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 | ||||||
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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. | ||||||
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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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