Lipase Catalyzed Esterification and Transesterification in Low Water Media with Microwave Assistance

Transcription

1 Proc Indian natn Sci Acad 78 No. 4 December 2012 pp Printed in India. Research Paper Lipase Catalyzed Esterification and Transesterification in Low Water Media with Microwave Assistance VEENA SINGH, KUSUM SOLANKI 1 and MUNISHWAR NATH GUPTA* Department of Chemistry, Indian Institute of Technology Delhi, New Delhi , India (Received 19 March 2012; Accepted 20 March 2012) The esterification of palmitic acid and transesterification of ethyl oleate with n-propanol were studied in different solvents at 45 C using lipase Novozyme 435. Both microwave assistance and conventional heating were employed to maintain the temperature. It was found that log P of the organic solvent correlated well with the ratio of the initial rates obtained with microwave assistance over those obtained with conventional heating in case of esterification reaction of palmitic acid with n-propanol. The correlation was not so good in the transesterification reaction between ethyl oleate and n-propanol. The results are discussed in the context of complex set of parameters involved during microwave assisted enzymatic reactions in dry organic solvents. Key Words: Biocatalysis; Biotransformation; Biosurfactants; Enzyme Activity; Lipase; Microwave Assisted Reactions 1. Introduction The microwave assisted enzymatic reactions in organic media continue to attract considerable attention [1-8]. The existence of specific microwave effects which have often been called non thermal effects continues to be debatable. In recent years, Yadav and Lathi reported the enhancement of initial rates in the range of for a Novozyme 435 catalyzed transesterification reaction by microwaves over conventional heating [4]. Interestingly, this range is of the same order as the corresponding range reported from our laboratory for α-chymotrypsin catalyzed esterification/ transesterification [2]. Similar results have been reported by Huang et al. for Lipozyme RMIM catalyzed esterification [9]. Réjasse et al. [5] and Leadbeater et al. [6] on the other hand find that microwave assistance showed no noticeable effect on the initial reaction rates. Both groups used lipase B from Candida antarctica. While in the former case, solvent free conditions were employed, in the latter case the choice of the reaction medium was limited to toluene which does not absorb microwaves [10]. de Souza et al. [11] also found no non-thermal effect of microwave irradiation during the kinetic resolution of rac-1-phenylethanol. Most of studies have employed limited range of organic solvents as reaction media in each case. Some limited studies on the effect of nature of the reaction medium have been, however, made [9 & 12]. Why such diametrically opposite views have been expressed on the issue of significance of microwave assistance of enzyme catalyzed reactions in organic solvents? One reason can be that we do not yet understand how various parameters influence this effect. It should be appreciated that our understanding of such parameters in the context of nonaqueous enzymology in general has been slow to develop and is perhaps less than complete even now. The second reason could be that design of proper experiment for this comparison (between microwave *Author for Correspondence : munishwar48@yahoo.co.uk; Tel: ; Present Address: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA

2 630 Veena Singh et al. assisted vs. conventional heating) is very difficult. For example, how do we control/monitor a w in microwave assisted reactions [13]. The present work makes an attempt to evaluate the effect of the nature of the organic solvent used as a reaction medium in the case of microwave assisted enzyme catalyzed reactions. The system we have chosen is the Novozyme 435 catalyzed esterification for propyl palmitate synthesis and transesterification for propyl oleate synthesis. Such esters are widely used as biosurfactants [14]. 2. Materials and Methods 2.1 Materials Novozyme 435, an immobilized preparation of lipase from Candida antarctica was a kind gift from Novozymes, South Asia Pvt. Ltd., Bangalore, India. The organic solvents: n-octane, n-propanol, n-hexane, toluene, cyclopentyl methyl ether (CPME), 2- pentanone, acetone, dioxane, dimethyl formamide (DMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the solvents used in this work were of anhydrous grade and were also further dried by a gentle shaking with 3 Å molecular sieves obtained from E- Merck (Mumbai, India). The substrates palmitic acid and ethyl oleate were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA) respectively. 2.2 Microwave Reactor for Enzymatic Reaction The enzymatic reactions were performed in a monomode microwave reactor (CEM Microwave, Model Discover, CEM Co., Matthew, NC). This microwave has an operating frequency of 2.5 GHz and a maximum power of 300 W. The quartz glass tube (10 ml capacity) containing the reaction mixture was placed in the cavity of the monomode microwave reactor (CEM Discover). The tube was then capped with a pressure regulator accessory (CEM Benchmate). The temperature of the reaction mixture was monitored with non-contact infrared sensor, which was located beneath the reaction vessel. The reaction mixture was stirred at medium speed using a teflon coated magnetic bar which moves due to inbuilt rotating magnetic plate inside the cavity of the microwave reactor. The set reaction temperature was maintained by using a sensor controlled magnetron. During the microwave irradiation, compressed air was passed on the outer vessels of the reaction tube through a pipe connected to the air compressor [3 and 15]. This helped in pumping in more microwave on the reaction mixture for maintaining the set temperature (45 C in our case). Though the power was set at 20 W before starting the reaction, it varied between 8 to 20 W (depending upon the polarity of the organic solvent used as a reaction medium) to maintain the temperature at 45 C. 2.3 Esterification of Palmitic Acid with n-propanol The reaction mixture contained palmitic acid (0.25 g, mmoles) and n-propanol (365 µl, 4.87 mmoles) in the molar ratio of 1:5. The reaction was carried out in both solvent free medium as well as in the presence of a solvent as a reaction medium. For the reaction to be carried out with an organic solvent as a reaction medium, 1ml of the organic solvent was added to the above reaction mixture. Following solvents were tried: n-octane, n-hexane, toluene, cyclopentyl methyl ether, 2-pentanone, acetone, dioxane and dimethylformamide. The reaction was carried out both by (a) conventional heating in an orbital shaker set at temperature of 45 C with constant shaking at 200 rpm and (b) in the microwave reactor (magnetically stirred at medium speed) set at temperature of 45 C. When the temperature of the reaction medium reached 45 C, the reaction was started by adding Novozyme 435 lipase (12.5 mg, 5 % w/w of palmitic acid). The reaction was then monitored by taking out aliquots (15 µl) periodically and analyzing them for the amount of product formation by gas chromatography. All the experiments were done in duplicates and in all the cases readings within the duplicate set differed by less than 5 %. 2.4 Transesterification Reaction of Ethyl Oleate with n-propanol The reaction mixture contained ethyl oleate (350 µl, mmoles) and n-propanol (368 µl, 4.87 mmoles)

