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Functional Expression and Stabilization of Horseradish Peroxidase by Directed

Summary: Functional Expression and Stabilization
of Horseradish Peroxidase by Directed
Evolution in Saccharomyces cerevisiae
Birgit Morawski, Sara Quan, Frances H. Arnold
Division of Chemistry and Chemical Engineering 210-41, California Institute
of Technology, Pasadena, CA 91125; telephone: (626) 395-4162; fax: (626)
568-8743; e-mail: frances@cheme.caltech.edu
Received 11 November 2000; accepted 3 March 2001
Abstract: Biotechnology applications of horseradish per-
oxidase (HRP) would benefit from access to tailor-made
variants with greater specific activity, lower Km for per-
oxide, and higher thermostability. Starting with a mutant
that is functionally expressed in Saccharomyces cerevi-
siae, we used random mutagenesis, recombination, and
screening to identify HRP-C mutants that are more active
and stable to incubation in hydrogen peroxide at 50C. A
single mutation (N175S) in the HRP active site was found
to improve thermal stability. Introducing this mutation
into an HRP variant evolved for higher activity yielded
HRP 13A7-N175S, whose half-life at 60C and pH 7.0 is


Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology


Collections: Chemistry; Biology and Medicine