Bioprocess Biosyst Eng (2008) 31:87–94 DOI 10.1007/s00449-007-0149-5 ORIGINAL PAPER Studies on crystallization and cross-linking of lipase for biocatalysis Akhila Rajan Æ T. Emilia Abraham Received: 15 June 2007 / Accepted: 13 July 2007 / Published online: 11 August 2007
Springer-Verlag 2007 Abstract The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonalshaped crystals of 2–5 lm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using b cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate. Keywords CLEC lipase
Thermal stability
Crystallization
Cross-linking
Organic solvent Introduction Enzymes are recognized as useful tools for accomplishing chemical reactions in a stereo-, regio- and chemoselective manner [1]. The development of robust biocatalysts with increased stability and catalytic activity in organic media is A. Rajan
T. Emilia Abraham (&) Chemical Science and Technology Division, NIST (Regional Research Laboratory) CSIR, Trivandrum 695 019, India e-mail: emiliatea@yahoo.com a major challenge in industrial biocatalysis. A major breakthrough in this field was the development of crosslinked enzyme crystals (trademarked as CLEC1), which combine the features of essentially pure protein with high specific activity and high stability in organic solvents [2]. CLECs are prepared by controlled precipitation of enzymes into micro crystals followed by cross-linking using bifunctional reagents to form strong covalent bond between e-amino groups of lysine residues [3]. CLECs retain their activity in environments that are normally incompatible with enzyme function such as prolonged exposure to high temperature, extreme pH and non-aqueous solvents. The biocatalytic processes depend on the stability and activity of the enzyme under sub-optimal conditions and crosslinked enzyme crystals can be used. It is mainly useful in the manufacture of chiral compounds, having high commercial value as fine chemicals, pharmaceuticals and agro chemicals, flavors, cosmetics, peptides, tailor made fats, and in therapy. Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3.) are versatile enzymes that catalyze the hydrolysis of ester linkages, primarily in neutral lipids such as triglycerides. They hydrolyze the acyl chains either at primary [4–6] or secondary positions. Lipase catalyzes ester synthesis and transesterification under micro-aqueous condition. Lipases have been used to resolve the kinetic resolution of racemic compounds through esterification [7, 8]. Crude lipase mixtures suffer from stability problems in organic solvents and they contain contaminating side activities that have unexpected synthetic properties affecting the enzyme catalysis [9–12]. Soluble pure lipases cannot be used in synthesis as a result of low stability against high temperatures and organic solvents [9, 14, 15]. Cross-linked lipase crystals are considerably more stable in the presence of organic solvents than the soluble crude lipase preparations 123 88 Bioprocess Biosyst Eng (2008) 31:87–94 [9–13]. Cross-linked crystals of Candida rugosa lipase and Candida antarctica lipase B have been used as catalysts for the resolution of chiral esters. No reports are available for the Burkholderia cepacia Lipase CLEC. Here we are reporting a method to produce micro crystals of CLEC lipase, which is thermostable, active in both polar and non polar solvents and can be even stored at room temperature. Experimental Materials Lipase PS from Burkholderia cepacia (E.C 3.1.1.3) obtained from Amano, Japan was used for the study. 2 methyl-2, 4-pentane diol (MPD) and glutaraldehyde (50% solution) was purchased from Sigma (St Louis, USA). Poly ethylene glycol (PEG-6000) and ammonium sulfate (Enzyme grade) was purchased from SISCO Laboratories, India. Iso propanol was procured from BDH, Mumbai, India. All other reagents used were of analytical grade. Optimization of lipase crystallization and cross-linking Crystallization of lipase PS Crystallization was done in different combinations using poly ethylene glycol (PEG-6000), ammonium sulfate, MgCl2 and 2 methyl-2, 4–25%pentane diol (MPD) and isopropanol. The various combinations used were 2 M to 50% (NH4)2SO4, 0.2 M MgCl2 or 5–10% PEG and 10– 30% MPD or 1–10% isopropanol. Crystallization of 20 mL of enzyme extract was done in a glass beaker using a Teflon-coated magnetic bar of 1 cm length with a stirring speed of around 80 rpm. Cross-linking of lipase crystals The enzyme crystals were cross-linked using 0.5–5.0% glutraldehyde (v/v) in