title: 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.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: L
authors.initials: L
authors.lastname: Dai
authors.forename: A M
authors.initials: AM
authors.lastname: Klibanov
mesh_terms: D000363:Agaricales / Q000201:enzymology; D002384:Catalysis; D004156:Catechol Oxidase / Q000378:metabolism*; D002711:Chloride Peroxidase / Q000378:metabolism*; D003470:Culture Media; D004789:Enzyme Activation; D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology; D010544:Peroxidases / Q000378:metabolism*; D012996:Solutions; D012997:Solvents; D013025:Glycine max / Q000201:enzymology
chemical_list: D003470:Culture Media; D012996:Solutions; D012997:Solvents; D004156:Catechol Oxidase; D006735:Horseradish Peroxidase; D010544:Peroxidases; D002711:Chloride Peroxidase