The activity of enzymes to form a peptide bond in organic solvent was greatly influenced by observed pH and water content. The precursors of two sweeteners, P-Asp-Xaa-OR (P = Z or For, Xaa-OR = Phe-OMe or Ala-OcHex), were synthesized by enzyme, and the reaction conditions were studied systematically. Z-Asp-OH was coupled with H-Phe-OMe or H-Ala-OcHex by thermolysin in tert-amyl alcohol. The best coupling results were obtained when the optimized observed pH was 8 or 9, and the water content was about 6% (V/V). The protecting group Z is better than For under the reaction conditions and H-Phe-OMe is a better nucleophile than H-Ala-OcHex. The expected optically pure peptides were obtained when the racemic amino acids were used as amino components in the starting materials. The physical constants of P-Asp-Xaa-OR synthesized by thermolysin are identical with those of peptides synthesized by chemical method.