isopropanol for 20 min at 25 C. Coating the crystals and lyophilization Methods The crystals were coated with different surfactants such as b cyclodextrin, Tween 20, Tween 80, Triton X-100, Aerosol OT and PEG 1000. Enzyme assay measurement Lipase assay was done using olive oil as substrate in Tris– HCl buffer pH 8.5 at 30 ± 2 C [16]. One unit (U) of Lipase activity is defined as the amount of the enzyme, which liberated 1 lmol of free fatty acid per min under the assay conditions. To 10 mL buffer and 10 mL olive oil emulsion, 1 mL enzyme was added. Acetone was added to stop the reaction. It was then titrated against 0.05 M NaOH. Enzyme activity ðTest titre  blank titreÞ  Normality of NaOH ¼ 20  volume of lipase used  1000 ð1Þ Thermal stability of CLEC at 70 C Thermal stability of CLEC lipase was carried out at 70 C at different time intervals. Solvent stability in different organic solvents Solvent stability of CLEC lipase was studied after incubation with organic solvent–water (50%) mixtures for 24 h. Both polar and nonpolar solvents were used. Crystal morphology Protein estimation Lowry’s method was followed for protein estimation [17]. BSA was used as the standard. The crystal morphology was observed under X-ray diffraction (X-ray diffractometer, Philips) and scanning electron microscope (JEOL, Japan) at 10 kV accelerating voltage, after sputtering with gold. Effect of pH and temperature Enantioselective esterification of ibuprofen The enzyme activity was checked at different pH (3–9) and at different temperatures (10–60 C) keeping all the other parameters constant. 123 CLEC was used for enantioselective esterification of Ibuprofen with n-amyl alcohol. Racemic mixture of (R, S)- Bioprocess Biosyst Eng (2008) 31:87–94 Table 1 Crystallization using combination of salt and alcohol Table 2 Effect of different combination of PEG and MPD on crystallization 89 Salt (w/v) Alcohol(v/v) Weight of crystal (g) 50%(NH4)2SO4 15% MPD No crystallization 50%(NH4)2SO4 25% MPD 1.95 2 M (NH4) 2SO4 30% MPD 0.2 21.46 2 M (NH4) 2SO4 5% Isopropanol 1.45 22.20 0.2 M MgCl 30% MPD 0.6 14.50 2 Activity yield (%) 28.50 PEG-6000 (% w/v) MPD (%v/v) Total activity before crystallization (U/mL) Total enzyme activity in crystals (U/mL) Activity yield (%) 20 15 1,082.4 168 15.52 20 20 865 390 45.08 20 25 865 336 38.84 15 30 1,516.5 475.5 31.35 Ibuprofen was dissolved in isooctane. To this solution namyl alcohol and CLEC Lipase were added. The suspension was stirred at room temperature (28 ± 2 C) for 24 h for the synthesis of n-amyl ibuprofen. The water activity of the reaction mixture was controlled using molecular sieves [18]. found to be the best. The protein concentration of the extract was 32.79 mg/mL and was used directly for crystallization. Crystallization of lipase PS Synthesis of ethyl butyrate Ethanol was mixed with vinyl butyrate in equimolar ratio (1:1) for the synthesis of ethyl butyrate. All the reagents were dried over molecular sieve before the reaction. Ester synthesis was carried out in screw-cap bottles incubated at 45 C under constant agitation for 4 h. A control tube without lipase was prepared and incubated under the same conditions. GC–MS analysis Samples were analyzed on a GC–MS (Shimadzu QP-2010) fitted with a 50 m · 0.2 mm DB-1 of 0.17-lm-thick fused silica capillary column with EI mode, electron impact ionizing voltage 70 eV, source temperature 1,500C, electron multiplier voltage 2,000 eV with helium as the carrier gas (2 mL/min) with scan speed of 6,000 amu/s and scan range 40–500 amu. Results and discussions Extraction of Lipase PS was done in buffer having different pH and pH 7.0–8.0 phosphate buffer for 1 h from the commercial lipase powder (0.2 g/mL concentration) was Crystallization of lipase PS was attained in 16–18 h at 4 C. Crystallization using ammonium sulfate/MgCl2 and MPD/isopropanol Various proportions of ammonium sulfate/MgCl2 and MPD/isopropanol were added to the enzyme solution to get good micro crystals at the shortest time. The optimum combination was found to be 50% (NH4)2SO4 and 25% MPD, which gave maximum an activity yield of 28.50% for the resultant crystals (Table 1). Crystallization using poly ethylene glycol and 2 methyl-2, 4-pentane diol (MPD) Maximum activity yield of 45.08% for the crystals was obtained with 20% PEG-6000 and 20% MPD combination (Table 2). The active site of the lipase contains serine, histidine and glutamic acid and this is called the catalytic triad and a peptide flap shields the active site. When PEG is used for the crystallization of lipase, the active site is only partially opened , which in turn may reduce the enzyme activity with bigger substrates. Hence these crystals were not used for further studies. 