Biomacromolecules 2004, 5, 249-254
Improving the Activity of Lipases from Thermophilic Organisms at Mesophilic Temperatures for Biotechnology Applications Jose M. Palomo, Rosa L. Segura, Cesar Mateo, Roberto Fernandez-Lafuente,* and Jose M. Guisan* Departamento of Biocatalisis. Instituto of Catalisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain Received August 25, 2003 Revised Manuscript Received November 11, 2003
Introduction One of the most straightforward ways to solve the enzyme stability problem is the selection of enzymes produced by thermophilic organisms. The enzymes from thermophilic microorganisms have been described to be thermo-stable and resistant to the action of organic solvents.1-8 Thus, these enzymes can be used as interesting biocatalysts for biotechnological applications.9-12 However, in general, these enzymes usually present very low activity at temperatures below 30 °C, conditions where most of the interesting compounds in fine chemistry are stable. Lipases, which are very useful enzymes in organic chemistry,13-15 present a complex mechanism of action, suffering critical conformational changes between a closedinactive structure and an open-active one.16-18 These changes are more difficult to have happen at temperatures far from their natural working temperature, increasing the problem of the low activity at mesophilic temperature. Cellular components of members of the genus Thermus are generally more resistant to the most common protein denaturants than their counterparts among mesophilic bacteria, which makes them extremely interesting.19-20 In this paper, we have performed an initial characterization of the lipases from Thermus thermophilus (TTL) and from Thermus aquaticus (TAL), studying their stability and activity of the free and immobilized enzyme. The immobilization has been performed with the use of a new protocol designed to specifically immobilize lipases, based upon the catalytic mechanism of lipases, i.e., the use of hydrophobic support at low ionic strength. This support mimics the natural substrate (drops of oil) and has permitted the one step purification, immobilization, and hyperactivation of several mesophilic lipases21-23 (Scheme 1). Experimental Section Materials. The lipases from TTL (62296) and TAL (62293) were supplied by Fluka. Octyl-agarose 4BCL was purchased from Pharmacia Biotech (Uppsala, Sweden). Octadecyl-Sepabeads were generously donated by Resindion Srl (Mitsubishi Chem. Coorp.) (Milan, Italy). Triton X-100, * To whom correspondence should be addressed. (J.M.G./R.F.-L.) Telephone: +34 91 585 48 09. Fax: +34 -91 585 47 60. E-mail: jmGuisa´
[email protected];
[email protected].
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butyl butyrate and ethyl butyrate (1) were obtained from Sigma. (()-Glycidyl butyrate [(()-3] was kindly donated by Dr M. Terreni (Pavia, Italy). Methods. All experiments were carried out in triplicate, and the experimental error was never over 5%. Activity Determination Assay. The assay was performed using a pHstat-instrument (Mettler Toledo D50) by measuring the release of butyric acid promoted by the enzymatic hydrolysis of butyl butyrate dissolved at different temperatures in 25 mM sodium phosphate buffer at pH 7 to a substrate concentration of 30 mM. 150 mM NaOH was used as titrating agent. The assay temperature was 45 °C for TTL and 65 °C for TAL. Immobilization of Lipases on Octadecyl-Sepabeads Support. Lipase commercial extracts were dissolved to give 10 mg of extract/mL in 5 mM sodium phosphate, submitted to gentle stirring during 2 h at 25 °C and pH 7, and centrifuged at 12 000 rpm during 30 min. The protein concentration in the commercial preparation of the different lipases was determined by the Bradford method.24 The TTL contained 0.95 mg of protein/mL and the TAL presented 0.5 mg of protein/mL. In a standard experiment, 10 mL of octadecyl-Sepabeads were added to a solution of 10 mL (to TAL) or 20 mL (to TTL) of lipase preparation in 90 mL of 5 mM sodium phosphate buffer at pH 7 and 25 °C to obtain immobilized preparations with a final enzyme loading of 1 mg/mL (Scheme 2). A blank suspension was prepared by adding 1 mL of octadecyl-Sepabeads. Periodically, the suspensions and supernatants activity was analyzed using the previous butyl butyrate assay. In all of the cases, more than 95% of the available enzyme was immobilized. After immobilization, the adsorbed lipase preparation was thoroughly washed with distilled water. SDS-PAGE Analysis. 