Striking activation of oxidative enzymes suspended in nonaqueous media

The catalytic activity of four lyophilized oxidative enzymes-horseradish pe roxidase, soybean peroxidase, Caldariomyces fumago chloroperoxidase, and mu shroom polyphenol oxidase-is much lower when directly suspended in organic solvents containing little water than when they are introduced into the sam e largely nonaqueous media by first dissolving them in water acid then dilu ting with anhydrous solvents. The lower the water content of the medium, th e greater this discrepancy becomes. The mechanism of this phenomenon was fo und to arise from reversible denaturation of the oxidases on lyophilization : because of its conformational rigidity, the denatured enzyme exhibits ver y limited activity when directly suspended in largely nonaqueous media but renatures and thus yields much higher activity if first redissolved in wate r. Two independent means were discovered for dramatically minimizing the ly ophilization-induced inactivation, both involving the addition of certain t ypes of excipients to the aqueous enzyme solution before lyophilization, Th e first group of excipients consists of phenolic and aniline substrates as well as other hydrophobic compounds; these presumably bind to the hydrophob ic pocket of the enzyme active site, thereby preventing its collapse during dehydration. The second group consists of general lyoprotectants such as p olyols and polyethylen glycol that apparently preserve the overall enzyme s tructure during dehydration. The activation effects of such excipients can reach into the tens and hundreds of fold. Moreover, the activations afforde d by the two excipient groups are additive, resulting in up to a complete p rotection against lyophilization-induced inactivation when representatives of the two are present together.