123 90 Table 3 Crystallization with ammonium sulfate Bioprocess Biosyst Eng (2008) 31:87–94 Ammonium sulfate concentration (w/v) Total activity before crystallization (U/mL) Total activity in crystals (U/mL) Weight of crystals (g) Activity yield (%) 60% 1,415 532.6 1.05 37.63 70% 1,415 546.3 1.13 38.60 80% 1,302 412.5 1.10 31.68 2M 1,326 346 2.1 26.29 Table 4 Activity of surfactant-coated crystals Additives Aerosol-OT CLEC Coating the crystals with surfactant and lyophilization Enzyme activity (U/mL) 7 b Cyclodextrin CLEC 19.25 Tween 20 CLEC 13.75 PEG 6000 CLEC 17 PEG 1000 CLEC Control CLEC 6.25 21.25 Crystallization using ammonium sulfate alone (NH4)2SO4 salt was added to the enzyme solution in small amounts at equal intervals of time with constant stirring for crystallization. The solution was kept under incubation at 4 C overnight, when the protein crystallizes out. The protein concentration before crystallization was 33.8 mg/ mL. The maximum activity (38.60%) and quantity yield (1.13 g) of crystals was obtained with 60–70 % (NH4)2SO4 salt (Table 3). Crosslinking with glutaraldehyde The enzyme crystals were cross-linked using 0.5–5.0% glutraldehyde (v/v) in isopropanol for 20 min at 25 C. After cross-linking it was washed with 0.1 M phosphate buffer of pH 7.5% glutraldehyde retained maximum activity yield after cross-linking in 20 min time. Lower concentrations of glutaraldehyde were not effective in cross-linking. Fig. 1 Crystals of Lipase PS 123 The crystals were coated with different surfactants like Tween 20, Tween 80, Triton X-100 and Aerosol OT and Brij. There was a slight reduction in the activity due to the masking of the active site due to the coating. However, the maximum enzyme activity (19.25 U/mL) was retained in b cyclodextrin coated CLEC (Table 4). Crystal morphology Scanning electron micrograph studies showed very small crystals of 10–20 lm size (Figs. 1, 2) and the X-ray diffraction crystal structure showed a 2h-angle diffraction at ´ 16.5 and 33.5 and d spacing of 5.30 and 2.65 Å, respectively (Fig. 3). Solvent stability of CLEC lipase CLEC lipase had higher activity retention of 97.14, 65.71 and 57.14%, respectively in organic solvents like ethanol, hexane and ethyl acetate (Table 5). The increase in stability of CLEC lipase in organic solvents is due to the number of covalent bonds between enzyme molecules created by the glutaraldehyde cross-linking. Cross-linking increases the rigidity of the enzyme molecules and hence reduces the unfolding of the three-dimensional structure of the protein by the organic solvents. Bioprocess Biosyst Eng (2008) 31:87–94 91 Fig. 2 Crosslinked crystals of Lipase PS Fig. 3 X-ray diffraction pattern of Crystal structure Thermal stability of CLEC lipase Soluble Lipase PS was found to be thermostable up to 70 C. Hence the thermostability of the CLEC lipase was also conducted at the same temperature. The half-life of CLEC lipase was not reached even after 6 h of incubation at 70 C, whereas soluble enzyme got inactivated rapidly at this temperature (Fig. 4). The increased thermal stability of CLEC lipase offers major advantages to the organic chemist, so as to perform the lipase-catalyzed reactions at higher temperature, thereby increasing the reaction rate and productivity. Table 5 Stability of CLEC lipase after incubation with organic solvent–water (50%) mixtures for 24 h (retention of activity %) Solvent Dielectric constant The crystalline enzyme maintains its native conformation at elevated temperature and having lower tendency to aggregate. This is because in CLECs, the enzyme molecules are symmetrically arranged and hence their native conformation is stabilized. When an enzyme forms a crystal, a very large number of stabilizing contacts are formed between individual enzyme molecules [19]. The increased thermal stability of CLEC lipase may be due to the preordered arrangement of the molecules by inter and intramolecular cross-links between the crystals, and hence the rigidity of the three-dimensional arrangement of molecules [20]. Energy must be introduced into the system in log P Polarity Activity retention (%) 65.71 Hexane 1.9 4.0 Non polar Ethyl acetate 6.0 0.73 Dipolar aprotic 57.14 Chloroform 4.8 1.97 Non polar 37.14 Isopropanol 19.9 0.05 Polar protic 34.28 