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Synthesis of delicious peptide fragments catalyzed by immobilized proteases
Heterogeneous Catalysis and Fine Chemicals IV H.U. Blaser, A. Baiker and R. Prins (editors) © 1997 Elsevier Science B.V. All rights reserved.
Synthesis of delicious immobilized proteases.
peptide
657
fragments
catalyzed
by
Romero, M.D.; Aguado, J.; Guerra, M.J.; Alvaro, G.*; Navarro, R.; Rubio, E. Chemical Engineering Department. Faculty of Chemistry. Complutense University of Madrid, 28040 Madrid, Spain * Chemical Engineering Department. Faculty of Sciences. Autonoma University of Barcelona, 08193 Bellaterra, Barcelona, Spain 1. ABSTRACT The synthesis of benzyloxycarbonyl-lysine-glycine methyl ester (CBZLys-Gly-OMe) and benzyloxycarbonyl-serine-leucine methyl ester (CBZ-SerLeu-OMe) have been carried out in aqueous organic systems catalyzed by immobilized trypsin and thermolysin respectively. The dipeptides originated by the elimination of benzyloxycarbonyl and methoxy groups are two fragments of the delicious peptide, which is an octapeptide of industrial interest with a taste profile umami/sour. In both synthesis we have studied the influence of pH, temperature and substrate concentration on the yield and the rate of synthesis. In the optimum conditions, the synthetic yields were 80% for CBZ-Lys-Gly-OMe and 100% for CBZ-Ser-Leu-OMe. Both synthesis present an inhibition effect by the acyl donor when the concentrations of CBZ-Lys and CBZ-Ser are higher than 20 mM. In the synthesis of CBZ-Ser-Leu-OMe an anomalous role of thermolysin has been observed forming oligopeptides of higher molecular weight by addition of new molecules of aminoacid Leu-OMe. 2. INTRODUCTION In recent years there has been an increasing interest in the isolation and characterization of biologically active peptides and flavor peptides of medical, pharmaceutical and food interest (1). Although the chemical peptide synthesis have been successfully applied for products of biological and industrial interest, this synthetic methodology is still in need of innovations. The use of enzymes provide an alternative to chemical peptide synthesis, for which the most notable advantage lies on the stereo and regiospecifically peptide bond formation without the need of group protection and very mild operating conditions (2).
658 In protease-mediated peptide synthesis, the enzymatic specificity prevents the formation of by-products often formed in the course of conventional chemical synthesis. Short oligopeptides play an important role in the sensorial appreciation of food and much attention has been paid to the relationship between the structure of peptides and their taste, based on four basic taste sensations (sweet, bitter, sour and salty). The delicious peptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala, is an octapeptide which was isolated from the gravy of beef and its primary structure was proposed in 1978 (3). It possesses a taste profile umami/sour (4), and it has a great interest in the food industry. Three fragments of the delicious peptide sequence: lysine-glycine, serine-leucine-alanine and aspartic acid-glutamic acid-glutamic acid possess separately umami/salty, bitter and sour taste respectively but mixtures or combinations of them produce a similar taste of that corresponding to the complete octapeptide. The synthesis of CBZ-Lys-Gly-OMe and CBZ-Ser-LeuOMe were carried out using immobilized trypsin and thermolysin. We have studied the influence of reaction medium (pH, temperature and substrate concentration) on the yield and initial reaction rate of synthesis. 3. EXPERIMENTAL 3.1. Materials Agarose gels lOB-CL were supplied free of charge by Hispanagar S.A. (Burgos, Spain). Commercial thermolysin from Bacillus thermoproteolyticus rokko and bovine trypsin Type III from SIGMA Co. were used as the source of enzymes. Benzyloxycarbonyl-lysine, glycine methyl ester, benzoylarginine ethyl ester (BAEE), benzyloxycarbonyl-serine, leucine methyl ester and furyl-acryloyl-glycil-leucinamide (FAGLA), were purchased from Sigma Co. Analytical grades of other reagent and solvents were used. 3.2. Activation of agarose gels Agarose gel lOB-CL containing 200 |Limol aldehyde/ml gel was prepared by etherification of agarose gels with glycidol and further oxidation with periodate as described in the literature (5). 3.3. Immobilization of trypsin on the activated gels Immobilized trypsin-agarose was prepared as reported elsewhere (6). Trypsin immobilization was carried out in presence of borate buffer (50 mM) containing benzamidine (75 mM) at pH 10 and 25°C. After 72 hours, the trypsin-agarose was reduced with NaBH4 (Img/ml) for 30 minutes. Finally, the immobilized preparation was washed with water and stored at 4°C. The derivative obtained had around 320 units/ml gel using benzoyl-arginine ethyl ester (BAEE) as substrate. One unit of enzyme activity is defined as
