cover illustration We developed a PCR technique for focused directed evolution that randomizes one or several cassettes in any given position of a gene. Over 95% mutations incorporated independently of their position within the gene, and more than one spot could be mutated simultaneously. The method was tested by creating focused libraries of / Pseudomonas fluorescens /esterase I variants. This led to the identification of a mutant within the OSCARR library with a 10-fold higher catalytic efficiency towards medium chain /p/-nitrophenyl esters. The modeled F125I mutant shown in the cover (docked with /p/-nitrophenyl octanoate) displays a wider entrance to the active site (shown in blue). For further details please see Hidalgo et al, pp. 567-576.
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