- Email: [email protected]
Carboxypeptidase A-catalyzed dipeptide synthesis in organic media
Journal of Biotechnology 66 (1998) 75 – 82
Carboxypeptidase A-catalyzed dipeptide synthesis in organic media Ade´l Ve´rtesi, L. Maria Simon * Department of Biochemistry, Jo´zsef Attila Uni6ersity, Ko¨ze´pfasor 52, H-6726 Szeged, Hungary Received 6 January 1998; received in revised form 2 April 1998; accepted 23 April 1998
Abstract The carboxypeptidase A-catalyzed syntheses of dipeptides from L-amino acids (Phe, Tyr, Trp, Leu and Ile) were studied in various water-miscible (acetone, acetonitrile, ethanol, methanol and 1,4-dioxane) organic solvents. The highest yield (43%) was achieved in acetonitrile with L-Phe as substrate, after a 24-h incubation. The optimal conditions of Phe–Phe synthesis in acetonitrile were determined. For maximal conversion 1.2 mM L-Phe, 1.4 mg ml − 1 enzyme and about 10% water are needed in buffered aqueous acetonitrile (pH 5.5) at 30°C. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carboxypeptidase A; Catalysis; Synthesis; Dipeptides; L-Amino acids; Organic solvents
1. Introduction In recent years, enzyme-catalyzed reactions in organic media have attracted considerable attention because of their many potential advantages, including the increased solubility of hydrophobic substrates, the shifting of thermodynamic equilibria in the synthetic direction, and the ease of both product and enzyme recovery (Dordick, 1989). However, it has been demonstrated that the enzyme properties, e.g. the catalytic parame-
* Corresponding szeged.hu
author.
E-mail:
[email protected]
ters, the substrate specificity (Kawashiro et al., 1997) and the enantioselectivity (Wescott and Klibanov, 1993), can be altered by the nature of the reaction medium. Peptides are an important class of biologically active molecules that find use in agricultural chemicals (Duke and Abbas, 1995), pharmaceuticals and hormones (Taylor and Amidon, 1995). Solid-phase chemical techniques have predominated in the preparation of peptides and peptide derivatives for use in drug discovery (Pinilla et al., 1995). However these procedures involve several drawbacks, such as the need for the protection and deprotection of amino acid functional groups, racemization and the lack of stereoselectivity.
0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00159-X
76
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
The use of enzymes allows these limitations to be overcome because of the mild reaction conditions and high specificity. Studies were earlier reported of the enzymatic syntheses of peptides through the use of various proteases (including a-chymotrypsin, trypsin, subtilisins, papain, etc.) in organic media (Clape´s et al., 1990; Hwang et al., 1995). Among the carboxypeptidases, the potential for peptide synthesis of carboxypeptidase Y from yeast has been investigated (Kunugi et al., 1997). The aim of the present work was to study the use of bovine carboxypeptidase A (CPA) for the syntheses of dipeptides in organic media, and to establish the optimal conditions of Phe – Phe synthesis.
2. Materials and methods
2.1. Materials Bovine pancreas CPA (peptidyl-L-amino acid hydrolase, EC 3.4.17.1) was obtained from Fluka AG, Switzerland. The specific activity of the enzyme was 53.8 U mg − 1. Hippuryl-L-phenylalanine and L-amino acids (Phe, Tyr, Trp, Leu and Ile) were from Sigma-Aldrich, Hungary. The precoated silica plates for TLC analysis were from Merck, Germany. Organic solvents and all other chemicals were commercial products of Reanal, Hungary.
2.2. Methods 2.2.1. Assay of enzyme acti6ity The activity of CPA was determined spectrophotometrically with hippuryl-L-phenylalanine as substrate, via the change in absorbance at 254 nm at 25°C (Folk and Schirmer, 1963). The reaction mixture (3 ml) contained 55 mM Tris – HCl buffer (pH 7.5), 500 mM NaCl and 0.5 mM hippuryl-L-phenylalanine. The reaction was initiated by the addition of 100 ml of enzyme (5 U). One unit of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 mmol of hippuryl-L-phenylalanine per min under the assay conditions.
