Kinetics of enzymatic solid-to-solid peptide synthesis: intersubstrate compound, substrate ratio, and mixing effects

Area of Science:

• Biocatalysis
• Enzyme kinetics
• Peptide synthesis

Background:

• Thermolysin is an enzyme used in peptide synthesis.
• Solid-to-solid synthesis offers advantages in specific applications.
• Understanding kinetics in low-water environments is crucial for optimizing biocatalytic processes.

Purpose of the Study:

• To investigate the kinetics of thermolysin-catalyzed solid-to-solid peptide synthesis.
• To explore the impact of highly concentrated substrates and low water content (10-20% w/w).
• To elucidate the factors influencing reaction rates in this system.

Main Methods:

• Systematic kinetic studies using Z-Gln and Leu-NH2 as model substrates.
• Melting point experiments to identify intermediate compounds.
• Development of a kinetic model correlating substrate ratio with liquid phase composition and pH.

Main Results:

• Preheating and ultrasonic treatments showed no significant effect.
• Discovery of substrate salt formation, impacting reaction kinetics.
• Kinetics demonstrated a strong dependence on substrate ratio, with optimal rates observed at specific compositions (e.g., 60% Leu-NH2, 40% Z-Gln).
• A model successfully explained experimental rates based on substrate ratio-dependent liquid phase properties.

Conclusions:

• Substrate ratio is a critical parameter in thermolysin-catalyzed solid-to-solid peptide synthesis.
• Salt formation and its influence on local pH are key factors affecting reaction kinetics.
• The developed model provides a basis for further optimization and scale-up of this enzymatic synthesis.