title: Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae.
abstract: Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C. 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 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide.
authors.affiliation: Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: B
authors.initials: B
authors.lastname: Morawski
authors.forename: S
authors.initials: S
authors.lastname: Quan
authors.forename: F H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D015870:Gene Expression; D014644:Genetic Variation; D006207:Half-Life; D006735:Horseradish Peroxidase / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D007532:Isoleucine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase