Sialic acid aldolases (E.C.4.1.3.3) catalyze the reversible aldol cleavage of N-acetyl-D-neuraminic acid (Neu5Ac) to from N-acetyl-D-mannosamine (ManNAc) and pyruvate. In this study, a sialic acid aldolase (PdNAL) from Peptoclostridium difficile NAP08 was expressed in Escherichia coli BL21 (DE3). This homotetrameric enzyme was purified with a specific activity of 1-8.34 U/mg for the cleavage of Neu5Ac. The optimal pH and temperature for aldol addition reaction were 7.4 and 65 degrees C, respectively. PdNAL was quite stable at neutral and alkaline pH (6.0-10.0) and maintained about 89% of the activity after incubation at pH 10.0 for 24 h. After incubation at 70 degrees C for 15 min, almost no activity loss was observed. The high thermostability simplified the purification of this enzyme. Interestingly, substrate profiling showed that PdNAL not only accepted ManNAc but also short chain aliphatic aldehydes such as acetaldehyde, propionaldehyde and n-butyraldehyde as the substrates. This is the first example that a sialic acid aldolase is active toward aliphatic aldehyde acceptors with two or more carbons. The amino acid sequence analysis indicates that PdNAL belongs to the NAL subfamily rather than 4-hydroxy-2-oxopentanoate (HOPA) aldolase, but it is interesting that the enzyme possesses the activity of HOPA aldolase. (C) 2016 Elsevier Inc. All rights reserved.