**4. Metabolism of acrylamide**

**Microorganisms Source Conditions Reference**

*Pseudomonas chlororaphis* B23 Soil Aerobic (Enzymatic degradation) [46] *Arthrobacter* sp. J-1 Soil Aerobic (Enzymatic degradation) [56] *Rhodococcus rhodochrous* J1 Soil Aerobic (Free cells) [47] *Pseudomonas* sp. Soil Aerobic (Free cells) [48]

*Rhodococcus* sp. Soil Aerobic (Enzymatic degradation) [50] *Rhodococcuserythropolis* MP50 Soil Aerobic (Enzymatic degradation) [64] *Rhodococcus* sp. Soil Aerobic (Immobilized cells) [51]

*Rhodopseudomonas palustris* Bovine slaughterhouse Photoheterotropic (Free cells) [57]

*Bacillus cereus* DRY135 Soil Aerobic (Free cells) [53] *Pseudomonas* sp. DRYJ7 Antarctic soil Aerobic (Free cells) [54]

*Ralstonia eutropha* AUM-01 Soil Aerobic (Free cells) [59]

*Pseudomonas aeruginosa* DS-4 Soil Aerobic (Free cells) [55]

food and beverage industries

*Pseudomonas aeruginosa* Soil Aerobic (Free and immobilized

Soil Aerobic (Immobilized cells) [49]

cells)

cells)

Domestic wastewater Aerobic (Free cells) [61]

Soil Aerobic (Free cells) [62]

Aerobic (Free cells) [52]

Anaerobic (Free cells) [58]

Aerobic and anaerobic (Free cells) [69]

Aerobic (Free cells) [64]

[3]

[60]

**Bacteria**

*Pseudomonas* sp. *Xanthomonas maltophilia*

*Kluyvera georgiana Klebsiella pneumoniae Enterococcus faecalis*

AUT-01

**Fungi**

*Geobacillus thermoglucosidasius*

*Pseudomonas stutzeri* Wastewater treatment

104 Applied Bioremediation - Active and Passive Approaches

*Ralstonia eutropha* TDM-3 Wastewater treatment

Natural microbial populations Rocky Ford Highline Canal,

*Aspergillus oryzae* KBN 1010 Filamentous fungi used in

**Table 1.** Acrylamide-degrading microorganisms.

system

system

Colorado USA

*Enterobacter aerogenes* Domestic wastewater Aerobic (Free and immobilized

Until now, we can not deny possible routes for acrylamide other than deamination via amidase [50, 59, 62, 64, 67]. The subsequent fate of acrylate is not well understood but probably involves pathways and enzymes that have been characterized to various degrees for other acrylate utilizing bacteria (Figure 1). Acrylate metabolism is believed to proceed via hydroxylation to β-hydroxypropionate, then oxidized to CO2 [48] or reduced to propionate [57]. Another plausible pathway for mineralization of acrylamide is via formation of acrylyl CoA which eliminates lactate as a final product [48].

A powerful tool that also enables unraveling acrylamide metabolic pathways is the sequential induction of catabolic enzymes and intermediatary metabolites. Further, insight into degra‐ dative pathways is also provided from assaying the probable key proteins that are synthesized at sufficient levels when acrylamide is present. Using proteome analysis, fifteen proteins differentially expressed from *Enterobacter aerogenes* grown on acrylamide were identified. Six protein homologues with amidohydrolase, urease accessory protein, quaternary ammonium compound resistance proteins, dipeptide transport protein, Omp36 osmoporin and large conductance mechanosensitive channel proteins (MscL) are seemingly involved in acrylamide stress response and its degradation. Five proteins identified as GroEL-like chaperonin, ArsRtranscriptional regulator, Ts- and Tu-elongation factor and trigger factor and four proteins (phosphoglycerate kinase, ATP synthase β-subunit, malate dehydrogenase and succinyl-CoA synthetase α-subunit) are expected to be relevant to adaption of *E. aerogenes*in the presence of acrylamide [70]. Based on the results, Charoenpanich and Tani have proposed acrylamide may be assimilated using Omp36 osmoporin and dipeptide transport proteins. Acrylamide is toxic, indeed lethal, to most microorganisms, however some bacteria have adapted their metabolism to use this substance as an energy source. Important to this adaptation is the evolution of genes that encode amidohydrolase (amidase) and other synthesis proteins that deaminate acryla‐ mide to acrylic acid and ammonium [48, 50-51, 60-61]. With this, acrylic acid can be changed to propionate and subsequently succinyl CoA [57, 71-72] to generate energy. Potentially harmful ammonium is detoxified and with MscL protein and released from the cell [70].

