Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display.
We report a method for in vitro selection of catalytically active enzymes from large libraries of variants displayed on the surface of the yeast S. cerevisiae. Two libraries, each containing approximately 2 x 10(6) variants of horseradish peroxidase (HRP), were constructed; one involved error-prone PCR that sampled mutations throughout the coding sequence, whereas the other involved complete combinatorial enumeration of five positions near the active site to non-cysteine residues. The enzyme variants displayed on the yeast surface were allowed to modify it with a fluorescently labeled substrate. A combination of positive and negative selection applied to the active-site-directed library resulted in variants with up to an 8-fold altered enantioselectivity, including its reversal, toward L/D-tyrosinol. In contrast, the library constructed by using error-prone PCR yielded no HRP variants with a significantly improved enantioselectivity.
Lipovsek
Dasa
D
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Antipov
Eugene
E
Armstrong
Kathryn A
KA
Olsen
Mark J
MJ
Klibanov
Alexander M
AM
Tidor
Bruce
B
Wittrup
K Dane
KD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry; D015723:Gene Library; D005815:Genetic Code; D014644:Genetic Variation / Q000235:genetics*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013237:Stereoisomerism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D005452:Fluoresceins; D014443:Tyrosine; D006735:Horseradish Peroxidase; D003545:Cysteine