1 mL of enzyme solution or 0.7 g of enzymatic preparations were suspended in 1 mL of 0.125 mM Tris, containing bromophenol, 10% (v/v) mercaptoethanol, 40% glycerol, and 4% (w/v) SDS. This treatment desorb any protein physically adsorbed on the support.21 SDS-PAGE electrophoresis was performed according to the Laemli’s method25 in a SE 250-Mighty Small II electrophoretic unit (Hoefer Co.) with the use of gels of 12% polyacrylamide in a separation zone of 9 cm × 6 cm and a concentration zone of 5% polyacrylamide. The gels were stained following the silver staining method.26 Molecular weight markers were the LMW kit (14 400-94 000 Da) from Pharmacia (Figure 1). Stability Assay of the Different Lipase Preparations. The different immobilized preparations were incubated under the conditions aforementioned (pH, T). Samples of these inactivating reactions were periodically withdrawn and their activity was assayed as previously described. Temperature-Enzyme Activity Profile of Different Lipases Preparations. The effect of the temperature on the enzyme activity was checked during the hydrolysis of butyl
10.1021/bm034317d CCC: $27.50 © 2004 American Chemical Society Published on Web 12/19/2003
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Notes
Scheme 1. Interfacial Activation of Lipases on Octadecyl-Sepabeads
Scheme 2. Different Organic Compounds Hydrolyzed by Lipases
butyrate dissolved in 25 mM sodium phosphate buffer at pH 7 to a substrate concentration of 30 mM. The buffer was preincubated to reach the desired temperature before adding the butyl butyrate and the enzyme. The initial rates of preparations were calculated at each temperature to avoid effects due to the thermal denaturation of the enzyme. Synthesis of 2-O-Butyryl-2-phenylacetic Acid [(()-2]. 1H and 13C NMR spectra were obtained with TMS (tetra-
methylsilane) as an internal standard using a Bruker AC300 (1H-250 MHz and 13C-75.5 MHz) spectrometer. A solution of butyryl chloride (2131 mL, 20 mmol) in diethyl ether (100 mL) was added dropwise over a stirred solution of mandelic acid (3042 g, 20 mmol) in diethyl ether (200 mL) with NEt3 (2.88 mL, 20 mmol). The mixture was stirred at 25 °C for 4 h (it was monitored by HPLC, as described bellow). The mixture was extracted with water and diethyl ether. The organic phase was dried over Na2SO4, filtered and concentrated under reduced pressure. Yield: 50%, light yellow oil, Pm: 222.1, 1H-RMN (CDCl3): δ (ppm): 0.95 (m, 3H), 1.7 (septete, 2H), 2.4 (m, 2H), 5.9 (s, 1H, CH), 7.4 (m, 4H), 13C NMR (CDCl3): 173 (CdO), 170 (CdO), 131.8 (C), 127.8 (CH), 127.3 (CH), 126.1 (CH), 76.018 (CH), 34.24 (CH2), 16.76 (CH2), 12.065 (CH3). Enzymatic Hydrolysis of Esters. The activities of TTL and TAL preparations were analyzed during the hydrolysis reaction of different esters (Scheme 2). Compound 1 was dissolved in 100 mL of 25 mM sodium phosphate buffer at pH 7 to a final substrate concentration of 10 mM, and 0.1 g of immobilized preparation were added. Compound (()-2 was dissolved in a 3 mL solution of 25 mM sodium acetate buffer (pH 5) or 25 mM sodium phosphate buffer (at pH 7) at 25 °C to a substrate concentration of 0.5 mM, and 0.5 g of immobilized preparation were added. Substrate (()-3 was dissolved in a 10 mL solution of 25 mM sodium phosphate buffer to a concentration of 5 mM at pH 7 under different conditions (pH, T, presence of acetonitrile), and 0.05 g of immobilized preparation were added.
Figure 1. SDS-page gel of immobilized T. aquaticus lipase (TAL) on octadecyl-Sepabeads support at 25 °C and pH 7. Lane 1: molecular weight markers. Lane 2: commercial TAL preparation. Lane 3: lipase adsorbed on octadecyl-Sepabeads support. Experiments were performed as described in the Experimental Section.
