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Chem Biol. 2007 Oct;14(10):1176-85.

Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display.

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1
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

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.

PMID:
17961829
DOI:
10.1016/j.chembiol.2007.09.008
[Indexed for MEDLINE]
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