Enzymatic peptide synthesis in frozen aqueous systems: influence of modified reaction conditions on the peptide yield.

The alpha-chymotrypsin (EC 3.4.21.1)-catalyzed reaction of Mal-Phe-OMe with H-Leu-NH2 has been studied under a range of reaction conditions, for example various cryogenic reagents for shock-freezing, addition of dimethyl sulfoxide (DMSO) and decreased reaction temperatures down to 213 K. It has been shown that the peptide yield is independent of the method of shock-freezing. The optimal reaction temperature was between 263 K and 248 K. Lower temperatures result in clearly retarded reactions. Addition of DMSO leads to decreasing peptide yields. It is certain that the peptide bond formation is catalyzed by the active enzyme, since unspecific protein surface catalysis gave no peptide yields at all.