The effect of ultrasonic pre-treatment on the catalytic activity of lipases in aqueous and non-aqueous media

Background Ultrasound has been used to accelerate the rates of numerous chemical reactions, however its effects on enzymatic reactions have been less extensively studied. While known to result in the acceleration of enzyme-catalysed reactions, ultrasonication has also been shown to induce enzyme inactivation. In this study we investigated the effects of ultrasonic pretreatment on lipases in both aqueous and non-aqueous media. Results Our results show that the ultrasonic pre-irradiation of lipases (from Burkholderia cepacia and Pseudomonas fluorescens) in aqueous buffer and organic solvents enhanced enzymic activities. In addition, we report the enhancement of hydrolytic (esterase) and transesterification activities. On using pre-irradiated enzyme, we found that the conversion rate for the transesterification of ethyl butyrate to butyl butyrate, increased from 66% to 82%. Similarly, a 79% conversion of Jatropha oil to biodiesel was observed upon employing pre-irradiated enzyme, in contrast to a 34% conversion with untreated enzyme. CD spectra showed that while the enzyme's secondary structure remained largely unaffected, the microenvironments of aromatic amino acids were altered, with perturbation of the tertiary structure having also occurred. SEM analysis demonstrated significant morphological changes in the enzyme preparation as a result of ultrasonication. Conclusion In contrast to the effects of ultrasonic irradiation on other enzymes, for the lipases focused upon in this study, we report an enhancement of biocatalytic activity, which is thought to originate from morphological changes on the macro and molecular levels.


Background
Ultrasound has been used to accelerate the rates of numerous of chemical reactions [1][2][3]. These rate enhancements, mediated by cavitation, are believed to originate from the build up of high local pressures (up to 1000 atm) and temperatures (up to 5000 K), as well as increased catalytic surface areas.
The effects of ultrasound on enzymatic reactions, however, have been less extensively studied [4][5][6][7][8][9]. The few studies that have been carried out can be categorised into two main groups. The first approach involves using ultrasound as an enzymic pretreatment to reduce particle size. This is especially relevant when using enzyme powders to catalyse reactions in organic media [10][11][12]. In such cases, the reduction in particle size and consequent increase in the catalytic surface area are thought to reduce mass trans-fer limitations. The second approach involves the use of ultrasound throughout the reaction. Here the cavitation energy is thought to accelerate the reaction rate, yet the mechanism by which this occurs is unclear. It may be that by increasing the movement of liquid molecules, the substrate's access to the active site is increased. Other mechanisms have also been suggested [5].
While it has been shown that the second of these approaches can accelerate enzymatic reactions [4,6], other reports have demonstrated enzyme inactivation [7][8][9]. In general, enzymes are known to be more stable in nearlyanhydrous organic solvents [10][11][12], therefore it is not surprising that all the reported cases of rate enhancements resulting from ultrasonic treatment are those involving enzymic catalysis in organic media [4,6].
By focusing on lipases, which have extensive applications in biotransformations [10][11][12], the aim of this study was to look at the effect of ultrasonic pretreatment on catalytic performance.
We placed lipases in both aqueous as well as organic media during ultrasonic irradiation. The pretreated lipases were evaluated for both esterase (in water) and transesterification (in organic solvent) activities. Lastly, ultrasonically pre-irradiated lipase was also used in the transesterification of Jatropha oil to biodiesel. Biodiesel production is an interesting application of lipase-catalysed transesterification [13,14], for which Jatropha oil can be considered an economically sound choice as a starting material [14,15].