2.2.2. Dipeptide syntheses and analyses The standard reaction mixture consisted of one or other of the above-listed L-amino acids and CPA in a total volume of 2.4 ml. First, 2.5 mM amino acid was dissolved in buffered (50 mM potassium phosphate buffer pH 5.5) aqueous organic solvent (usually acetonitrile). The reaction was started by the addition of 0.15 ml of a crystalline suspension of enzyme (190 U), and the mixture was stirred at 450 rpm for 6 h at 30°C. After appropriate incubation periods, samples were withdrawn and analyzed by TLC. The water content in the reaction mixture was determined by Karl-Fischer titration (Hansen, 1966). The precoated silica plates were developed in n-butanol/acetic acid/water (4:1:1 v/v) and dried. The plates were sprayed with ninhydrin reagent (2%) and placed in an oven at 70°C for 30 min. The spots were identified by means of standards and quantified by use of the Gelbase Pro and GelBase/GelBlot Pro computer program (UVP, UK).
3. Results
3.1. Effects of organic sol6ents The syntheses of dipeptides with CPA were carried out in different water-miscible (acetonitrile, acetone, ethanol, methanol and 1,4-dioxane) organic solvents. The water content of the reaction mixture was 8%. The results for Phe–Phe synthesis are summarized in Table 1. During a 24-h incubation, in acetone and 1,4-dioxane, LPhe underwent similar conversions, but the highest yield of dipeptide, 26.8%, was achieved in Table 1 Phe–Phe synthesis of carboxypeptidase A in different organic solvents Solvent
Log P
Yield after 24 h (%)
Acetone Ethanol Acetonitrile Methanol 1,4-Dioxane
−0.23 −0.24 −0.33 −0.76 −1.10
3.3 7.82 26.8 9.62 2.46
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
77
Fig. 1. Rates of dipeptide synthesis from different L-amino acids with CPA at 30°C. The reaction mixture (2.4 ml) contained 5 mM acid, 2 mg ml − 1 enzyme and 10% water in acetonitrile (pH 5.5).
L-amino
acetonitrile. The yield of the dipeptide synthesis did not correlate with the log P values of the solvents.
3.2. Syntheses of different dipeptides in acetonitrile For the syntheses of different dipeptides, L-Phe, and L-Ile were used in concentrations of 2.5 and 5 mM. The results of dipeptide synthesis at amino acid concentrations of 5 mM are shown in Fig. 1. The reactions were followed for 6 h at 30°C. The highest yield (12.0%) was achieved with L-Phe and the lowest with L-Tyr, for which no dipeptide formation was found. In further experiments, the conditions for Phe –Phe synthesis were studied in acetonitrile.
L-Tyr, L-Trp, L-Leu
3.3. Optimization of Phe– Phe synthesis with CPA in acetonitrile 3.3.1. Effects of water content, temperature and pH The synthesis of Phe – Phe with CPA in acetoni-
trile was performed for 6 h at different water contents (Fig. 2A) at pH 5.5, and 30°C. To achieve the highest conversion of L-Phe, a water content of about 10% was needed in the reaction mixture. The synthesis of Phe–Phe with CPA was analysed for 6 h in acetonitrile in the temperature range 0–60°C. The water content in the reaction mixture was 10%. The highest yield was achieved at 25–30°C (Fig. 2B). Effects of pH on Phe–Phe synthesis were studied in buffered aqueous acetonitrile solvent at 30°C, at 10% water content. The buffers applied were 50 mM sodium acetate buffer (pH range 5.0–5.6) or 50 mM potassium phosphate buffer (pH range 5.5–8.0). The reaction time was 6 h. The results for the pH range 5–6 are presented in Fig. 2C. The dipeptide synthesis exhibited a marked dependence on pH. The highest conversion was achieved at about pH 5.5.
3.3.2. Effects of substrate concentration The effects of the substrate concentration on the synthesis of Phe–Phe with CPA from L-Phe
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A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
Fig. 2. Effects of water content (A), temperature (B), and pH (C) on Phe – Phe synthesis with CPA in acetonitrile. The reaction was performed for 6 h.
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
79
Fig. 3. Effects of L-Phe concentration on Phe–Phe synthesis with CPA in acetonitrile at pH 5.5 and 30°C.
were studied in the L-Phe concentration range 0.67 –10 mM at pH 5.5 and 30°C. The water content in the reaction mixture was 10%. The initial rates at different L-Phe concentrations are shown in Fig. 3. At 1.2 mM L-Phe was measured the highest rate. At higher concentrations the peptide synthesis decreased.