During the reaction, the pH value was maintained constant using a pH-stat Mettler Toledo DL50 graphic. The enzymatic activity was defined as µmol of substrate hydrolyzed per minute per mg of immobilized protein. The degree of hydrolysis was analyzed by reverse-phase HPLC (Spectra Physic SP 100 coupled with an UV detector Spectra Physic SP 8450). For these assays, a Kromasil C18 (25 × 0.4 cm) column was used, a mobile phase acetonitrile/10 mM ammonium phosphate buffer at pH 2.95 (35:65, v/v) at 1.5 mL/min, and UV detection was performed at 225 nm (compounds 1 and 3) and 254 nm (compound 2).
Notes
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Figure 3. Reaction temperature effect on the hydrolytic activity of T. thermophilus lipase. Experiments were performed using butyl butyrate at pH 7, as described in the Experimental Section; octadecylSepabeads (rhombus), soluble enzyme (squares).
Figure 2. Immobilization course of Thermus lipases on octadecylSepabeads support at 25 °C and pH 7. (a) T. thermophilus lipase; (b) T. aquaticus lipase Suspension (triangles), supernatant (rhombus), soluble enzyme (squares). Experiment was performed as described in the Experimental Section.
Determination of Enantiomeric Excess and Enantioselectivity. The enantiomeric excess (ee) of the released acid of compound (()-2 was analyzed by the Chiral Reverse Phase HPLC. The column was a Chiracel OD-R, and the mobile phase was an isocratic mixture of acetonitrile: NaClO4/HClO4 0.5 M, (5:95 v/v) with a final pH of 2.3, at a flow of 0.5 mL/min and UV detection was performed at 225 nm. The enantiomeric excess (ee) of the remaining ester of compound (()-3 was analyzed by the Chiral Phase HPLC. The column was a Chiracel OD, with a mobile phase being an isocratic mixture of hexane and 2-propanol (5:95 v/v) at a flow of 0.5 mL/min and UV detection was performed at 225 nm. The enantiomeric ratio (E) was calculated in all cases using the equations reported by Chen et al.27 Results and Discussion Immobilization of Lipases from Thermus Microorganisms. The lipases from TTL and from TAL were immobilized on octadecyl-Sepabeads. In the SDS-PAGE, it may be observed that the lipase from T. aquaticus was the only enzyme adsorbed on the support from the crude extract at very low ionic strength (Figure 1). In fact, native electrophoresis only reveals one band with esterase activity. Therefore, this may be a very simple method to purify lipases.21-23 Figure 2 shows the immobilization course for TTL and TAL on octadecyl-Sepabeads support. TTL was adsorbed very quickly on this hydrophobic matrix, with more than 95% of the available enzyme immobilized in 1 h. Moreover, the lipase increased its activity by a 4-fold factor at 45 °C. However, TAL did not exhibit a significant increase in the specific activity after its adsorption on octadecyl-Sepabeads at the assay temperature (65 °C), maintaining the initial one.
The immobilization of these lipases via other methodologies, such as glyoxyl-agarose (covalent attachment) or DEAE (ionic exchange), did not produce this increase in the activity but promoted a decrease of up to 20-30% of the initial value (results not shown). Activity-T Profile of the Different Lipase Preparations. The evolution of the enzymatic activity related to the temperature for free and immobilized TTL is shown in Figure 3. It can be observed that the lipase in free form at temperatures below 30 °C presented very low activity or even no detectable activity, whereas after its adsorption on octadecyl-Sepabeads support, the lipase showed an increase in the activity of more than 50-fold factor at 30 °C. This increment in the activity at low temperatures may increase the uses of these lipases and suggests that the low activity of the free enzyme may be the result of a small percentage of “open lipases” at low temperatures. Moreover, although the optimal temperature was not significantly altered, the residual activity of the free enzyme at high temperature decreases rapidly, becoming almost inactive at 56 °C, whereas the immobilized enzyme showed essentially no increase in its activity over the range from 25 to 56 °C, and after this point, the activity significantly increased. TAL presented a behavior similar to TLL at temperatures below 30 °C where the immobilized lipase increased its activity by more than 10 times with respect to the soluble enzyme activity. The soluble enzyme reached a maximum of activity around 60 °C, whereas the maximum activity of octadecyl-Sepabeads-TAL preparation was reached at 70 °C (Figure 4). Furthermore, another interesting phenomenon of this enzyme was that, after adsorption on octadecyl-Sepabeads, it was possible to observe two different activation energies; the immobilized enzyme almost did not increase the activity in the range from 25 to 56 °C, and at this point, its activity strongly increases. Therefore, the immobilization of these thermophilic lipases on this hydrophobic support allows their utilization at low temperatures when previously it was pretty difficult or even impossible when thermophilic lipases or immobilized lipases on other supports were used due to their low activity. This way of improving the enzyme activity at low temperature may generally apply to other thermophilic lipases. Effect of Immobilization on Enzyme Stability. Figure 5 shows the thermal inactivation of TAL at 70 °C. The
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Notes
Figure 4. Reaction temperature effect on the hydrolytic activity of T. aquaticus lipase. Experiments were performed using butyl butyrate at pH 7, as described in the Experimental Section; octadecylSepabeads (squares), soluble enzyme (triangles).