energy requirements and, therefore, low costs. Major limitations are the bioavailability of the organic matter and the finding of efficient biodegraders. Physico-chemical environmental conditions also greatly influence the rate and extent of degradation. In general, degradation efficiency is dependent on three overall factors (i) microorganisms that can degrade the specific chemical structure (ii) environmental conditions that allow the microorganisms to grow and express their degradation enzymes and (iii) good physical contact between the organic

Removal of Acrylamide by Microorganisms http://dx.doi.org/10.5772/56150 107

Rapid degradation of acrylamide coupled with growth requires not only amidase or micro‐ organism producing amidase, but also a whole pathway, i.e. a set of enzymes that are differ‐ entially synthesized in the presence of acrylamide. Although a complete catabolic pathway for acrylamide does not exist, recombination and mutation processes and exchange of genetic information between microorganisms may lead to the development of organisms with improved catabolic activities. Alternatively, microorganisms can cooperate by combining their catabolic potential in mixed cultures and in this way may completely mineralize acrylamide. Wang and Lee elucidated the effectiveness of *Ralstonia eutropha* TDM-3 and mixed cultures of wastewater from the manufacture of polyacrylonitrile fiber in treating acrylamide in synthetic wastewater. They found that mixed culture and *R. eutropha* TDM-3 can jointly consume acrylamide up to concentrations of 1446 mg/l and completely remove acrylamide with a sufficient supply of nitrate as electron acceptors [58]. A similar result has been found in *E. aerogenes*. If grown with mixed cultures from a municipal wastewater treatment plant, they can completely and rapidly convert acrylamide to acrylic acid [68]. Acrylamide up to 100 mg/ L can efficiently be removed from amended canal water and sediment slurries under aerobic conditions. Using natural nitrate-reducing microorganisms in a canal environment, potential

Microorganisms typically require sufficient water, inorganic nutrients, carbon sources, and trace elements for maintenance and growth. Besides growth substrates, other specific organic compounds such as vitamins or other growth factors are essential for some microorganisms. Monosaccharides like glucose and fructose have been reported as support elements for the growth and degradation potential of acrylamide-degrading bacteria [53-54]. However, in some cases supplementation of acrylamide containing growth medium with glucose or succinate as additional carbon source demonstrated a severe repression in degrading ability [48, 71-75]. Addition of glutamate or ammonium sulfate as an additional nitrogen source to the growth medium demonstrated an increase in degradation potential compared to the cells grown only on acrylamide [48]. One interesting study found that *Pseudomonas aeruginosa* DS-4 isolated from lipid wastewater required salad oil for growth and acrylamide degradation [55].

Toxic compounds (e.g. heavy metals) should not be present at high concentrations, since they can inactivate essential enzymes. As explained in [51] iron (<10 mM) enhanced the rates of acrylamide degradation of *Rhodococcus* sp. but copper, cobalt and nickel inhibited the degra‐ dation. Mercury and chromium inhibited acrylamide degradation by *Pseudomonas aeruginosa* while nickel at lower concentrations (200 and 400 ppm) improved the degrading ability [3]. Optimum conditions for acrylamide biodegradation are achieved if pH and temperature are in the range of pH 6-8 and mesophilic temperature (15-30ºC), respectively [3, 45-48, 53-55].

substrate and the organism.

fate of acrylamide (70.3-85%) was found after 60 days [69].

**Figure 1.** Possible biological fates of acrylate produced from acrylamide deamidation.

#### **5. Bioremediation of acrylamide and future prospects**

Bioremediation is viewed as a sustainable process for wastewater treatment, which under appropriate conditions, can promote an efficient reduction of organic matter with minimal energy requirements and, therefore, low costs. Major limitations are the bioavailability of the organic matter and the finding of efficient biodegraders. Physico-chemical environmental conditions also greatly influence the rate and extent of degradation. In general, degradation efficiency is dependent on three overall factors (i) microorganisms that can degrade the specific chemical structure (ii) environmental conditions that allow the microorganisms to grow and express their degradation enzymes and (iii) good physical contact between the organic substrate and the organism.

mide to acrylic acid and ammonium [48, 50-51, 60-61]. With this, acrylic acid can be changed to propionate and subsequently succinyl CoA [57, 71-72] to generate energy. Potentially harmful ammonium is detoxified and with MscL protein and released from the cell [70].