3 Lipase Catalyzed Esterification and Transesterification in Low Water Media 631 in the molar ratio of 1:5. For the reaction to be carried out with an organic solvent as a reaction medium, 1ml of the organic solvent was added to the above reaction mixture. Following solvents were tried: n- octane, toluene, cyclopentyl methyl ether, 2- pentanone, acetone and dioxane. The reaction was carried out again both by conventional heating as well as in the microwave reactor as described above for the esterification reaction. In this case, the reaction was started by adding Novozyme 435 lipase (15 mg, 5 % w/w of ethyl oleate). This reaction was also monitored in an identical way as done in esterification reaction. 2.5 Gas Chromatographic Analysis The alkyl esters were analyzed on Agilent Technologies 6890 N network GC systems, USA with a flame ionization detector (FID). The capillary column EQUITY TM 5 of length 30 m, internal diameter of 0.25 mm with nitrogen as the carrier gas at a constant pressure of 4 kg/cm 2 was used. The column oven temperature was programmed in the range of 100 C to 200 C at 30 C min 1 and 200 C to 250 C at 20 C min 1 with injector and detector temperatures at 240 C and 250 C, respectively. 3. Results and Discussion Lipase catalyzed reactions have been the dominating subject of previous investigations [6 and 16]. The immobilized version of Candida antarctica lipase B has been the enzyme which is used in few previous studies [4 and 5]. We decided to work with Novozyme 435 which is the immobilized version of Candida antarctica lipase B. Unlike CAL B which has to be added as a solution in water, Novozyme 435 is available as a dry powder and is easier to use in anhydrous conditions. While it is generally appreciated that absorption of the microwaves by the solvent medium is an issue [4], less attention has been paid to the nature of the substrate. To start with, we decided to work with palmitic acid (for esterification) and ethyl oleate (for transesterification) which are fairly non polar and as such would not play a role in microwave effects. The other substrate, alcohol was chosen to be n-propanol which is a relatively more non-polar option than shorter chain alcohols methanol and ethanol. Also, its effect (in terms of a microwave absorbing constituent) was evaluated by carrying out the esterification reaction in solvent free medium. Higher molar ratio of alcohol: acid (up to 10:1 has been studied) has been shown to facilitate esterification [16]. As a trade-off between obtaining higher reaction rates and process economy, a ratio of 5:1 was chosen in the present investigation. Table 1 summarizes the results on esterification of palmitic acid with n-propanol in different dry organic solvents. The reaction was carried out at 45 C (the choice of this temperature was somewhat arbitrary). Higher temperature was not chosen as that precludes the use of low boiling point solvents like acetone. Also, Novozyme 435 is quite stable at 45 C under low water conditions [16] and the temperature was maintained either by conventional heating or microwave irradiation. The initial rates for the esterification reaction were consistently higher in all the solvents when microwaves were used (instead of conventional heating) (Table 1). In general, the effect of microwave assistance was more pronounced in polar solvents. While the ratio of initial rates obtained by microwave assistance to initial rates obtained under conventional heating was 1.7 in n-octane, it was 2.2 in 1,4 dioxane (Table 1). Under solvent free conditions when the substrates were not diluted, the ratio increased to 2.7. This could be the synergistic effect of polarity of medium (n-propanol is fairly polar) and mass action due to high substrate concentration. In case of polar solvents, the amount of microwaves required to maintain the desired temperature is less than that required in non polar solvents (see later for a discussion on this in terms of tan ä of the reaction medium). Microwaves seem to have a harmful effect on the enzymes so less amount of microwave energy means less exposure for the enzyme and this may translate into higher initial rates in polar solvents (with microwaves as a heating mode). This effect was also observed by Réjasse et al. where they reported that the Candida antarctica lipase B is more stable in polar solvents under microwave irradiations [5].

