Biocatalysis directed evolution

The complexity of today s pharmaceutical compounds and an increasing awareness of the environmental impact of traditional chemical syntheses have opened the door to biocatalysis. Directed evolution is an integral tool in the development of synthetic enzymes, ensuring they are suitable for use in an industrial setting. The past success of this approach indicates that it will continue to provide many examples of safe and efficient production of chemical intermediates and medical compounds. 

The cholesterol-lowering drug atorvastatin, marketed as Lipitor, is an example where biocatalysis research has been applied extensively and is in industrial use. The enzyme 2-deoxyribose-5-phosphate aldolase (DERA) has been a target of directed evolution for the production of atorvastatin intermediates [8,9,71]. DeSantis and coworkers [8,9] used structure-based... 

Zhao, H.M., Chockalingam, K. and Chen, Z.L. (2002) Directed evolution of enzymes and pathways for industrial biocatalysis. Current Opinion in Biotechnology, 13, 104—110. 

Jaeger, K.E. and Eggert, T. (2004) Enantioselective biocatalysis optimized by directed evolution. Current... 

One drawback of biocatalysis is that enzymes are not available in both enantiomeric forms. Particularly where a class of enzymes whose natural substrates are optically active, such as nucleosides, it can be difficult if not impossible to find an alternative enzyme that will accept the unnatural substrate enantiomer. This is not insurmountable if directed-evolution approaches are used, but it can be prohibitively expensive, especially when the desired product is in an early stage of development or required for use only as an analytical reference or standard. 

Turner, N., Directed evolution of enzymes for applied biocatalysis Trends BiotechnoL, 2003,21, 474-478 Johannes, T.W. and Zhao, H., Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol., 2006, 9, 261-267. 

Thus far only three reports regarding the directed evolution of enantioselective EHs have appeared 95,96,143), notwithstanding the fact that these enzymes, even as wild types, constitute important catalysts in synthetic organic chemistry 7-12,144,145). Indeed, since two EHs became commercially available recently, this type of biocatalysis offers exciting prospects for the practicing organic chemist. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

Reetz, M. T. Jaeger, K. E. Superior biocatalysts by directed evolution. Topics in Current Chemistry 1999, 200 (Biocatalysis From Discovery to Application), 31-57. 

Williams GJ, Nelson AS, Berry A (2004) Directed evolution of enzymes for biocatalysis and the life sciences. Cell Mol Life Sci 61 3034—3046... 

Bomscheuer, U.T. 2001. Directed evolution of enzymes for biocatalytic applications. Biocatalysis and Biotransformation, 19 85-97. 

Keywords Directed evolution. Random mutagenesis, DNA shuffling. Biocatalysis, Combinatorial libraries, Enantioselectivity. 

Biocatalysis is the study of biological catalysts with regard to their kinetics, mechanisms, specificity, and application in synthesis and analysis. In addition to the traditional study of mechanistic enzymology, biocatalysis is concerned with the use of recombinant DNA technology, site-specific mutagenesis, directed evolution, pathway engineering, substrate design, and structure-based approaches as tools for the development of novel catalysts and reactions. 

The power of directed evolution is now well documented. These methods are robust and are able to improve industrial enzymes in reasonably short times. The first laboratory-evolved enzymes are now used commercially in laundry detergents12011 other commercial applications are on the horizon. Directed evolution may well help move biocatalysis from an enabling tool to a lowest cost approach . It also offers new opportunities to engineer multi-enzyme pathways and even whole microbes [69- 224> 2251, which will lead to straightforward single-pot, multi-enzyme bioconversions and new fermentation processes based on green resources such as glucose or inexpensive waste materials. 

Despite the progress biocatalysis has made in the last few years its potential is still increasing. By improved screening methods new catalysts will be detected and made available in large amounts by cloning and overexpression. Directed evolution will be used to improve properties such as stability or selectivity(118> 119). Metabolic engineering will be used to analyze and remove bottlenecks in the metabolism or to create novel biocatalysts[1Z01. 

Turner, N.). (2003) Directed evolution of enzymes for applied biocatalysis. Trends Biotech-nol. 21, 474-478. 

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