Biotechnology Letters 23: 1033–1037, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
1033
Enantioselective production of levofloxacin by immobilized porcine liver esterase Sang-Yoon Lee1 , Byung-Hyuk Min1, Sung-Ho Hwang1 , Yoon-Mo Koo1 , Choul-Kyun Lee1 , Seong-Won Song2 , Sun-Young Oh2 , Sang-Min Lim2 , Sang-Lin Kim2 & Dong-II Kim1,∗ 1 Department
of Biological Engineering, Inha University, Incheon 402-751, Korea Research Institute, Boryung Pharmaceutical Co., Ansan 425-120, Korea ∗ Author for correspondence (Fax: +82-32-875-0827; E-mail:
[email protected]) 2 Central
Received 25 January 2001; Revisions requested 5 February 2001/28 March 2001; Revisions received 16 March 2001/27 April 2001; Accepted 27 April 2001
Key words: immobilized enzyme, levofloxacin, ofloxacin, porcine liver esterase
Abstract Porcine liver esterase, which cleaves ofloxacin butyl ester enantioselectively to levofloxacin, was successfully immobilized in calcium alginate and polyacrylamide gel. Immobilized esterase in 5% (w/v) calcium alginate exhibited 58% immobilization efficiency and could be reused five times without severe loss of enzyme activity. On the other hand, entrapped esterase in polyacrylamide gel, composed of 20% of total monomer and 8.3% of cross-linking agent, could be reused 10 times, and 51% of enzyme activity remained after the 10th batch without decrease of enantioselectivity. Compared with entrapped methods, significant reduction of enzyme activity was found in the case of physical adsorption on to QAE-Sephadex.
Introduction Since expensive enzymes can be reused and easily separated after the reaction, immobilized enzymes have been used in various areas (Tischer & Wedekind 1999). Usually immobilized enzymes have a shift in the optimum pH and temperature and these changes could result in a positive effect on productivity (Liu et al. 1999). In addition, improvements in stability through immobilization are well known (Cao et al. 1996, Fernandez-Lafuente et al. 1995). Utilization of the entrapment method using calcium alginate or polyacrylamide gel is a typical method of enzyme immobilization (Das et al. 1998). Ofloxacin, one of the fluoroquinolone antibiotics, has focused much attention as the next generation antibiotics. Levofloxacin, the L-form of ofloxacin, is a more effective antibiotic than D-ofloxacin or ofloxacin (Martinez-Martinez et al. 1999). Therefore, levofloxacin is of much higher commercial value than D -ofloxacin. In general, when only one component of a mixture of enantiomers is active, the other com-
ponent is regarded as an impurity since it lowers the activity or may also be toxic in the long term (Kim & Lee 1996). Thus, the selective isolation of the active isomer is important. Many researches are being conducted using enantioselective enzymes or cells expressing with enzymes since the isolations are often carried out under mild condition with high stereospecifivity compared to chemical methods (Vicenzi et al. 1997). In addition to the kinetic (biocatalytic) resolution, one of the most used techniques for the development of chiral compounds, stereoselective assimilation from racemate for the optical resolution has also been reported (Kim et al. 2000). Levofloxacin can be produced selectively from ofloxacin butyl ester using an esterase (Lee et al. 2000). For the enantioselective production of levofloxacin, immobilization is necessary to decrease production costs. In this study, porcine liver esterase was immobilized and used for the enantioselective production of levofloxacin. Immobilization efficiency and the decrease of activity during the repeated use of the immobilized enzyme were studied by comparing cal-
1034 cium alginate and polyacrylamide gel as matrices for entrapment and QAE-Sephadex as an adsorbent. As far as we are aware, this is the first report regarding the use of immobilized esterase in the enantioselective production of levofloxacin.
Materials and methods Chemicals and enzyme reaction Porcine liver esterase and all the chemicals used in this study were obtained from Sigma Chemical Co. The activity of esterase was 20 units per mg solid. One unit hydrolyzes 1 µmol ethyl butyrate to butyric acid and ethanol per min at pH 8 at 25 ◦ C. For enzyme reaction, 2 g esterase l−1 and 5 g ofloxacin butyl ester l−1 were added into 0.1 M phosphate buffer, pH 6.8. The enzyme reaction was carried out in a 100 ml flask containing 20 ml reaction solution shaken at 200 rpm and 30 ◦ C. Enzyme activity was measured as follows. A 0.1 ml sample was held at 100 ◦ C for 5 min. After adding 0.1 ml methanol, the mixture was centrifuged at 5000 g for 10 min and 0.1 ml supernatant was taken to quantify the amount of product.