659 the amount of enzyme that hydrolyzes 1 |imol/min of substrate under the assay conditions. 3.4. Immobilization of thermolysin on the activated gels. Immobilized thermolysin-agarose was prepared using the method described above. Thermolysin immobilization was carried out in the presence of borate buffer (50 mM) containing CaCk (10 mM) at pH 10 and 25°C (7), (8). After 7 hours, the thermolysin-agarose was reduced with NaBH4 (1 mg/ml) during 30 minutes. Finally the immobilized thermolysin was washed with CaCk (10 mM) and stored at 4°C. The derivative obtained had around 550 units/ml gel using furyl-acryloyl-glycyl-leucinamide (FAGLA) as substrate (9). One unit is the amount of enzyme that hydrolyzes 1 jamol/min of substrate under the assay conditions. 3.5. Synthesis of N-benzyloxycarbonyl-lysine-glycine methyl ester. All the experiments were carried out in stoppered flasks with 25 ml of capacity placed in a thermostatic bath. The reaction medium, 0.1 M phosphate/butanediol/dioxane (1:3:6) (v/v) containing the substrates was adjusted to the appropriate pH. The reactions were started by adding 640 units of immobilized trypsin. Aliquots were taken at different times and analyzed by HPLC equipped with an octadecyl silica 150x46 mm column and UV-Vis detector at X, = 254 nm. Phosphate buffer 0.1 M (pH = 2.3) and acetonitrile (80:20) (v/v) was used as eluent at a flow rate of 1 ml/min. 3.6. Synthesis of N-benzyloxyearbonyl-serine-leucine methyl ester. The synthesis was also carried out in similar stoppered flasks placed in a thermostated bath. The reaction medium (aqueous pH 7 and biphasic acetate buffer 0.1 M, CaCb 10 mM/ethyl acetate (1:1) (v/v)), containing the substrates were adjusted to the desired pH. Reactions were started by adding 1100 units of immobilized thermolysin. Aliquots were taken at different times and analyzed by HPLC as above mentioned. In this case the eluent was phosphate buffer 0.1 M (pH = 2.3) and acetonitrile (62:38) (v/v). 4. RESULTS AND DISCUSSION 4.1. Synthesis of N-benzyloxycarbonyl-lysine-glycine methyl ester. The presence of high concentration of organic solvents is essential for achieving high synthetic yields in peptide synthesis under thermodynamically controlled conditions. CBZ-Lys-Gly-OMe has been synthesized from CBZ-Lys and Gly-OMe in a mixture system 0.1 M phosphate/butanediol/dioxane (1:3:6) (v/v) and catalyzed by immobilized trypsin on agarose gels. We have observed that some reaction conditions, such as pH, temperature and substrate concentration, have a great influence on enzyme activity and product yields.
660 Effect of P H Figure 1 shows the influence of the solution pH on the yield and initial rate of synthesis of CBZ-Lys-Gly-OMe at a temperature of 25°C and [CBZ-Lys] = [Gly-OMe] = 20 mM. The maximum yield is achieved for pH values around 6-6.5. Under thermodynamically controlled conditions, the peptide synthesis occurs between the non-ionic forms of the acyl-donor (CBZ-Lys) and the nucleophile (Gly-OMe). The concentration of these nonionic forms depends on the pH, since an intermediate value between both pK (pHopt = V^2[pKa +pKb]) is needed in order to achieve high synthetic yields. On the other hand, the reaction rate increases up to pH 7, which is in agreement with the results obtained in the synthesis of the peptide benzoylarginine-leucinamide catalyzed by immobilized trypsin (10), where the authors suggest the nucleophilic attack of the non-ionic form of the nucleophile on the acyl-enzyme complex as the controlling step of the peptide reaction. Effect of temperature As it was expected, the reaction rate increases with increasing temperature (figure 2). On the other hand, since the peptide synthesis is generally an exothermic reaction (2), the peptide yield slightly decreases as the temperature is increased.
"20 3Cr THVIPB^-RJRETO
Figure 1. Effect of pH on the synthesis of CBZ-Lys-Gly-OMe. T= 25°C, [CBZ-Lys]=[Gly-OMe]= 20 mM.
