Blotechnol. Prog. 1994, 10, 398-402
398
Modification of Lipase with Various Synthetic Polymers and Their Catalytic Activities in Organic Solvent Yoshihiro Ito,' Hajime Fujii, and Yukio Imanishi* Department of Polymer Chemistry, Faculty of Engineering, Kyoto University, Sakyo-ku, Kyoto, Japan 606-01
A new enzyme modification has been devised where various vinyl monomers were graftpolymerized using enzyme-containing aliphatic azo groups as initiators. Thus, a n enzyme, lipase, which is not soluble in organic solvents, was made soluble in organic solvents. The modified enzyme catalyzed the esterification reaction in chloroform. Catalytic activity in chloroform was obtained by modification with amphiphilic polymers, such as poly(N-vinylpyrrolidone),as well as hydrophobic polymers, such as polystyrene and poly(methy1 methacrylate). The catalytic activities of the hybrid enzymes increased linearly with increased solubility in chloroform. The hybrid enzymes were thermally stable and used repeatedly.
Introduction The use of enzymes in organic solvents greatly expands their potential applications in organic synthesis (Roberts, 1990; Sneider, 1985; Jones, 1986; Whitesides and Wong, 1985). To accomplish this, enzymes have been suspended in organic media, immobilized on insoluble matrices, encapsulated in reverse-phase micelles, suspended in biphasic systems, complexed with lipids, gene-engineered, alkylated, and hybridized with synthetic polymers (as reviewed by us: Ito et al. (1992a, 1993a)l. Although using the enzymes in the suspension state is simple, the catalytic reaction rate is low because of its heterogeneity. It takes several days to complete the reaction and even more time for high molecular weight substrates. Hybridization with polymers has the advantage of complete solubilization of the enzyme in organic media. Therefore, the reaction rate is usually enhanced 10-100 times, although the final yield of the product in the solvated state is the same as that under suspension. However, the modification polymers have been limited to amphiphilic polymers, such as poly(oxyethylene), carbohydrate-based polymers, and their derivatives. It was believed that these grafted amphiphilic polymers provide a waterlike microenvironment for the enzymes, as shown in Figure 1. Recently, we devised a new method of synthesizing vinyl polymer/enzyme hybrids, in which various vinyl polymers can be selected, by using lipoprotein lipase (LPL) (Ito et al., 1990, 1992a,b) or trypsin (Ito et al., 1993a,b) coupled with an aliphatic azo compound as the initiator for graft polymerization. We then investigated catalysis by the hybrid enzyme in organic solvents. This method was applied to various functional polymers, including those that are hydrophobic and hydrophilic. Materials and Methods Materials and Reagents. Lipoprotein lipase (Pseudomonas fluorescens origin), 4,4'-azobis(4-cyanovaleric acid) (ACV), and the water-soluble carbodiimide (WSC),1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride, were purchased from Amano Pharm. (Nagoya, Japan), Wako Pure Chem. Ind. (Osaka, Japan), and Dojin Lab. (Kumamoto, Japan), respectively. Monomers and solvents were purified by conventional methods and were used without further purification. Buffers of pH 4-6 6-8, and 8-10 were prepared from 0.05 M (N8756-7938/94/3010-0398$04.50/0
Amphipathic polymer
Hydrophobic polymer
Figure 1. Schematic drawing of polymer-modified enzymes working in organic media. It is believed that the water molecules in amphiphilic polymers are important for the catalytic activity of the modified enzymes.
