5 to 1% (Data not shown). At higher concentrations significant reduction of growth was observed probably caused by the increase in osmotic pressure or in the reduction of water activity. Water effluxes from the cytosol into the extracellular environment may dehydrate the cell and damage the plasma membrane. The consequences are, the cell usually becomes metabolically inactive and ceases to grow^[37]. The growth rate of bacteria usually increases when carbon sources and other nutrients are added to a minimal medium. Previous study showed that Klebsiella pneumoniae NCTR 1^[35,13] demonstrated optimum growth at 1% glucose supplemented in the medium as a carbon source.  Kotlova et al.,^[31] reported a much higher concentration of glucose at 20% as optimum. In contrast, Egorova et al.,^[30] used soluble starch as the carbon source of choice for the growth of Pseudonocardia thermophilia.

Effect of Acrylamide Concentrations

This experiment was carried out to determine the optimum concentration of acrylamide for the growth of Strain DR.Y27. Different concentrations of acrylamide ranging from 100 to 5000 mg L^-1 were used in this study. Figure 6 shows bacterial growth increased when acrylamide concentrations were also increased. Highest growth was obtained at acrylamide concentrations of between 100 to 2000 mg L^-1. Since acrylamide concentrations as a pollutant in the environment can reached as high as 1000 mg L^-1 the ability of this strain to degrade high starting concentration of acrylamide is an advantage. Growth is inhibited at 2000 mg L^-1 and above. This is probably due to the inhibitory effect of acrylamide on thiol groups of proteins^[38]. A previous study reported that Pseudomonas stutzeri had an optimum growth at 440 mg L^-1 ^[12]. Figure 7 shows that Strain DR.Y27 could degrade 850 mg L^-1 of acrylamide after ten days of incubation with concomitant cell growth. Growth increase up to 2 log units at day 2 before it reaches a plateau. For the purpose of bioremediation, immobilization of bacterial cells has been reported to increase degradative capacity of microbes. Nawaz et al.,^[32] studied the comparison of the degradation of acrylamide by immobilized and free cells of Rhodococcus sp. indicate that immobilized cells of Rhodococcus sp., are faster than free cells in degrading acrylamide to acrylic acid and ammonia. The rapid transformation of acrylamide to acrylic acid and ammonia is due to the fact that immobilized cells contain a high concentration of cells at their peak catabolic activity whereas free cells take time to multiply. Our current work includes immobilization of the strain for bioremediation of acrylamide in soils. 

The Effects of Different Amides on Growth of Bacterium

Figure 8 shows the result of the effects of different amides on growth of this strain. The result shows that 2-chloroacetamide, methacrylamide, nicotinamide, acrylamide, acetamide, propionamide and urea supported growth with increasing assimilative capability from 2-chloroacetamide to urea. Skouloubris et al.,^[29] reported that it is common for amide-degrading bacteria to be able to use several short-chain aliphatic amides aside from acrylamide. Of all of these substrates, acrylamide ranks as number one in terms of pollution and toxicity^[39].

Figure 5. Effect of different carbon sources on the growth of Strain DR.Y27. The error   bars represent the mean ± standard deviation for three replicates 

Figure 6. Effect of different concentrations of acrylamide (substrate) on the growth of Strain DR.Y27.  The error bars represent the mean  standard deviation for three replicates

Figure 7. Acrylamide degradation () and cellular growth () of Strain DR.Y27 under optimized conditions. The error bars represent the mean  standard deviation for three replicates

Figure 8. Growth of Strain DR.Y27 on different aliphatic amides at 100 mg L^-1. Other parameters were held constant. The error bars represent the mean  standard deviation for three replicates

CONCLUSION

In conclusion, we have isolated and characterized an acrylamide-degrading bacterium from local soil. Our works sought to increase the repertoire of bacterial species able to degrade acrylamide. Knowledge of the various optimized parameters would facilitate an easy and more effective translation of the laboratory results to the fields. 

We have studied the effect of various parameters on acrylamide degradation from this bacterium. From the present study, all of these parameters play a crucial role for the bacterial growth and acrylamide degradation. Understanding acrylamide degradation is vital not only in the microbiological point of view, but the prospect of lowering acrylamide concentrations via bioremediation can lower the potential of acrylamide presenting itself as a pollutant and contaminant.  In our work we screen for indigenous microbes that is able to degrade acrylamide because introducing a foreign micro organism into a soil ecosystem is not only sometimes unsuccessful but could damage the ecosystem. Foreign microbes would have a difficult time adapting to a local environment since the indigenous microbes is adapted to the specific environment and will out compete any foreign microbes. A good example of the banning of the importation of foreign micro organism for the purpose of bioremediation is in the study on the bioremediation of pentachlorophenol in New Zealand. Preliminary studies by Zyzak et al.,^[8] showed that a wood-rot; P. chrysosporium could degrade pentachlorophenol both in vitro and in soil. However since it is not native to New Zealand and its release has not been permitted under the Hazardous Substances and New Organisms Act of New Zealand, they have begun screening for indigenous white rot fungi to be used in bioremediation. Malaysia is on the move to impose laws on the banning of importation of foreign microbes and GMOs for bioremediation of xenobiotics and thus we view our current local isolate as the future for the bioremediation of polluted land and aquatic bodies in Malaysia. Currently, work is underway to conduct soil bioremediation studies and to purify the amidase from this bacterium. 

