KINETICS OF SURFACE HYDROLYSIS OF POLY[(R)3-HYDROXYBUTYRATE] FILM BY PHB DEPOLYMERASE FROM ALCALIGENES-FAECALIS T1

The kinetics and mechanism of surface hydrolysis of poly[(R)-3-hydroxybutyrate] (P(3HB)) film have been studied with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. The PHB depolymerase was stable at temperatures up to 40 degrees C in aqueous solution at pH values of 5.0-8.0. The HPLC analysis of water-soluble products showed that the primary product of the enzymatic hydrolysis of P(3HB) film was the dimer of 3-hydroxybutyric acid. The dimer was subsequently hydrolyzed to monomer by the PHB depolymerase in the aqueous solution. A kinetic study of the enzymatic hydrolysis of P(3HB) film was carried out at temperatures in the range 25-37 degrees C and pH values in the range 6.0-8.0 in 0.1-1.0 M potassium phosphate buffer. The kinetic data were accounted for in tersm of a two-step enzymatic reaction involving surface hydrolysis of the P(3HB) film which takes place via the two steps of adsorption and hydrolysis by the PHB depolymerase with the binding and catalytic domains. The heat of adsorption of the enzyme on the P(3HB) surface was as large as 43 kJ/mol, indicative of a strong interaction between the binding domain of the enzyme and the hydrophobic surface of the P(3HB) film. The activation energy of hydrolysis of the P(3HB) chain by the adsorbed enzyme was determined as 82 kT/mol.