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Materials Science and Engineering C 13 Ž2000. 109–116 www.elsevier.comrlocatermsec
Peptide synthesis catalyzed by a-chymotrypsin immobilized in the poly žN-isopropylacrylamideracrylamide/ gel Norihiro Kato ) , Ayumi Oishi, Fujio Takahashi Department of Applied Chemistry, Faculty of Engineering, Utsunomiya UniÕersity, 7-1-2 Yoto, Utsunomiya 321-8585, Japan
Abstract The peptide syntheses were performed with a-chymotrypsin immobilized in the thermosensitive poly Ž Nisopropylacrylamideracrylamide. ŽNIPAAmrAAm. gels. In the reaction between N-acetylphenylalanine ethyl ester ŽAc-Phe-OEt. and alaninamide ŽAla-NH 2 ., the product ratio of Ac-Phe–Ala-NH 2rAc-Phe-OH increased with lowering temperature. The highest ratio was 3.3 in the reaction with immobilized enzyme at y108C, while that was around 1.4 with free enzyme at 348C. On the contrary, CBZ-Phe–Leu-NH 2 obtained from N-carbobenzoxyphenylalanine ŽCBZ-Phe. and leucinamide ŽLeu-NH 2 . increased with increasing temperature Žconversion: 13% at 108C, 34% at 358C. in Tris buffer without the organic solvent. The gel of NIPAAm copolymerized with AAm ŽNIPAAmrAAms 80r20. was effective as compared with the polyŽNIPAAm. gel for the peptide production catalyzed by a-chymotrypsin using the ester substrate as the acyl donor. The reaction mechanism was discussed in connection with the hydrophobic surroundings inside the deswollen thermosensitive polymer gel. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Peptide synthesis; Thermosensitive gel; N-Isopropylacrylamide; a-Chymotrypsin; Immobilized enzyme
1. Introduction The thermally sensitive polymers or polymer gels, possessing a lower critical solution temperature ŽLCST., have been applied to the carrier of immobilized enzyme as new intelligent systems w1–11x. Since the polymers precipitate above the temperature of the LCST, the enzyme immobilized in the polymer can be separated from the solution containing the products. The system consisting of thermosensitive gels immobilized enzyme is advantageous for the product separation, but the slow diffusion rate of the substrate and the product delays the enzymatic reaction w12–14x. On the other hand, it was reported that the apparent activity of the enzyme immobilized in the gel increased by introducing the process of repeating the deswelling–swelling of the thermosensitive gels due to temperature change w15,16x. In our previous report, the poly Ž N-isopropylacrylamideracrylamide. gel ŽpolyŽNIPAAmrAAm. gel. immobilized the ferromagnetic powder, and the b-galactosidase was utilized as the magnetically activated reactor w17x.
) Corresponding author. Tel.: q81-28-689-6157; fax: q81-28-6896009.
It is considered that the rate of peptide bond formation, which is catalyzed by the immobilized enzyme, should be controlled by the amount of water inside the gel. The application of the enzyme immobilized in the thermosensitive gel is expected to enhance the peptide synthesis. a-Chymotrypsin, which is well-known for hydrolyzing peptide bond, could also catalyze the reverse reaction as peptide bond formation between two amino acids. The reverse reactions for some kinds of proteases are classified into two types according to the principles w18x: Ž1. the equilibrium-controlled reaction will produce only peptide from amino acid possessing free –COOH as the substrate; Ž2. the kinetically controlled reaction will produce peptide and hydrolyzed product of ester from amino acid possessing –COOR as the substrate. The schemes for both reactions are shown in Fig. 1a and b, respectively. N-Carbobenzoxyphenylalanine ŽCBZ-Phe. and leucinamide ŽLeu-NH 2 . are used as the model substrates for the equilibrium-controlled peptide synthesis, and N-acetylphenylalanine ethyl ester ŽAc-Phe-OEt. and alaninamide ŽAla-NH 2 . are used as the respective model substrates of acyl donor ester and a nucleophile acceptor for the kinetically controlled reaction. This paper presents the results on peptide syntheses catalyzed by a-chymotrypsin immobilized in the
0928-4931r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 0 . 0 0 1 8 4 - 3
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was prepared as follows. NIPAAm Ž652 mg., AAm Ž102 mg., N, N X-methylenebisacrylamide Ž46 mg. were dissolved into 3 ml of 0.1 M Tris–maleate buffer ŽpH 7 or 8., and then nitrogen gas was bubbled into the solution. A total of 30,000 units of a-chymotrypsin, dissolved into 3 ml of buffer, was added to the pre-gel solution. The cylindrical gel rod was prepared in a silicone tube ŽB s 2 mm. using ammonium peroxydisulfate as the initiator and N, N, N X , N X-tetramethylethylene-diamine as the accelerator. The gel rods, pulled out from the mold, were cut into 10 mm each, and washed with buffer solution. Then the gel rods were freeze-dried. In the case of the kinetically controlled reaction, 0.1 M carbonate buffer solution ŽNa 2 CO 3rNaHCO 3 buffer: pH 9–10.5., abbreviated as CBS, was used instead of the Tris–maleate buffer. The other gels wNIPAAmrAAms 100r0 and 90r10x were prepared in the same manner. 2.3. Deswelling profiles of the gel
Fig. 1. Schemes for the peptide syntheses with a-chymotrypsin ŽCT.. Ža. The equilibrium-controlled reaction as coupling between CBZ-Phe and Leu-NH 2 . Žb. The kinetically controlled reaction as coupling between Ac-Phe-OEt and Ala-NH 2 .
polyŽNIPAAmrAAm. gel. We discuss the relationship between the apparent enzyme activity and the hydrophobic surroundings inside the thermosensitive gel. The temperature dependency on the conversion ratio of peptider byproduct is studied in connection with swelling–deswelling of the thermosensitive gel.