Figure 6. pH-stability of T. aquaticus lipase on octadecyl-Sepabeads support. Inactivation was performed at 25 °C and different pHs. (a) pH 9, (b) pH 5. Octadecyl-Sepabeads-TAL (squares), soluble enzyme (circles). Figure 5. Thermostability of T. aquaticus lipase on octadecylSepabeads support. Inactivation was performed at pH 7 and 70 °C. Octadecyl-Sepabeads-TAL (squares), soluble enzyme (circles).
octadecyl-Sepabeads-TAL preparation maintained 100% of its activity after 70 h of incubation under those conditions, whereas the soluble enzyme activity decreased up to 20%. A similar stabilization was found using TTL (results not shown). With regard to the stability at different pHs, the activity of octadecyl-Sepabeads-TAL preparation was fully maintained after 5 incubation days at pH 9 and at 25 °C, whereas the soluble enzyme lost more than 50% of the initial activity (Figure 6a). Moreover, when the preparations were incubated at pH 5 and at 25 °C for 5 days, the octadecyl-SepabeadsTAL retained 100% of its activity, whereas the soluble enzyme maintained around 60% of its initial activity, being more stable than at pH 9 (Figure 6b). These results show that the lipase presented an important improvement in its stability under different conditions after interfacial adsorption, allowing the use of these lipases in very extreme reaction conditions. Specificity of Immobilized Preparations of Thermus Lipases at 25 °C. The initial activity displayed by the lipases adsorbed on octadecyl-Sepabeads in the hydrolysis of different substrates, a simple aliphatic ester (compound 1) and two chiral compounds with the stereogenic center in the acyl chain (compounds 2 and 3) are shown in Table 1.
As for the octadecyl-Sepabeads-TTL preparation, the highest activity was found using ethyl butyrate (1). The activity of this immobilized preparation was more than 150 times higher with compound 1 than with compound 2 at pH 7. In relation to the activity of the TLL immobilized preparation in the hydrolysis of compound 2, the decrease in the pH value from 7 to 5 promoted a slight reduction in the enzymatic activity by a factor of 2. The activity of TAL adsorbed on octadecyl-Sepabeads proved how glycidyl butyrate (3) was the best substrate for the enzyme, with the immobilized preparation being more than 2 times more active with this substrate than with compound 1 at pH 7. Moreover, the octadecyl-SepabeadsTAL presented a very low activity with compound 2, more than 400 times lower than with compound 1 at pH 7. When the pH changed from 7 to 5 in the hydrolysis of compound 2, the enzymatic activity of octadecyl-Sepabeads-TAL decreased more than 7 times, whereas this decrease in the pH did not have a significant effect on the activity of the immobilized preparation in the hydrolysis of compound 3. Enzymatic Resolution of Compounds (()-2 and (()-3. Table 2 shows the enantioselectivity of both lipases adsorbed on octadecyl-Sepabeads support in the hydrolytic resolution of (()-2 at 25 °C and different pHs (Scheme 3). Both preparations presented the highest enantiomeric ratio at pH 7 and at 25 °C, (E ) 9.2 for TTL and 6.5 for TAL
Table 1. Specific Activity of Immobilized Preparations of Thermus Lipases in the Hydrolysis Reaction of Different Esters (Specific Activity (SA): µmol/h mg of prot)a enzyme TTL TAL a
support octadecyl-sepabeads octadecyl-sepabeads
nd: no determined.