ACKNOWLEDGEMENTS 

This project was supported by funds from The Ministry of Science, Technology and Innovation, Malaysia (MOSTI) under IRPA-EA grant no: 09-02-04-0758-EA001.

REFERENCES

1.	V. A. Myagchenkov and V. E. Proskurina, Kinetics of flocculation and densification in ocher suspension in the presence of polyacrylamide poly ethylene oxide and their 1: 1 mixture, Colloid journal of the Russian Academy of Sciences, 62(5): 588-593, (2000).  
2.	IPCS, Acrylamide: The International Programme on Chemical Safety, J. Environ Health Crit., 49, (2003).
3.	T. K. F. Franzen, Garshol, and N. Tomisawa, Sprayed concrete for final linings: ITA working group report, Journal of Tunnelling Underground Space Technology, 16(4): 295-309, (2001).  
4.	K. Svensson, L. Abramsson, W. Becker, A.  Glynn, K. E. Hellenäs, Y. Lind, and J.  Rosén, Dietary intake of acrylamide in Sweden, Journal of Food Chemical and Toxicology,  41(11): 1581-1586, (2003).
5.	S. M. Rogacheva, and  O. V. Ignatov, The Respiratory Activity of Rhodococcus rhodochrous M8 Cells Producing Nitrile-Hydrolyzing Enzymes, Journal of Applied Biochemistry and Microbiology, 37: 282-286, (2001).
6.	Auditor-General’s Report, Activities and special studies of Sarawak Ministerial/Department/Agency Putrajaya Malaysia, 2005. 
7.	E. A. Smith, Prues, S. L and F. W.  Oehme, Environmental degradation of polyacrylamide,  1. Effects of artificial Environmental conditions: Temperature, Light, and pH,  Ecotoxicology and Environmental Safety, 35: 121-135, (1996).
8.	D. V. Zyzak, R. A. Sanders, M. Stojanavic, D. H. Tallmadge, B. L. Eberhart, D. K. Ewald, D. C. Gruber, T. R. Morsch, M. A. Strothers, G. P. Rizzi  and M. D. Villagran, Acrylamide formation mechanism in heated foods, Journal of Agricultural and Food Chemistry, 51: 4782-4787, (2003).
9.	L. Brown, M. M. Rhead, K. C. C. Bancroft, and N. Allen, Model studies on the degradation of acrylamide monomer,  Water Research, 14:775-778, (1980).
10.	A. B. Cherry, A. J. Gabaccia, H. W.  Senn, The assimilation behavior of certain toxic organic compounds in natural water, Journal of  Sewage Ind Wastes, 28: 1137-1146, (1956).
11.	H. Yamada, Y. Asano, T. Hino, Y. Tani, Microbial utilisation of acrylonitrile,  Journal of Fermentation Technology, 57(1): 8-14, (1979).
12.	C. C. Wang and C. M. Lee,    Denitrification with acrylamide in pure culture of bacteria isolated from acrylonitrile-butadiene-styrene resin manufactured for wastewater treatment, Chemosphere, 44: 1047-1053, (2001).
13.	L. M. Ciskanik, J. M. Wilczek, R. D. Fallon, Purification and characterization of an enantioselective amidase from Pseudomonas chlororaphis B23, Applied and Environmental Microbiology, 61(3): 998-1003, (1995).
14.	M. J. Caulfield, X. Hao, G. G. Qiao, and D. H. Salomon, Degradation on polyacrylamides, Part I, Linear polyacrylamide, Polymer, 44: 1331-1337, (2003).
15.	R. Devereux, and S. S.  Wilkinson, Amplification of ribosomal RNA sequences In: Akkermans ADL van Elsas JD de Bruijn FJ eds Molecular Microbial Ecology Manual, 2^nd ed, Netherlands, Kluwer Academic Publishing, 2004, 1–17.
16.	D. Higgins, J. Thompson,  T. Gibson, J. D.  Thompson, D. G.  Higgins, T. J.  Gibson,   CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting position-specific gap penalties and weight matrix choice, Journal of Nucleic Acids Research, 22: 4673–4680, (1994).
17.	T. H. Jukes, and C. R. Cantor, Evolution of protein molecules, In H. N. Munro, ed., Mammalian Protein Metabolism, Academic Press, New York, 1969, 21-132.
18.	N. Saitou, and M. Nei, The neighbor-joining method: a new method for reconstructing phylogenetic trees,  Molecular Biology and Evolution, 4: 406.(1987).
19.	J. Felsenstein, Confidence limits on phylogenies: An approach using the bootstrap, Evolution, 39: 783-791, (1985).
20.	T. Margush,  and F. R. McMorris, Consensus n-trees, Bulletin of Mathematical Biology, 43: 239-244, (1981).
