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.

Automated summary

In this study, a potential enzyme sequence with the function of carnosine synthesis was screened out using the ASR technique. The enzymatic properties of the enzyme LUCA-DmpA were characterized, including its melting temperature, denaturation enthalpy, and secondary structure composition. The enzyme exhibited optimal temperature and pH, as well as remarkable pH tolerance and promotion of activity by organic solvents. These findings demonstrate the potential of LUCA-DmpA for industrial applications.