(CH3CH2CH2CH-CO-S-CoA) <sup>β</sup>-Hydroxy-propionyl CoA

Bioremediation is viewed as a sustainable process for wastewater treatment, which under appropriate conditions, can promote an efficient reduction of organic matter with minimal

H2O H2O Acrylyl CoA (CH2=CH-CO-S-CoA)

Acrylic acid (CH2=CH-COOH)

+

Ammonia (NH3)

(HOCH2-CH2-CO-S-CoA)

Malonyl CoA (HOOC-CH2-CO-S-CoA)

> Acetyl CoA (CH3-CO-S-CoA)

CO2

**Figure 1.** Possible biological fates of acrylate produced from acrylamide deamidation.

**5. Bioremediation of acrylamide and future prospects**

ATP + CoASH

H2O

Acrylamide

106 Applied Bioremediation - Active and Passive Approaches

(CH2=CH-CONH2) Amidase

ADP + H2O + Pi

CO2 + H2O

Lactate (CH3CH-OH-COOH)

Lactoyl CoA

H2O -Hydroxypropionate

Propionate (CH3-CH2-COOH)

2H+

(HOCH2-CH2-COOH)


> Acetate Acetyl

Rapid degradation of acrylamide coupled with growth requires not only amidase or micro‐ organism producing amidase, but also a whole pathway, i.e. a set of enzymes that are differ‐ entially synthesized in the presence of acrylamide. Although a complete catabolic pathway for acrylamide does not exist, recombination and mutation processes and exchange of genetic information between microorganisms may lead to the development of organisms with improved catabolic activities. Alternatively, microorganisms can cooperate by combining their catabolic potential in mixed cultures and in this way may completely mineralize acrylamide. Wang and Lee elucidated the effectiveness of *Ralstonia eutropha* TDM-3 and mixed cultures of wastewater from the manufacture of polyacrylonitrile fiber in treating acrylamide in synthetic wastewater. They found that mixed culture and *R. eutropha* TDM-3 can jointly consume acrylamide up to concentrations of 1446 mg/l and completely remove acrylamide with a sufficient supply of nitrate as electron acceptors [58]. A similar result has been found in *E. aerogenes*. If grown with mixed cultures from a municipal wastewater treatment plant, they can completely and rapidly convert acrylamide to acrylic acid [68]. Acrylamide up to 100 mg/ L can efficiently be removed from amended canal water and sediment slurries under aerobic conditions. Using natural nitrate-reducing microorganisms in a canal environment, potential fate of acrylamide (70.3-85%) was found after 60 days [69].

Microorganisms typically require sufficient water, inorganic nutrients, carbon sources, and trace elements for maintenance and growth. Besides growth substrates, other specific organic compounds such as vitamins or other growth factors are essential for some microorganisms. Monosaccharides like glucose and fructose have been reported as support elements for the growth and degradation potential of acrylamide-degrading bacteria [53-54]. However, in some cases supplementation of acrylamide containing growth medium with glucose or succinate as additional carbon source demonstrated a severe repression in degrading ability [48, 71-75]. Addition of glutamate or ammonium sulfate as an additional nitrogen source to the growth medium demonstrated an increase in degradation potential compared to the cells grown only on acrylamide [48]. One interesting study found that *Pseudomonas aeruginosa* DS-4 isolated from lipid wastewater required salad oil for growth and acrylamide degradation [55].

Toxic compounds (e.g. heavy metals) should not be present at high concentrations, since they can inactivate essential enzymes. As explained in [51] iron (<10 mM) enhanced the rates of acrylamide degradation of *Rhodococcus* sp. but copper, cobalt and nickel inhibited the degra‐ dation. Mercury and chromium inhibited acrylamide degradation by *Pseudomonas aeruginosa* while nickel at lower concentrations (200 and 400 ppm) improved the degrading ability [3].