morpho1ino)ethanesulfonicacid, 0.05 M phosphoric acid, and 0.05 M boric acid, respectively. The pH was adjusted with HC1 or NaOH. Synthesis of Hybrid Lipase. The hybrid lipase was synthesized as previously reported (Ito et al., 1992a,b, 1993a,b). ACV (12 mg) and WSC (8 mg) were added to a buffer solution (10mL) of agiven pH value. The mixture was stirred for 2 h at 4 "C. After the addition of lipase (5 mg), the mixture was stirred further for 24 h at 4 "C. Unreacted ACV and WSC were removed by dialysis, and the product was freeze-dried from 0.05 M aqueous phosphate buffer (pH 7.4). The amount of ACV introduced to the enzyme was determined by measuring the residual amino groups of lipase by 2,4,6-trinitrobenzenesulfonic acid, as reported previously (Habeeb, 1966). The azo-containinglipase (ACV-lipase, 5 mg) was added to undiluted sytrene (ST) or methyl methacrylate (MMA), to a toluene solution (60 or 80 vol 5%) of ST or MMA, or to an aqueous solution (5 or 10 vol ?4) of N-vinylpyrrolidone (VP) or acrylonitrile (AN). After it was purged with nitrogen gas, the mixture was photoirradiated for 12 h with a 120-W mercury lamp. After photoinitiated graft polymerization, the reaction product was recovered and purified as follows. In the graft polymerization of ST,the polystyrene (PST)-lipase hybrid (PST-LPL) is soluble in toluene or ST. Therefore, insoluble, unused ACV-lipase was filtered, and the filtrate was poured into a cyclohexane/
0 1994 American Chemical Society and American Instkute of Chemical Engineers
Biotechnol. Prog., 1994, Vol. 10, No. 4
acetone (Ul,v/v) mixture. The precipitated PST-LPL was recovered. In the graft polymerization of MMA, insoluble, unused ACV-lipase was also filtered and the filtrate was poured into acetone. The precipitated poly(methyl methacrylate) (PMMA)-lipase (PMMA-LPL) was recovered. In the graft polymerization of VP, soluble, unused ACV-lipase was removed by ultrafiltration using an Amicon PM-30 membrane. The complete removal of unreacted initiator was confirmed by gel filtration with Sephadex G-100. The residual solution was poured into ethanol to precipitate and recover the poly(N4nylpyrrolidone) (PVP)-lipase hybrid (PVP-LPL). In the graft polymerization of AN, poly(acrylonitri1e) (PAN) and the PAN-lipase hybrid (PAN-LPL) precipitated during polymerization. The precipitate was collected, and unused ACV-lipase was removed with water. PAN was extracted with nitromethane. Analysis of Vinyl Polymer-Lipase Hybrids. The amount of grafted vinyl polymer was calculated on the basis of the vinyl polymer versus the lipase weight ratio, as determined by the intensity ratios of the characteristic absorptions in Fourier transform infrared (FT-IR)spectra. The solubility of hybrid LPLs in chloroform was also determined by FT-IR spectroscopy. A hybrid LPL (10 mg) was dissolved in chloroform (500 pL), shaken for 15 min, and then centrifuged. The surpernatant (20 pL) was cast into a film on a Ge plate. The film was dried in vacuo and subjected to FT-IR spectroscopy to determine the amount of lipase. Enzymatic Activity of Hybrid LPLs. The hydrolytic activity of aqueous hybrid LPL was determined using the “Lipase kit S” (Dainihon Seiyaku, Tokyo, Japan), which measures the absorbance a t 410 nm and is based upon the 2-nitrobenzoic acid thioanion produced by a combination of 5,5’-dithiobis(2-nitrobenzoicacid) and 2,3-dimercaptopropanol, which arises from the hydrolysis of 2,3dimercaptopropanol tributyrate. The esterification activity of hybrid LPL in chloroform was measured as follows. Hybrid LPL (5 mg) was added to chloroform (1mL) containing n-amyl alcohol (0.46M) and n-caprylic acid (0.32 M), and the mixture was incubated at 37 “C. After various incubation periods, the amount of ester produced in the chloroform solution was determined by HPLC (Cosmosil-packed column 5SL; 4.6 mm diameter X 150 mm length; moving phase, CHCld n-hexanelisopropyl alcohol (99/99/2, v/v); velocity, 1 mL/ min) .
Results and Discussion Introduction of an Aliphatic Azo Group into Lipase. ACV was coupled to amino groups of LPL under different pH values. More ACV was introduced to the enzyme under more acidic conditions, as shown in Figure 2. The enzymatic activity of lipase gradually decreased with the increase of the coupling ratio to 80% and dropped sharply with higher ACV coupling ratios. Graft Polymerization Initiated by ACV-Lipase. Since the graft polymerization of vinyl monomers by ACVlipase was achieved under irradiation with a mercury lamp, the influence of UV irradiation on the enzymatic activity of ACV-lipase was investigated in the solvents used, namely, water and toluene. Although the hydrolytic activity of ACV-lipase decreased slightly with an increased irradiation period, the nature of the solvent (water or toluene) did not affect it. Prolonged irradiation had a marked effect. Therefore, the samples were prepared by a 12-h irradiation. UV irradiation of the monomer solution containing ACV-lipase induced polymerization of ST, MMA, VP,
300
100
3
4
5
6
7
8
9
1011
PH
Figure 2. Dependence of the rate of ACV coupling to lipase on
the pH value of the reaction solution.