Figure 2. Effect of temperature on the synthesis of CBZ-Lys-GlyOMe. pH= 6.5, [CBZ-Lys]=[GlyOMe]= 20 mM.
Kinetic analysis Figure 3 shows the initial reaction rate in experiments carried out at pH 6.5, temperature of 30°C and [Gly-OMe] = 20 mM when the CBZ-Lys concentration changes in the range 2-40 mM. Inhibition is observed by this aminoacid at concentrations higher than 20 mM. However, when the concentration of Gly-OMe is changed, while keeping constant CBZ-Lys concentration, a Michaelis-Menten kinetic behaviour is observed. Figure 4 shows the results of these experiments.
661
20 • 30 • 40 CBZ-LYaNE(rTM)
Figure 3. Effect of CBZ-Lys concentration on the synthesis of CBZ-Lys-Gly-OMe. T=30°C, pH= 6.5, [Gly-OMe]= 20 mM.
20 30 40 aYCINEMETl-YLE5TER(rTiVI)
Figure 4. Effect of Gly-OMe concentration on the synthesis of CBZ-Lys-Gly-OMe. T=30°C, pH= 6.5, [CBZ-Lys]= 4 mM.
4.2. Synthesis of N-benzyloxycarbonyl-serine-leucine methyl ester. CBZ-Ser-Leu-OMe synthesis has been carried out starting from CBZSer and Leu-OMe, using free thermolysin in a monophasic system, and thermolysin immobilized-stabilized on agarose gels, in a biphasic system. The influence of different variables such as : pH, polarity of the reaction medium, temperature and substrate concentrations on the dipeptide yield and reaction rate has been studied. Synthesis of CBZ-Ser-Leu-OMe with free thermolysin. The dipeptide synthesis with free thermolysin (2 mg/ml) was carried out in water 10 mM of CbCa at pH 6,8 and 20 °C. The yield of peptide was below 10%, due to some reaction byproducts coming from the hydrolysis of methyl ester group of the peptide and consequent product hydrolysis as is shown in the following reactions : CBZ-Ser-Leu-OMe
CBZ-Ser-Leu + MeOH
[Reaction 1]
CBZ-Ser-Leu
CBZ-Ser + Leu
[Reaction 2]
The low yield obtained in this reaction needs the use of biphasic media to obtain high yields of the desired product avoiding the ester hydrolysis. Synthesis of CBZ-Ser-Leu-OMe with thermolysin immobilized Thermolysin immobilized on agarose gels as described in the experimental section was used as catalyst in a biphasic reaction system (acetate buffer 0,1 M and 10 mM CaCk/ethyl acetate (1:1) (v/v)). This medium could prevent the secondary reactions [1] and [2], since the dipeptide is more soluble in the organic phase than in water, and it allows to achieve high synthetic yields by removing the product from the aqueous phase.
662 Effect of pH Figure 5 shows the yields of CBZ-Ser-Leu-OMe and the reaction rate obtained at 20°C and substrates concentration of 25 mM for a range of pH from 5.5 to 8. The maximum reaction rate is observed at pH 7 which is in agreement with the optimum conditions for this enzyme (11). Also a yield increase is observed as the pH is decreased, a feature possibly explained taking into account the influence of pH on partition coefficients (12) of both substrates between two phases aqueous/organic. Thus, an optimum pH is expected to be around 5.5-6. Effect of temperature Figure 6 shows the influence of temperature in the range 20-40°C when the synthesis is carried out at pH 7. An increase of temperature increases the reaction rate and decreases slightly the synthesis yields due to the exothermicity of these reactions, as mentioned above.
"20 30 m~ TBVIFe^TURETO
Figure 5. Effect of pH on the synthesis of CBZ-Ser-Leu-OMe. T= 20°C, [CBZ-Ser]=[Leu-OMe]= 25 mM.
Figure 6. Effect of temperature on the synthesis of CBZ-Ser-LeuOMe. pH=7.0, [CBZ-Ser]= [LeuOMe]= 25 mM.
