Characteristic Interactions between Poly(hydroxybutyrate)

Enzymatic degradation of poly[(R)-3-hydroxybutyrate] (P(3HB)) film by the poly(hydroxybutyrate) (PHB) depolymerase from Ralstonia picketti T1 was studied in 0.01 M phosphate buffer solution (pH 7.4) at 37°C by using a quartz crystal microbalance (QCM) technique. Enzymatic degradation of P(3HB) film was quantitatively followed by QCM as a positive frequency shift. While, the amount of depolymerases adsorbed on the film could be evaluated as a negative frequency shift by using a mutant enzyme which had no hydrolytic activity in a catalytic site. The degradation rate increased with enzyme concentration to reach a maximum value at 1.0 g*mL-1, and then the rate decreased at higher enzyme concentration. This enzyme concentration dependence could be quantitatively explained in terms of a change of coverage of the film surface by the adsorbed enzyme. When the wild-type enzyme solution in a QCM cell was replaced with the mutant enzyme solution in the middle of the reaction, the degradation rate was reduced markedly, indicating that the wild-type enzyme adsorbed on the P(3HB) surface is easily substituted by the mutant enzyme in the solution. On the other hand, replacement of the wild-type enzyme solution with other proteins or buffer solutions did not affect the degradation rate at all, suggesting that the adsorbed enzyme was not desorbed from the film surface. Thus, the adsorbed PHB depolymerase is released from the P(3HB) surface only by interaction with the same depolymerase in solution.