Asn at position 285 (N285) in the catalytic domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 most likely participates in the cleavage of ester bonds as revealed by our previous evolutionary engineering study using the error-prone polymerase chain reaction (PCR) method. To exhaustively examine the effects of mutations at that position, we conducted site-directed saturation mutagenesis at that position and the resultant mutant enzymes (N285X) were evaluated in p-nitrophenyl ester (pNPCn) hydrolysis and PHB degradation. Kinetic studies demonstrated that the PHB-degrading activities of N285X were reciprocally related to their pNPCn-hydrolyzing activities, with the exception of N285A and N285G, and that His residue could functionally substitute for Asn285 on PHB degradation.