Abstract Various lipases were screened for their hydro- lytic efficiency towards methyloleate. Lipase from. Chromobacterium viscosum gave highest hydrolysis ...
Bioprocess Engineering 19 (1998) 475±478 Ó Springer-Verlag 1998
Hydrolysis of methyloleate using immobilized lipase from Chromobacterium viscosum M.S.R.C. Murthy, Z. Aslam Basha, T. Swaminathan
Abstract Various lipases were screened for their hydrolytic ef®ciency towards methyloleate. Lipase from Chromobacterium viscosum gave highest hydrolysis ef®ciency of 92% in 24 h. Different cation exchange resins were screened to immobilize lipase from Chromobacterium viscosum. A weakly acidic macroreticular type resin, IRC-50 having carboxyl end group functionality gave highest activity yield of 18.8%. Strongly acidic cation exchange resins with sulphonic functionality and macroreticular type did not give much activity yield when compared to weakly acidic non macroreticular type resins. It was observed that end group functionality and structure of the matrices plays an important role in obtaining highest activity yield. For a speci®c water concentration, the hydrolysis ratio reached 85% in less than 7 h when the substrate to enzyme ratio was 4. As the ratio is increased above 4, the availability of water at the interface has become a limitation for obtaining maximum hydrolysis.
1 Introduction Lipases are characterized by their ability to rapidly catalyze the hydrolysis of ester bonds at the interface between the insoluble substrate phase and the aqueous phase in which the enzyme is soluble. The ability of lipases to catalyze the hydrolysis of insoluble fatty acid esters distinguishes them from other esterases which catalyze the hydrolysis of water soluble esters in preference to insoluble esters [1]. Lipases being reversible hydrolytic enzymes with low Gibb's free energy difference between forward and backward reactions, the equilibrium can be shifted towards synthesis by suitably changing the reaction environment [2]. This particular principle is the basis for lipase catalyzed esteri®cation, transesteri®cation both alcoholysis and acidolysis, and interesteri®cation reactions. The use of lipases in organic solvents or microaqueous systems offers many advantages over reactions in
Received: 12 January 1998
M.S.R.C. Murthy, Z. Aslam Basha, T. Swaminathan Biotechnology Research Centre, Department of Chemical Engineering, Indian Institute of Technology, Madras 600 036, India Correspondence to: T. Swaminathan
aqueous environment [3]. Since then the utility of lipases has been increasing at a tremendous rate. Numerous reports have been published in literature on using free/soluble and immobilized lipase for different applications in aqueous, biphasic and reverse micellar media [5±12]. Immobilization of lipase by glutaraldehyde enhanced adsorption, and by covalent bonding on hydrophilic, hydrophobic and cation exchange resins have been studied depending upon the application and substrate of interest. Recently some reports have been published on using Accurel as a carrier for immobilizing lipase by adsorption [13]. The cost of the lipase has become a major limitation for its commercial exploitation which has made the enzymatic fat splitting process unable to compete with conventional chemical fat splitting Colgate-Emeray process. One possible solution is reusing the enzyme by immobilization. Besides providing large surface area, immobilization offers improved dispersivity, mass transfer, activity, and stabilization of the enzyme. It has been observed that the rate of enzyme catalyzed reactions is higher when the enzyme is immobilized on a carrier than when free enzyme is used [13]. In majority of the cases oils and fats are the main substrates in lipase catalyzed reactions. The disadvantages of using oils and fats are they are highly hydrophobic, require additives for emulsi®cation, require heat to melt the solid substrate, and result in clump formation in the reaction mixture. The major disadvantage from the industrial point of view is the dif®culty of ¯uid transportation due to their high viscosity. The advantages of soap making from methyl and ethylesters, derived from vegetable oils and fats, over conventional oils and fats has been discussed by Ogoshi et al. [14]. Recently it has been found that methyl and ethylesters of vegetable oils and fats can be used as a substitute for diesel. This alternative fuel has been named as ``Biodiesel'' [15±18]. Their low viscosity and less hydrophobic nature is responsible for utilizing them as substitute for fossil fuel in compression ignition (diesel) engines. They are less hydrophobic than oils and fats which makes them comparatively easily miscible with hydrophilic solvents. In the present work lipases from three different sources were screened for their hydrolytic ef®ciency towards methyloleate. With the most active lipase enzyme various resins were screened for immobilization by covalent coupling using carbodiimide. The hydrolysis of methyloleate was further studied with the most stable immobilized enzyme.
