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.

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution

“…Because only one in 10 5 mutants were active in the first round, obtaining such a large reservoir would be impossible using a robotic screen, where the maximum throughput is ∼10 5 samples per day. Indeed, a plate-based directed evolution study to improve HRP (27) finds only a single non-wild-type active mutant in the first round of screening of ∼10 4 reactions, in accord with these observations. The effectiveness of the large reservoir of potentiating mutations in bringing about adaptive change underscores the advantage of the ultrahigh-throughput microfluidic screening platform.…”

“…The ability to screen libraries of >10 7 in just a few hours at a cost of only a few dollars will be of enormous benefit for directed evolution. There has already been some success screening small libraries that yield only modest improvements, and then performing repeated rounds of mutation and screening (27). However, when selecting for the binding activity of proteins, a clear relationship between library size and the affinity of the selected proteins is observed experimentally (7): Using antibody V-genes from nonimmunized donors, small phage-antibody libraries of <10 8 genes yield only antibodies with K d ∼ 10 −6 affinities, whereas larger libraries of >10 10 yield K d ∼ 10 −9 .…”

Effects of mutations in the helix G region of horseradish peroxidase

“…Directed evolution has been used to alter rHRP thermal stability [20] but we have found no reports of HRP stability manipulation via rational, site-directed mutagenesis.…”

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…by construction of an improved luminol 270 binding site [72]. Directed evolution stabilised HRP against thermal denaturation [39] 271 and has endowed it with increased H 2 O 2 tolerance and increased catalytic activity 272 [40]. Further targeted directed evolution, focussing on the substrate access channel 273 and binding pocket, could allow HRP to accept an increased variety of substrates [73], 274 and promote further diversification of HRP applications in organic synthesis [41].…”