Directed evolution of a fungal peroxidase

Cherry, Joel R.; Lamsa, Michael; Schneider, Palle; Vind, Jesper; Svendsen, Allan; Jones, Aubrey; Pedersen, Anders H.

doi:10.1038/7939

Cited by 292 publications

(170 citation statements)

References 19 publications

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“…In this respect, the CiP peroxidase promises to be a highly interesting biocatalyst. The enzyme is available on a large scale (from Novo Nordisk), and it has already been shown that mutants may be obtained via directed evolution techniques (53). This may also be the way to obtain an improved biocatalyst for application in epoxidation reactions.…”

Section: Conversions Of the Different Styrene Derivatives By Cip And Mpomentioning

confidence: 99%

Enantioselective Epoxidation and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase

Tuynman¹,

Spelberg²,

Kooter³

et al. 2000

Journal of Biological Chemistry

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We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H 2 O 2 had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H 2 O 2 (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-␣-C-␤ bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-␣-C-␤ cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carboncarbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.

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Section: Conversions Of the Different Styrene Derivatives By Cip And Mpomentioning

confidence: 99%

Enantioselective Epoxidation and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase

Tuynman¹,

Spelberg²,

Kooter³

et al. 2000

Journal of Biological Chemistry

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“…Directed evolution experiments can indeed yield synthetic catalysts that are thermostable and active either at high (77) or low (78,79) temperatures. However, when random mutants of mesophilic and thermophilic enzymes were only screened for high activity at low temperatures during directed evolution (80)(81)(82)(83)(84)(85), the evolved enzymes generally display a concomitant reduced thermostability.…”

Section: Figmentioning

confidence: 99%

Extreme catalysts from low-temperature environments

Hoyoux

Blaise

Collins

et al. 2004

Journal of Bioscience and Bioengineering

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Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0°C. To maintain metabolic rates and to prosper in cold environments, these extremophilic organisms have developed a vast array of adaptations. One main adaptive strategy developed in order to cope with the reduction of chemical reaction rates induced by low temperatures is the synthesis of cold-adapted or psychrophilic enzymes. These enzymes are characterized by a high catalytic activity at low temperatures associated with a low thermal stability. A study of protein adaptation strategies suggests that the high activity of psychrophilic enzymes could be achieved by the destabilization of the active site, allowing the catalytic center to be more flexible at low temperatures, whereas other protein regions may be destabilized or as rigid as their mesophilic counterparts. Due to these particular properties, psychrophilic enzymes offer a high potential not only for fundamental research but also for biotechnological applications.[Key words: psychrophile, extremophiles, cold adaptation, enzyme kinetics, flexibility strategy]Life under low-temperature conditions was identified as early as 1840 by Hooker, who observed that algae were associated with sea ice. In 1887, Forster was the first who reported that microorganisms isolated from fish could grow well at 0°C (1). The term "psychrophilic" was first used in 1902 by Schmidt-Nielsen to describe such cold-adapted organisms (2). Psychrophile is defined as an organism, prokaryotic or eukaryotic, living permanently at temperatures close to the freezing point of water in thermal equilibrium with the medium. Thus psychrophiles are numerous, including a large range of species of gram-positive and gram-negative bacteria, yeast, algae, marine invertebrates, insects and polar fish, and are widely distributed (3). Psychrophiles have developed mechanisms of adaptation to temperature including a huge range of structural and physiological adjustments in order to cope with the deleterious effect of low temperatures. Indeed, they display metabolic fluxes at low temperatures that are more or less comparable to those exhibited by closely related mesophiles living at moderate temperatures (4-6). This is explained by the capability of these psychrophilic organisms to produce "cold-adapted" enzymes which are able to cope with the reduction of chemical reaction rates induced by low temperatures. However, most cellular adaptations to low temperatures and the underlying molecular mechanisms are not fully understood and are still being investigated. Moreover, a study of proteins and enzymes from cold-adapted organisms is not only useful in the understanding of some general processes related to the protein structure and function but also in protein folding investigations. In addition, cold-active and heat-labile psychrophilic enzymes possess an interesting biotechnological potential.

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“…The heme peroxidase from Coprinus cinereus was subjected to multiple rounds of directed evolution in an effort to produce a mutant stable in laundry detergent conditions: pH 10.5 at 50ºC and 5-10 mM peroxide concentration [237]. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of the wild-type enzyme.…”

Section: Evolved Enzymesmentioning

confidence: 99%

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hernández

Berenguer-Murcia²,

Rodrigues³

et al. 2012

COC

140

107

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Hydrogen peroxide is a substrate or side-product in many enzyme-catalyzed reactions. For example, it is a side-product of oxidases, resulting from the re-oxidation of FAD with molecular oxygen, and it is a substrate for peroxidases and other enzymes. However, hydrogen peroxide is able to chemically modify the peptide core of the enzymes it interacts with, and also to produce the oxidation of some cofactors and prostetic groups (e.g., the hemo group). Thus, the development of strategies that may permit to increase the stability of enzymes in the presence of this deleterious reagent is an interesting target. This enhancement in enzyme stability has been attempted following almost all available strategies: site-directed mutagenesis (eliminating the most reactive moieties), medium engineering (using stabilizers), immobilization and chemical modification (trying to generate hydrophobic environments surrounding the enzyme, to confer higher rigidity to the protein or to generate oxidation-resistant groups), or the use of systems capable of decomposing hydrogen peroxide under very mild conditions. If hydrogen peroxide is just a side-product, its immediate removal has been reported to be the best solution. In some cases, when hydrogen peroxide is the substrate and its decomposition is not a sensible solution, researchers coupled one enzyme generating hydrogen peroxide "in situ" to the target enzyme resulting in a continuous supply of this reagent at low concentrations thus preventing enzyme inactivation.This review will focus on the general role of hydrogen peroxide in biocatalysis, the main mechanisms of enzyme inactivation produced by this reactive and the different strategies used to prevent enzyme inactivation caused by this "dangerous liaison". Outlook

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Directed evolution of a fungal peroxidase

Cited by 292 publications

References 19 publications

Enantioselective Epoxidation and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase

Enantioselective Epoxidation and Carbon–Carbon Bond Cleavage Catalyzed by Coprinus cinereus Peroxidase and Myeloperoxidase

Extreme catalysts from low-temperature environments

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

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