Preparation and Characterization of an Ancient Aminopeptidase Obtained from Ancestral Sequence Reconstruction for L-Carnosine Synthesis

As a biologically active peptide, L-carnosine has been widely used in the pharmaceutical, cosmetic and health care industries due to its various physiological properties. However, relatively little research is available regarding L-carnosine's enzymatic synthesis function. In this study, a potential enzyme sequence with the function of carnosine synthesizing was screened out using the ancestral sequence reconstruction (ASR) technique. Identified with L-carnosine synthesis activity, this enzyme was further confirmed using autoproteolytic phenomenon via Western blot and N-terminal sequencing. After purification, the enzymatic properties of LUCA-DmpA were characterized. The melting temperature (T-m) and denaturation enthalpy (Delta H) of LUCA-DmpA were 60.27 +/- 1.24 degrees C and 1306.00 +/- 26.73 kJ center dot mol(-1), respectively. Circular dichroism (CD) spectroscopy results showed that this ancestral enzyme was composed of alpha-helix (35.23 +/- 0.06%), beta-sheet (11.06 +/- 0.06%), beta-turn (23.67 +/- 0.06%) and random coil (32.03 +/- 0.06%). The enzyme was characterized with the optimal temperature and pH of 45 degrees C and 9.0, respectively. Notably, LUCA-DmpA was also characterized with remarkable pH tolerance based on the observation of more than 85% remaining enzymatic activity after incubation at different pH buffers (pH = 6-11) for 12 h. Additionally, rather than being improved or inhibited by metal ions, its enzymatic activity was found to be promoted by introducing organic solvent with a larger log P value. Based on these homology modeling results, the screened LUCA-DmpA is suggested to have further optimization potential, and thereafter to be offered as a promising candidate for real industrial applications.