Optimum conditions for acrylamide biodegradation are achieved if pH and temperature are in the range of pH 6-8 and mesophilic temperature (15-30ºC), respectively [3, 45-48, 53-55]. Most microorganisms consume considerably less energy for the maintenance of basic functions under neutral conditions. This means that more energy is available for growth. It has been known that metabolic activity of tropical soils typically is high and fosters several processes such as carbohydrate fermentation and carbon dioxide production leading to the lowering of pH. Thus, for successful bioremediation of pollutants including acrylamide pH control may be essential. Addition of an inexpensive chemical such as calcium carbonate to neutralize soil pH during bioremediation can optimize remediation [76].

these enzymes involving the reduction of organic nitrogen compounds and ammonia pro‐ duction exhibited several conserved motifs. One of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis. Sequence conservation over the entire length of these enzymes, as well as the similarity in the reactions constitutes a definite family which points to a common catalytic mechanism [88]. Chemical mutagenesis and X-ray crystallography have been analyzed for three-dimensional structures of amidases. Only a few crystal structures of nitrilase-related amidases have been reported with *Pseudomonas aerugi‐ nosa* amidase the first [89-90]. The three dimensional-structures showed a conserved α-β-β-α sandwich fold resembling the conserved structural fold of the nitrilase superfamily structures. Analysis of the three dimension-structures identified E59, K134, and C166 as a catalytic triad [89]. Similar catalytic triad residues were also reported in the three dimensional structural models of amidase from *Rhodococcus erythropolis*, *Helicobacter pylori*, and *Bacillus stearothermo‐ philus* [89] and also in the amidase of novel acrylamide-degrading *Enterobacter aerogenes* [91]. The crystal structure of *Xanthomonas campestris* XC1258 amidase showed a monomeric structure of globular α/β protein comprising mainly six α helices and two six-stranded β-sheet (Figure 2). This is the typical nitrilase-superfamily α-β-β-α fold. The hexamer preserving the eight-layered α-β-β-αα-β-β-α structure in holoenzyme across an interface has also been reported [92]. The analysis of small asymmetric catalytic site of the *Geobacilus pallidus* RAPc8 amidase suggested that access of a water molecule to the catalytic triad (C, E, K) side chains would be impeded by the formation of the acyl intermediate. The conserved E142 in the catalytic site acts as a general base to catalyze the hydrolysis of this intermediate [93]. This confirmed the conservation of the E, K, C catalytic triad across the nitrilase superfamily members and also supported the classification of the amidases in the nitrilase superfamily.

Removal of Acrylamide by Microorganisms http://dx.doi.org/10.5772/56150 109

**Figure 2.** (a) The monomeric tertiary structure of amidase from *Xanthomonas campestris* XC1258, color-coded from blue (N-terminal) to red (C-terminal), and (b) the primary sequence of XC1258 amidase. Reprinted from Ref. [92].

Studies on acrylamide biodegradation are mainly concerned with the isolation and identifi‐ cation of suitable microbial strains. Most studies use either free or immobilized cells for acrylamide removal. Of these, immobilized cells are advantageous because the immobilized cells are less likely than free cells to be adversely affected by predators, toxin, or parasites [77-78]. Additionally, they can be reused, saving resources and time. However, the imple‐ mentation of immobilized cells may be sensitive to pH, temperature and acrylamide concen‐ tration. Moreover, large accumulations of the metabolic intermediate, acrylic acid, may affect some microbial activity [3, 51, 60]. Hence, the attempt to biotransform acrylamide with amidase or nitrile-converting enzymes via hydrolysis.

Microbial degradation of nitriles proceeds through two enzymatic pathways. Nitrilase (EC 3.5.5.1) catalyzes the direct cleavage of nitriles to the corresponding acids and ammonia, and nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides. Both nitrile-converting enzymes have increasingly attracted attention as catalysts for processing many organic chemicals [79-81]. Nitrile hydratase is commonly used as the catalyst in the production of acrylamide and is known as one of the most important industrial enzymes [82-83]. Generally, the gene operon of nitrile hydratase consists of the genes for the alpha and beta subunits of NHase, the NHase activator and amidase. The amides produced by NHase are degraded to their corresponding free carboxylic acids and ammonia by the action of amidases [84]. Thus, nitrile-converting enzymes are of broad use as alternatives for acrylamide biotransformation.

Acrylic acid, the intermediate product in acrylamide catabolism, is a commodity chemical with an estimated annual production capacity of 4.2 million metric tons [85]. Acrylic acid and its esters can be used in paints, coatings, polymeric flocculants, paper and so on. It is conven‐ tionally produced from petrochemicals. Currently, most commercial acrylic acid is produced by partial oxidation of propene which produces undesirable by-products and large amount of inorganic wastes [86]. Currently, there is an innovative manufacturing method using nitrileamide converting enzymes. For acrylamide degraders, it is initially degraded to ammonia and acrylic acid (acrylate), a process catalyzed by amidase. Then acrylate is reduced to generate energy for growth. Until now, the acrylate-utilizing enzyme has not been well characterized but believed to be acrylate reductase [48, 57]. The identification of the gene encoding this enzyme remains a challenge. Moreover, from an economic aspect, the acrylate reductasedeficient strains created by a gene-disruption method, lead to acrylic acid accumulation in wastewater and are recommended for acrylamide bioremediation in the future.