3.3.3. Effects of enzyme concentration The synthesis of Phe – Phe with CPA was carried out in acetonitrile at pH 5.5 and 30°C at enzyme concentrations in the range 0.5 – 3.5 mg ml − 1. The incubation period was 6 h. At a water content of 10%, optimum was achieved at about enzyme concentration 1.4 mg ml − 1 (Fig. 4). 3.3.4. Dipeptide synthesis under optimized conditions The CPA-catalyzed synthesis of Phe – Phe was in acetonitrile at pH 5.5, with 1.2 mM L-Phe, 2 mg ml − 1 CPA and 10% water content studied. The results are shown in Fig. 5. The highest
conversion (43%) was achieved after 24 h and no significant increase was measured during longer incubations.
4. Discussion Little has been published on peptide synthesis via peptidases in organic solvents. Carboxypeptidase Y-catalyzed reactions (Kunugi et al., 1997) have been thoroughly investigated. Studies on peptide syntheses with proteases have focused on transpeptidation and transesterification reactions and very few authors have examined condensation reactions. Activated amino acids or N-protected amino acid derivatives have been used for synthetic reactions (Clape´s et al., 1997; Mitin et al., 1997). From a structural aspect, ten water molecules (not in contact with the surrounding solvent) are involved in the active conformation of CPA (Hartsuck and Lipscomb, 1970; Sebastian et al., 1996). Accordingly, it appeared interesting
80
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
Fig. 4. Dependence of Phe–Phe synthesis with CPA on enzyme concentration. For details, see the text.
to investigate synthetic reactions at low water content. The CPA-catalyzed condensation of LPhe was studied in different water-miscible organic solvents. The highest yield of dipeptide (43%) was achieved in acetonitrile. In neutrasecatalyzed peptide synthesis, acetonitrile was likewise used as solvent, as one of the best for peptide synthesis (Clape´s et al., 1990). The effects of organic solvents on a-chymotrypsin-catalyzed dipeptide synthesis were studied by Lozano et al. (1995). The solvent hydrophilicity was found to be a key parameter as concerns increase of the synthetic activity of chymotrypsin. The highest activity was observed in the most hydrophilic solvent (DMS). A study of the effect of substrate concentration on the CPA-catalyzed condensation reaction indicated that 1.2 mM L-Phe was required for maximum conversion. On further increase of the substrate concentration, the yield of dipeptide decreased, probably because of substrate inhibition (Hartsuck and Lipscomb, 1970). An apparent substrate inhibition has also been observed for carboxypeptidase Y-catalyzed peptide condensation reactions (Kunugi et al., 1992). A drastic
reduction of reaction yield was found with more than 30 mM of carboxylic component for pepsincatalyzed peptide synthesis in a biphasic system (Bemquerer et al., 1994). A relatively high amount of water (10%) and 1.4 mg ml − 1 carboxypeptidase A were necessary to achieve maximum conversion at 30°C. The hydrophobicity and water activity relationship of water-miscible aprotic solvents was studied for a-chymotrypsin-catalyzed peptide synthesis from activated amino acids and maximum synthetic activity was measured at a water activity higher than 0.8 in all solvents (Lozano et al., 1997). To obtain the maximum synthetic activity, an appropriate concentration of uncharged carboxyl groups of L-Phe is probably needed. The CPAcatalyzed peptide synthesis was maximum in a slightly acidic milieu (pH 5.5). Change of the pH greatly influences the ionization state of the enzyme. This might explain why the synthetic reactions with proteases were carried out between pH 3.2 and pH 7, depending on the enzymes and substrates (Wayne and Fruton, 1983; Mitin et al., 1997).
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
81
Fig. 5. Phe–Phe synthesis with CPA under optimized conditions. For details, see the text.
Our preliminary results on the rate of the CPAcatalyzed condensation reaction under optimized conditions reveal an apparent equilibrium with a highest yield of 43% dipeptide, which did not increase during longer incubation, probably because of the inactivation of the enzyme. In the carboxypeptidase Y-catalyzed dipeptide syntheses from Cbz-Phe and Gly-NH2, dioxane in 50% concentration proved the best cosolvent. The yield of dipeptide condensation in the different solvents with a reaction time of 5 h at pH 6.3 and 30°C varied between 10 and 50% (Kunugi et al., 1997). The yields of peptide synthesis from N-protected and activated L-amino acids with proteolytic enzymes in organic media ranged from 57 to 99% for different incubation times (24 – 72 h).