Effect of presence of ammonium sulphate in the reaction medium. The presence of this salt enhances the yields as well as the reaction rate up a concentration 2.8 mM. Table 1 shows both the yield and reaction rate in peptide synthesis of CBZ-Ser-Leu-0-Me at pH 7, 20°C and [CBZ-Ser] = [Leu-OMe] = 50 mM at different salt concentrations. Table 1. Effect of Ammonium sulphate concentration (mM) 0 1 2 Vox 104 1.63 1.83 2.83 (nmol/min/UFAGLA) 32.2 40.2 50.3 Yield (%)
2.8 21.01
3.56 6.27
96.0
70.4
663 It is noteworthy the favourable effect on the peptide yield produced by the ammonium sulphate up concentration 2.8 mM. The presence of this salt seems to improve the hydrophobic adsorption of the nucleophile on the active centre of thermolysin. Effect of substrate concentration Figure 7 shows the synthesis yields and reaction rates at pH 6.0, 30°C and constant concentration of Leu-OMe, 25 mM, varying [CBZ-Ser] up to 100 mM. CBZ-Ser concentration exerts a strong inhibition on the reaction rate for concentration higher than 25 mM, while the reaction yields decrease continuously. On the other hand, figure 8 shows the increase in the reaction rate in all the range of Leu-OMe concentration, reaching yields values around 100% for [Leu-OMe] = 150 mM.
40 CBZ-SBRINE(rTiVI)
Figure 7. Effect of CBZ-Ser concentration on the synthesis of CBZ-Ser-Leu-OMe. T=30°C, pH=: 6.0, [Leu-OMe]= 25 mM.
80
•
1^0
1 ^
LELX:iNEMETH'LE5THR (nM)
Figure 8. Effect of Leu-OMe concentration on the synthesis of CBZ-Ser-Leu-OMe. T=30°C, pH= 6.0, [CBZ-Ser]= 25 mM.
For reaction times longer than 50 hours, the formation of reaction byproducts with high molecular weight, probably oligopeptides, has been detected. This observation could support the yield decrease observed in figure 7 where the reaction rate is very low and therefore the equilibrium yield can not be reached. The formation of oligopeptides come probably from the attack of a molecule of nucleophile (Leu-OMe) to a dipeptide previously formed, to give tripeptides or another oligopeptides. This role of thermolysin has been described by Morihara (13) in the synthesis of CBZ-Leu-Leu, CBZ-Phe-Leu, CBZ-Gly-Leu and CBZ-Leu-Leu-Leu-Leu-NH2. 5. CONCLUSIONS The synthesis of CBZ-Lys-Gly-OMe and CBZ-Ser-Leu-OMe could be carried out in aqueous organic systems using immobilized trypsin and
664 thermolysin respectively. The obtained yields were 80% for CBZ-LysGlyOMe and 100% for CBZ-Ser-LeuOMe in mild conditions, temperature 30°C, pH 6,0-6,5 and in the presence of organic solvents. An inhibition effect was observed with both acyl donors (CBZ-Lys and CBZ-Ser) for concentrations higher than 20 mM, whereas the nucleophiles (Gly-OMe and Leu-OMe) show a kinetic behaviour without inhibition.
REFERENCES 1. Gill, R. Lopez-Fandino, X. Jorba, N. Vulfson, Enzyme Microb. Technol. 18 (1996) 162. 2. W. KuUmann, Enzymatic peptide synthesis. CRC Press Inc. Eds., Florida. USA. (1987). 3. Y. Yamasaki, K. Maekawa, Agric. Biol. Chem. 42 (1978) 1761 4. Y. Yamasaki, K. Maekawa, Agric. Biol. Chem. 44 (1980) 93. 5. J.M. Guisan, Enzyme Microb. Technol. 10 (1988) 375 6. R.M. Blanco, J.J. Calvete, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 353 7. J.M. Guisan, G. Alvaro, J. Aguado, M.D. Romero, M.J. Guerra, E. Polo, Rev. Real. Acad. C.C. Ex. Fis. YNat. 88 (1994) 8. M.D. Romero, J. Aguado, J.M. Guisan, M.J. Guerra, E. Pardo, Proceedings of the Specialized Catalysis Group Congress, (1995) Peniscola. Spain 9. J. Feder, Biochem. and Biophys. Res. Commun, 32 (1968) 326 10. R.M. Blanco, G. Alvaro, J.C. Tercero, J.M. Guisan, Journal of Molecular Catalysis, 73(1992)97 11. S. Kunugi, H. Hirohara, N. Ise, Eur. J. Biochem. 124 (1982) 157 12. K. Nakanishi, Y. Kimura, R. Matsuno, Eur. J. Biochem. 161 (1986) 541 13. K. Morihara, H. Tsuzuki, T. Oka, Biochem. Biophys. Res. Commum. 84 (1978) 95
Synthesis of delicious immobilized proteases.