2. Materials and methods 2.1. Materials a-Chymotrypsin from bovine pancreas ŽEC 3.4.21.1. was purchased from Wako. CBZ-Phe, Ac-Phe-OEt, LeuNH 2 P HCl, and Ala-NH 2 P HCl were purchased from Sigma. N-Acetylphenylalanine ŽAc-Phe-OH. was purchased from Kanto Chemical. CBZ-Phe–Leu-NH 2 and Ac-Phe–Ala-NH 2 were kindly synthesized by Dr. T. Uyehara and Dr. M. Karikomi of Utsunomiya University. N-Isopropylacrylamide was purchased from Wako. All other chemicals were of guaranteed grade or the best commercially available.
The freeze-dried gel rod Ž L22 s 60 mm at 228C. was re-swollen in Tris–maleate buffer solution. The equilibrium length of the gel rod Ž L. was measured at different temperatures. The values of L were given with increasing the temperature. Ž LrL22 . 3 was calculated as the volume change of the gel. Similarly, the volume change of the gel rod re-swollen in the CBS was evaluated as Ž LrL0 . 3 , where L0 is the length of the gel rod just after polymerization. 2.4. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 using the free enzyme An amount of 0.9 ml of Tris–maleate buffer solution containing CBZ-Phe and Leu-NH 2 was mixed with 0.1 ml of the enzyme solution Ž1500 units. at desired temperature Ž10–608C.. The initial concentrations of CBZ-Phe and Leu-NH 2 were 23.8 and 25.0 mM, respectively. 2.5. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 using the immobilized enzyme Ten freeze-dried gel rods Ž L0 s 10 mm. were re-swollen in 950 ml of CBZ-Phe buffer solution at 48C. After full swelling, the gel rods in the CBZ-Phe solution were incubated at the desired temperature Ž10–508C.. Then, 50 ml of Leu-NH 2 buffer solution was added in order to start the enzyme reaction at the same temperature. The initial concentrations of the CBZ-Phe and Leu-NH 2 were 23.8 and 25.0 mM, respectively.
2.2. Polymerization of poly(NIPAAmr AAm) gel immobilized a-chymotrypsin
2.6. HPLC analysis for CBZ-Phe and CBZ-Phe–Leu-NH2
PolyŽNIPAAmrAAm. gel wNIPAAmrAAms 80r20 Žmolrmol.x, used for the equilibrium-controlled reaction,
Precipitates, which are composed of peptides, increased in the solution with time. After heating at 808C for the
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denaturation of enzyme, ethanol was added in order to dissolve the precipitates at 228C. CBZ-Phe and CBZ-Phe– Leu-NH 2 were analyzed using a reverse phase HPLC column of Asahipak ODP-50 ŽShimazu, B 6.0 mm, 250 mm long.. The eluent, a mixed solvent of phosphate buffer solution of pH 2 Ž100 mM KH 2 PO4rH 3 PO4 . and acetonitrile with a volume ratio of 55:45, was pumped into the column at a flow rate of 1 mlrmin at 358C. Each component was monitored spectrophotometrically at 260 nm. The conversion was determined from the ratio of the peak height as compared with pure reagent. 2.7. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 using the free enzyme a-Chymotrypsin in CBS was added to the mixed solution of acetonitrile and CBS ŽpH 8., which dissolved Ac-Phe-OEt and Ala-NH 2 , in order to start the enzymatic reaction. The initial concentrations of Ac-Phe-OEt, AlaNH 2 and a-chymotrypsin were 16.0 mM, 80.0 mM and 1500 unitsrml, respectively. 2.8. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 using the immobilized enzyme Ten freeze-dried gel rods Ž L0 s 10 mm. were re-swollen in 900 ml of Ala-NH 2 solution at 48C. After full swelling, the gel rods in Ala-NH 2 solution were incubated at the desired temperature Žy108C to 508C.. Then 80 ml of Leu-NH 2 buffer solution was added in order to start the enzymatic reaction at the same temperature. The initial concentrations of Ac-Phe-OEt, and Ala-NH 2 were 16.0 and 80.0 mM, respectively. 2.9. HPLC analysis for Ac-Phe–Ala-NH2 , Ac-Phe-OEt, and Ac-Phe-OH
Fig. 3. The conversion of CBZ-Phe and Leu-NH 2 into CBZ-Phe–LeuNH 2 with time. a-Chymotrypsin immobilized was incubated with the substrates ŽCBZ-Phe, Leu-NH 2 . in 0.1 M Tris–maleate buffer ŽpH 8. at 358C Žv . and 228C Ž`..