Enzyme immobilization Sodium alginate and 2 g enzyme l−1 were dissolved in 0.1 M Tris/HCl buffer at pH 6.8 to make the final concentration of alginate 3 and 5% (w/v), respectively. The mixture was slowly dripped into 0.7 M CaCl2 solution using a peristaltic pump and slowly agitated for 1 h at room temperature to harden the bead. 0.1 M Tris/HCl buffer was used to wash the beads and they were dehydrated under vacuum. For the estimation of the immobilization efficiency, protein amounts in the CaCl2 solution as well as in washing solution were quantified by the Bradford method. For the formation of polyacrylamide gel, 0.04 g enzyme dissolved in 1.25 ml of 0.5 M Tris/HCl buffer (pH 6.8) was added in 3.33 ml stock solution containing 0.275 g acrylamide l−1 and 0.025 g N,N methylenebisacrylamide l−1 . After mixing, 0.42 ml distilled water was added to make the final volume 5 ml. To start the polymerization reaction, 25 µl ammonium persulfate stock solution (0.1 g ml−1 ) and 2.5 µl of N,N,N ,N -tetramethylethylenediamine (TEMED) were added. Gelling of acrylamide was performed on a glass plate with the width of 1 mm. After
Fig. 1. Effects of alginate concentration on the immobilization efficiency and enzyme activity during the repeated use. Relative activity is the activity of immobilized enzyme relative to the activity of free enzyme.
gel formation, the gel was cut at the size of 2 × 2 mm and used for the experiment. For the enzyme reaction, 0.1 M phosphate buffer at pH 6.8 was used. For the physical adsorption of enzyme, 1 g QAESephadex was pretreated with 200 ml distilled water in a 500 ml beaker by agitating at 120 rpm and 25 ◦ C for 24 h. After the pretreatment, 0.04 g enzyme and the adsorbent were placed in 0.1 M phosphate buffer (pH 6.8) and stirred for 2 h at 4 ◦ C. The adsorbent was washed with excess buffer and collected for the use in enzyme reaction. Analytical method The quantitative analysis of levofloxacin was by HPLC using a Capcell pak column (4.6×250 mm, Shiseido Co.) with detection of the eluate at 330 nm. The mobile phase was water/methanol (85:15 v/v) with the addition of 1.21 g L-isoleucine l−1 and 1.07 g CuSO4 · 5H2 O l−1 . The flow rate was 1.0 ml min−1 .
Results and discussion Entrapment in alginate beads The physical characteristics of alginate beads are affected by the concentration of alginate and by the type and concentration of metal ion used for gelling, which made the diffusion characteristics in alginate beads different (Tanaka et al. 1984). This diffusion behavior plays a key role in the effective supply of the substrate
1035
Fig. 2. Changes of enantioselectivity during the repeated enzyme reaction by porcine liver esterase entrapped in calcium alginate gel beads obtained at different concentration. Enantioselectivity is defined as [(L-form – D-form)/(L-form + D-form)] × 100.
and the excretion of the product from enzyme reaction out of the beads (Martinsen et al. 1992). Therefore, effects of alginate concentration on the immobilization efficiency and the retention efficiency with repeated use were investigated and the results are shown in Figure 1. Although the initial immobilization efficiency was 58% for the enzyme in both 3% and 5% alginate beads, the former beads readily lost activity with repeated use whereas the latter beads did not. This may be due to the effusion of the enzyme at low concentration of alginate (Martinsen et al. 1989) and it was confirmed that there was continuous effusion in the washing solution for 3% alginate beads. However, when the enzyme was entrapped in 5% alginate, a loss of enzyme activity was not observed after using five times. Figure 2 shows the changes of enantioselectivity during the reaction with immobilized enzyme in alginate. Free esterase when reacted with L-ofloxacin butyl ester and D-ofloxacin butyl ester at the molar ratio of 4:1 gave an enantioselectivity of 60%. However, when the enzyme was immobilized in 5% alginate bead, it initially had an enantioselectivity of 53% and the value remained after repeated use. Whereas the enzyme immobilized in 3% alginate showed the same value as that of free enzyme until the 4th batch. The reason for the lower enantioselectivity in 5% alginate is probably due to diffusion limitations at the high alginate concentration. The drastic decrease of enantioselectivity for the 2nd batch can be explained by the clogging of the pores (Puri et al. 1996). Increase of
Fig. 3. Effects of the concentration of cross-linking agent on the activity of porcine liver esterase entrapped in polyacrylamide gel.
enantioselectivity from the 3rd batch seems to be originated from the damage of some part of the pores and increase of the width, due to the changes in internal structure of alginate bead (Goncalves et al. 1996). As a result, the enzyme immobilized in 3% alginate did not affect the diffusion but the effusion of the enzyme. In 5% alginate, stable retention of the enzyme could be maintained, but the reduction in enantioselectivity according to diffusional limitation could be deduced. Entrapment by polyacrylamide gel The esterase was immobilized in 20% acrylamide gel with N,N -methylenebisacrylamide as the crosslinking agent. Since the ratio of acrylamide to crosslinking agent was known to affect the hardness and porosity of the gel (Palmer 1995), effect of the percentages of N,N -methylenebisacrylamide in total monomers was observed. As shown in Figure 3, the optimal value for maintaining enzyme activity over repeated runs was 8.3%. According to Pizarro et al. (1996), the activity of immobilization enzyme changed as the amount of cross-linking agent increased. When the added amount is small, the enzyme activity is reduced because of the enzyme loss. In contrast, when excess amount of cross-linking agent was added, contact between enzyme and substrate is limited due to the hardness of the gel. The result is also continued in this study. When the cross-linking agent used at 8.3%, even after the repeated use of 10 times, the enzyme activity was maintained at 51%. In addition, the reduction of enantioselectivity was not observed (Figure 4).