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2 Materials and methods
476
2.1 Lipases from Chromobacterium viscosum, and Pseudomonas ¯uoroscens bio type-1 were kind gifts from Dr. Y. Kosugi, NIBH, Japan. Lipase from Candida cylindracea (Sigma, USA). Cationic Ion Exchange Resins, Amberlite50, 252, 76 (Japan Organo Co. Ltd., Tokyo, Japan). 1-Cyclohexyl-3-(2-Morpholino-Ethyl) Carbodiimide Metho-p-Toluenesulfonate (Sigma, USA). Methyloleate (Wako Pure Chemical Company, Japan), and all other chemicals and reagents were of analytical grade. 2.2 Screening lipases 1 cm3 of 10U per mg of protein of each lipase in 0.05 M phosphate buffer (pH 7.0) was added to 100 mM methyloleate and incubated on rotary shaker (180 rpm Orbitek, India) at room temperature 30 °C 2). The samples taken at speci®ed intervals of time were analyzed by HPLC for methyloleate and oleic acid. 2.3 Pretreatment and screening of cationic exchange resins for immobilization The pretreatment of the resin was necessary to activate the resin and enhance the covalent bonding between the resin and the enzyme. The pretreatment was initiated by adding 1 g of cationic exchange resin to a freshly prepared solution of Carbodiimide (50 mg per 10 cm3). The mixture was shaken at room temperature in reciprocating shaker at 180 rpm for about 4 to 6 hours until the pH of the mixture was stabilized between 4 or 5. The resin was ®ltered under vacuum and washed thoroughly with distilled water to remove excess carbodiimide. 150 mg of crude lipase was dissolved in 20 cm3 of 0.1 M phosphate buffer pH 7.0±7.2. 5 cm3 of this solution was added to 1 g pretreated resin in 50 cm3 conical ¯ask. Immobilization was carried out overnight by shaking at 4 °C after which the resin was ®ltered and washed with 15 cm3 phosphate buffer. 2.3 Hydrolysis of methyloleate The hydrolysis of methyloleate was studied at different concentrations of substrate keeping the concentrations of enzyme (0.25 g), and water (1 g), constant. Hydrolysis was carried out by mixing the enzyme and the substrate in a 100 cm3 Erlenmayer ¯asks and incubating the mixture on a rotary shaker at room temperature. Samples taken at regular intervals were diluted appropriately with 1:1 v/v acetone:acetonitrile (9:1 acetonitrile: 5 mM phosphoric acid). The samples were ®ltered through Millipore's MIllex±GV 0.22 lm ®lter to avoid chocking column before injection. The hydrolysis ef®ciency is de®ned as 100 times the ratio of amount of oleic acid present to the amount of methyloleate present.
2.4 Analytical methods Protein was estimated by Lowry method [20] using Folin Ciocalteau reagent. 2.4.1 Lipase activity was measured by following the procedure of Kosugi et al. [19]. To 75 cm3 of 2% (w/v) PVA solution, 25 cm3 of methyloleate was added and emulsi®ed for 5 minutes. This emulsion was used as the substrate for measuring lipase activity. To 1 cm3 of lipase solution, 5 cm3 of substrate emulsion, and 4 cm3 of 0.05 M phosphate buffer pH 7.0 were added. The assay mixture was incubated at 30 °C for 20 minutes. After incubation, 20 cm3 of 1:1 acetone:methanol mixture was added to stop the reaction. The whole mixture was titrated against 0.05N NaOH using the phenolphthalein indicator. One unit of lipase activity was de®ned as one micromole of fatty acid released per min. 2.4 Substrate and product analysis by HPLC The substrate methyloleate and the hydrolysis product oleic acid were analyzed by Shimadzu CTO-10A HPLC, using RP Shim-Pack CLC-ODS (M) column and SPD-10A UV-Visible detector at 205 nm. The mobile phase 9:1 (v/v) acetonitrile:5 mM phosphoric acid was passed at a constant ¯ow rate of 2 ml/min. 3 Results and discussion Since lipases differ in their speci®city to substrate, lipases from different sources were ®rst screened for their activity towards methyloleate as substrate. The results are given in Table 1. Lipase from Chromobacterium viscosum gave the highest hydrolysis ratio with methyloleate. Lipases from Candida cylindracea showed relatively low activities in the early stages (up to 12 hours) but its activity increased rapidly later. Though the lipases from Pseudomonas ¯uorescens and Chromobacterium viscosum showed similar activities at 6 hours, the activity of Chromobacterium viscosum lipase was higher as re¯ected in the hydrolysis ratios at later periods. This behaviour of lipases can be attributed to the speci®cities of substrates and the relative accessibility of the aqueous substrate for the enzyme hydrolysis in the initial stages. It is known from literature [21] that the lipase from Chromobacterium viscosum is Table 1. Screening lipases Source of the lipase