peptide
657
fragments
catalyzed
by
Romero, M.D.; Aguado, J.; Guerra, M.J.; Alvaro, G.*; Navarro, R.; Rubio, E. Chemical Engineering Department. Faculty of Chemistry. Complutense University of Madrid, 28040 Madrid, Spain * Chemical Engineering Department. Faculty of Sciences. Autonoma University of Barcelona, 08193 Bellaterra, Barcelona, Spain 1. ABSTRACT The synthesis of benzyloxycarbonyl-lysine-glycine methyl ester (CBZLys-Gly-OMe) and benzyloxycarbonyl-serine-leucine methyl ester (CBZ-SerLeu-OMe) have been carried out in aqueous organic systems catalyzed by immobilized trypsin and thermolysin respectively. The dipeptides originated by the elimination of benzyloxycarbonyl and methoxy groups are two fragments of the delicious peptide, which is an octapeptide of industrial interest with a taste profile umami/sour. In both synthesis we have studied the influence of pH, temperature and substrate concentration on the yield and the rate of synthesis. In the optimum conditions, the synthetic yields were 80% for CBZ-Lys-Gly-OMe and 100% for CBZ-Ser-Leu-OMe. Both synthesis present an inhibition effect by the acyl donor when the concentrations of CBZ-Lys and CBZ-Ser are higher than 20 mM. In the synthesis of CBZ-Ser-Leu-OMe an anomalous role of thermolysin has been observed forming oligopeptides of higher molecular weight by addition of new molecules of aminoacid Leu-OMe. 2. INTRODUCTION In recent years there has been an increasing interest in the isolation and characterization of biologically active peptides and flavor peptides of medical, pharmaceutical and food interest (1). Although the chemical peptide synthesis have been successfully applied for products of biological and industrial interest, this synthetic methodology is still in need of innovations. The use of enzymes provide an alternative to chemical peptide synthesis, for which the most notable advantage lies on the stereo and regiospecifically peptide bond formation without the need of group protection and very mild operating conditions (2).
658 In protease-mediated peptide synthesis, the enzymatic specificity prevents the formation of by-products often formed in the course of conventional chemical synthesis. Short oligopeptides play an important role in the sensorial appreciation of food and much attention has been paid to the relationship between the structure of peptides and their taste, based on four basic taste sensations (sweet, bitter, sour and salty). The delicious peptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala, is an octapeptide which was isolated from the gravy of beef and its primary structure was proposed in 1978 (3). It possesses a taste profile umami/sour (4), and it has a great interest in the food industry. Three fragments of the delicious peptide sequence: lysine-glycine, serine-leucine-alanine and aspartic acid-glutamic acid-glutamic acid possess separately umami/salty, bitter and sour taste respectively but mixtures or combinations of them produce a similar taste of that corresponding to the complete octapeptide. The synthesis of CBZ-Lys-Gly-OMe and CBZ-Ser-LeuOMe were carried out using immobilized trypsin and thermolysin. We have studied the influence of reaction medium (pH, temperature and substrate concentration) on the yield and initial reaction rate of synthesis. 3. EXPERIMENTAL 3.1. Materials Agarose gels lOB-CL were supplied free of charge by Hispanagar S.A. (Burgos, Spain). Commercial thermolysin from Bacillus thermoproteolyticus rokko and bovine trypsin Type III from SIGMA Co. were used as the source of enzymes. Benzyloxycarbonyl-lysine, glycine methyl ester, benzoylarginine ethyl ester (BAEE), benzyloxycarbonyl-serine, leucine methyl ester and furyl-acryloyl-glycil-leucinamide (FAGLA), were purchased from Sigma Co. Analytical grades of other reagent and solvents were used. 3.2. Activation of agarose gels Agarose gel lOB-CL containing 200 |Limol aldehyde/ml gel was prepared by etherification of agarose gels with glycidol and further oxidation with periodate as described in the literature (5). 3.3. Immobilization of trypsin on the activated gels Immobilized trypsin-agarose was prepared as reported elsewhere (6). Trypsin immobilization was carried out in presence of borate buffer (50 mM) containing benzamidine (75 mM) at pH 10 and 25°C. After 72 hours, the trypsin-agarose was reduced with NaBH4 (Img/ml) for 30 minutes. Finally, the immobilized preparation was washed with water and stored at 4°C. The derivative obtained had around 320 units/ml gel using benzoyl-arginine ethyl ester (BAEE) as substrate. One unit of enzyme activity is defined as
