Striking activation of oxidative enzymes suspended in nonaqueous media

Abstract

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...

Salt‐activation of nonhydrolase enzymes for use in organic solvents

“…Another general approach is the co-lyophilization of enzyme with molecules such as sugars, alcohols, or polymers that are hypothesized to protect the enzyme against freeze-drying or organic solvents (Dabulis and Klibanov, 1993;Laszlo et al, 2001;Murakami et al, 2001). Other investigators have added small hydrophobic molecules with the idea of protecting the hydrophobic pocket of the enzyme from collapsing (Dai and Klibanov, 1999). Recently, a few groups have shown that crown ethers, oligoamines, and cyclodextrins each are capable of protecting proteases (Broos et al, 1995;Hasegawa et al, 2003;Kudryashova et al, 2003;Montanez-Clemente et al, 2002;van Unen et al, 2002).…”

“…This process has been optimized for numerous combinations of salts and lyophilization times (Ru et al, 1999(Ru et al, , 2000. Furthermore, the addition of general protectants against protein denaturation during freeze-drying has also been shown to enhance enzyme activity in organic solvents (Dai and Klibanov, 1999;Ru et al, 2001), albeit to a lesser extent than the inclusion of activating salts.…”

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

“…To counter this limitation, many methods have been introduced leading to improved enzyme activity in organic solvents. For example, control of the pH value (Yang et al, 1993), colyophilization with lyoprotectants (Dabulis and Klibanov, 1993) and salts (Khmelnitsky et al, 1994;Ru et al, 1999), addition of water-mimicking agents Kitaguchi et al, 1990), imprinting with substrates and substrate analogs (Rich and Dordick, 1997;Russell and Klibanov, 1988), immobilization (Orsat et al, 1994;Petro et al, 1996;Ruiz et al, 2000), solubilization Okahata et al, 1995a,b;Paradkar and Dordick, 1994;Wangikar et al, 1997;Xu et al, 1997), mutagenesis (Chen and Arnold, 1993), and solvent precipitation (Dai and Klibanov, 1999) represent methods that have been successful for improving the catalytic activity of enzymes. One of the most successful groups of activating additives identified thus far are macrocyclic compounds, which includes cyclodextrins Ooe et al, 1999;Santos et al, 1999) and crown ethers (Broos et al, 1995a;Engbersen et al, 1996;Itoh et al, 1996;Reinhoudt et al, 1989;van Unen, 2000;van Unen et al, 1998van Unen et al, , 2001.…”

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…The major problems of 278 substrate solubility and unwanted side reactions promoted by water are also overcome 279 during organic solvent based synthesis. Additionally, in some anhydrous solvents 280 peroxidase (HRP and SBP) activity was actually increased [75], with additional 281 methods, such as salt activation [76] and excipient aided lyophilisation [77] also 282 resulting in increased peroxidase activity. However, in some low water solvents, 283 peroxidases can lose their confirmational structure [78]; although recent advances in 284 peroxidase encapsulation in amphiphilic matrices [79], the use of reverse micelles 285 [80] and oil emulsions [81] allow for peroxidase activity in an extended range of 286 anhydrous solvents.…”