The development and preparation of novel enzymes for use in biological remediation or for the industrial solubilisation of acti-vated sludge remains a key challenge and a safe and economic alternative to commonly, and perhaps now redundant, physico-chemical strategies. There are perhaps two approaches: rational and evolutionary. With the former, amino acid sequences, func-tional properties and structural features of different enzymes are compared, combined, then tested to see if the desired effect is accomplished. In the evolutionary design, a large library of ran-dom mutations in proteins is made followed by a selection of enzymes that work well with a particular contaminant. In prin-cipal, multiple environmental factors would ‘select’ enzymes to meet these challenges. Molecular evolution[157,158]is a useful tool for evolving enzymes with extended substrate specificities for any recalcitrant pollutant. Furthermore, this technology is more likely to ‘succeed’ then rational approaches as the latter requires multiple sets of structural and biochemical informa-tion on every enzyme involved. Sequences encoding specific enzymes can be retrieved direct from environmental samples
312 C.G. Whiteley, D.-J. Lee / Enzyme and Microbial Technology 38 (2006) 291–316
thereby circumventing the process of isolating and screening wild-type organisms. Degenerate primers can be used to amplify central segments from these genes by PCR and inserted into the original functional gene. Such an approach allows rapid exploitation of the natural sequence diversity already present in the environment for creation of novel hybrid enzymes[159].
With the advent of molecular engineering the principle of devel-oping a new “designer” enzyme and the creation of micro assem-blers or microchips with the role of the computer as a delivery vehicle cannot be to far into the future.
One major feature to consider is to generate a new novel structure for use in activated floc solubilisation. In view of the extreme conditions that the activated sludge digesters may oper-ate, the new enzyme molecules often have to be stable and active under unusual and extreme conditions of temperature, acidity, solvents, chemicals and pH. Enzyme properties, can be exploited to engineer active-site topology, to enlarge binding pockets and to alter the substrate specificity and stability. Con-sequently, the ability to modify a protein or structure to make it more stable to such conditions, or make it more resistant to self destruction, or make it target directed and functional in the presence of other toxic elements creates enormous challenges for enzymologists. Over the next 20 years, the enzyme–floc model will be exploited at a molecular level from a ratio-nal design to specific delivery of enzymes to the active areas disguised in vectors called nanoparticles. These will be the tools and scientific technological platforms for the investigation and transformations of any activated wastewater or biological system.
Under these pretexts biological remediations can only be rationalised by specific finite measurements, for each floc enzyme in the study, of maximal enzymatic rate (Vmax), sub-strate specificity (Km), turnover number (kcat), enzyme effi-ciency (kcat/Km).
Acknowledgements
The authors wish to thank the National Science Council, Taiwan and National Taiwan University, Taipei for financial sup-port.
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