659 the amount of enzyme that hydrolyzes 1 |imol/min of substrate under the assay conditions. 3.4. Immobilization of thermolysin on the activated gels. Immobilized thermolysin-agarose was prepared using the method described above. Thermolysin immobilization was carried out in the presence of borate buffer (50 mM) containing CaCk (10 mM) at pH 10 and 25°C (7), (8). After 7 hours, the thermolysin-agarose was reduced with NaBH4 (1 mg/ml) during 30 minutes. Finally the immobilized thermolysin was washed with CaCk (10 mM) and stored at 4°C. The derivative obtained had around 550 units/ml gel using furyl-acryloyl-glycyl-leucinamide (FAGLA) as substrate (9). One unit is the amount of enzyme that hydrolyzes 1 jamol/min of substrate under the assay conditions. 3.5. Synthesis of N-benzyloxycarbonyl-lysine-glycine methyl ester. All the experiments were carried out in stoppered flasks with 25 ml of capacity placed in a thermostatic bath. The reaction medium, 0.1 M phosphate/butanediol/dioxane (1:3:6) (v/v) containing the substrates was adjusted to the appropriate pH. The reactions were started by adding 640 units of immobilized trypsin. Aliquots were taken at different times and analyzed by HPLC equipped with an octadecyl silica 150x46 mm column and UV-Vis detector at X, = 254 nm. Phosphate buffer 0.1 M (pH = 2.3) and acetonitrile (80:20) (v/v) was used as eluent at a flow rate of 1 ml/min. 3.6. Synthesis of N-benzyloxyearbonyl-serine-leucine methyl ester. The synthesis was also carried out in similar stoppered flasks placed in a thermostated bath. The reaction medium (aqueous pH 7 and biphasic acetate buffer 0.1 M, CaCb 10 mM/ethyl acetate (1:1) (v/v)), containing the substrates were adjusted to the desired pH. Reactions were started by adding 1100 units of immobilized thermolysin. Aliquots were taken at different times and analyzed by HPLC as above mentioned. In this case the eluent was phosphate buffer 0.1 M (pH = 2.3) and acetonitrile (62:38) (v/v). 4. RESULTS AND DISCUSSION 4.1. Synthesis of N-benzyloxycarbonyl-lysine-glycine methyl ester. The presence of high concentration of organic solvents is essential for achieving high synthetic yields in peptide synthesis under thermodynamically controlled conditions. CBZ-Lys-Gly-OMe has been synthesized from CBZ-Lys and Gly-OMe in a mixture system 0.1 M phosphate/butanediol/dioxane (1:3:6) (v/v) and catalyzed by immobilized trypsin on agarose gels. We have observed that some reaction conditions, such as pH, temperature and substrate concentration, have a great influence on enzyme activity and product yields.
660 Effect of P H Figure 1 shows the influence of the solution pH on the yield and initial rate of synthesis of CBZ-Lys-Gly-OMe at a temperature of 25°C and [CBZ-Lys] = [Gly-OMe] = 20 mM. The maximum yield is achieved for pH values around 6-6.5. Under thermodynamically controlled conditions, the peptide synthesis occurs between the non-ionic forms of the acyl-donor (CBZ-Lys) and the nucleophile (Gly-OMe). The concentration of these nonionic forms depends on the pH, since an intermediate value between both pK (pHopt = V^2[pKa +pKb]) is needed in order to achieve high synthetic yields. On the other hand, the reaction rate increases up to pH 7, which is in agreement with the results obtained in the synthesis of the peptide benzoylarginine-leucinamide catalyzed by immobilized trypsin (10), where the authors suggest the nucleophilic attack of the non-ionic form of the nucleophile on the acyl-enzyme complex as the controlling step of the peptide reaction. Effect of temperature As it was expected, the reaction rate increases with increasing temperature (figure 2). On the other hand, since the peptide synthesis is generally an exothermic reaction (2), the peptide yield slightly decreases as the temperature is increased.
"20 3Cr THVIPB^-RJRETO
Figure 1. Effect of pH on the synthesis of CBZ-Lys-Gly-OMe. T= 25°C, [CBZ-Lys]=[Gly-OMe]= 20 mM.
