N-Succinylamino acid racemase (NSAAR) with N-acylamino acid racemase (NAAAR) activity together with a D- or L-aminoacylase allows the total transformation of N-acetylamino acid racemic mixtures into optically pure D- or L-amino acids, respectively. In this work we have cloned and expressed the N-succinylamino acid racemase gene from the thermophilic Bacillus-related species Geobacillus kaustophilus CECT4264 in Escherichia coli BL21 (DE3). G. kaustophilus NSAAR (GkNSAAR) was purified in a one-step procedure by immobilized cobalt affinity chromatography and showed an apparent molecular mass of 43 kDa in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 150 kDa, suggesting that the native enzyme is a homotetramer. Optimum reaction conditions for the purified enzyme were 55 degrees C and pH 8.0, using N-acetyl-D-methionine as substrate. GkNSAAR showed a gradual loss of activity at preincubation temperatures over 60 degrees C, suggesting that it is thermostable. As activity was greatly enhanced by Co(2+), Mn(2+) and Ni(2+) but inhibited by metal-chelating agents, it is considered a metalloenzyme. The Co(2+)-dependent activity profile of the enzyme was studied with no detectable inhibition at higher metal ion concentrations. GkNSAAR showed activity towards both aliphatic and aromatic N-acetylamino acids such as N-acetylmethionine and N-acetyl-phenylalanine, respectively, with k(cat)/K(m) values ranging from 1 x 10(3) to 9 x 10(3) s(-1) M(-1). Kinetic parameters were better for N-acetyl-D-amino acids than for N-acetyl-L-Specific ones. (C) 2009 Elsevier Ltd. All rights reserved.