Acetone 20.7 0.24 Dipolar aprotic 17.14 Ethanol 24.6 0.30 Polar protic 97.14 Methanol 32.7 0.74 Polar protic 45.71 Acetonitrile 37.5 0.34 Dipolar aprotic 17.14 Water 80.2 – polar aprotic 123 92 Bioprocess Biosyst Eng (2008) 31:87–94 Enzyme Activity (U/ml) 12 11 10 9 8 7 6 5 0 30 60 90 120 150 180 240 300 360 Time (min) Fig. 4 Thermal stability of CLEC lipase order to disrupt these new contacts, so that additional energy is required to break the covalent cross-links before the CLEC begins to dissolve and then denature. Storage study The shelf life of lipase was improved by surfactant coating. b-CD coated CLEC lipase was even active at 28 ± 2 C for 4 months (Table 6). Soluble lipase preparations are not stable at this temperature. Room temperature storage stability for an enzyme is highly desirable in the industry. Application of CLEC lipase The CLEC was successfully used for the following two biotransformations 1. 2. Kinetic resolution of ibuprofen and Transesterification reaction for the synthesis of ethyl butyrate. ibuprofen. The acid and ester can conveniently be separated by simple extraction. CLECs are recovered by filtration. The pH of the reaction mixture is adjusted to between 2 and 3, and then extracted with diethyl ether. The combined ether extracts are extracted with saturated pH-9.5 sodium carbonate, and then the combined aqueous layers are back extracted with diethyl ether. The combined ether layers are washed with saturated sodium chloride and dried over anhydrous sodium sulfate and the solvent is evaporated under reduced pressure to give (R) ibuprofen methyl ester as colorless oil. The acid is precipitated from the combined sodium carbonate extracts by adjusting the pH of the aqueous layer to 2 with 6 N HCl, saturating with sodium chloride and then dried over anhydrous sodium sulfate and the ether is evaporated under reduced pressure to give (S)-ibuprofen as a crystalline white solid [21]. The MS data shows that the molecular ion at 276 is the amyl ester of ibuprofen and the molecular ion at 206 is the ibuprofen and at 161 is isopropyl benzene the most stable molecular ion. The daughter fragments shows aromatic ring structures formed. The MS data was 276 (M+), 161 (M+– 45) 119, 91, 71 (Fig. 6). CLEC in flavor synthesis Transesterification of ethyl butyrate and reusability. Transesterification of vinyl butyrate and ethanol was carried out in screw-cap bottles incubated at 45 C under constant agitation. The presence of product formed was analyzed using GCMS. Thirty-four percent of ethyl butyrate was formed after the reaction (Fig. 7). Synthesis of ethyl butyrate was continued for ten cycles. CLEC was active even after ten cycles of reaction. After the tenth cycle, 23% of ethyl butyrate was formed. The quantification of ethyl butyrate was taken from GC analysis. Enantioselective esterification of ibuprofen using CLEC lipase (Fig. 5) Conclusions CLEC lipase added to (R, S)-ibuprofen dissolved in isooctane and n-amyl alcohol. The suspension is stirred at room temperature for 24 h for the production of n-amyl Table 6 Storage stability of CLEC lipase at 30 C 123 Months Activity of the CLEC (U/mL) 1 22.5 2 18.6 3 14.5 4 10.2 Crystallization and cross-linking conditions were optimized for the production of enzyme crystals. Effect of pH, surfactants, co solvents and different precipitants were studied. The maximum yield of crystals was observed with 70% ammonium sulfate concentration. The crystals were cross-linked with 5% glutaraldehyde in isopropanol for 20–30 min. SEM studies showed small hexagon-shaped crystals having a size of 2–5 lm. Thermostability and solvent stability was improved for CLEC. The cross-linked crystals were coated using the additive b cyclodextrin for improving the storage and reuse stability. CLEC lipase was used for enantioselective esterification Bioprocess Biosyst Eng (2008) 31:87–94 93 Fig. 5 Reaction scheme of ibuprofen using CLEC lipase OH CLEC Lipase O (R,S)-Ibuprofen n-Amyl alcohol in isooctane O O (S)-Amyl Ibuprofen OH O (R)-Ibuprofen Fig. 6 GC MS of esterification of Ibuprofen Fig. 7 GC MS of ethyl butyrate of ibuprofen and synthesis of ethyl butyrate. Reusability of CLEC lipase was 23% after the tenth cycle in the synthesis of ethyl butyrate. Acknowledgments The authors acknowledge the help extended by the Director, RRL for providing the necessary facilities and thank the Department of Biotechnology for funding the project. References 1. 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