Sequence similarities have been identified using computer methods for database searches and multiple alignment, between several nitrilases, cyanide hydratase, β-alanine synthase and the first type of aliphatic amidases which hydrolyze only short-chain aliphatic amides [87]. All these enzymes involving the reduction of organic nitrogen compounds and ammonia pro‐ duction exhibited several conserved motifs. One of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis. Sequence conservation over the entire length of these enzymes, as well as the similarity in the reactions constitutes a definite family which points to a common catalytic mechanism [88]. Chemical mutagenesis and X-ray crystallography have been analyzed for three-dimensional structures of amidases. Only a few crystal structures of nitrilase-related amidases have been reported with *Pseudomonas aerugi‐ nosa* amidase the first [89-90]. The three dimensional-structures showed a conserved α-β-β-α sandwich fold resembling the conserved structural fold of the nitrilase superfamily structures. Analysis of the three dimension-structures identified E59, K134, and C166 as a catalytic triad [89]. Similar catalytic triad residues were also reported in the three dimensional structural models of amidase from *Rhodococcus erythropolis*, *Helicobacter pylori*, and *Bacillus stearothermo‐ philus* [89] and also in the amidase of novel acrylamide-degrading *Enterobacter aerogenes* [91]. The crystal structure of *Xanthomonas campestris* XC1258 amidase showed a monomeric structure of globular α/β protein comprising mainly six α helices and two six-stranded β-sheet (Figure 2). This is the typical nitrilase-superfamily α-β-β-α fold. The hexamer preserving the eight-layered α-β-β-αα-β-β-α structure in holoenzyme across an interface has also been reported [92]. The analysis of small asymmetric catalytic site of the *Geobacilus pallidus* RAPc8 amidase suggested that access of a water molecule to the catalytic triad (C, E, K) side chains would be impeded by the formation of the acyl intermediate. The conserved E142 in the catalytic site acts as a general base to catalyze the hydrolysis of this intermediate [93]. This confirmed the conservation of the E, K, C catalytic triad across the nitrilase superfamily members and also supported the classification of the amidases in the nitrilase superfamily.

Most microorganisms consume considerably less energy for the maintenance of basic functions under neutral conditions. This means that more energy is available for growth. It has been known that metabolic activity of tropical soils typically is high and fosters several processes such as carbohydrate fermentation and carbon dioxide production leading to the lowering of pH. Thus, for successful bioremediation of pollutants including acrylamide pH control may be essential. Addition of an inexpensive chemical such as calcium carbonate to neutralize soil

Studies on acrylamide biodegradation are mainly concerned with the isolation and identifi‐ cation of suitable microbial strains. Most studies use either free or immobilized cells for acrylamide removal. Of these, immobilized cells are advantageous because the immobilized cells are less likely than free cells to be adversely affected by predators, toxin, or parasites [77-78]. Additionally, they can be reused, saving resources and time. However, the imple‐ mentation of immobilized cells may be sensitive to pH, temperature and acrylamide concen‐ tration. Moreover, large accumulations of the metabolic intermediate, acrylic acid, may affect some microbial activity [3, 51, 60]. Hence, the attempt to biotransform acrylamide with amidase

Microbial degradation of nitriles proceeds through two enzymatic pathways. Nitrilase (EC 3.5.5.1) catalyzes the direct cleavage of nitriles to the corresponding acids and ammonia, and nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides. Both nitrile-converting enzymes have increasingly attracted attention as catalysts for processing many organic chemicals [79-81]. Nitrile hydratase is commonly used as the catalyst in the production of acrylamide and is known as one of the most important industrial enzymes [82-83]. Generally, the gene operon of nitrile hydratase consists of the genes for the alpha and beta subunits of NHase, the NHase activator and amidase. The amides produced by NHase are degraded to their corresponding free carboxylic acids and ammonia by the action of amidases [84]. Thus, nitrile-converting enzymes are of broad use as alternatives for acrylamide biotransformation. Acrylic acid, the intermediate product in acrylamide catabolism, is a commodity chemical with an estimated annual production capacity of 4.2 million metric tons [85]. Acrylic acid and its esters can be used in paints, coatings, polymeric flocculants, paper and so on. It is conven‐ tionally produced from petrochemicals. Currently, most commercial acrylic acid is produced by partial oxidation of propene which produces undesirable by-products and large amount of inorganic wastes [86]. Currently, there is an innovative manufacturing method using nitrileamide converting enzymes. For acrylamide degraders, it is initially degraded to ammonia and acrylic acid (acrylate), a process catalyzed by amidase. Then acrylate is reduced to generate energy for growth. Until now, the acrylate-utilizing enzyme has not been well characterized but believed to be acrylate reductase [48, 57]. The identification of the gene encoding this enzyme remains a challenge. Moreover, from an economic aspect, the acrylate reductasedeficient strains created by a gene-disruption method, lead to acrylic acid accumulation in