References Bemquerer, M.P., Adlercreutz, P., Tominaga, M., 1994. Pepsin catalyzed peptide synthesis in organic media: studies with free and immobilized enzyme. Int. J. Pept. Protein Res. 44, 448 – 456.
Clape´s, P., Adlercreutz, P., Mattiasson, B., 1990. Enzymatic peptide synthesis in organic media: a comparative study of water miscible and water immiscible solvent systems. J. Biotechnol. 15, 323 – 338. Clape´s, P., Pera, E., Torres, J.L., 1997. Peptide bond formation by the industrial protease, neutrase in organic media. Biotechnol. Lett. 19, 1023 – 1026. Dordick, J.S., 1989. Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol. 11, 194 – 211. Duke, S.D., Abbas, H.K., 1995. Natural products with potential use as herbicides. ACS Symp. Ser. 582, 348 – 362. Folk, J.E., Schirmer, E.W., 1963. The porcine pancreatic carboxypeptidase A system. J. Biol. Chem. 238, 3884 – 3890. Hansen, H., 1966. Hoppe-Seyler/Thierfelder Handbuch der physiologisch- und pathologisch-chemischen Analyse. Springer, Berlin, ch. 6, p. 84. Hartsuck, J.A., Lipscomb, N., 1970. The Enzymes, 3rd ed. Academic Press, New York, pp. 1 – 56. Hwang, K.A., Lee, S.B., Lee, K.H., 1995. Effect of water activity on thermolysin-catalyzed peptide synthesis in organic solvents. Biotechnol. Lett. 17, 71 – 76. Kawashiro, K., Sugahara, H., Sugiyama, S., Hayashi, H., 1997. Effect of organic solvents on enantioselectivity of protease catalysis. Biotechnol. Bioeng. 53, 26 – 31. Kunugi, S., Suzuki, N., Yokoyama, M., Nomura, A., 1992. Carboxypeptidase Y reactions in organic solvents. Ann. NY Acad. Sci. 672, 323 – 328.
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Kunugi, S., Suzuki, N., Nishimoto, S., Morisawa, T., Yoshida, M., 1997. Kinetic study of carboxypeptidase Y-catalyzed peptide condensation reactions in aqueous-organic solvent. Biocatal. Biotransform. 14, 205–217. Lozano, P., de Diego, T., Iborra, J.L., 1995. Effect of watermiscible aprotic solvents on kyotorphin synthesis catalyzed by immobilized a-chymotrypsin. Biotechnol. Lett. 17, 603 – 608. Lozano, P., de Diego, T., Iborra, J.L., 1997. Hydrophobicity and water activity relationship of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by a-chymotrypsin. Biotechnol. Lett. 19, 1005–1009. Mitin, Y.V., Braun, K., Kuhl, P., 1997. Papain catalyzed synthesis of glyceryl esters of N-protected amino acids and peptides for the use of trypsin catalyzed peptide synthesis. Biotechnol. Bioeng. 54, 287–290.
Pinilla, C., Appel, L., Blondelle, S., Dooley, C., Dorner, B., Eichler, J., Ostresh, J., Houghten, R.A., 1995. A review of the utility of soluble peptide combinatorial libraries. Biopolymers 37, 221 – 240. Sebastian, J.F., Liang, G., Jabarin, A., Thomas, K., Bonnie Wu, H., 1996. Effect of enzyme-substrate interactions away from the reaction site on carboxypeptidase A catalysis. Bioorg. Chem. 24, 290 – 303. Taylor, M.D., Amidon, G.L., 1995. Peptide Based Drug Design: Controlling Transport and Metabolism. ACS, Washington, DC. Wescott, C.R., Klibanov, A.M., 1993. Solvent variation inverts substrate specificity of enzyme. J. Am. Chem. Soc. 115, 1629 – 1631. Wayne, S.I., Fruton, J.S., 1983. Thermolysin-catalyzed peptide bond synthesis. Proc. Natl. Acad. Sci. USA 80, 3241 – 3244.
.
