Peptide Synthesis of Aspartame Precursor Using Organic-Solvent-Stable PST-01 Protease in Monophasic Aqueous-Organic Solvent Systems

Abstract

The PST-01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water-soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Cbz-Asp-Phe-OMe) synthesis from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester (Phe-OMe) in the presence of water-soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz-Asp-Phe-OMe were attained by the PST-01 protease when 30 mM Cbz-Asp and 500 mM Phe-OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 degrees C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz-Asp-Phe-OMe using the PST-01 protease attained 83% in the presence of 50% (v/v) DMSO.

An Oxidant- and Organic Solvent-Resistant Alkaline Metalloprotease from Streptomyces olivochromogenes

“…Additionally, although solventand/or detergent-stable proteolytic enzymes have been extensively reported from various bacteria and fungi, these enzymes from actinomycetes are rare. Characteristics of SOMP such as solvent, oxidant, and detergent stability along with pH and temperature profiles are different from those of stable proteases reported from actinomycetes strains [3,20,31] and from other sources (Tables 2 and 5); therefore, it might be a novel proteolytic enzyme. …”

Activity and Stability of Proteases in Hydrophilic Solvents

“…The effect of DMSO on the initial reaction rate of a protease PST -01 -catalyzed synthesis of Z -aspartame in a mixture of DMSO and Tris -HCl buffer at pH 8 was investigated by Tsuchiyama et al [36] . The initial reaction rate decreased with increasing DMSO concentration (20 -70% (v/v)).…”