title: Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display.
abstract: 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.
authors.affiliation: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Dasa
authors.initials: D
authors.lastname: Lipovsek
authors.forename: Eugene
authors.initials: E
authors.lastname: Antipov
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Armstrong
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Olsen
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Klibanov
authors.forename: Bruce
authors.initials: B
authors.lastname: Tidor
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: 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
chemical_list: D005452:Fluoresceins; D014443:Tyrosine; D006735:Horseradish Peroxidase; D003545:Cysteine