Poly(3-hydroxybutyrate) [P(3HB)] was prepared by the ring-opening polymerization of (R,S)-beta-butyrolactone (BL) using two types of PHB depolymerase with or without substrate-binding domains (SBD) as the catalyst. The SBD lacking PHB depolymerase exhibited better catalytic activities for the ring-opening polymerization of BL.