ODP y 50 ŽShimazu, B 6.0 mm, 250 mm long.. The eluent, a mixed solvent of phosphate buffer solution of pH 2 Ž20 mM, NaH 2 PO4rH 3 PO4 ., acetonitrile, and methanol with a volume ratio of 65:25:10, was pumped into the column at a flow rate of 1 mlrmin at 358C. Each component was monitored spectrophotometrically at 260 nm. Peaks of ester substrate, peptide and byproduct were separated clearly. The conversion was determined from the ratio of the peak height as compared with pure reagent.
3. Results 3.1. Deswelling profiles of poly(NIPAAmr AAm) gels
Ac-Phe–Ala-NH 2 , Ac-Phe-OEt, and Ac-Phe-OH were analyzed using a reverse phase HPLC column of Asahipak
Almost the same deswelling profiles were shown on the gels in Tris–maleate buffer ŽpH 7 or 8. as compared with that on the gels in water in Fig. 2.
Fig. 2. Deswelling profiles for the polyŽNIPAAmrAAm. gel wNIPAAmrAAm s80r20 Žmolrmol.x in water Ž'., and 0.1 M Tris– maleate buffer wpH 7 Že. and pH 8 Ž`. x.
Fig. 4. The pH dependency on the conversion for CBZ-Phe–Leu-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates ŽCBZPhe, Leu-NH 2 . in 0.1 M Tris–maleate buffer ŽpH 7–9. for 24 h at 358C.
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shown in Fig. 4. The optimum pH was at around 8 for the immobilized enzyme, and hereafter, the reaction was carried out using Tris–maleate buffer ŽpH 8.. The temperature dependency on the conversion was obtained for comparing the free and the immobilized enzymes ŽFig. 5.. Each maximum conversion for the free and the immobilized enzymes appeared at 348C and 388C, respectively. 3.3. Deswelling profiles of gels in the solÕent containing acetonitrile
Fig. 5. Temperature dependency on the conversion for CBZ-Phe–LeuNH 2 using free Ža. and immobilized enzyme Žb.. The reaction was carried out in 0.1 M Tris–maleate buffer ŽpH 8. for 24 h.
3.2. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 The reaction between CBZ-Phe and Leu-NH 2 was carried out with immobilized a-chymotrypsin at pH 8 at 358C and 228C. The result on the conversion with time was shown in Fig. 3. The pH profile was obtained from the 24 h reaction with the same composite mixture at 358C as
The relationship between the temperature and the gel volume was determined in water, CBS and the mixed solvent wCBSracetonitriles 80r20x ŽFig. 6.. The increase of the AAm content in the polymer made the LCST higher in water and CBS ŽFig. 6a,b.. The gel of the polymer containing AAm could swell more than polyŽNIPAAm. gel at lower than 208C ŽFig. 6a,b.. The deswelling profile in the mixed solvent was different from that in water or CBS ŽFig. 6c.. The thermosensitive property of the polyŽNIPAAm. gel diminished in the mixed solvent containing the organic solvent. 3.4. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 Since the ester substrate did not dissolve in the aqueous buffer solution, the reaction was carried out in the aqueous solution containing acetonitrile. The ester substrate remained after 24 h reaction in the aqueous solution containing above 30% of acetonitrile at 228C as shown in Fig. 7. The maximum conversion into the peptide appeared around 20% of acetonitrile and at pH 9 of the mixed solvent wacetonitrilerbuffers 20r80x at 228C ŽFig. 8..
Fig. 6. Deswelling profiles for the polyŽNIPAAmrAAm. gel in water Ža., 0.1 M CBS wpH 8x Žb., and mixed solvent wCBSracetonitriles 80r20x Žc.. NIPAAmrAAms 100r0 Ž`., 90r10 Žl., and 80r20 Ž^..
N. Kato et al.r Materials Science and Engineering C 13 (2000) 109–116
Fig. 7. Solvent dependency on the reaction between Ac-Phe-OEt and Ala-NH 2 . Acetonitrile was added into CBS ŽpH 8. to make the mixture of acetonitrilerCBS ŽpH 8. between 10r90 and 50r50: Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že.. The conversion was obtained after 24 h at 228C.
Fig. 9 showed the time dependency on the conversion in the mixed solvent of acetonitrile and CBS ŽpH 9. with a ratio of 20:80 at 228C. The ester substrate was consumed completely within 2 h under above described condition. The temperature dependency on the conversion was shown in Fig. 10. The peptide ŽAc-Phe–Ala-NH 2 . and the byproduct ŽAc-Phe-OH. decreased above 308C in all cases. These results indicate that the ester substrate remains in the solution after 24 h above 308C. The ratio Ž P . of peptiderbyproduct was approximately 1:1 for polyŽNIPAAm. gel at the temperature between y108C and 208C. On the other hand, the concentration of the byprod-
Fig. 8. The pH dependency on the conversion for Ac-Phe–Ala-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates ŽAc-PheOEt, Ala-NH 2 . in the mixed solvent wbufferracetonitriles80r20x for 24 h at 228C: pH 7 and 8 for 0.1 M phosphate buffer. pH 9, 10, and 10.5 for CBS. Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
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Fig. 9. The conversion of Ac-Phe-OEt and Ala-NH 2 into Ac-Phe–AlaNH 2 with time. a-Chymotrypsin immobilized in the gel wNIPAAmrAAm s100r0x was incubated with the substrates ŽAc-PheOEt, Ala-NH 2 . in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h at 228C: Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
uct was far lower than that of the peptide at y108C for the copolymer gels wNIPAAmrAAms 90r10, and 80r20x.
