Dipeptide synthesis by an aminopeptidase from Streptomyces septatus TH-2 and its application to synthesis of biologically active peptides

Abstract

Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.

FIG. 1.

HPLC profile of product synthesized by SSAP. (A) HPLC of dipeptide synthesis by SSAP. Free phenylalanine at 50 mM and Phe-OMe at 50 mM were used as substrates. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 3 h. (B) HPLC of unknown product. The separated unknown product was treated with alkali or SSAP and then analyzed by HPLC. PhePhe was detected by the HPLC of the product treated with alkali, and free phenylalanine and a small amount of Phe-OMe were detected by treatment of the product with SSAP.

FIG. 2.

Investigation of arrangement of dipeptide synthesized by SSAP. (A and B) HPLC and MS of product synthesized with free phenylalanine and Trp-OMe as substrates (A) and HPLC of this product treated with SSAP (B). The components of this product treated with SSAP were free phenylalanine and Trp-OMe. (C and D) HPLC and MS of product synthesized with free tryptophan and Phe-OMe as substrates (C) and HPLC of this product treated with SSAP (D). Free phenylalanine and Trp-OMe were detected with the treatment of this product with SSAP. In HPLC profiles, the peaks of phenylalanine and Phe-OMe are very small because the absorption coefficient of tryptophan at 210 nm is approximately fourfold higher than that of phenylalanine in this investigation.

FIG. 3.

Effect of substrate concentration on rate of dipeptide synthesis by SSAP. (A) Effect of acyl acceptor concentration. Free phenylalanine at 50 mM and Phe-OMe at 0 to 160 mM were used as the acyl donor and acyl acceptor, respectively. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 2 h. (B) Effect of acyl donor concentration. Free Phe at 0 to 32 mM and Phe-OMe at 50 mM were used as the acyl donor and acyl acceptor, respectively. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 2 h. Each value is the average of three independent experiments ± the standard deviation.

FIG. 4.

Effects of temperature on stability and rate of dipeptide synthesis by SSAP. (A) Thermal stability of SSAP in aqueous solution and 98% MeOH. Each value is the average of five independent experiments ± the standard deviation. (B) Effect of temperature on dipeptide synthetic activity. Free phenylalanine at 20 mM and Phe-OMe at 50 mM were used as the acyl donor and acyl acceptor, respectively. The reaction was performed by using 20 μg of SSAP with vigorous shaking at an appropriate temperature for 1 h. Each value is the average of three independent experiments ± the standard deviation.

FIG. 5.

Investigation of reaction equilibrium. (A) Investigation of reaction equilibrium when 20 mM free phenylalanine and 50 mM Phe-OMe were used as substrates. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 1, 6, 22, and 50 h. (B) HPLC profile of product when 50 mM free phenylalanine and 50 mM Phe-OMe were used as substrates for dipeptide synthesis by SSAP. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 22 h. Intens., intensity; arb., arbitrary.

FIG. 6.

Stability of SSAP in 98% MeOH. The enzyme sample (20 μg/ml protein) was incubated in 98% MeOH at 25°C for an appropriate time. Residual activity was measured under the conditions described in Materials and Methods. Each value is the average of five independent experiments ± the standard deviation.

FIG. 7.

Substrate specificity of dipeptide synthesis by SSAP. (A) Specificity of SSAP toward acyl donor. (B) Specificity of SSAP toward acyl acceptor. In all cases, 20 mM free amino acid and 50 mM aminoacyl-OMe were used as the acyl donor and acyl acceptor, respectively. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 3 h. Each value is the average of three independent experiments ± the standard deviation.

FIG. 8.

Synthesis of biologically active dipeptides AspPhe-OMe (A), ValTyr-OMe (B), AlaTyr-OMe (C), and AlaDOPA-OMe (D) by SSAP. In all cases, 20 mM free amino acid and 50 mM aminoacyl-OMe were used as the acyl donor and acyl acceptor, respectively. All panels show the HPLC profiles of the reaction mixture with (w/SSAP) and without (w/o SSAP) the enzyme for the comparison of dipeptide synthesis by SSAP with a negative control. The reaction was performed by using 20 μg of SSAP with vigorous shaking at 25°C for 24 h. (B, C and D) Under the HPLC conditions described in Materials and Methods, the DOPA-OMe and Tyr-OMe peaks formed a shoulder. The hydroxyl groups of the side chain of the substrates may be the cause of the shoulders near these peaks. Intens., intensity; arb., arbitrary.