Biotechnol. Prog. 1003, 9, 128-130
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Catalytic Peptide Synthesis by Trypsin Modified with Polystyrene in Chloroform Yoshihiro Ito, Hajime Fujii, and Yukio Imanishi' Department of Polymer Chemistry, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto, Japan, 606-01
Trypsin was modified with a hydrophobic synthetic polymer, polystyrene. The attachment was achieved by first coupling free amines in the trypsin with azobis(4cyanovaleric acid) and then irradiating the product in the presence of styrene. Decomposition of the azo structure provided free radical initiation of styrene polymerization, with the trypsin thus becoming part of the end group for the polymer chain initiated. T h a t the modified trypsin retained catalytic activity when dissolved in chloroform was demonstrated by using it to catalyze the formation of a peptide linkage between amino acids. The catalytic reaction was stereoselective.
Introduction The use of enzymes in organic solvents greatly expands their potential applications in organic synthesis. To facilitate this use, enzymes were suspended in organic media, immobilized on insoluble matrices, encapsulated in a reversed-phase micelle,suspended in biphasic systems, complexed with a lipid, mutated site specifically, and hybridized with a synthetic polymer (see Ito et al., 1992). Some researchers reported alkylation of enzymes for this use (Arseguelet al., 1990). Although use in the suspension state is the simplest, the catalytic reaction rate is very low. On the other hand, the hybridization with a polymer has an advantage in the solubilization of enzymes in organic media. However, the modification polymers have been limited to amphiphilic polymers, such as polyethylene glycol (Inada et al., 1986) and polysaccharide-based polymers (Wakselman and Cararet, 1987; Wang et al., 1992). It was believed that these grafted amphiphilic polymers provided a waterlike microenvironment for the enzymes and that this microenvironment is indispensable for enzymatic activity in organic media. However, we recently developed a general method to modify enzymes with various vinyl polymers and found that an enzyme lipase modified with hydrophobic polymers had catalytic activity (Ito et al., 1992). In this article, the new method was applied to another enzyme, trypsin, and the catalytic activity of the modified enzyme was investigated. Materials and Methods Materials. Bovine pancreas trypsin, 4,4'-azobis(4cyanovaleric acid) (ACV, 11, and l-ethyl-3-[3-(dimethy1amino)propyllcarbodiimide hydrochloride (water-soluble carbodiimide, WSC) were purchased from Sigma (St. Louis, MO), Wako (Osaka, Japan),and Dojin (Kumamoto, Japan), respectively. Buffered solutions of pH 4-6,6-8,
HOOCCH,CHzC(CN)(CH,)N=NC(CN)(CH,)CH,CH,COOH 1
and 8-11 were prepared from 0.05 M N-morpholinesulfonic acid, 0.05 M phosphoric acid, and 0.05 M boric acid, respectively. The pH was adjusted by 1 N NaOH. Styrene, toluene, and chloroform were purified by conventional methods. Tos-Lys-OMe.HC1was purchased from Peptide Institute (Osaka, Japan) and desalted by 8756-7938/93/3009-0128$04.00/0
PH Figure 1. pH dependence of the coupling reaction of 4,4'azobis(4-cyanovaleric acid) onto trypsin.
5 5% NaHC03. L-Phe-NH2.HC1 and L-Leu-NHrHC1 were purchased from Kokusan (Tokyo, Japan). They were neutralized by 1N NaOH and extracted with chloroform. D-Phe-NHZwas synthesized from D-Phe-OMe-HC1,which was purchased from Kokusan, by incubation in a 4 N methanol solution of ammonia for 4 days at room temperature. Synthesis of Grafted Trypsin. Trypsin was grafted with polystyrene as was lipase previously (Ito et al., 1992). ACV (12 mg) and WSC (8mg) were mixed in the buffered solution, and the mixture was stirred for 2 h at 4 OC. Trypsin (5 mg) was added to the mixture and stirring continued for 24 h at 4 "C. Nonreacted ACV and WSC were removed by dialysis, and the product (ACV-coupled trypsin) was freeze-dried. The ACV-coupled trypsin was then added to styrene or a toluene solution of styrene (60vol % ). After the mixture was sealed under a nitrogen atmosphere, it was irradiated with a mercury lamp (120 W) for 12 h. Finally the nonsoluble part (ACV-coupledtrypsin) was removed, and polystyrene-grafted trypsin was precipitated in cyclohexaneiacetone (1i1 v/v). Polystyrene was soluble in the mixed solvent. Characterization of ModifiedTrypsin. The content of the amino groups was measured by an aqueous solution of 2,4,6-trinitrobenzenesulfonicacid (0.17% ) as previously reported (Habeeb, 1966). The content of the graft polymer
0 1993 American Chemical Society and American Institute of Chemical Engineers
B b t M .