1036 acrylamide was much better for the immobilization of porcine liver esterase. Since this is the first report regarding the use of immobilized porcine liver esterase for the enantioselective production of levofloxacin, the process should be optimized further. Covalent immobilization of this enzyme may also be possible. In addition, development of processes and bioreactors for the use of immobilized porcine liver esterase are necessary for the commercial application.
Acknowledgement
Fig. 4. Changes of enantioselectivity during the repeated enzyme reactions by porcine liver esterase entrapped in polyacrylamide gel obtained at various concentrations of cross-linking agents.
This work was supported by the Center of Advanced Bioseparation Technology, Inha University. The support is deeply appreciated.
References
Fig. 5. Relative activity of porcine liver esterase adsorbed on to QAE-Sephadex compared to free enzyme during the repeated use.
Adsorption The same amount of the enzyme used in the previous experiment was immobilized through the adsorption on to QAE-Sephadex and the enzyme reaction was performed for comparison. The results are shown in Figure 5. Initial activity of the immobilized enzyme was 27%, which was significantly lower than that in the entrapped methods with calcium alginate and polyacrylamide. In addition, the reduction of enzyme activity due to desorption was observed during the repeated use. Considering this result, it was apparent that the stable reuse of enzyme is not possible with the adsorption method. Therefore, it was concluded that the entrapment method using calcium alginate or poly-
Cao SG, Yang H, Ma L, Guo SQ (1996) Enhancing enzymatic properties by the immobilization method. Appl. Biochem. Biotechnol. 59: 7–14. Das N, Kayastha AM, Malhotra OP (1998) Immobilization of urease from pigeonpea (Cajanus cajan L.) in polyacrylamide gels and calcium alginate beads. Biotechnol. Appl. Biochem. 27: 25–29. Fernandez-Lafuente R, Cowan DA, Wood ANP (1995) Hyperstabilization of a thermophilic esterase by multipoint covalent attachment. Enzyme Microbiol. Tech. 17: 366–372 Goncalves APV, Cabral JMS, Aires-Barros MR (1996) Immobilization of a recombinant curtinase by entrapment and by covalent binding: kinetic and stability studies. Appl. Biochem. Biotechnol. 60: 217–228. Kim BY, Hwang KC, Song HS, Chung NH, Bang WK (2000) Optical resolution of RS(±)-mandelic acid by Pseudomonas sp. Biotechnol. Lett. 22: 1871–1875. Kim MG, Lee SB (1996) Enzymatic resolution of racemic ibuprofen by lipase-catalyzed esterification reaction: effects of water content and solid supports. J. Ferment. Bioeng. 81: 269–271. Lee SY, Min BH, Hwang SH, Koo YM, Lee CG, Song SW, Oh SY, Lim SM, Kim SL, Kim DI (2000) Enantioselective production of levofloxacin from ofloxacin butyl ester by porcine liver esterase. Kor. J. Biotechnol. Bioeng. 15: 313–317. Liu BL, Jong CH, Tzeng YM (1999) Effect of immobilization on pH and thermal stability of Aspergillus ficuum phytase. Enzyme Microbiol. Tech. 25: 517–521. Martinez-Martinez L, Pascual A, Suarez AI, Perea EJ (1999) Invitro activity of levofloxacin, ofloxacin and D-ofloxacin against coryneform bacteria and Listeria monocytogenes. J. Antimicrobiol. Chemother. 43: 27–32. Martinsen A, Skjåk-Bræk G, Smidsrød O (1989) Alginate as immobilization material: I. Correlation between chemical and physical properties of alginate gel beads. Biotechnol. Bioeng. 33: 79–89. Martinsen A, Storrø I, Skjåk-Bræk G (1992) Alginate as immobilization material: III. Diffusional properties. Biotechnol. Bioeng. 39: 186–194. Palmer T (1995) Understanding Enzymes, 4th edn. New York: Practice Hall/Ellis Horwood.
1037 Pizarro C, Fernandez-Torroba MA, Benito C, Gonzalez-Saiz JM (1997) Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment. Biotechnol. Bioeng. 53: 497–506. Puri M, Marwaha SS, Kothari RM (1996) Studies on the applicability of alginate-entrapped naringinase debittering of kinnow juice. Enzyme Microbiol. Tech. 18: 281–285. Tanaka H, Matsumura M, Veliky IA (1984) Diffusion characteristics of substrates in Ca-alginate gel beads. Biotechnol. Bioeng. 26: 53–58.
Tischer W, Wedekind F (1999) Immobilized enzymes: methods and applications. Topics Curr. Chem. 200: 95–126. Vicenzi JT, Zmijewski MJ, Reinhard MR, Landen BE, Muth WL, Marler PG (1997) Large-scale stereoselective enzymatic ketone reduction with in situ product removal via polymeric adsorbent resins. Enzyme Microbiol. Tech. 20: 494–499.