and AN. However, no polymerization occurred in the presence of natural lipase. Therefore, we concluded that the polymerization was initiated by the ACV group introduced to lipase. The reaction product in the graft polymerization was recovered and purified. Peaks characteristic of LPL and the synthetic polymers were observed in the FT-IR spectra of PST-LPL and PVP-LPL. By assuming that all of the ACV groups coupled to lipase were used to initiate the graft polymerization, the molecular weight (MW) of the graft chain was calculated and is shown in Table 1 along with other results. In all graft polymerizations of the four monomers, the MW of the graft chain decreased with a decreasing concentration of feed monomer and an increasing initiator concentration, that is, increasing ACV content in the ACV-lipase. The conversion of the monomer and the grafting efficiency were lower in the graft polymerizations of ST and MMA, which were homogeneous, than those in the graft polymerizations of VP and AN, which were heterogeneous. The yield of hybrids increased with increasing synthetic polymer content in the modified LPL. Solubility of Vinyl Polymer/Lipase Hybrids. The solubility in chloroform of vinyl polymer/lipase hybrids with a constant number of various graft chains (ACV coupling, 60%)is shown in Figure 3. PST-LPL, PMMALPL, and PVP-LPL solubility in chloroform increased with increasing amounts of vinyl polymers, while PANLPL was nearly insoluble, which was not influenced by the PAN content. The solubilities in chloroform of PST-LPL with different numbers of graft chains are shown in Figure 4.All of the curves turn upward, indicating that increasing the chain length of the graft chains increases the solubility of hybrid LPL. Comparison of PST-LPLs with a constant amount of graft chains showed that a few, long graft chains are more effective in increasing the solubility in chloroform than many short chains. Catalytic Activity of Hybrid LPLs in the Esterification Reaction in Chloroform. Catalytic activities in the condensation reaction of n-amyl alcohol and n-caprylic acid of LPL hybridized with various vinyl polymers and PST-LPL with various numbers of graft chains are shown in Figure 5. High catalytic activities in the esterification reaction in chloroform were observed with LPL hybridized with hydrophobic PST or PMMA, as well as with LPL hybridized with amphiphilic PVP. The catalytic activity increased with the content of graft
400
Biotechnol. Prog., 1994, Vol. 10, No. 4
Table 1. Graft Polymerization with Modified Lipase modification monomer initiator conversion MW grafting yield concn (vol %) concn (nmol/L) (%)a ( X I@)* efficiency(%)c (%)d no. sample ration (%) 1 PST-LPL 80 100 8.10 8.2 5.9 30.4 93 2 80 8.10 6.3 4.9 34.4 92 3 60 8.10 6.3 3.9 29.8 74 4 60 100 6.14 4.5 7.4 32.8 69 5 80 6.14 3.9 5.8 31.7 58 6 60 6.14 3.1 4.1 21.7 34 7 40 100 4.14 2.6 7.9 18.5 32 8 80 4.14 2.8 6.7 17.5 30 9 60 4.14 1.9 5.4 19.8 21 10 20 100 2.10 1.9 12 11.2 19 11 80 2.10 1.3 9.8 10.4 12 12 60 2.10 1.5 7.8 12.4 15 13 PMMA-LPL 60 100 6.14 18 16 41.4 91 14 60 6.14 8.2 7.1 19.4 49 15 PW-LPL 60 10 6.14 41 4.0 58.3 70 5 16 6.14 44 2.7 50.1 66 17 PAN-LPL 60 10 6.14 55 12 63.0 88 18 5 6.14 45 11 60.4 35 Conversion = [(weight of polymerized monomer)/(total weight of monomer added)] X 100. Molecular weight of a graft chain = (moles of monomer unit grafted)/(moles of azo group added) X (molecular weight of vinyl monomer). e Grafting efficiency (%) = (weight of grafted monomer)/(weight of polymerized monomer)] X 100. Yield (%) was based on the protein obtained.
6 4 -
2 -
E
v
n
I
70
80
90
100
Vinyl Polymers in hybrid LPL ( w t % ) Figure 3. Dependence of hybrid LPL solubility in chloroform on the content of vinyl polymers in hybrid LPL: (B) PST-LPL; (0) PMMA-LPL; (0) PAN-LPL; ( 0 )PVP-LPL.
0" 70
I
75
'
"
80
I
'
85
I
90
.
