Halogenation and Oxidation Reactions with Haloperoxidases

Franssen, M.C.R.

doi:10.3109/10242429409065220

Cited by 60 publications

(24 citation statements)

References 72 publications

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“…fumago chloroperoxidase (23,24) was also found to be much more active in 99.4% isopropyl alcohol when added via water than when directly suspended in that medium (v avw ͞v ds ϭ 190). As in the case of the two plant peroxidases, this ratio could be dropped 27-fold to 7.0 if the enzyme was lyophilized in the presence of the substrate o-hydroxybenzyl alcohol.…”

Section: Resultsmentioning

confidence: 99%

Striking activation of oxidative enzymes suspended in nonaqueous media

Dai

Klibanov

1999

Proc. Natl. Acad. Sci. U.S.A.

101

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The catalytic activity of four lyophilized oxidative enzymes-horseradish peroxidase, soybean peroxidase, Caldariomyces fumago chloroperoxidase, and mushroom polyphenol oxidase-is much lower when directly suspended in organic solvents containing little water than when they are introduced into the same largely nonaqueous media by first dissolving them in water and then diluting with anhydrous solvents. The lower the water content of the medium, the greater this discrepancy becomes. The mechanism of this phenomenon was found to arise from reversible denaturation of the oxidases on lyophilization: because of its conformational rigidity, the denatured enzyme exhibits very limited activity when directly suspended in largely nonaqueous media but renatures and thus yields much higher activity if first redissolved in water. Two independent means were discovered for dramatically minimizing the lyophilization-induced inactivation, both involving the addition of certain types of excipients to the aqueous enzyme solution before lyophilization. The first group of excipients consists of phenolic and aniline substrates as well as other hydrophobic compounds; these presumably bind to the hydrophobic pocket of the enzyme active site, thereby preventing its collapse during dehydration. The second group consists of general lyoprotectants such as polyols and polyethylen glycol that apparently preserve the overall enzyme structure during dehydration. The activation effects of such excipients can reach into the tens and hundreds of fold. Moreover, the activations afforded by the two excipient groups are additive, resulting in up to a complete protection against lyophilization-induced inactivation when representatives of the two are present together.When placed in nonaqueous solvents instead of the natural, aqueous milieu, enzymes exhibit remarkable new properties, including the ability to catalyze reactions impossible in water, enhanced thermostability, molecular ''memory,'' and radically altered selectivity (1). This holds a promise of substantially broadening the biotechnological utility of enzymes (1).The main drawback of enzymes functioning in organic solvents is their drastically reduced catalytic activity compared with that in water (2). Ironically, one of the principal causes of this inactivation is not an adverse effect of the organic solvent itself but rather denaturation of the enzyme brought about by its prior dehydration (2), usually through lyophilization (3). Although a number of approaches have been proposed to minimize this denaturation, they have been tested only with relatively simple, hydrolytic enzymes (2). And yet, the greatest practical potential rests with more complex, oxidative enzymes, e.g., peroxidases (4, 5).In the present work, we have expanded the scope of both the mechanistic investigation of the lyophilization-induced inactivation and the search for effective remedies to oxidative enzymes, including peroxidases. Their catalytic performance in organic solvents containing from Ͻ1% to a few percent...

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Section: Resultsmentioning

confidence: 99%

Striking activation of oxidative enzymes suspended in nonaqueous media

Dai

Klibanov

1999

Proc. Natl. Acad. Sci. U.S.A.

101

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“…The enzymebound intermediates formed during this reaction are very unstable and hypohalous acid ultimately forms, which leads to unspecific halogenation reactions which are not in agreement with expectations from enzyme-catalyzed reactions. [4,5] Nonetheless, there are situations in which unspecific halogenation reactions play important roles in nature, such as the defence mechanism of mammals against infections. Here, for example, myeloperoxidase has an important function.…”

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confidence: 99%

Purification and Partial Characterization of Tryptophan 7-Halogenase (PrnA) fromPseudomonas fluorescens

Keller

Wage

Hohaus

et al. 2000

Angew. Chem. Int. Ed.

181

165

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“…CPO catalyzes a variety of synthetically useful selective oxidations with hydrogen peroxide without the need for cofactors. The oxidation of indole to oxindole (Corbett and Chipko, 1979), sulfoxidation (Colonna et al, 1992;Colonna et al, 1990;Fu et al, 1992), epoxidation (Allain et al, 1993;Colonna et al, 1993;Dexter et al, 1995;Geigert et al, 1986;Hager and Allain, 1994), hydroxylation of aromatic systems (Miller et al, 1995;Zaks and Dodds, 1995), N-demethylation (Kedderis and Hollenberg, 1984;Kedderis and Hollenberg, 1983;Kedderis et al, 1980), the oxidation of alcohols to aldehydes (Geigert et al, 1983), and halogenation reactions (Franssen, 1994) have been performed, generally with high selectivity. CPO also retains its activity in aqueous t-butyl alcohol media, which is an advantage with non-polar organic substrates (Deurzen et al, 1994).…”

Section: Introductionmentioning

confidence: 99%