Carboxypeptidase A-catalyzed dipeptide synthesis in organic media Ade´l Ve´rtesi, L. Maria Simon * Department of Biochemistry, Jo´zsef Attila Uni6ersity, Ko¨ze´pfasor 52, H-6726 Szeged, Hungary Received 6 January 1998; received in revised form 2 April 1998; accepted 23 April 1998
Abstract The carboxypeptidase A-catalyzed syntheses of dipeptides from L-amino acids (Phe, Tyr, Trp, Leu and Ile) were studied in various water-miscible (acetone, acetonitrile, ethanol, methanol and 1,4-dioxane) organic solvents. The highest yield (43%) was achieved in acetonitrile with L-Phe as substrate, after a 24-h incubation. The optimal conditions of Phe–Phe synthesis in acetonitrile were determined. For maximal conversion 1.2 mM L-Phe, 1.4 mg ml − 1 enzyme and about 10% water are needed in buffered aqueous acetonitrile (pH 5.5) at 30°C. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Carboxypeptidase A; Catalysis; Synthesis; Dipeptides; L-Amino acids; Organic solvents
1. Introduction In recent years, enzyme-catalyzed reactions in organic media have attracted considerable attention because of their many potential advantages, including the increased solubility of hydrophobic substrates, the shifting of thermodynamic equilibria in the synthetic direction, and the ease of both product and enzyme recovery (Dordick, 1989). However, it has been demonstrated that the enzyme properties, e.g. the catalytic parame-
* Corresponding szeged.hu
author.
E-mail:
[email protected]
ters, the substrate specificity (Kawashiro et al., 1997) and the enantioselectivity (Wescott and Klibanov, 1993), can be altered by the nature of the reaction medium. Peptides are an important class of biologically active molecules that find use in agricultural chemicals (Duke and Abbas, 1995), pharmaceuticals and hormones (Taylor and Amidon, 1995). Solid-phase chemical techniques have predominated in the preparation of peptides and peptide derivatives for use in drug discovery (Pinilla et al., 1995). However these procedures involve several drawbacks, such as the need for the protection and deprotection of amino acid functional groups, racemization and the lack of stereoselectivity.
0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0168-1656(98)00159-X
76
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
The use of enzymes allows these limitations to be overcome because of the mild reaction conditions and high specificity. Studies were earlier reported of the enzymatic syntheses of peptides through the use of various proteases (including a-chymotrypsin, trypsin, subtilisins, papain, etc.) in organic media (Clape´s et al., 1990; Hwang et al., 1995). Among the carboxypeptidases, the potential for peptide synthesis of carboxypeptidase Y from yeast has been investigated (Kunugi et al., 1997). The aim of the present work was to study the use of bovine carboxypeptidase A (CPA) for the syntheses of dipeptides in organic media, and to establish the optimal conditions of Phe – Phe synthesis.
2. Materials and methods
2.1. Materials Bovine pancreas CPA (peptidyl-L-amino acid hydrolase, EC 3.4.17.1) was obtained from Fluka AG, Switzerland. The specific activity of the enzyme was 53.8 U mg − 1. Hippuryl-L-phenylalanine and L-amino acids (Phe, Tyr, Trp, Leu and Ile) were from Sigma-Aldrich, Hungary. The precoated silica plates for TLC analysis were from Merck, Germany. Organic solvents and all other chemicals were commercial products of Reanal, Hungary.
2.2. Methods 2.2.1. Assay of enzyme acti6ity The activity of CPA was determined spectrophotometrically with hippuryl-L-phenylalanine as substrate, via the change in absorbance at 254 nm at 25°C (Folk and Schirmer, 1963). The reaction mixture (3 ml) contained 55 mM Tris – HCl buffer (pH 7.5), 500 mM NaCl and 0.5 mM hippuryl-L-phenylalanine. The reaction was initiated by the addition of 100 ml of enzyme (5 U). One unit of enzyme activity was defined as the amount of enzyme that hydrolyzed 1 mmol of hippuryl-L-phenylalanine per min under the assay conditions.
2.2.2. Dipeptide syntheses and analyses The standard reaction mixture consisted of one or other of the above-listed L-amino acids and CPA in a total volume of 2.4 ml. First, 2.5 mM amino acid was dissolved in buffered (50 mM potassium phosphate buffer pH 5.5) aqueous organic solvent (usually acetonitrile). The reaction was started by the addition of 0.15 ml of a crystalline suspension of enzyme (190 U), and the mixture was stirred at 450 rpm for 6 h at 30°C. After appropriate incubation periods, samples were withdrawn and analyzed by TLC. The water content in the reaction mixture was determined by Karl-Fischer titration (Hansen, 1966). The precoated silica plates were developed in n-butanol/acetic acid/water (4:1:1 v/v) and dried. The plates were sprayed with ninhydrin reagent (2%) and placed in an oven at 70°C for 30 min. The spots were identified by means of standards and quantified by use of the Gelbase Pro and GelBase/GelBlot Pro computer program (UVP, UK).