Hydrolysis ratio in 6 hours, %
Hydrolysis Hydrolysis ratio in ratio in 12 hours, % 24 hours, %
Candida cylindracea Pseudomonas ¯uorescens biotype±1 Chromobacterium viscosum
21
37.3
72.7
32
54.0
80.0
34
68
92
M.S.R.C. Murthy et al.: Hydrolysis of methyloleates using immobilized Lipase
alkalophilic and thermophilic. So this lipase can be successfully used for direct formation of salts of free fatty acids from corresponding esters or oils and fats. Since lipase from Chromobacterium viscosum has two isoelectric points at pI 3.0 and 7.2 the activity yield strongly depends on the pH of the buffer used for immobilization. In this study it was observed that the activity yield was very low at pH 3. Four cation exchange resins Amberlite 50, 76, 252 were chosen for immobilization studies based on their availability and their characteristics are given in Table 2. The immobilization was carried out at 4 °C and the results are presented in Table 3. Amberlite IRC-50, a weakly acidic cation exchanger of macroreticular type gave the highest activity yield with methyloleate when compared with strongly acidic macroreticular type Amberlite 252 and weakly acidic non macroreticular type IRC-76. Also carriers with carboxyl functionality gave higher activity yield than carriers with sulphonic functionality. This ®nding gives us an idea that the nature of the carrier and end group functionality plays an important role during the formation of covalent coupling with lipase. A possible explanation to this observation is, that during the pretreatment of the carrier for activation, the carboxyl functionality present on the carrier was prone to complex formation with carbodiimide easily than the sulphonic functionality which facilitates a uniform morphological orientation for peptide bond formation with the lipase. Since the pretreatment was done in the pH range of 4±5, ionization of the end group functionalities is of considerable importance during immobilization. In the present work, since the immobilization was done in the pH range 7.0±7.2, which is very close to the isoelectric point of lipase, the formation of covalent coupling is highly feasible with weak carboxyl functionalities than with strong sulphonic functionality. The hydrolysis ratio is also a function of the ratio of substrate to enzyme (Fig. 1). It started decreasing rapidly as the ratio was increased, from above 85% within 7 hours at substrate to enzyme ratio of 4 to well below 30% at higher ratios. This is more clearly seen in Fig. 2, where the hydrolysis ratio is relatively high at low substrate to enzyme ratio. Since the water concentration was held constant in all the experiments, at low substrate concentrations the interface available for catalytic activity of the enzyme is than at high substrate concentration. As the
Table 3. Screening cation exchange resinb Type of Resin
Protein adsorbed, mg/g of support
Activity of the IMEa U/g of support
Speci®c activity, U/mg of protein
Activity yield, measured against methyloleate
Activity of the crude enzyme IRC-50 IRC-76 Amberlit e-252
±
±
406
±
0.81 1.10 0.64
62.0 53.0 21.0
76.5 48.2 32.8
18.8 11.9 8.0
a
Immobilized enzyme Amberlite 200C was not considered for immobilization as it was not stable during pretreatment b
ratio was increased, more and more amount of substrate gradually occupies the interface and ®nally the composition of the reaction mixture becomes two phase which is not an ideal condition for lipase to exert its catalytic activity. In Fig. 2, A represents the zone of high interface availability and which starts decreasing as we move to zone B.
Fig. 1. Effect of substrate concentration on hydrolysis ef®ciency
Table 2. Characteristics of the carriers used for immobilization Amberlite Grade
200C
252
IRC-50
Type
Strongly acidic cation exchanger, Macroreticular type
Strongly acidic cation exchanger, Macroreticular type
Weakly acidic cation exchanger, Macroreticular type
Functional Structure Ionic forms available Physical form Densities (g/ml) Effective operating pH range Effective size (mm)
±SO3M Na+ Beads 1.26 0 to 14 0.5 to 0.65
±SO3M Na+ Beads 1.27 0 to 14 0.4 to 0.50
±COOM H+ Beads 1.25 5 to 14 0.33 to 0.50
IRC-76 Weakly acidic cation exchanger, non macroreticular type ±COOM H+ Beads 1.14 5 to 14 0.36 to 0.44
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References
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