wastewater and are recommended for acrylamide bioremediation in the future.

Sequence similarities have been identified using computer methods for database searches and multiple alignment, between several nitrilases, cyanide hydratase, β-alanine synthase and the first type of aliphatic amidases which hydrolyze only short-chain aliphatic amides [87]. All

pH during bioremediation can optimize remediation [76].

or nitrile-converting enzymes via hydrolysis.

108 Applied Bioremediation - Active and Passive Approaches

**Figure 2.** (a) The monomeric tertiary structure of amidase from *Xanthomonas campestris* XC1258, color-coded from blue (N-terminal) to red (C-terminal), and (b) the primary sequence of XC1258 amidase. Reprinted from Ref. [92].

Acrylamide amidases have similar sequences with nitrilases and seem to have descended from a common ancestry along with members of the sulfhydryl enzyme family. In these amidases an invariant cysteine residue was reported to act as the nucleophile in the catalytic mechanism and is confirmed by the three dimensional structural model of the amidase of *Pseudomonas aeruginosa*. This was built by comparative modeling using the crystal structure of the worm nitrilase fusion protein, NitFhit as the template. The putative catalytic triad C-E-K is conserved in all members of the nitrilase superfamily [89]. The signature amidases possesses two real active site residues D191 and S195 among the various conserved residues within the signature sequence common to all enantioselective amidases. D191N and S195A substitutions in *Rhodococcus* amidase has been shown to completely suppress amidase activity [94-95]. These sequences are also present within the active site sequences of aspartic proteinases. Thus, amide bond cleaving enantioselective amidases that are coupled with nitrile hydratases are evolu‐ tionary related to aspartic proteinases. Further structural characterization of the amidase produced by acrylamide-degrading bacteria should reveal what other differences are present. It may be possible to use this information to aid protein engineering of the enzymes in order to improve their efficiency and specificity.

of acrylamide depends on the ability of microbes to adapt to new environmental conditions and the availability of active and stable chemical degrading bacteria. Indigenous predators, parasites and toxicants are known to severely restrict biodegradation and should be a concern.

Removal of Acrylamide by Microorganisms http://dx.doi.org/10.5772/56150 111

The author is grateful to Dr. N. Kurukitkoson for his encouragement to write this review and

Department of Biochemistry, Faculty of Science, Burapha University, Bangsaen, Chonburi,

[1] IARCSome Chemicals Used in Plastics and Elastomers. In: IARC Monographs on the evaluation of carcinogenic risk of chemicals to human. France: International Agency

would like to thank F.W.H. Beamish for proofreading the manuscript.

**Nomenclature**

Amino acids

K: Lysine C: Cysteine

S: Serine A: Alanine

E: Glutamic acid

D: Aspartic acid N: Asparagine

**Acknowledgements**

**Author details**

Thailand

**References**

Jittima Charoenpanich\*

Address all correspondence to: jittima@buu.ac.th

for Research on Cancer; (1986). , 403.

Development of thermostable amidase is also important. Based on the three-dimensional structure of amidase, additional disulfide bridges can be engineered by site-directed muta‐ genesis for enzyme stabilization. Novel amidases that show broad substrate specificity may be developed to biodegrade the toxic environmental pollutants, acrylamide and amides. Random approaches such as directed evolution, reverse engineering and site-directed mutagenesis could be applied to achieve such ends.

Our understanding of the biochemistry and molecular biology of amidase is advancing rapidly and already providing information that is of use today. Moreover, recent developments in amidase studies have broadened the scope of potential applications of the enzyme in acryla‐ mide bioremediation as well as that of acrylic acid production. I predict that these develop‐ ments combined with progress in genetic engineering and enzyme crystallography will have a major effect on the practical applications of acrylamide bioremediation.