3. Results
3.1. Effects of organic sol6ents The syntheses of dipeptides with CPA were carried out in different water-miscible (acetonitrile, acetone, ethanol, methanol and 1,4-dioxane) organic solvents. The water content of the reaction mixture was 8%. The results for Phe–Phe synthesis are summarized in Table 1. During a 24-h incubation, in acetone and 1,4-dioxane, LPhe underwent similar conversions, but the highest yield of dipeptide, 26.8%, was achieved in Table 1 Phe–Phe synthesis of carboxypeptidase A in different organic solvents Solvent
Log P
Yield after 24 h (%)
Acetone Ethanol Acetonitrile Methanol 1,4-Dioxane
−0.23 −0.24 −0.33 −0.76 −1.10
3.3 7.82 26.8 9.62 2.46
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
77
Fig. 1. Rates of dipeptide synthesis from different L-amino acids with CPA at 30°C. The reaction mixture (2.4 ml) contained 5 mM acid, 2 mg ml − 1 enzyme and 10% water in acetonitrile (pH 5.5).
L-amino
acetonitrile. The yield of the dipeptide synthesis did not correlate with the log P values of the solvents.
3.2. Syntheses of different dipeptides in acetonitrile For the syntheses of different dipeptides, L-Phe, and L-Ile were used in concentrations of 2.5 and 5 mM. The results of dipeptide synthesis at amino acid concentrations of 5 mM are shown in Fig. 1. The reactions were followed for 6 h at 30°C. The highest yield (12.0%) was achieved with L-Phe and the lowest with L-Tyr, for which no dipeptide formation was found. In further experiments, the conditions for Phe –Phe synthesis were studied in acetonitrile.
L-Tyr, L-Trp, L-Leu
3.3. Optimization of Phe– Phe synthesis with CPA in acetonitrile 3.3.1. Effects of water content, temperature and pH The synthesis of Phe – Phe with CPA in acetoni-
trile was performed for 6 h at different water contents (Fig. 2A) at pH 5.5, and 30°C. To achieve the highest conversion of L-Phe, a water content of about 10% was needed in the reaction mixture. The synthesis of Phe–Phe with CPA was analysed for 6 h in acetonitrile in the temperature range 0–60°C. The water content in the reaction mixture was 10%. The highest yield was achieved at 25–30°C (Fig. 2B). Effects of pH on Phe–Phe synthesis were studied in buffered aqueous acetonitrile solvent at 30°C, at 10% water content. The buffers applied were 50 mM sodium acetate buffer (pH range 5.0–5.6) or 50 mM potassium phosphate buffer (pH range 5.5–8.0). The reaction time was 6 h. The results for the pH range 5–6 are presented in Fig. 2C. The dipeptide synthesis exhibited a marked dependence on pH. The highest conversion was achieved at about pH 5.5.
3.3.2. Effects of substrate concentration The effects of the substrate concentration on the synthesis of Phe–Phe with CPA from L-Phe
78
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
Fig. 2. Effects of water content (A), temperature (B), and pH (C) on Phe – Phe synthesis with CPA in acetonitrile. The reaction was performed for 6 h.
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
79
Fig. 3. Effects of L-Phe concentration on Phe–Phe synthesis with CPA in acetonitrile at pH 5.5 and 30°C.
were studied in the L-Phe concentration range 0.67 –10 mM at pH 5.5 and 30°C. The water content in the reaction mixture was 10%. The initial rates at different L-Phe concentrations are shown in Fig. 3. At 1.2 mM L-Phe was measured the highest rate. At higher concentrations the peptide synthesis decreased.
3.3.3. Effects of enzyme concentration The synthesis of Phe – Phe with CPA was carried out in acetonitrile at pH 5.5 and 30°C at enzyme concentrations in the range 0.5 – 3.5 mg ml − 1. The incubation period was 6 h. At a water content of 10%, optimum was achieved at about enzyme concentration 1.4 mg ml − 1 (Fig. 4). 3.3.4. Dipeptide synthesis under optimized conditions The CPA-catalyzed synthesis of Phe – Phe was in acetonitrile at pH 5.5, with 1.2 mM L-Phe, 2 mg ml − 1 CPA and 10% water content studied. The results are shown in Fig. 5. The highest
conversion (43%) was achieved after 24 h and no significant increase was measured during longer incubations.