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Table I. Graft Polymerization onto Trypsin conversion monomer sample no. concn (vol 5% ) (%)"
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polystyrene solubility content (5%) (mg of trypsinlmL of CHCla) 4.0 1.7 84 8.2 100 1 3.8 1.2 79 6.3 60 2 Calculated molecular weight of graft chains. c Grafting Conversion(% ) = [(weightof polymerized monomer)/(weightof used monomer)]100. efficiency(%) = [(weight of grafted monomer)/(weight of polymerized monomer)] 100. MW (x104P
grafting efficiency 58.9 62.9
(I
Table 11. Catalytic Peptide Synthesis by Polystyrene-Grafted Trypsin (Sample 1) in Chloroform substrate catalytic activity carboxy terminal amino terminal (pmolihlmg of trypsin) L-P~~-NH~ 8.0 f 1.7 Tos-Lys-OMe L-Leu-NH? 6.9 f 2.0 Tos-Lys-OMe D-P~~-NH~ 0.7 f 0.2 Tos-Lys-OMe
1800 17501700 1650 1600 15% 1500 1454
1400
WAVENUMBER ( C W 1 )
F i g u r e 2. FT-IR spectra of (a) nonmodified trypsin, (b) polystyrene, and (c) polystyrene-grafted trypsin.
in the grafted trypsin was determined by FT-IR measurement of the absorbance ratio of graft polymer to trypsin. The solubility was measured as follows. Ten milligrams of grafted trypsin was added to 500 pL of chloroform, and the solution was stirred for 15 min. After centrifuging, the supernatant was cast onto a germanium plate and the sample was dried in a vacuum. The amount of dried sample was determined by FT-IR measurement of the driedsample. A calibration curve was obtained by casting a known weight of the sample on the plate. The measurement was repeated five times, and the averaged values were calculated with the standard deviation. Catalytic Activity Measurements. Catalytic peptide synthesis was measured by using amino acids. Five milligrams of grafted trypsin was added to a solution containing 1 mL of chloroform, an amino terminal (LPhe-NH2, L - L ~ u - N Hor ~ , ~-phe-NHz,150 mM), and a carboxyterminal (Tos-Lys-OMe,50 mM), and the solution was incubated at 37 O C . After 10 min, 20 p L of the solution was taken and the amount of formed peptide was determined by HPLC (Cosmosil packed column 5SL; size, 4.6 X 150 mm; eluent, chloroform/methanol/acetic acid 751
2015; rate, 0.6 mL/min). The conversion was only known at the single time. The measurement was repeated five times, and the averaged values were calculated with the standard deviation.
Results and Discussion Coupling of Azo Groups (ACV) onto Trypsin. The percent modification of amino groups by ACV was dependent on the reaction solution pH, as shown in Figure 1. Trypsin contains 15 primary amino groups. In this report, the modified trypsin, in which ca. 50 % of the amino groups were coupled to ACV, was used for further experiments. Graft Polymerization. Evidence that graft polymerization occurred at the ACV site was obtained by using native trypsin containing no ACV. The trypsin did not initiate polymerization. The FT-IR spectra of the modified trypsin are shown in Figure 2. By assuming that all of the ACV introduced was used for initiation, the length of each graft chain was calculated and is shown in Table I. The molecular weight of the graft chains and the conversion ratio were low when the monomer concentration was low. However, the difference did not significantly affect solubility in chloroform. Peptide Syntheses by Grafted Trypsin in Chloroform. Table I1 shows that the polystyrene-grafted trypsin catalyzed peptide linkage formation. The polystyrenemodified lipase catalyzed esterification as did both the poly(N-vinylpyrro1idone)-modified (Ito et al., 1992) and the poly(ethy1ene glycol)-modified (Inada et al., 1986) lipases. Enzymes modified with not only amphiphilic polymers but also hydrophobic polymers in organic solvents catalyzed chemical reactions which are the reverse of those in aqueous solutions. The D isomer was not catalyzed by the grafted trypsin as shown in Table XI. In general, enzymes placed in organic solvents retain their stereoselectivity when used in suspended states, although this depends on the nature of the enzymes and solvents (Zaks, 1991). The grafted trypsin also retained its stereoselectivity in the transparent solution. Many types of enzymes in organic solvents catalyze various reactions in a suspended state. However, in this case no significant activity was found in the nonmodified trypsin. Solubilization of enzymes into organic solvents remarkably enhanced the reaction rates. This study shows that modification by hydrophobic polymers is one of the most useful methods so far to enhance the solubility of enzymes into organic solvents for catalytic reactions. Literature Cited Arseguel, D.; Lattes, A.; Baboulene, M. Enzymes in organic synthesis VII-Enzymatic activity of H R P after chemical modification of the carbohydrate moiety. Biocatalysis 1990, 3,227-233. Habeeb, A. F. S. A. Determination of free aminogroups in proteins by trinitrobenzenesulfonicacid. Anal. Biochem. 1966,14,32& 336. Inada, Y.; Takahashi, K.; Yoshimoto, T.;Ajima, A.; Mataushima, A.; Saito, Y. Application of polyethylene glycol-modified
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enzymes in biotechnological processes: organic solvent-soluble enzymes. Trends Biotechnol. 1986, 4 , 190-194. Ito, Y.; Fujii, H.; Imanishi, Y. Enzyme hybridization with synthetic polymers for use in organic media. Makromol. Chem. Rapid Commun. 1992, 13, 315-319. Wakselman, M.; Cararet, D. In Biocatalysis in organic media; Laane, C.,Tramper, J.,Lilly, M. D., Eds.; Elsevier: Amsterdam, 1987; pp 253-260.
Biotechnol. h g . , 1993, Vol. 9,No. 2
Wang, P.; Hill, T. G.; Wartchow, C. A.; Huston, M. E.; Oehler, L. M.; Smith, M. B.; Bednarski, M. D.; Callstrom, M. R. New carbohydrate-based materials for stabilization of proteins. J . Am. Chem. SOC.1992, 114, 378-380. Zaks, A. In Biocatalysts for Industry; Dordick, J. S.,Ed.; Plenum: New York, 1991; pp 161-180. Accepted November 24, 1992.
