Gerichtete Evolution und Biokatalyse

Directed Evolution of Enantioselective Enzymes: Iterative Cycles of CASTing for Probing Protein‐Sequence Space

“…Other researchers8 have also contributed to this new area of asymmetric catalysis. Most directed evolution studies either begin with one or more cycles of error‐prone polymerase chain reactions (epPCRs)9 to introduce more or less random mutations that cover the whole gene (and thus enzyme) or a recombinant method such as DNA shuffling10 is applied.…”

“…However, it is not, in itself, an evolutionary process because only the initial libraries are considered. In our original paper,16 we suggested that, for future work, Darwinistic character, which is central to directed evolution,1, 2, 10, 11, 15 can be introduced through the performance of further rounds of mutagenesis/screening. This can be performed by using any one of the conventional methods, such as epPCR or DNA shuffling after CASTing, which may lead to remote effects 1.…”

Artificial Metalloenzymes for Enantioselective Catalysis: Recent Advances

“…2 Homogeneous and enzymatic catalyses are in many respects complementary in terms of substrate and reaction scope, operating conditions, enantioselection mechanism, etc 3. In terms of performance optimization, directed evolution methodologies outperform combinatorial ligand libraries 4–8…”

Assembly of Designed Oligonucleotides as an Efficient Method for Gene Recombination: A New Tool in Directed Evolution

“…About 25 of these clones turned out to be active catalysts. This result (about 1 % active clones) is to be expected in experiments of this kind, which involve the recombination of family genes, and is expected in traditional DNA shuffling experiments 1c, 9–12. The active clones (hits) were then used as catalysts in the hydrolytic desymmetrization of 1,4‐diacetoxycyclopent‐2‐ene ( 1 ) to form the enantiomers ( S , R )‐ 2 and ( R , S )‐ 2 (Scheme ).…”

“…For example, ligation was first described as a method to assemble DNA sequences from oligos in the 1970s,6 followed by the FokI method of gene synthesis7 and the process of self‐priming PCR 8. Another advancement was made by the introduction of DNA shuffling, a method of in vitro recombination that results in the formation of gene libraries from random fragments, which in turn are generated by partial DNaseI digestion of appropriate “starting” genes 1c, 9. Relatively high diversity is achieved, although reassembly relies on homologous recombination during the PCR step, which precludes the creation of crossovers between genes at loci of low homology.…”