Directed evolution of Candida antarctica lipase A using an episomaly replicating yeast plasmid.
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).
Sandström
Anders G
AG
Department of Organic Chemistry, Stockholm University, Stockholm, Sweden.
Engström
Karin
K
Nyhlén
Jonas
J
Kasrayan
Alex
A
Bäckvall
J-E
JE
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D020134:Catalytic Domain; D020650:Combinatorial Chemistry Techniques; D019020:Directed Molecular Evolution; D008049:Lipase / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010843:Pichia / Q000235:genetics*; D010957:Plasmids
D019151:Peptide Library; D008049:Lipase