I
95
"
100
PST i n PST-LPL ( w t % ) Figure 4. Dependence of PST-LPL solubility in chloroform on the content of PST in PST-LPL with rates of ACV coupling of 20% (o), 40% (0),60% (o),and 80% (B). polymers and varied with the kind, number, and length of graft chains for a given amount of graft polymers. The catalytic activities during the esterification reaction in chloroform of LPL hybridized with various vinyl
0 70
75
80
85
90
95
100
Vinyl Polymers in hybrid LPL ( w t % ) Figure 5. Dependence of hybrid LPL catalytic activities in chloroform on the content of graph polymers. Hybrid LPLs upon PST; ( 0 )PMMA (0) PVP; the rate of 60% ACV coupling: (0) (m) PAN.
polymers or with different numbers of PST were plotted against their solubility in chloroform (Figure 6). The catalytic activity increased linearly with the solubility in chloroform. These results indicate that the catalytic activity of LPL enhanced by hybridization with vinyl polymers is not due to substrate affinity by the graft chains but is due to the accessibility of the substrate to the enzyme, as has been explained by Zaks and Klibanov (Zaks and Klivanov, 1985; Zaks, 1991). The effect of N,N-dimethylformamide (DMF) addition on the catalytic activity of hybrid LPLs in the esterification reaction in chloroform was investigated, and the results are shown in Figure 7. The catalytic activity decreased in the presence of increasing DMF and ultimately disappeared at over 40 76 (v/v) DMF. The deteriorating effect of DMF was slightly stronger with PVP-LPL than with PST-LPL. This difference should be due to the slightly higher solubility of PVP-LPL than PST-LPL in DMF. Amphiphilic poly(oxyethy1ene) has often been used for enzyme modification. The use of poly(oxyethy1ene) has been rationalized on the basis that the amphiphilicpolymer chains hold the water needed to maintain the native conformation of the enzyme (Takahashi et al., 1984; Yoshimoto et al., 19841, as shown in Figure 1. This
Bbtechml. Rog., 1994, Vol. 10, No. 4
h
t
40 I
I
8 -
6 -
2 3 4 5 Solubility (mg LPL/mL chloroform) Figure 6. Relationshipbetween catalyticactivity and solubility of hybrid LPLs in chloroform. Hybrid LPLs: ( 0 )PST-LPL; (m) PMMA-LPL; (0) PAN-LPL; (0) PVP-LPL. The number indicates the sample number shown in Table 1. 0
30 40
1
1oop
I
h
.-*
Ic)
CI
v
10 20 30 40 50 Amount of DMF in DMF/CHC13 mixture (~01%) Figure 7. Decrease in catalytic activities of hybrid LPLs in chloroform after the addition of DMF [(O)PST-LPL and (0) PVP-LPL], with an ACV coupling rate of 60%.
0
consideration has been supported by the fact that the catalytic activity of the hybrid enzyme decreases sharply with the addition of a water-miscible organic solvent, such as dimethylformamide. However, this investigation showed that LPL modified with hydrophobic polymers containing no water exhibited catalytic action in organic solvents and that the activity was by the addition of dimethylformamide. These results demonstrate that water bound in the part of the enzyme, not in the part of the modification polymer, maintains the catalytic activity of the hybrid, as shown in Figure 1. Repeated Use and Thermal Stability of Hybrid LPLs in Organic Solvents. PST-LPL (ACV coupling, 60%; PST content, 90.4 wt 7%) and PVP-LPL (ACV coupling, 60%; PVP content, 83.7 wt 5%) were recovered from esterification solutions by precipitation with methanol and cyclohexane, respectively. The catalytic activities of PST-LPL and PVP-LPL decreased slightly after repeated use, but maintained 60-70 % of the original values after being used five times. The behavior of the two modified LPLs was similar. Improved stability after repeated use has been reported for modification with poly(oxyethylene) (Yoshimoto et al., 1984). The same PST-LPL and PVP-LPL were incubated in water or chloroform at a specified temperature for 15min,
5 0 60 7 0 8 0 90 100 Temperature ( C)
Figure 8. Thermal effect on the catalytic activities of hybrid LPLs under various conditions: (e) LPL in water; (a) PVPLPL in water; ( 0 )PST-LPL in chloroform, (0) PVP-LPL in chloroform.
and the hydrolytic activities in an aqueous solution and the esterification activities in chloroformwere investigated. Figure 8 shows that the native LPL lost catalytic activity completely after incubation in water at 85 "C for 15 min, but that PVP-LPL is more thermally resistant under the same conditions. In the esterification reaction in chloroform, PST-LPL and PVP-LPL maintained relative catalytic activities of about 50% after incubation at 85 "C for 15 min. PVP-LPL seemed to be more thermally resistant in chloroform than in water, although a strict comparison cannot be made between esterification and hydrolysis. Improved thermal stability of enzymes by modification with synthetic polymers has been reported (Hiroto et al., 1992). In addition Zaks and Klibanov (1986)previously reported that the enzyme was more stable in organic media at high temperatures. The method of enzyme modification, which we reported here, effectively improved the thermal stability and allowed repeated use. The stabilization of enzyme by modification with synthetic polymers, particularly in organic solvents, may have resulted from the enzyme assuming a rigid conformation after modification.