4. Discussion Little has been published on peptide synthesis via peptidases in organic solvents. Carboxypeptidase Y-catalyzed reactions (Kunugi et al., 1997) have been thoroughly investigated. Studies on peptide syntheses with proteases have focused on transpeptidation and transesterification reactions and very few authors have examined condensation reactions. Activated amino acids or N-protected amino acid derivatives have been used for synthetic reactions (Clape´s et al., 1997; Mitin et al., 1997). From a structural aspect, ten water molecules (not in contact with the surrounding solvent) are involved in the active conformation of CPA (Hartsuck and Lipscomb, 1970; Sebastian et al., 1996). Accordingly, it appeared interesting
80
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
Fig. 4. Dependence of Phe–Phe synthesis with CPA on enzyme concentration. For details, see the text.
to investigate synthetic reactions at low water content. The CPA-catalyzed condensation of LPhe was studied in different water-miscible organic solvents. The highest yield of dipeptide (43%) was achieved in acetonitrile. In neutrasecatalyzed peptide synthesis, acetonitrile was likewise used as solvent, as one of the best for peptide synthesis (Clape´s et al., 1990). The effects of organic solvents on a-chymotrypsin-catalyzed dipeptide synthesis were studied by Lozano et al. (1995). The solvent hydrophilicity was found to be a key parameter as concerns increase of the synthetic activity of chymotrypsin. The highest activity was observed in the most hydrophilic solvent (DMS). A study of the effect of substrate concentration on the CPA-catalyzed condensation reaction indicated that 1.2 mM L-Phe was required for maximum conversion. On further increase of the substrate concentration, the yield of dipeptide decreased, probably because of substrate inhibition (Hartsuck and Lipscomb, 1970). An apparent substrate inhibition has also been observed for carboxypeptidase Y-catalyzed peptide condensation reactions (Kunugi et al., 1992). A drastic
reduction of reaction yield was found with more than 30 mM of carboxylic component for pepsincatalyzed peptide synthesis in a biphasic system (Bemquerer et al., 1994). A relatively high amount of water (10%) and 1.4 mg ml − 1 carboxypeptidase A were necessary to achieve maximum conversion at 30°C. The hydrophobicity and water activity relationship of water-miscible aprotic solvents was studied for a-chymotrypsin-catalyzed peptide synthesis from activated amino acids and maximum synthetic activity was measured at a water activity higher than 0.8 in all solvents (Lozano et al., 1997). To obtain the maximum synthetic activity, an appropriate concentration of uncharged carboxyl groups of L-Phe is probably needed. The CPAcatalyzed peptide synthesis was maximum in a slightly acidic milieu (pH 5.5). Change of the pH greatly influences the ionization state of the enzyme. This might explain why the synthetic reactions with proteases were carried out between pH 3.2 and pH 7, depending on the enzymes and substrates (Wayne and Fruton, 1983; Mitin et al., 1997).
A. Ve´rtesi, L.M. Simon / Journal of Biotechnology 66 (1998) 75–82
81
Fig. 5. Phe–Phe synthesis with CPA under optimized conditions. For details, see the text.
Our preliminary results on the rate of the CPAcatalyzed condensation reaction under optimized conditions reveal an apparent equilibrium with a highest yield of 43% dipeptide, which did not increase during longer incubation, probably because of the inactivation of the enzyme. In the carboxypeptidase Y-catalyzed dipeptide syntheses from Cbz-Phe and Gly-NH2, dioxane in 50% concentration proved the best cosolvent. The yield of dipeptide condensation in the different solvents with a reaction time of 5 h at pH 6.3 and 30°C varied between 10 and 50% (Kunugi et al., 1997). The yields of peptide synthesis from N-protected and activated L-amino acids with proteolytic enzymes in organic media ranged from 57 to 99% for different incubation times (24 – 72 h).
References Bemquerer, M.P., Adlercreutz, P., Tominaga, M., 1994. Pepsin catalyzed peptide synthesis in organic media: studies with free and immobilized enzyme. Int. J. Pept. Protein Res. 44, 448 – 456.
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