Peroxidase directed evolution

Chemical modification of surface residues of HRP is one method which may offer some improvement in thermal or long-term stability of the enzyme. The -amino groups of the six surface Lys residues can be modified by reaction with carboxylic anhydrides and picryl sulfonic acid (296). In this example the number of sites modified was found to be more significant than the chemical nature of the modification, at least as a criterion for improved stability. Other methods explored include the use of bifunctional crosslinking reagents to couple surface sites on the enzyme (297). Future developments are likely to be concerned with the selection of site-directed mutants of HRP C that show enhanced thermal stability. Dramatic increases in thermal stability of up to 190-fold have been reported recently for mutants of Coprinus cinereus peroxidase (CIP) generated using a directed evolution approach (298). 

The reaction has found little application so far however, one of the first examples of an asymmetric synthesis using peroxidases was the highly regio- and diastereo-selective halohydration of glycals to 2-deoxy-2-halo sugars in the presence of CPO [263]. Some examples of selective halogenation reactions of aromatic compounds using CPO optimized by directed evolution approaches are also known [264]. 

J. Vind, A. Svendsen, A. Jones, and A. H. Pedersen, Directed evolution of a fungal peroxidase, Nature Biotechnology 1999, 27, 379-384. 

Garcia E, Martmez MJ, Ruiz-Duenas FJ et al (2009) High redox-potential peroxidases from directed evolution (Peroxidasas de elevado potential redox disenadas por evolution dirigida). Patent (Spain) application P200930157... 

Morawski B, Quan S, Arnold FH (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol Bioeng 76 99-107... 

Rai GP, Sakai S, Florez AM, Mogollon L, Hager LP (2001) Directed evolution of chloro-peroxidase for improved epoxidation and chlorination catalysis. Adv Synth Catal 343 1-8... 

Iffland A, Gendreizig S, Tafemeyer P, Johnsson K (2001) Changing the substrate specificity of cytochrome c peroxidase using directed evolution. Biochem Biophys Res Commun 286 126-132... 

Cherry JR, Lamsa MH, Schneider P et al (1999) Directed evolution of a fungal peroxidase. Nat Biotechnol 17 379-384... 

P450s are generally less stable than peroxidases. Recently a naturally thermostable P450 (stable up to 85 °C) was identified and characterized [25, 100 -103], Both P450s and peroxidases are inactivated during catalysis, via heme alkylation by terminal olefins (see Section 10.8.3) and oxidative damage by peroxides. Eukaryotic P450s are associated with cell membranes and are therefore insoluble and difficult to use outside the cell. We believe that many of these limitations can be addressed by directed evolution. 

Our laboratory is also studying the directed evolution of a new type of potentially ligninolytic peroxidase classified as unspecific peroxygenase, UPO (EG 1.11.2.1). UPO was initially defined as a heme-thiolate peroxidase, exhibiting both per-oxidative and peroxygenative activities toward aromatic compounds (aromatic peroxygenase, also referred to as APO [26, 75]). However, more recent studies have described the monooxygenase activity of UPO toward ahphatic compounds (UPO,... 

Directed evolution of the peroxidase activity of a novo-designed protein. 

TL. Garda-Ruiz, E., Gonzalez-Perez, D., Ruiz-Duenas, F.J., Martinez, A.T., and Alcalde, M. (2012) Directed evolution of a temperature, peroxide and alkaline pH tolerant versatile peroxidase. Biochem.., 441, 487-498. 

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