UBC Theses and Dissertations
Application of optimization for the enzymatic synthesis of sweet aspartyl dipeptide analogs catalyzed by thermolysin Lee, Grace Ilah
The enzymatic peptide synthesis of two sweet dipeptide analogs (aspartyl derivatives), Z-L-Asp-Tyr-OMe and Z-L-Asp-Met-OMe, was attempted using thermolysin as a catalyst. These two dipeptides are analogs of Z-L-Asp-Phe-OMe, the precursor to aspartame, which has already been successfully synthesized enzymatically using thermolysin. Random Centroid Optimization was employed to obtain optimal conditions for maximum yield. Seven factors were optimized for each peptide and they included: reactant concentration (only the amino component for Z-L-Asp-Tyr-OMe but both carboxyl and amino components for Z-L-Asp-Met- OMe), solvent concentration, buffer concentration, enzyme concentration, calcium chloride concentration, pH and temperature. Factor ranges were entered into the Random Centroid Optimization program, which generated the conditions for the experiments. Organic solvents were used in order to shift the equilibrium towards synthesis. A mixture of two solvents, water-miscible dimethylformamide (DMF), and water-immiscible ethyl acetate (EA), were used for both the first and second cycles in the optimization of Z-L-Asp-Tyr-OMe. For Z-LAsp- Met-OMe, glycerol, a polyol, was used in the first cycle but in the second cycle, another polyol, ethanediol was substituted because glycerol appeared to have an inhibitory effect on the enzyme. The maximum yield of Z-L-Asp-Tyr-OMe was 2.13 ± 0.46% (Mean ± S.D., n=3) under the following conditions: 80mM Z-L-Asp, 154mM Tyr-OMe, 2.3/9.7/88 DMF/EA/buffer, 39μM thermolysin, 7mM CaCl2, pH 6.3, at 46°C for 24 hours. The optimum yield of Z-L-Asp-Met-OMe was 9.13 ± 1.22% (Mean ± S.D., n=3) under the following conditions: 53mM Z-L-Asp, 96mM Met-OMe, 2/98 ethanediol/buffer, 35μM thermolysin, 3.8mM CaCl2, pH 5.1, at 49°C for 24 hours. Random Centroid Optimization was shown to be a useful method for attaining the conditions of maximum peptide synthesis, especially with the Z-L-Asp-Met-OMe. However, there were few clear indications of which factors most affected the yield. In general, it was found that the type of solvent used definitely had a great impact on the synthesis of both peptides. Z-L-Asp-Tyr-OMe synthesis was not very successful since the DMF:EA solvents did not sufficiently solubilize the reactants. In addition, high concentrations of solvents which are normally used in other systems could not be used for this synthesis. This indicates that the effect of the solvent on the enzyme must be examined further. It was apparent that not only the solvent had an effect on the synthesis, but that other factors were also important, although the nature of these other factors could not be deduced from this study.
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