Figure 2. Effect of temperature on the synthesis of CBZ-Lys-GlyOMe. pH= 6.5, [CBZ-Lys]=[GlyOMe]= 20 mM.
Kinetic analysis Figure 3 shows the initial reaction rate in experiments carried out at pH 6.5, temperature of 30°C and [Gly-OMe] = 20 mM when the CBZ-Lys concentration changes in the range 2-40 mM. Inhibition is observed by this aminoacid at concentrations higher than 20 mM. However, when the concentration of Gly-OMe is changed, while keeping constant CBZ-Lys concentration, a Michaelis-Menten kinetic behaviour is observed. Figure 4 shows the results of these experiments.
661
20 • 30 • 40 CBZ-LYaNE(rTM)
Figure 3. Effect of CBZ-Lys concentration on the synthesis of CBZ-Lys-Gly-OMe. T=30°C, pH= 6.5, [Gly-OMe]= 20 mM.
20 30 40 aYCINEMETl-YLE5TER(rTiVI)
Figure 4. Effect of Gly-OMe concentration on the synthesis of CBZ-Lys-Gly-OMe. T=30°C, pH= 6.5, [CBZ-Lys]= 4 mM.
4.2. Synthesis of N-benzyloxycarbonyl-serine-leucine methyl ester. CBZ-Ser-Leu-OMe synthesis has been carried out starting from CBZSer and Leu-OMe, using free thermolysin in a monophasic system, and thermolysin immobilized-stabilized on agarose gels, in a biphasic system. The influence of different variables such as : pH, polarity of the reaction medium, temperature and substrate concentrations on the dipeptide yield and reaction rate has been studied. Synthesis of CBZ-Ser-Leu-OMe with free thermolysin. The dipeptide synthesis with free thermolysin (2 mg/ml) was carried out in water 10 mM of CbCa at pH 6,8 and 20 °C. The yield of peptide was below 10%, due to some reaction byproducts coming from the hydrolysis of methyl ester group of the peptide and consequent product hydrolysis as is shown in the following reactions : CBZ-Ser-Leu-OMe
CBZ-Ser-Leu + MeOH
[Reaction 1]
CBZ-Ser-Leu
CBZ-Ser + Leu
[Reaction 2]
The low yield obtained in this reaction needs the use of biphasic media to obtain high yields of the desired product avoiding the ester hydrolysis. Synthesis of CBZ-Ser-Leu-OMe with thermolysin immobilized Thermolysin immobilized on agarose gels as described in the experimental section was used as catalyst in a biphasic reaction system (acetate buffer 0,1 M and 10 mM CaCk/ethyl acetate (1:1) (v/v)). This medium could prevent the secondary reactions [1] and [2], since the dipeptide is more soluble in the organic phase than in water, and it allows to achieve high synthetic yields by removing the product from the aqueous phase.
662 Effect of pH Figure 5 shows the yields of CBZ-Ser-Leu-OMe and the reaction rate obtained at 20°C and substrates concentration of 25 mM for a range of pH from 5.5 to 8. The maximum reaction rate is observed at pH 7 which is in agreement with the optimum conditions for this enzyme (11). Also a yield increase is observed as the pH is decreased, a feature possibly explained taking into account the influence of pH on partition coefficients (12) of both substrates between two phases aqueous/organic. Thus, an optimum pH is expected to be around 5.5-6. Effect of temperature Figure 6 shows the influence of temperature in the range 20-40°C when the synthesis is carried out at pH 7. An increase of temperature increases the reaction rate and decreases slightly the synthesis yields due to the exothermicity of these reactions, as mentioned above.
"20 30 m~ TBVIFe^TURETO
Figure 5. Effect of pH on the synthesis of CBZ-Ser-Leu-OMe. T= 20°C, [CBZ-Ser]=[Leu-OMe]= 25 mM.
Figure 6. Effect of temperature on the synthesis of CBZ-Ser-LeuOMe. pH=7.0, [CBZ-Ser]= [LeuOMe]= 25 mM.
