The demand on thermostable D-lactate dehydrogenases (D-LDH) has been increased for D-lactic acid production but thermostable D-DLHs with industrially applicable activity were not much explored. To identify a thermostable D-LDH, three D-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three D-LDHs (D-LDH3) exhibited higher optimal reaction temperature (50 degrees C) than the others. The T-50(10) value of this thermostable D-LDH3 was 48.3 degrees C, much higher than the T-50(10) values of the others (42.7 and 42.9 degrees C) and that of a commercial D-lactate dehydrogenase (41.2 degrees C). The T-m values were 48.6, 45.7 and 55.7 degrees C for the three D-LDHs, respectively. In addition, kinetic parameter (k(cat)/K-m) of D-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 degrees C, implying that D-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the D-LDH3 characterized in this study a good candidate for the microbial production of D-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial. Crown Copyright (c) 2012 Published by Elsevier Ltd. All rights reserved.