Strategies for discovery and improvement of enzyme function: state of the art and opportunities.
Bottom Line: Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non-conventional media, using new substrates for old enzymes, active-site imprinting, altering temperature, etc.Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem.The present review attempts to highlight some of these achievements and potential opportunities.
Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi - 110 016, India.Show MeSH
Mentions: Enzymes obtained by screening approaches (traditional or metagenome) do not necessarily fulfil all process requirements and need further fine tuning for adaptability to industrial scale production. Often inadequacies such as low stability, substrate and/or product inhibition, narrow substrate spectrum, enantioselectivity, etc. limit the application of the enzyme at hand for practical purposes. Using the tools of protein engineering, researchers are now able to reprogram the enzyme characteristics thus providing opportunities for tailoring enzymes for specific reactions. A practical approach for protein design is to use the three‐dimensional enzyme structure and rational design to systematically alter specific amino acid residues in the active site. This approach although more pragmatic, requires detailed knowledge of the structure and function of the protein to make desired changes. One of the important landmark studies using this approach was reported recently for production of the antidiabetic drug Sitagliptin (Savile et al., 2010). Chemical methods (rhodium‐catalysed hydrogenation) used for the production of the drug are highly undesirable and offer problems such as product contamination, inadequate stereoselectivity, etc. This prompted search for a transaminase mediated alternative using prositagliptin ketone (precursor for the product) as a substrate (Fig. 6). However, initial screening suggested that no enzyme activity could be obtained for the bulky substrate. By using the approach of structure‐based rational protein design and directed evolution, it was possible to alter the substrate specificity and stability of transaminase from Arthrobacter sp. (Koszelewski et al., 2008) to obtain variants that could not only accept the bulky ketone as substrate at high concentrations, but also tolerate elevated temperatures and high solvent concentrations (to enhance solubility of the insoluble bulky ketone). Using the transaminase variants it has been possible to produce various trifluoromethyl amines and phenylethylamines with electron rich substituents with high enantioselectivity, a feat that was impossible to achieve using the existing chemical methods. The process advancement efforts (developed jointly by Merck and Codexis) were also recognized by awarding the prestigious Presidential Green Chemistry Award from US Environmental Protection Agency (EPA) in June 2010.
Affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi - 110 016, India.