Biocatalyst directed evolution techniques

But there is still another point, not yet discussed but with considerable potential, which may also impact eventually on technical asymmetric catalysis. Even though biocatalysts are efficient, active, and selective, there still remains one big disadvantage At present, there is not yet an appropriate enzyme known or available for every given chemical reaction. It is estimated that about 25 000 enzymes exist in Nature, and 90% of these have still to be discovered [28, 29]. New biocatalysts are made available nowadays not only from screening known organism but also via screening metagenomic libraries and directed evolution techniques [30]. 

Various examples were described, including whole cell-biocatalyzed reductions [4], especially employing baker s yeast [la,5]. However, there was always the problem that several active enzymes with different selectivities were present, although in particular cases excellent conversions and stereoselectivities were achieved. Later on, by directed evolution techniques [6], improvement of stereoselectivities and substrate specificities of enzymes was achieved, as well as several enzymatic properties (e.g., thermal stability) were modified. Moreover, large amounts of biocatalysts have been produced by fermentation of recombinant bacteria that express the gene, when their corresponding DNA has been doned. The drawback of the expensive cofactor recyding has also been overcome by the devdopment of effident techniques [7]. 

Further advancement of biocatalysis will require the use of directed evolution to bridge the functional gap between wild-type and desired biocatalyst properties. These studies underscore the power of directed evolution to create artificial enzymes derived from wild-type enzymes with the desired catalytic activity. Directed evolution techniques will continue to fulfill the promise of biocatalysis for industrial applications. 

All these techniques create genetic diversity by recombination and point mutations and are well developed. However, insertions and deletions (indels) are also important types of mutation which are probably underrepresented in many conventional mutagenesis strategies. Methods for incorporation of indels in predefined positions in a combinatorial manner have been developed.Although there are some published studies on their use in the directed evolution of biocatalysts,the full potential of these newer methods of gene mutation for enzyme improvement are yet to be demonstrated. 

Any or all of these conditions may limit productivity, and whichever of the previously listed sequences occur in a particular process, the biocatalytic conversion needs to be selected and designed to cope with these issues. Such issues may be solved by process techniques (using purification or a change of medium/catalyst between the reactions) or potentially by alteration of the properties of the biocatalyst via selection or directed evolution. A further approach is to combine operations in a single-pot operation using process techniques, biocatalyst modification, and a degree of compromise. 

DNA shuffling offers an alternative technique for directed evolution of biocatalysts. Compared with point mutagenesis using epPCR, DNA shuffling... 

In particular, recombinant variants of the monoamine oxidase N (MAO-N) originating from Aspergillus niger have been established over the past years as excellent biocatalysts for the deracemization and stereoinversion of diverse primary, secondary, and even tertiary amines by Turner and coworkers [18]. A series of tailored variants were created by an intensive study and successive optimization of each catalyst by the combination of directed evolution, rational protein design, and novel techniques of high-throughput screenings [95,129]. 

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