Notation ACV
4,4'-azobis(4-cyanovaleric acid) wsc water-soluble carbodiimide LPL lipoprotein lipase ST styrene PST polystyrene MMA methyl methacrylate PMMA poly(methy1 methacrylate) VP N-vinylpyrrolidone PVP poly (N-vinylpyrrolidone) AN acrylonitrile PAN poly(acrylonitri1e) PST-LPL polystyrene-modified lipase PMMA-LPL poly(methy1 methacrylate)-modifiedlipase PVP-LPL poly(N-vinylpyrro1idone)-modified lipase poly(acrylonitri1e)-modifiedlipase PAN-LPL Supplementary Material Available: FT-IR spectra and plots indicating the catalytic activities of various polystyrene-
Blotechnol. frog,, 1994,Vol. 10,No. 4
402
modified lipases (3pages). Ordering information is given on any current masthead page.
Literature Cited Habeeb, A. F. S. A. Determination of free aminogroup in proteins by trinitrobenzenesulfonicacid. Anal. Biochem. 1966,14,328336. Hiroto, M.; Matsushima, A,; Kodera, Y.; Shibata, Y.; Inada, Y. Chemical modification of lipase with a comb-shaped synthetic copolymer of polyoxyethylenealkyl methyl diether and maleic anhydride. Biotechnol. Lett. 1922,14, 559-564. Ito, Y.; Fujii, H.; Imaniehi, Y. In Protein Engineering Protein Design in Basic Research, Medicine, and Industry; Ikehara., M., Ed.; Springer-Verlag: Tokyo, 1990; pp 249-252. Ito, Y.; Fujii, H.; Imanishi,Y. Enzyme hybridizationwith synthetic polymers for use in organic media. Makromol. Chem. Rapid Commun. 1992a, 13,315-319. Ito, Y.; Fujii, H.; Imanishi, Y. Lipase modification by various synthetic polymers for use in chloroform. Biotechnol. Lett. 1992b,14, 1149-1152. Ito, Y.; Fujii, H.; Imanishi, Y. Catalytic peptide synthesis by trypsin modified with polystyrene in chloroform. Biotechnol. Prog. 1993a,9,128-130. Ito, Y.; Kotoura, M.; D. J. Chung; Imanishi, Y. Synthesis of vinyl polymer/trypsin hybrid with variable solubilities in response to external signal. Bioconjugate Chem. 1993b, 4, 358-361. Jones, J. B.Enzymes in organic synthesis. Tetrahedron 1986, 42,3351-3403.
Roberta, S. M. In Microbial enzymes and biotechnology, 2nd ed.;Fogarty, W .M., Kelly, C. T., Eds.; Elsevier: London, 1990; pp 396-422. Sneider, M. D. Enzymes as catalysts in organic synthesis; D. Reidel Publishers: Dordrecht, The Netherlands, 1985. Takahashi, K.; Nishimura, H.; Yoshimoto, T.; Okada, M.; Ajima, A.; Matsushima, A.; Tamura, Y.; Inada, Y. Polyethylene glycolmodified enzymestrap water on their surfaceand exert enzymic activity in organic solvents. Biotechnol. Lett. 1984,6, 765770. Whitesides, G. M.; Wong, C. H. Enzymes as catalysts in synthetic organic chemistry. Angew. Chem., Int. Ed. Engl. 1985, 24, 617-718. Yoshimoto,T.; Takahashi, K.; Nishimura, H.; Ajima, A.; Tamura, Y.; Inada, Y. Modified lipase having high stability and various activities in benzene, and its re-use by recovering from benzene solution. Biotechnol. Lett. 1984,6 , 337-340. Zaks, A. In Biocatalysts for industry; Dordick, J. S., Ed.; Plenum: New York, 1991;pp 161-180. Zaks, A,; Klibanov, A. M. Enzymatic catalysis in organic media at 100 OC. Science 1984,224,1249-1251. Zaks, A.;Klibanov, A. M. Enzyme-catalyzed processes in organic solvents. h o c . Natl. Acad. Sci. U.S.A. 1985,82,3192-3196. Accepted November 23, 1993.' 0 Abstract published in Advance ACS Abstracts, February 15, 1994.