Effect of presence of ammonium sulphate in the reaction medium. The presence of this salt enhances the yields as well as the reaction rate up a concentration 2.8 mM. Table 1 shows both the yield and reaction rate in peptide synthesis of CBZ-Ser-Leu-0-Me at pH 7, 20°C and [CBZ-Ser] = [Leu-OMe] = 50 mM at different salt concentrations. Table 1. Effect of Ammonium sulphate concentration (mM) 0 1 2 Vox 104 1.63 1.83 2.83 (nmol/min/UFAGLA) 32.2 40.2 50.3 Yield (%)
2.8 21.01
3.56 6.27
96.0
70.4
663 It is noteworthy the favourable effect on the peptide yield produced by the ammonium sulphate up concentration 2.8 mM. The presence of this salt seems to improve the hydrophobic adsorption of the nucleophile on the active centre of thermolysin. Effect of substrate concentration Figure 7 shows the synthesis yields and reaction rates at pH 6.0, 30°C and constant concentration of Leu-OMe, 25 mM, varying [CBZ-Ser] up to 100 mM. CBZ-Ser concentration exerts a strong inhibition on the reaction rate for concentration higher than 25 mM, while the reaction yields decrease continuously. On the other hand, figure 8 shows the increase in the reaction rate in all the range of Leu-OMe concentration, reaching yields values around 100% for [Leu-OMe] = 150 mM.
40 CBZ-SBRINE(rTiVI)
Figure 7. Effect of CBZ-Ser concentration on the synthesis of CBZ-Ser-Leu-OMe. T=30°C, pH=: 6.0, [Leu-OMe]= 25 mM.
80
•
1^0
1 ^
LELX:iNEMETH'LE5THR (nM)
Figure 8. Effect of Leu-OMe concentration on the synthesis of CBZ-Ser-Leu-OMe. T=30°C, pH= 6.0, [CBZ-Ser]= 25 mM.
For reaction times longer than 50 hours, the formation of reaction byproducts with high molecular weight, probably oligopeptides, has been detected. This observation could support the yield decrease observed in figure 7 where the reaction rate is very low and therefore the equilibrium yield can not be reached. The formation of oligopeptides come probably from the attack of a molecule of nucleophile (Leu-OMe) to a dipeptide previously formed, to give tripeptides or another oligopeptides. This role of thermolysin has been described by Morihara (13) in the synthesis of CBZ-Leu-Leu, CBZ-Phe-Leu, CBZ-Gly-Leu and CBZ-Leu-Leu-Leu-Leu-NH2. 5. CONCLUSIONS The synthesis of CBZ-Lys-Gly-OMe and CBZ-Ser-Leu-OMe could be carried out in aqueous organic systems using immobilized trypsin and
664 thermolysin respectively. The obtained yields were 80% for CBZ-LysGlyOMe and 100% for CBZ-Ser-LeuOMe in mild conditions, temperature 30°C, pH 6,0-6,5 and in the presence of organic solvents. An inhibition effect was observed with both acyl donors (CBZ-Lys and CBZ-Ser) for concentrations higher than 20 mM, whereas the nucleophiles (Gly-OMe and Leu-OMe) show a kinetic behaviour without inhibition.
REFERENCES 1. Gill, R. Lopez-Fandino, X. Jorba, N. Vulfson, Enzyme Microb. Technol. 18 (1996) 162. 2. W. KuUmann, Enzymatic peptide synthesis. CRC Press Inc. Eds., Florida. USA. (1987). 3. Y. Yamasaki, K. Maekawa, Agric. Biol. Chem. 42 (1978) 1761 4. Y. Yamasaki, K. Maekawa, Agric. Biol. Chem. 44 (1980) 93. 5. J.M. Guisan, Enzyme Microb. Technol. 10 (1988) 375 6. R.M. Blanco, J.J. Calvete, J.M. Guisan, Enzyme Microb. Technol. 11 (1989) 353 7. J.M. Guisan, G. Alvaro, J. Aguado, M.D. Romero, M.J. Guerra, E. Polo, Rev. Real. Acad. C.C. Ex. Fis. YNat. 88 (1994) 8. M.D. Romero, J. Aguado, J.M. Guisan, M.J. Guerra, E. Pardo, Proceedings of the Specialized Catalysis Group Congress, (1995) Peniscola. Spain 9. J. Feder, Biochem. and Biophys. Res. Commun, 32 (1968) 326 10. R.M. Blanco, G. Alvaro, J.C. Tercero, J.M. Guisan, Journal of Molecular Catalysis, 73(1992)97 11. S. Kunugi, H. Hirohara, N. Ise, Eur. J. Biochem. 124 (1982) 157 12. K. Nakanishi, Y. Kimura, R. Matsuno, Eur. J. Biochem. 161 (1986) 541 13. K. Morihara, H. Tsuzuki, T. Oka, Biochem. Biophys. Res. Commum. 84 (1978) 95