Fig. 10. Temperature dependency on the reaction between Ac-Phe-OEt and Ala-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h. The ratios of NIPAAmrAAm were 100r0 ŽA., 90r10 ŽB., and 80r20 ŽC.: Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
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Fig. 11. Temperature dependency on P as the peptiderbyproduct. Free ŽA. and immobilized ŽB. a-chymotrypsin was incubated with the substrates ŽAc-Phe-OEt, Ala-NH 2 . in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h: ŽA. free Ž`., ŽB. the ratio of NIPAAmrAAm s100r0 Ž^., 90r10 Že., and 80r20 Žv ..
The ratios Ž P . were calculated from data in Fig. 10 and plotted against temperature on the reaction with the free enzyme as well as the immobilized enzyme as shown in Fig. 11. The maximum P Ž1.4. was obtained at 348C with the free enzyme. On the other hand, the P values reached the highest at y108C with the immobilized enzyme. The highest P was 3.3 when the gel containing 20% of AAm was used.
4. Discussion 4.1. Selection of the model substrates for the reÕerse reaction of enzyme Many kinds of hydrolases such as trypsin, papain, thermolysin, etc. w19–27x, are known to catalyze the reverse reaction. a-Chymotrypsin also is reported to play a role to form peptide bonds. a-Chymotrypsin-catalyzed hydrolysis proceeds in two processes: the first process is acylation of serine-195 residue, and the second is a subsequent de-acylation. If the nucleophile molecule is not water but amino acid in the second process, the deacylation of enzyme occurs with the formation of peptide w28,29x. Hydrophobic surroundings are the necessary condition for the reverse reaction. In general, the equilibrium-controlled reaction was carried out in the solution consisting of two phases between
water and water-immiscible organic solvent in order to exhaust the hydrophobic product into the organic phase w30–33x. Since the thermosensitive polymer gel dehydrated with deswelling, surroundings of the enzyme inside the deswollen gel are hydrophobic. This is advantageous for the reverse reaction of the enzyme. However, there has been no report concerning the enzyme immobilized in the thermosensitive gels in order to carry out the reverse reaction for the peptide synthesis. The peptide synthesis under the kinetically controlled condition was usually carried out in the mixed solvent between water and the water-miscible organic solvent, in which the substrates and the produced peptide were dissolved. The peptide synthesis without the organic solvent was possible in the case of coupling between CBZ-Phe and Leu-NH 2 using Tris buffer w20x. The peptide bond formation of hydrophobic Leu-NH 2 was much easier than that of Gly-NH 2 or Ala-NH 2 w20x. Since CBZ is a hydrophobic protecting group substituted on the N-terminal of Phe, the peptide produced exhibits the hydrophobic property to precipitate easily from the Tris buffer. Similarly, the peptide produced from Leu-NH 2 as a nucleophile precipitated easily because the leucine residue in Leu-NH 2 exhibited the hydrophobic property. Therefore, we applied abovedescribed compounds for the reaction of enzyme immobilized in the polyŽNIPAAmrAAm. gel. 4.2. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 The result on the temperature dependency in Fig. 5 showed that the free and immobilized enzymes were deactivated above 348C or 388C, at which temperature the gels were in deswollen state. It was supposed from the apparent activity of enzyme that the immobilized enzyme became heat-resistant in the gel. The immobilized enzyme was considered to be located in hydrophobic surroundings inside the deswollen gel due to dehydration. CBZ-Phe, including a hydrophobic CBZ group, is able to react with enzyme to form the acyl–enzyme complex in the hydrophobic surroundings. According to the previous report w28,29x, the acylation for producing the intermediate as acyl–enzyme complex makes the rate-determining step in the sequential reactions for peptide synthesis. It is plausible that CBZ-Phe and Leu-NH 2 may react to form the peptide in the process, as shown in Fig. 1a. 4.3. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 The temperature dependency has been reported concerning the peptide synthesis catalyzed by a-chymotrypsin immobilized in agarose w33x. The paper describes that the yield of the kinetically controlled peptide synthesis in-
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creases with decreasing temperature. Our results in Fig. 10B,C show that the conversion into Ac-Phe–Ala-NH 2 is much different from that into Ac-Phe-OH at lower than 58C. The P temperature profile on free enzyme is different from that on immobilized enzyme as shown in Fig. 11. In the case of the free enzyme, the conversion of Ac-Phe– Ala-NH 2 is smaller than that of Ac-Phe-OH at lower temperature as the P is smaller than 1. In the case of the immobilized enzyme, the polymer may exclude water surrounding the acyl–enzyme complex, which is possible to react with Ala-NH 2 rather than water. On the other hand, Fig. 10 indicates that AAm may influence the hydration in the polyŽNIPAAmrAAm. gel. It is speculated that AAm may have stronger affinity of hydration than NIPAAm. Therefore, the immobilized a-chymotrypsin could enhance to catalyze the peptide synthesis as compared with the free enzyme at subzero temperature. The content of AAm in the polyŽNIPAAmrAAm. gel controls the conversion into the peptide, too.
Acknowledgements The authors are indebted to Mr. Kazutoshi Nagaoka and Mr. Hiroyuki Oiwa for their assistance during the experiment.
