Combinatorial multiple-cassette mutagenesis

Finally, the region of accessible protein sequence space was extended by developing a modified version of Stemmer s combinatorial multiple-cassette mutagenesis (CMCM)... 

Figure 2.9 Schematic summary of the directed evolution of enantioselective lipase variants originating from the WT PAL used as catalysts in the hydrolytic kinetic resolution of ester rac-1. CMCM = Combinatorial multiple-cassette mutagenesis [8c,22],...

Fig. 15. Extended combinatorial multiple-cassette mutagenesis in the evolution of an (S)-selective lipase-variant from P. aeruginosa as a catalyst in the kinetic resolution of 1 (A position 20 A position 161 A position 234 O position 53 position 180 position 272) (49).

Subsequent directed evolution work on Pseudomonas aeruginosa demonstrates that protein sequence space with respect to enantioselectivity is best explored by a three-step procedure (Reetz, 2001) (i) generation of mutants by error-prone PCR at a high-mutation rate (ii) identification of hot regions and spots in the enzyme by error-prone PCR and substantiation by simplified combinatorial multiple-cassette mutagenesis (iii) extension of the process of combinatorial multiple-cassette mutagenesis to cover a defined region of protein sequence space. 

K. -E. Jaeger, Directed evolution of an enantioselective enzyme through combinatorial multiple-cassette mutagenesis, Angew. Chem. Inti Ed. 2001b, 40, 3589-3591. 

The starting point for further exploration of protein sequence space was the conjecture that Stemmer s method of Combinatorial Multiple Cassette Mutagenesis (CMCM) [74] can be applied in appropriately modified form. It is a special type of DNA-shuffling which can be used to generate mutant gene libraries in which cassettes composed of random or defined sequences and the wild-type are incorporated randomly. CMCM had been developed for use in the area of functional antibodies [74],... 

Fig. 11.16. Schematic representation of modified Combinatorial Multiple Cassette Mutagenesis of lipase genes [43],...

In the case of the most (S)-selective variant 1H8 observed so far, which was evolved by Combinatorial Multiple Cassette Mutagenesis [43] (see above), the analysis based on molecular modeling and molecular dynamics calculations has not been completed. However, based on the results of the sequencing experiments and the X-ray structure... 

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