SA-1 2.5 4.08
SA-2 pH 7 0.015 9.6 × 10-3
SA-2 pH 5 10-3
6.55 × 1.4 × 10-3
SA-3 pH 7
SA-3 pH 5
nd 9.4
nd 9.2
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Notes Table 2. Enantioselective Resolution of (()-2 Catalyzed by Thermus lipases Adsorbed on Octadecyl-Sepabeads Support at 25°Ca pH 5
Scheme 4. Enzymatic Resolution of (()-glycidyl butyrate [(()-3] at 25 °C
pH 7
immobilized preparation
Sp
ee
E
Sp
ee
E
octadecyl-Sepabeads-TTL octadecyl-Sepabeads-TAL
R S
21 7.6
1.5 1.2
R R
80 73
9.2 6.5
Sp: stereochemical preference; ee ) enantiomeric excess of the release product at 15% of conversion; E ) enantiomeric ratio. a
E value slightly decreased (E ) 3), hydrolyzing in both cases the R isomer quicker. Therefore, the results showed that these lipases adsorbed by interfacial activation on the hydrophobic support can be used in the resolution of different chiral compounds, even at temperatures below 30 °C. This is not possible by using the free enzyme because of its very low activity under these conditions. Conclusion
Figure 7. Evolution of enantiomeric excess of remaining substrate (ee) versus conversion in the hydrolysis of (()-2 catalyzed by octadecyl-Sepabeads-TTL immobilized preparation. Experiments were performed using concentration of substrate of 0.5 mM pH 7, 25 °C, as described in the Experimental Section. Scheme 3. Enantioselective Resolution of (()-2-O-Butyryl-2-phenylacetic Acid [(()-2] Catalyzed by Lipases from Thermus sp
Table 3. Enantioselective Resolution of (()-3 Catalyzed by Octadecyl-Sepabeads-TAL at Different pH Conditionsa pH condition T (°C) time (min) conversion (%) Sp ee (%) 7
25
5
25
30 120 60 160
17 52 23 53
R R R R
12 60 14 52
E 4 4.1 3 3
a Sp: stereochemical preference, ee ) enantiomeric excess of the remaining ester; E ) enantiomeric ratio.
toward the R isomer), showing a pronounced decrease at pH 5 (E value lower than 2). Strangely enough, TAL presented an inverted enantioselectivity at this pH 5 (E ) 1.2), favoring the S enantiomer. In this way, by hydrolyzing the racemic mixture of compound 2 with octadecyl-Sepabeads-TTL preparation at pH 7 and at 25 °C, a quite pure (S)-2 could be produced at 65% hydrolysis (with ee over 90%) (Figure 7). Table 3 shows the E value of the octadecyl-SepabeadsTAL immobilized preparation in the hydrolytic resolution of (()-3 under different reaction conditions (Scheme 4). The best enantioselectivity with this immobilized preparation was obtained at pH 7 and 25 °C (E ) 4.1), whereas at pH 5, the
The results state that soluble lipases from Thermus sp. presented very low activity at temperatures below 30 °C which can limit their biotechnological use. The interfacial activation of lipases on octadecyl-Sepabeads is an efficient and simple method to immobilize, purify, and increase the enzymatic activity of these lipases at temperatures below 30 °C in one single step (e.g., an increase in the activity of TTL by a factor of 4). Thus, after the adsorption on octadecyl-Sepabeads support, these lipases presented high activities (more than 10 times higher in relation with the initial activity) at temperatures between 25 and 30 °C, allowing them to be used in the resolution of different chiral esters at these temperatures, something not feasible with the soluble enzymes due to their very low activity under these conditions. Moreover, the interfacial activation on octadecyl-Sepabeads increased the stability of these lipases under different conditions of pH and temperature, even though they are very stable by nature. This stabilization was much more significant than the use of multipoint covalent attachment techniques, perhaps reflecting the stability of the adsorbed open form of the lipase.23 To conclude, these lipases could be used in some interesting resolutions. Slight changes in the reaction conditions (from pH 5 to 7) produced extreme changes in the E values, e.g., the enantioselectivity of octadecyl-Sepabeads-TTL varied from being almost negligible (1.5) at pH 5 to E ) 9.2 at pH 7 in the resolution of (()-2. Acknowledgment. This work has been sponsored by the Spanish CICYT (Projects BIO2001-2259 and PPQ 200201231). Authors thanks CAM for a Ph D fellowship for Mr Palomo. We thank Resindion Srl for donation of the octadecyl-Sepabeads, Dr. Terreni for the kind supply of compound 3 and Angel Berenguer and Alicia Palomo for the interesting suggestions. References and Notes (1) Owusu, R. K.; Cowan, D. A. Enzyme Microb. Technol. 1990, 12, 374-377. (2) Lee, S.; Rhee, J. Enzyme Microbiol. Technol. 1993, 15, 617-623. (3) Rathi, P.; Sapna, B.; Sexena, R.; Gupta, R. Biotechnol. Lett. 2000, 22, 495-498.