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Peptide synthesis catalyzed by a-chymotrypsin immobilized in the poly žN-isopropylacrylamideracrylamide/ gel Norihiro Kato ) , Ayumi Oishi, Fujio Takahashi Department of Applied Chemistry, Faculty of Engineering, Utsunomiya UniÕersity, 7-1-2 Yoto, Utsunomiya 321-8585, Japan
Abstract The peptide syntheses were performed with a-chymotrypsin immobilized in the thermosensitive poly Ž Nisopropylacrylamideracrylamide. ŽNIPAAmrAAm. gels. In the reaction between N-acetylphenylalanine ethyl ester ŽAc-Phe-OEt. and alaninamide ŽAla-NH 2 ., the product ratio of Ac-Phe–Ala-NH 2rAc-Phe-OH increased with lowering temperature. The highest ratio was 3.3 in the reaction with immobilized enzyme at y108C, while that was around 1.4 with free enzyme at 348C. On the contrary, CBZ-Phe–Leu-NH 2 obtained from N-carbobenzoxyphenylalanine ŽCBZ-Phe. and leucinamide ŽLeu-NH 2 . increased with increasing temperature Žconversion: 13% at 108C, 34% at 358C. in Tris buffer without the organic solvent. The gel of NIPAAm copolymerized with AAm ŽNIPAAmrAAms 80r20. was effective as compared with the polyŽNIPAAm. gel for the peptide production catalyzed by a-chymotrypsin using the ester substrate as the acyl donor. The reaction mechanism was discussed in connection with the hydrophobic surroundings inside the deswollen thermosensitive polymer gel. q 2000 Elsevier Science S.A. All rights reserved. Keywords: Peptide synthesis; Thermosensitive gel; N-Isopropylacrylamide; a-Chymotrypsin; Immobilized enzyme
1. Introduction The thermally sensitive polymers or polymer gels, possessing a lower critical solution temperature ŽLCST., have been applied to the carrier of immobilized enzyme as new intelligent systems w1–11x. Since the polymers precipitate above the temperature of the LCST, the enzyme immobilized in the polymer can be separated from the solution containing the products. The system consisting of thermosensitive gels immobilized enzyme is advantageous for the product separation, but the slow diffusion rate of the substrate and the product delays the enzymatic reaction w12–14x. On the other hand, it was reported that the apparent activity of the enzyme immobilized in the gel increased by introducing the process of repeating the deswelling–swelling of the thermosensitive gels due to temperature change w15,16x. In our previous report, the poly Ž N-isopropylacrylamideracrylamide. gel ŽpolyŽNIPAAmrAAm. gel. immobilized the ferromagnetic powder, and the b-galactosidase was utilized as the magnetically activated reactor w17x.
) Corresponding author. Tel.: q81-28-689-6157; fax: q81-28-6896009.
It is considered that the rate of peptide bond formation, which is catalyzed by the immobilized enzyme, should be controlled by the amount of water inside the gel. The application of the enzyme immobilized in the thermosensitive gel is expected to enhance the peptide synthesis. a-Chymotrypsin, which is well-known for hydrolyzing peptide bond, could also catalyze the reverse reaction as peptide bond formation between two amino acids. The reverse reactions for some kinds of proteases are classified into two types according to the principles w18x: Ž1. the equilibrium-controlled reaction will produce only peptide from amino acid possessing free –COOH as the substrate; Ž2. the kinetically controlled reaction will produce peptide and hydrolyzed product of ester from amino acid possessing –COOR as the substrate. The schemes for both reactions are shown in Fig. 1a and b, respectively. N-Carbobenzoxyphenylalanine ŽCBZ-Phe. and leucinamide ŽLeu-NH 2 . are used as the model substrates for the equilibrium-controlled peptide synthesis, and N-acetylphenylalanine ethyl ester ŽAc-Phe-OEt. and alaninamide ŽAla-NH 2 . are used as the respective model substrates of acyl donor ester and a nucleophile acceptor for the kinetically controlled reaction. This paper presents the results on peptide syntheses catalyzed by a-chymotrypsin immobilized in the
0928-4931r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 0 . 0 0 1 8 4 - 3
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was prepared as follows. NIPAAm Ž652 mg., AAm Ž102 mg., N, N X-methylenebisacrylamide Ž46 mg. were dissolved into 3 ml of 0.1 M Tris–maleate buffer ŽpH 7 or 8., and then nitrogen gas was bubbled into the solution. A total of 30,000 units of a-chymotrypsin, dissolved into 3 ml of buffer, was added to the pre-gel solution. The cylindrical gel rod was prepared in a silicone tube ŽB s 2 mm. using ammonium peroxydisulfate as the initiator and N, N, N X , N X-tetramethylethylene-diamine as the accelerator. The gel rods, pulled out from the mold, were cut into 10 mm each, and washed with buffer solution. Then the gel rods were freeze-dried. In the case of the kinetically controlled reaction, 0.1 M carbonate buffer solution ŽNa 2 CO 3rNaHCO 3 buffer: pH 9–10.5., abbreviated as CBS, was used instead of the Tris–maleate buffer. The other gels wNIPAAmrAAms 100r0 and 90r10x were prepared in the same manner. 2.3. Deswelling profiles of the gel
Fig. 1. Schemes for the peptide syntheses with a-chymotrypsin ŽCT.. Ža. The equilibrium-controlled reaction as coupling between CBZ-Phe and Leu-NH 2 . Žb. The kinetically controlled reaction as coupling between Ac-Phe-OEt and Ala-NH 2 .
polyŽNIPAAmrAAm. gel. We discuss the relationship between the apparent enzyme activity and the hydrophobic surroundings inside the thermosensitive gel. The temperature dependency on the conversion ratio of peptider byproduct is studied in connection with swelling–deswelling of the thermosensitive gel.
