Background: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization.
Results: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components.
Conclusion: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.
Keywords: MD simulation; bitterness; carboxypeptidase; catalytic efficiency; umami.
© 2024 Society of Chemical Industry.