4 632 Veena Singh et al. Table 1: Effect of microwave radiations on initial rates of esterification of palmitic acid with n-propanol catalyzed by Novozyme 435 as compared to conventional heating in different organic solvents as a reaction medium Solvent Initial rates (nmoles min 1 mg 1 ) Conventional Microwave Ratio of initial heating (a) heating (b) rates (b/a) n-octane n-hexane Toluene CPME Pentanone n-propanol Acetone Dioxane DMF Solvent free * Initial rates were based upon conversions obtained by GC analysis (protocol given in materials and method section) It is necessary to point that even with temperature controlled true power microwave oven used here (and others in previous investigations) [7 and 12], the power did not remain constant. It was fixed at maximum of 20 W. The microwave ovens equipped with temperature controlled accessory irradiate a sample to the extent necessary for maintaining the desired temperature. Thus the difference between ambient temperature and fixed temperature dictates the extent of exposure to the microwave irradiations. In an extreme case, if the fixed temperature is same as the ambient temperature, microwave will not irradiate the sample! In the present case when non polar solvents like octane/ hexane were used the power reduced back to W soon after the reaction was started. After about 10 minutes presumably as esterification generated some water, the efficiency of the microwave effect increased and power further went down to 8-9 W. Unfortunately, the power and the temperature cannot be fixed simultaneously in any available microwave reactor. Such a design may be impossible. Thus all studies on effects of microwave assistance carry an inherent element of uncertainty over how much microwave energy was pumped in the reaction system. This fact has been mostly overlooked by other workers. Table 2 gives the similar data for transesterification of ethyl oleate with n-propanol in different solvents. Again, higher initial rates were obtained when more polar solvents were used. In both cases (and as reported by some earlier workers) [2 and 6], the effect of microwave assistance was never dramatic. It has not been often appreciated by enzymologists that heating characteristics of the solvent by microwave irradiations are dependent upon the dielectric properties of the organic solvents. The loss tangent, tan δ = ε /ε where ε is the dielectric loss and ε is the dielectric constant describing the polarizability of the molecules in the electric field [17 and 18]. In general, non polar solvents have low tan δ, that is, these absorb the microwaves poorly. More polar solvents, with higher tan δ show more efficient absorption. So, the polar solvents have two advantages. They are better in converting microwave energy to heat energy. Simultaneously, as pointed out earlier, less amount of microwave irradiations may be conducive to enzyme stability. Unfortunately, as Table 2: Effect of microwave radiations on initial rates of transesterification reaction of ethyl oleate with n-propanol catalyzed by Novozyme 435 as compared to conventional heating in different organic solvents as a reaction medium Solvent Initial rates (nmoles min 1 mg 1 ) Conventional Microwave Ratio of initial heating (a) heating (b) rates (b/a) n- Octane Toluene CPME Pentanone Acetone Dioxane * Initial rates were based upon conversions obtained by GC analysis (protocol given in materials and method section)

