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Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae

Morawski, Birgit and Quan, Sara and Arnold, Frances H. (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 76 (2). pp. 99-107. ISSN 0006-3592. https://resolver.caltech.edu/CaltechAUTHORS:20180824-083058588

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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°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°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_2O_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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1002/bit.1149DOIArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2001 John Wiley & Sons, Inc. Received 11 November 2000; accepted 3 March 2001. S. Quan was supported by the SURF program at Caltech. The authors thank K. Mayer for help with the manuscript.
Funders:
Funding AgencyGrant Number
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Subject Keywords:stability; peroxide; Pichia pastoris
Issue or Number:2
Record Number:CaltechAUTHORS:20180824-083058588
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180824-083058588
Official Citation:Morawski, B. , Quan, S. and Arnold, F. H. (2001), Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol. Bioeng., 76: 99-107. doi:10.1002/bit.1149
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89124
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Aug 2018 16:47
Last Modified:03 Oct 2019 20:13

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