21.	R. D. M. Page, TREEVIEW: an application to display phylogenetic trees on personal computers, Journal of Computer Application in the Bioscience, 12: 357–358, (1996).
22.	J. E. Brown, and P. H. Clarke, Mutations in a regulator gene allowing Pseudomonas aeruginosa 8602 to grow on butyramide, Journal of  General Microbiology,64: 329-342, (1970).
23.	J. Alt and  K.  Krisch, Isolation of an inducible amidase from Pseudomonas acidovorans AEL J, Journal of  General  Microbiology, 87: 260-272, (1975).
24.	M. S. Nawaz, K. D. Chapatwala, J. H. Wolfram, Degradation of acetonitrile by Pseudomonas putida, Journal of  Applied and Environmental Microbiology, 55: 2267-2274, (1989).
25.	H. Komeda, H. Harada, S. Washika,  T. Sakamoto, M. Ueda,  and Y. Asano, S-Stereoselective piperazine-2- tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase, European Journal of  Biochemistry, 271(8): 1465–1475, (2004a).  
26.	K. D. Chapatwala, M. S. Nawaz, J. D. Richardson, and J. H. Wolfram, Isolation and characterization of acetonitrile utilizing bacteria, Journal of Industrial Microbiology, 5: 65-69, (1990).
27.	H. Komeda, H. Harada, S. Washika,  T. Sakamoto, M. Ueda,  and Y. Asano,  A novel R-stereoselective amidase from Pseudomonas sp. MC13434 acting on piperazine - 2 - tert - butylcarboxamide, European Journal of Biochemistry, 271(8):1580-1590, (2004b).
28.	C. Novo, R. Tata, A. Clemente, P. R.  Brown, Burkholderia genome analysis reveals new enzymes belonging to the nitrilase superfamily The amidase of Burkholderia cepacia  hospital isolate, International Journal of Biomolecular Macromolecule, 33:175–182, (2003).
29.	S. Skouloubris, A. Labigne and H. De Reuse, Identification and characterization of an aliphatic amidase in Helicobacter pylori, Journal of Molecular Microbiology, 25: 989–998, (2001).
30.	K. Egorova, H. Trauthwein, S. Verseck, and G. Antranikian, Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila, Applied Microbiology and Biotechnology, 65: 38-45, (2004).
31.	E. K. Kotlova, G. G. Chestukhina, O. B. Astaurova, T. E. Leonova, A. S. Yanenko, and V. G.  Debabov, Isolation and primary characterization of an amidase from Rhodococcus rhodochrous, Biochemistry 64(4): 384-389, (1999).  
32.	M. S. Nawaz, S. M. Billedeau and C. E.   Cerniglia, Influence of selected physical parameters on the biodegradation of acrylamide by immobilized cells of Rhodococcus sp.,  Biodegradation, 9: 381–387, (1998).  
33.	A. Postec, P. Pignet, V. Cueff-Gauchard, A. Schmitt, J. Querellou, and A. Godfroy,  Optimisation of growth conditions for continuous culture of the hyperthermophilic archaeon Thermococcus hydrothermalis and development of sulphur-free defined and minimal media, Research in Microbiology, 156: 82–87, (2005).  
34.	D. H. Baek, S. J. Kwon, S. P. Hong, M. S.  Kwak, M. H. Lee, , J. J. Song, S. G. Lee, K. H. Yoon, and M. H. Sung, Characterization of a thermostable stereospecific alanine amidase from Brevibacillus borstelensis BCS-1, Journal of Applied and Environmental Microbiology, 69:980-986, (2003).    
35.	M. S. Nawaz, A. A. Khan, D. Bhattacharayya, P. H. Siitonen, and C. E. Cerniglia, Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumonia NCTR 1, Journal of Bacteriology, 178(8): 2397-2401, (1996).
36.	M. S. Nawaz, A. A. Khan, J. E. Seng, J. E. Leakey, P. H. Siitonen, and C. E. Cerniglia, Purification and characteriazation of an acrylamide-degrading Rhodococcus sp., Applied and Environmental Microbiology, 60(9): 3343-3348, (1994).  
37.	G. Cappuccino and N. Sherman, Microbiology: A Laboratory Manual. Benjamin/Cumming Science Publishing California, 1998.
38.	J. F. Cavins and M. Friedman, Specific modification of sulfhydryl groups with P-unsaturated compounds, Journal of  Biological Chemistry, 243: 3357-3360, (1968).
39.	J. L. Kay-Shoemake, M. E. Watwood, R. D. Lentz, R. E. Sojka, Polyacrylamide as an organic nitrogen source for soil microorganisms with potential effects on inorganic soil nitrogen in agricultural soil, Soil Biology and Biochemistry, 30: 1045–1052, (1998).
1.

ISSN : 1978-628X