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PEDS Advance Access originally published online on June 9, 2009
Protein Engineering Design and Selection 2009 22(7):413-420; doi:10.1093/protein/gzp019
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid{dagger}

Anders G. Sandström, Karin Engström, Jonas Nyhlén, Alex Kasrayan and J.-E. Bäckvall1

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden

1 To whom correspondence should be addressed. E-mail: jeb{at}organ.su.se

We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).

Keywords: Candida antarctica/directed evolution/hydrolases/Pichia pastoris/yeast vector

Received April 8, 2009; revised May 11, 2009; accepted May 11, 2009.

{dagger} Dedicated to Professor Karl Hult on the occasion of his 65th birthday.


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