2. Materials and methods 2.1. Materials a-Chymotrypsin from bovine pancreas ŽEC 3.4.21.1. was purchased from Wako. CBZ-Phe, Ac-Phe-OEt, LeuNH 2 P HCl, and Ala-NH 2 P HCl were purchased from Sigma. N-Acetylphenylalanine ŽAc-Phe-OH. was purchased from Kanto Chemical. CBZ-Phe–Leu-NH 2 and Ac-Phe–Ala-NH 2 were kindly synthesized by Dr. T. Uyehara and Dr. M. Karikomi of Utsunomiya University. N-Isopropylacrylamide was purchased from Wako. All other chemicals were of guaranteed grade or the best commercially available.
The freeze-dried gel rod Ž L22 s 60 mm at 228C. was re-swollen in Tris–maleate buffer solution. The equilibrium length of the gel rod Ž L. was measured at different temperatures. The values of L were given with increasing the temperature. Ž LrL22 . 3 was calculated as the volume change of the gel. Similarly, the volume change of the gel rod re-swollen in the CBS was evaluated as Ž LrL0 . 3 , where L0 is the length of the gel rod just after polymerization. 2.4. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 using the free enzyme An amount of 0.9 ml of Tris–maleate buffer solution containing CBZ-Phe and Leu-NH 2 was mixed with 0.1 ml of the enzyme solution Ž1500 units. at desired temperature Ž10–608C.. The initial concentrations of CBZ-Phe and Leu-NH 2 were 23.8 and 25.0 mM, respectively. 2.5. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 using the immobilized enzyme Ten freeze-dried gel rods Ž L0 s 10 mm. were re-swollen in 950 ml of CBZ-Phe buffer solution at 48C. After full swelling, the gel rods in the CBZ-Phe solution were incubated at the desired temperature Ž10–508C.. Then, 50 ml of Leu-NH 2 buffer solution was added in order to start the enzyme reaction at the same temperature. The initial concentrations of the CBZ-Phe and Leu-NH 2 were 23.8 and 25.0 mM, respectively.
2.2. Polymerization of poly(NIPAAmr AAm) gel immobilized a-chymotrypsin
2.6. HPLC analysis for CBZ-Phe and CBZ-Phe–Leu-NH2
PolyŽNIPAAmrAAm. gel wNIPAAmrAAms 80r20 Žmolrmol.x, used for the equilibrium-controlled reaction,
Precipitates, which are composed of peptides, increased in the solution with time. After heating at 808C for the
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denaturation of enzyme, ethanol was added in order to dissolve the precipitates at 228C. CBZ-Phe and CBZ-Phe– Leu-NH 2 were analyzed using a reverse phase HPLC column of Asahipak ODP-50 ŽShimazu, B 6.0 mm, 250 mm long.. The eluent, a mixed solvent of phosphate buffer solution of pH 2 Ž100 mM KH 2 PO4rH 3 PO4 . and acetonitrile with a volume ratio of 55:45, was pumped into the column at a flow rate of 1 mlrmin at 358C. Each component was monitored spectrophotometrically at 260 nm. The conversion was determined from the ratio of the peak height as compared with pure reagent. 2.7. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 using the free enzyme a-Chymotrypsin in CBS was added to the mixed solution of acetonitrile and CBS ŽpH 8., which dissolved Ac-Phe-OEt and Ala-NH 2 , in order to start the enzymatic reaction. The initial concentrations of Ac-Phe-OEt, AlaNH 2 and a-chymotrypsin were 16.0 mM, 80.0 mM and 1500 unitsrml, respectively. 2.8. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 using the immobilized enzyme Ten freeze-dried gel rods Ž L0 s 10 mm. were re-swollen in 900 ml of Ala-NH 2 solution at 48C. After full swelling, the gel rods in Ala-NH 2 solution were incubated at the desired temperature Žy108C to 508C.. Then 80 ml of Leu-NH 2 buffer solution was added in order to start the enzymatic reaction at the same temperature. The initial concentrations of Ac-Phe-OEt, and Ala-NH 2 were 16.0 and 80.0 mM, respectively. 2.9. HPLC analysis for Ac-Phe–Ala-NH2 , Ac-Phe-OEt, and Ac-Phe-OH
Fig. 3. The conversion of CBZ-Phe and Leu-NH 2 into CBZ-Phe–LeuNH 2 with time. a-Chymotrypsin immobilized was incubated with the substrates ŽCBZ-Phe, Leu-NH 2 . in 0.1 M Tris–maleate buffer ŽpH 8. at 358C Žv . and 228C Ž`..
ODP y 50 ŽShimazu, B 6.0 mm, 250 mm long.. The eluent, a mixed solvent of phosphate buffer solution of pH 2 Ž20 mM, NaH 2 PO4rH 3 PO4 ., acetonitrile, and methanol with a volume ratio of 65:25:10, was pumped into the column at a flow rate of 1 mlrmin at 358C. Each component was monitored spectrophotometrically at 260 nm. Peaks of ester substrate, peptide and byproduct were separated clearly. The conversion was determined from the ratio of the peak height as compared with pure reagent.
