Involvement of catalytic amino acid residues in enzyme-catalyzed polymerization for the synthesis of polyesters.

Recently, a variety of aliphatic polyesters have been synthesized using hydrolases such as lipases and PHB depolymerases, and the reaction mechanism for these enzyme-catalyzed polymerization has been discussed. In this paper, we have studied the involvement of the catalytic amino acid residues of the hydrolase in enzyme-catalyzed polymerization with an extracellular PHB depolymerase from Alcaligenes faecalis T1. A wild-type PHB depolymerase and three kinds of site-specific mutants (catalytic amino acids were substituted) were prepared and their polymerization activities for the ring-opening polymerization of (R)-beta-butyrolactone (BL) were compared. BL was polymerized at 80 degrees C in bulk by the wild-type enzyme to yield polymers consisting of cyclic and linear structures in a high monomer conversion. In contrast, none of the mutant enzymes showed obvious polymerization activity. These results have clearly demonstrated that the catalytic triad is indeed responsible for the enzyme-catalyzed polymerization of BL.