The use of synthetic polymers for preventing enzyme thermal inactivation: Some comments on the paper by Alfani and coworkers

Abstract

Biotechnology Letters, June ]984 issue, contained the paper "The use of synthetic polymers for preventing enzyme thermal inactivation" by F.Alfani, M.Cantarella, G.Cirielli, V.Scardi, which deals with an enzyme stabilization technique, that has already received some attention in recent years (see Table I and references therein). The method consists in injecting the protein into an unstirred, plane membrane, ultrafiltration cell together with a linear-chain, soluble polymer. By suitable choice of the membrane molecular-weight cut-off, both the enzyme and the polymer are rejected and therefore accumulate immediately upstream from the membrane. Because of the high concentration levels attained, the linear-chain macromolecules arrange themselves as a tight polymeric network, surrounding the enzyme. A constraint is thus exerted onto the protein, that prevents its unfolding to some extent. This reduces the rate of enzyme activity decay. The paper by Alfani and coworkers deals with acid phosphatase thermal deactivation. The stabilizing polymers employed are polyvinyl alcohol and polyvinyl pyrrolidone. The validity of the enzyme stabilization technique by linear polymers, is by no means limited to this specific choice either of the protein or of the stabilizing macromolecules. Furthermore, the method has been applied successfully in inhibiting enzyme inactivation processes other than thermal, provided the activity loss is directly related to the unfolding of the protein structure . Table I reports the different systems we have analyzed since 1981. The results can be directly compared with those proposed in Alfani et al., 1984, since the experimental procedures are identical. Two points raised by the Authors in the analysis of their experimental results deserve some discussion. Alfani and coworkers performed preliminary kinetic runs on acid phosphatase, at rather high enzyme dilution (5~g/ml), in test-tube reactors. Under those conditions, the kinetic parameters in the hydrolysis of p-nitro-phenyl phosphate were determined, both in the presence and in the absence of stabilizing polymer. Concentrations of the latter were also small, ranging from 1.25 to 5 mg/ml. By not observing any variation in either Vmax or Km, the Authors reach the conclusion that the linear-chain polymer does not affect enzyme kinetics. This statement, though obviously true under the highly diluted conditions of the experiment, appears to be arbitrarily extended by the Authors to the stabilized enzyme situation. Indeed, the concentration attained by the polymer in the U.F. membrane cell is approximately two orders of magnitude larger than those used in the test-tube reactor runs discussed by Alfani and coworkers. Polymer concentration within the polarization layer, where the stabilization effect occurs, are calculated readily from an elementary mass-balance. Now, while