**Nomenclature**

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

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

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 huge demand for acrylamide as an ubiquitous monomer for industry led to its environ‐ mental presence, however the International Agency for Research on Cancer has classified this compound as a probable human carcinogen. Bioremediation seems to be the only efficient and environmentally friendly process to decompose this monomer. The first step in developing acrylamide bioremediation is to choose high potent microorganisms. Choice of microorgan‐ isms is challenging owing to the large scale degradation of acrylamide and elucidation of the intermediate in catabolic pathways is the first important step. Nevertheless, the main problem is the rapid conversion of intermediate acrylic acid to other metabolites. Research on the relationship between degradation mechanisms and membrane structure of acrylamideutilizing bacteria awaits further characterization. It is noteworthy that successful remediation

a major effect on the practical applications of acrylamide bioremediation.

to improve their efficiency and specificity.

110 Applied Bioremediation - Active and Passive Approaches

**6. Concluding remarks**

mutagenesis could be applied to achieve such ends.

Amino acids E: Glutamic acid K: Lysine C: Cysteine D: Aspartic acid N: Asparagine S: Serine A: Alanine