5 Lipase Catalyzed Esterification and Transesterification in Low Water Media 633 is well known in non aqueous enzymology, these polar solvents also strip off the necessary water layer from the enzyme molecules and hence give poor initial rates [19 and 20]. These complex effects are perhaps the reason why microwave effects are either not observed or are at best marginal. This also explains why solvent free medium gave best results for microwave assistance. Not only n-propanol is a substrate, it has a higher tan δ also. The interesting comparison is with 1,4 dioxane. With a permanent dipole moment it is more or less microwave transparent [18]. Hence, it gives less effect than acetone during transesterification (Table 2). During esterification (Table 1) it is a slightly better solvent under microwave conditions (as compared to acetone), presumably because of generation of water during the esterification reaction results in leveling off effect. So, there are many parameters, some operating in different directions from others. When esterification reaction was carried out at two different powers of 20 W and 30 W; both octane and dioxane gave similar initial rates at both powers. The rates were identical at 715 nmolesmin 1 mg 1 with octane (at both powers) and with dioxane, these were 498 nmolesmin 1 mg 1 and 476 nmolesmin 1 mg 1 at 20 and 30 W respectively (data not shown separately as figure/ table). This further emphasizes the fact that it is immaterial at what level initial power is fixed (as far as it is not very high!). It varies, depending upon the solvent (compositions) during the reaction. When it comes to the effect of solvents on the rate of reactions under dry/ low water conditions, log P is considered the best parameter for correlation purposes [19, 21 and 22]. Fig. 1 shows that the non thermal effect of microwave (measured as a ratio of initial rates obtained with microwave assistance over those obtained with conventional heating) also gave a good fit with log P in the case of the esterification reaction. Hence water stripping effect seems to dominate over the non thermal effect of microwave assistance. In the case of transesterification, wherein no water is generated, the correlation with log P is not so good (Fig. 2). Perhaps, with no water added or generated during the reaction, tan δ effect competed Fig. 1: Correlation of log P with the enhancement of initial rates of esterification reaction of palmitic acid with n-propanol catalyzed by Novozyme 435 by microwave radiations as compared to conventional heating in low water media. Palmitic acid (0.975 mmoles) and n- propanol (4.87 mmoles) were taken in the ratio of 1:5 (mol/mol). For the reaction to be carried out with organic solvent as a reaction medium, 1 ml of the organic solvent was added to the above reaction mixture. Following solvents were varied: n-octane, n-hexane, toluene, cyclopentyl methyl ether, 2-pentanone, acetone, dioxane and DMF. 5 % (w/w of palmitic acid) Novozyme 435 was added. These mixtures were incubated at 45 C either in a) in an orbital shaker at 200 rpm rotation and b) in a microwave reactor, magnetically stirred at medium speed. Aliquots were taken out periodically and were analyzed by GC. Each reaction set was run in duplicates and the deviations within the pair of experimental data were 5 % with water stripping effect. It is not surprising that Réjasse et al. observed that the nonclassical effect of the microwave heating on the initial rate of the enzymatic inactivation was thus dependant on the temperature of the incubation [5]. One of the factors would be how far away the incubation temperature was from the ambient temperature.

6 634 Veena Singh et al. in nearly dry media. Young et al. observed that such systems have yielded inconclusive results because of greatly reduced enzymatic activity [8]. This is especially so since microwave effect will be higher in polar solvents (or strictly speaking high tan δ solvents) but such solvents strip off essential water layer. The other difficulties are that both power and temperature cannot be fixed simultaneously. Hence, one is never very sure of how much microwave energy is being pumped in. There is also a suspicion that microwave irradiation can cause hot spots in the medium [23], so stability of enzymes is another parameter. 4. Conclusions Fig. 2: Correlation of log P with the enhancement of initial rates of transesterification reaction of ethyl oleate with n-propanol catalyzed by Novozyme 435 by microwave radiations in low water media. Ethyl oleate (0.975 mmoles) and n- propanol (4.87 mmoles) were taken in the ratio of 1:5 (mol/mol). For the reaction to be carried out with organic solvent as a reaction medium, 1 ml of the organic solvent was added to the above reaction mixture. Following solvents were varied: n- octane, toluene, cyclopentyl methyl ether, 2-pentanone, acetone and dioxane. 5 % (w/w of ethyl oleate) Novozyme 435 was added. These mixtures were incubated at 45 C (a) in an orbital shaker at 200 rpm rotation and (b) in a microwave reactor, magnetically stirred at medium speed. Aliquots were taken out periodically and were analyzed by GC. Each reaction set was run in duplicates and the deviations within the pair of experimental data were 5 % To sum up, it seems that most of the workers observe non thermal or non classical effects of the microwave irradiations when enzymes are used The exact effect of the microwave assistance for the enzymatic reactions in low water containing organic solvents is difficult to study as large numbers of variables are involved. The nature of the organic solvent as the reaction medium is an important parameter. The present work shows that the nonthermal effect of the microwave assistance correlates best with the log P of the organic solvent. While the reaction rates decrease with decrease in log P, the nonthermal effects increase. The understanding of this trade off should lead to better design of microwave assisted enzymatic catalysis in dry organic solvents. 5. Acknowledgements The work was supported by the Core Group Grant for Applied Biocatalysis, Department of Science and Technology (DST), Government of India. The funds provided by UK India Education Research Initiative (UKIERI), UK and The Department of Biotechnology (DBT), Govt. of India are gratefully acknowledged. One of the authors (KS) is thankful to the University Grants Commission, New Delhi for the Senior Research Fellowship. References 1. Carrillo-Munoz J R, Bouvet D, Guibé-Jampel E, Loupy A and Petit A J Org Chem 61 (1996) Roy I and Gupta M N Tetrahedron 59 (2003) Hayes B L Aldrichim Acta 37 (2004) Yadav G D and Lathi P S J Mol Catal A: Chem 223 (2004) Réjasse B, Lamare S, Legoy M D and Besson T Org Biomol Chem 4 (2006) 3703

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