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(4) Kulkurani, N.; Gadre, R. Biotechnol. Lett. 1999, 21, 897-899. (5) Klaus, D. Chem. Eng. Technol. 1998, 21, 278-281. (6) Dong-Woo, L.; You-Seok, K.; Ki Jun, K.; Byung-Chan, K.; HakJong, C.; Doo-sik, K.; Maggy, T.; Yu-ryang, P. FEMS Microbiol. Lett. 1999, 179, 393-400. (7) Haki, G. D.; Rakshit,S. K. Bioresource Technol. 2003, 89, 17-34. (8) van den Burg, B. Curr. Opin. Microbiology. 2003, 6, 213-218. (9) Pennisi, E. Science 1997, 276, 705-706. (10) Demirjian, D. C.; Morı´s-Varas, F.; Cassidy, C. S. Curr. Opin. Chem. Biol. 2001, 5, 144-51. (11) (a) Jaeger, K.-E.; Reetz, M. T. Trends Biotechnol. 1998, 16, 396403. (b) Zeikus, J. G.; Vielle, C.; Savchenko, A. Extremophiles 1998, 2, 179-183. (12) Niehaus, F.; Bertoldo, C.; Ka¨hler, M.; Antranikian, G. Appl. Microbiol. Biotechnol. 1999, 51, 711-729. (13) Palomo, J. M.; Ferna´ndez-Lorente, G.; Mateo, C.; Guisa´n, J. M.; Ferna´ndez-Lafuente, R. Tetrahedron: Asymmetry 2002, 13, 23752381. (14) Reetz, M. T. Curr. Opin. Chem. Biol. 2002, 6, 145-150. (15) Kazlauskas, R. J.; Bornscheuer, U. T. Biotransformations with Lipases In Biotechnology; 1998; pp 68-87. (16) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson. G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen, B.;
Notes Norskov, L.; Thim, L.; Menge, U. Nature 1990, 343, 767-770. (17) Norin, M.; Boutleje, J.; Holmberg, E.; Hult, K. Appl. Microb. Technol. 1988, 28, 527-530. (18) Derewenda, U.; Brzozowski, A. M.; Lawson, D. M.; Derewenda, Z. Biochemistry 1992, 31, 1532-1541. (19) Lasa, I.; Berenguer, J. Microbiol. SEM 1993, 9, 77-89. (20) Oshima, T.; Imahori, K. J. Syst. Bacteriol. 1974, 24, 102-112. (21) Bastida, A.; Sabuquillo, P.; Armisen, P.; Ferna´ndez-Lafuente, R.; Huguet, J.; Guisa´n, J. M. Biotechnol. Bioeng. 1998, 58, 486-493. (22) Ferna´ndez-lafuente, R.; Armise´n, P.; Sabuquillo, P.; Ferna´ndezLorente, G.; Guisa´n, J. M. Chem. Phys. Lipids 1998, 93, 185-197. (23) Palomo, J, M.; Mun˜oz, G.; Ferna´ndez-Lorente, G.; Mateo, C.; Ferna´ndez-Lafuente, R.; Guisa´n, J. M. J. Mol. Catal. B: Enzymatic 2002, 19-20, 279-286. (24) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (25) Laemmli, U. K. Nature 1970, 227, 680-685. (26) Switzer, R. C.; Merril, C. R.; Shifrin, S. Anal. Biochem. 1979, 98, 231-237. (27) Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104, 7294-7299.
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