3. Results 3.1. Deswelling profiles of poly(NIPAAmr AAm) gels
Ac-Phe–Ala-NH 2 , Ac-Phe-OEt, and Ac-Phe-OH were analyzed using a reverse phase HPLC column of Asahipak
Almost the same deswelling profiles were shown on the gels in Tris–maleate buffer ŽpH 7 or 8. as compared with that on the gels in water in Fig. 2.
Fig. 2. Deswelling profiles for the polyŽNIPAAmrAAm. gel wNIPAAmrAAm s80r20 Žmolrmol.x in water Ž'., and 0.1 M Tris– maleate buffer wpH 7 Že. and pH 8 Ž`. x.
Fig. 4. The pH dependency on the conversion for CBZ-Phe–Leu-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates ŽCBZPhe, Leu-NH 2 . in 0.1 M Tris–maleate buffer ŽpH 7–9. for 24 h at 358C.
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shown in Fig. 4. The optimum pH was at around 8 for the immobilized enzyme, and hereafter, the reaction was carried out using Tris–maleate buffer ŽpH 8.. The temperature dependency on the conversion was obtained for comparing the free and the immobilized enzymes ŽFig. 5.. Each maximum conversion for the free and the immobilized enzymes appeared at 348C and 388C, respectively. 3.3. Deswelling profiles of gels in the solÕent containing acetonitrile
Fig. 5. Temperature dependency on the conversion for CBZ-Phe–LeuNH 2 using free Ža. and immobilized enzyme Žb.. The reaction was carried out in 0.1 M Tris–maleate buffer ŽpH 8. for 24 h.
3.2. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 The reaction between CBZ-Phe and Leu-NH 2 was carried out with immobilized a-chymotrypsin at pH 8 at 358C and 228C. The result on the conversion with time was shown in Fig. 3. The pH profile was obtained from the 24 h reaction with the same composite mixture at 358C as
The relationship between the temperature and the gel volume was determined in water, CBS and the mixed solvent wCBSracetonitriles 80r20x ŽFig. 6.. The increase of the AAm content in the polymer made the LCST higher in water and CBS ŽFig. 6a,b.. The gel of the polymer containing AAm could swell more than polyŽNIPAAm. gel at lower than 208C ŽFig. 6a,b.. The deswelling profile in the mixed solvent was different from that in water or CBS ŽFig. 6c.. The thermosensitive property of the polyŽNIPAAm. gel diminished in the mixed solvent containing the organic solvent. 3.4. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 Since the ester substrate did not dissolve in the aqueous buffer solution, the reaction was carried out in the aqueous solution containing acetonitrile. The ester substrate remained after 24 h reaction in the aqueous solution containing above 30% of acetonitrile at 228C as shown in Fig. 7. The maximum conversion into the peptide appeared around 20% of acetonitrile and at pH 9 of the mixed solvent wacetonitrilerbuffers 20r80x at 228C ŽFig. 8..
Fig. 6. Deswelling profiles for the polyŽNIPAAmrAAm. gel in water Ža., 0.1 M CBS wpH 8x Žb., and mixed solvent wCBSracetonitriles 80r20x Žc.. NIPAAmrAAms 100r0 Ž`., 90r10 Žl., and 80r20 Ž^..
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Fig. 7. Solvent dependency on the reaction between Ac-Phe-OEt and Ala-NH 2 . Acetonitrile was added into CBS ŽpH 8. to make the mixture of acetonitrilerCBS ŽpH 8. between 10r90 and 50r50: Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že.. The conversion was obtained after 24 h at 228C.
Fig. 9 showed the time dependency on the conversion in the mixed solvent of acetonitrile and CBS ŽpH 9. with a ratio of 20:80 at 228C. The ester substrate was consumed completely within 2 h under above described condition. The temperature dependency on the conversion was shown in Fig. 10. The peptide ŽAc-Phe–Ala-NH 2 . and the byproduct ŽAc-Phe-OH. decreased above 308C in all cases. These results indicate that the ester substrate remains in the solution after 24 h above 308C. The ratio Ž P . of peptiderbyproduct was approximately 1:1 for polyŽNIPAAm. gel at the temperature between y108C and 208C. On the other hand, the concentration of the byprod-
Fig. 8. The pH dependency on the conversion for Ac-Phe–Ala-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates ŽAc-PheOEt, Ala-NH 2 . in the mixed solvent wbufferracetonitriles80r20x for 24 h at 228C: pH 7 and 8 for 0.1 M phosphate buffer. pH 9, 10, and 10.5 for CBS. Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
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Fig. 9. The conversion of Ac-Phe-OEt and Ala-NH 2 into Ac-Phe–AlaNH 2 with time. a-Chymotrypsin immobilized in the gel wNIPAAmrAAm s100r0x was incubated with the substrates ŽAc-PheOEt, Ala-NH 2 . in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h at 228C: Ac-Phe-OEt Ž^., Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
uct was far lower than that of the peptide at y108C for the copolymer gels wNIPAAmrAAms 90r10, and 80r20x.
Fig. 10. Temperature dependency on the reaction between Ac-Phe-OEt and Ala-NH 2 . a-Chymotrypsin immobilized was incubated with the substrates in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h. The ratios of NIPAAmrAAm were 100r0 ŽA., 90r10 ŽB., and 80r20 ŽC.: Ac-Phe–Ala-NH 2 Žv ., Ac-Phe-OH Že..
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Fig. 11. Temperature dependency on P as the peptiderbyproduct. Free ŽA. and immobilized ŽB. a-chymotrypsin was incubated with the substrates ŽAc-Phe-OEt, Ala-NH 2 . in the mixed solvent wCBS ŽpH 8.racetonitriles80r20x for 24 h: ŽA. free Ž`., ŽB. the ratio of NIPAAmrAAm s100r0 Ž^., 90r10 Že., and 80r20 Žv ..
The ratios Ž P . were calculated from data in Fig. 10 and plotted against temperature on the reaction with the free enzyme as well as the immobilized enzyme as shown in Fig. 11. The maximum P Ž1.4. was obtained at 348C with the free enzyme. On the other hand, the P values reached the highest at y108C with the immobilized enzyme. The highest P was 3.3 when the gel containing 20% of AAm was used.
4. Discussion 4.1. Selection of the model substrates for the reÕerse reaction of enzyme Many kinds of hydrolases such as trypsin, papain, thermolysin, etc. w19–27x, are known to catalyze the reverse reaction. a-Chymotrypsin also is reported to play a role to form peptide bonds. a-Chymotrypsin-catalyzed hydrolysis proceeds in two processes: the first process is acylation of serine-195 residue, and the second is a subsequent de-acylation. If the nucleophile molecule is not water but amino acid in the second process, the deacylation of enzyme occurs with the formation of peptide w28,29x. Hydrophobic surroundings are the necessary condition for the reverse reaction. In general, the equilibrium-controlled reaction was carried out in the solution consisting of two phases between
water and water-immiscible organic solvent in order to exhaust the hydrophobic product into the organic phase w30–33x. Since the thermosensitive polymer gel dehydrated with deswelling, surroundings of the enzyme inside the deswollen gel are hydrophobic. This is advantageous for the reverse reaction of the enzyme. However, there has been no report concerning the enzyme immobilized in the thermosensitive gels in order to carry out the reverse reaction for the peptide synthesis. The peptide synthesis under the kinetically controlled condition was usually carried out in the mixed solvent between water and the water-miscible organic solvent, in which the substrates and the produced peptide were dissolved. The peptide synthesis without the organic solvent was possible in the case of coupling between CBZ-Phe and Leu-NH 2 using Tris buffer w20x. The peptide bond formation of hydrophobic Leu-NH 2 was much easier than that of Gly-NH 2 or Ala-NH 2 w20x. Since CBZ is a hydrophobic protecting group substituted on the N-terminal of Phe, the peptide produced exhibits the hydrophobic property to precipitate easily from the Tris buffer. Similarly, the peptide produced from Leu-NH 2 as a nucleophile precipitated easily because the leucine residue in Leu-NH 2 exhibited the hydrophobic property. Therefore, we applied abovedescribed compounds for the reaction of enzyme immobilized in the polyŽNIPAAmrAAm. gel. 4.2. Peptide synthesis as the reaction between CBZ-Phe and Leu-NH2 The result on the temperature dependency in Fig. 5 showed that the free and immobilized enzymes were deactivated above 348C or 388C, at which temperature the gels were in deswollen state. It was supposed from the apparent activity of enzyme that the immobilized enzyme became heat-resistant in the gel. The immobilized enzyme was considered to be located in hydrophobic surroundings inside the deswollen gel due to dehydration. CBZ-Phe, including a hydrophobic CBZ group, is able to react with enzyme to form the acyl–enzyme complex in the hydrophobic surroundings. According to the previous report w28,29x, the acylation for producing the intermediate as acyl–enzyme complex makes the rate-determining step in the sequential reactions for peptide synthesis. It is plausible that CBZ-Phe and Leu-NH 2 may react to form the peptide in the process, as shown in Fig. 1a. 4.3. Peptide synthesis as the reaction between Ac-Phe-OEt and Ala-NH2 The temperature dependency has been reported concerning the peptide synthesis catalyzed by a-chymotrypsin immobilized in agarose w33x. The paper describes that the yield of the kinetically controlled peptide synthesis in-
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creases with decreasing temperature. Our results in Fig. 10B,C show that the conversion into Ac-Phe–Ala-NH 2 is much different from that into Ac-Phe-OH at lower than 58C. The P temperature profile on free enzyme is different from that on immobilized enzyme as shown in Fig. 11. In the case of the free enzyme, the conversion of Ac-Phe– Ala-NH 2 is smaller than that of Ac-Phe-OH at lower temperature as the P is smaller than 1. In the case of the immobilized enzyme, the polymer may exclude water surrounding the acyl–enzyme complex, which is possible to react with Ala-NH 2 rather than water. On the other hand, Fig. 10 indicates that AAm may influence the hydration in the polyŽNIPAAmrAAm. gel. It is speculated that AAm may have stronger affinity of hydration than NIPAAm. Therefore, the immobilized a-chymotrypsin could enhance to catalyze the peptide synthesis as compared with the free enzyme at subzero temperature. The content of AAm in the polyŽNIPAAmrAAm. gel controls the conversion into the peptide, too.
Acknowledgements The authors are indebted to Mr. Kazutoshi Nagaoka and Mr. Hiroyuki Oiwa for their assistance during the experiment.
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