Results and Discussion
The effect of ultrasonic pre-irradiation on the esterase activity of lipases As expected, continuous ultrasonic pre-irradiation of the enzyme solution/suspension led to an increase in temperature. In order to assess the thermal effect (as a stress factor for denaturation), we decided to expose the enzyme solution/suspension to ultrasound for 5 min at 40°C, before maintaining the solution/suspension at 40°C in an incubator for a further 10 min. This method of interrupted ultrasonic pre-irradiation succeeded in maintaining the sample at 40°C ± 1. Figure 1 outlines the effect of ultrasonic pre-irradiation on the stability of Burkholderia cepacia lipase in aqueous buffer (20 mM phosphate buffer, pH 7.0) and three different organic solvents of differing polarities. The stability was evaluated by taking aliquots periodically and assaying the esterase activity in aqueous media.
For enzymes suspended in organic solvents, the latter was removed by centrifugation. The centrifuged enzyme was then transferred to the aqueous assay (hydrolysis of p-nitrophenyl palmitate) system in which it dissolved as expected (note: there was no organic solvent present in the aqueous assay system). In all cases, enzymic activity was seen initially to increase before then decreasing as a result of ultrasonic pre-irradiation ( Figure 1). The effects were marginal for enzyme suspensions in organic solvents, but were quite significant for those in aqueous solution. The likeliest explanation for the trend seems to be that pretreatment caused a decrease in the particle size of the catalyst, with further treatment then inducing enzyme inactivation. It is noteworthy that for none of the solvents, except DMF (the most polar organic solvent), was the final activity (after ultrasonication for 4 h) less than the starting activity (without ultrasonication pretreatment).
Controls were also carried out in which the Burkholderia cepacia lipase was incubated for 4 h at 40°C (in a water bath, without irradiation) in various solvents. Here surviving activities of 75%, 100%, 100% and 53% for aqueous buffer, acetonitrile, octane and DMF respectively (data not shown) were obtained. Therefore ultrasonic pretreatment compensated effectively for thermal inactiva- The effect of ultrasonic pre-irradiation on the stability of Bur-kholderia cepacia lipase in various solvents Figure 1 The effect of ultrasonic pre-irradiation on the stability of Burkholderia cepacia lipase in various solvents. pH tuned Burkholderia cepacia lipase (1 mg) was suspended in different solvents (100 μl each) and ultrasonicated at 110W continually. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be maintained at 40 ± 1°C throughout. The value on the xaxis denotes the total time during which the ultrasonicator was 'on'. Samples were extracted at various intervals (i.e. 1, 2, 3, and 4 h) and an esterase assay was performed using pNPP as a substrate. The experiments were carried out in triplicate. The error bar reflects the reproducibility for each data point. tion, with pretreatment in aqueous buffer being more effective, in terms of observed enzyme activity, than in organic solvents. Presumably, cavitation energy and its dispersion differ in aqueous and organic media.
It is relevant to compare these results with those reported by Vulfson et al [4] on subtilisin-catalysed transesterification. In that study a similar power output was employed (150 W), with the temperature maintained using a thermostatically-controlled glass reaction vessel. Ultrasonic treatment of subtilisin in phosphate buffer led to inactivation of the enzyme (50% in around out 2 h), while no equivalent effect was observed in t-amyl alcohol (containing 1% vv -1 buffer). Sinisterra [5] has also reported that the ultrasonic inactivation of subtilisin is more pronounced in aqueous media than under biphasic conditions (t-amyl alcohol, 1% phosphate buffer).
Another study of interest is that of Özbek and Ulgen [8], who recently investigated the effect of ultrasound on six enzymes (four dehydrogenases, alkaline phosphatase and β-galactosidase) in aqueous buffers. Apart from alkaline phosphatase, all other enzymes showed (variable) inactivation profiles (although ultrasonic treatment was carried out at 5°C). Higher ultrasonication times or power outputs resulted in greater inactivation. In addition, it was observed that on increasing the viscosity of the media by addition of glycerol increased the ultrasonic inactivation. Interestingly, none of these three studies reported enzyme activation. However, two studies by Ishimori et al. and [16] Sakakibara et al. [17], do report enhanced reaction rates resulting from the application of ultrasound to enzymatic reactions in aqueous buffers. The first of these reported the acceleration of α-chymotrypsin activity, whilst the second demonstrated that the activity of invertase was promoted by ultrasound at low substrate concentration. While the V max remained unaltered, K m roughly halved upon ultrasonication (i.e. affinity for the substrate increased).
Thus to recapitulate, as indicated by figure 1, ultrasonic pretreatment resulted in the aqueous enzyme being more active in aqueous buffer. These data are unusual and have not been previously observed.

The effect of ultrasonic pre-irradiation on lipase activity in organic solvents
We decided to apply our experiences with the ultrasonic pre-irradiation of lipases to a biotransformation in organic media. The transesterification (ethyl butyrate with butanol in anhydrous octane) activity was evaluated after 'drying', that is, removing bulk water from the pre-irradiated enzyme sample. (It has been reported that drying is more efficient when carried out through precipitation with an organic solvent rather than lyophilisation [18,19].) The initial rate of transesterification increased from 0.53 to 1.18 μmoles -1 mg -1 min at the optimum preirradiation time of 3 h (Figure 5a). The consequence of the enhanced initial rate was also reflected in the percentage conversions obtained (Figure 5b): for enzyme pre-irradi- The effect of ultrasonic pre-irradiation on the stability of Pseudomonas fluorescens lipase in various solvents Figure 2 The effect of ultrasonic pre-irradiation on the stability of Pseudomonas fluorescens lipase in various solvents. pH tuned Pseudomonas fluorescens lipase (1 mg) was suspended in different solvents (100 μl each) and ultrasonicated at 110W periodically. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be maintained at 40 ± 1°C throughout. The value on the x-axis denotes the total time during which the ultrasonicator was 'on'. Samples were extracted at various intervals (i.e. 1, 2, 3, and 4 h) and an esterase assay was performed out using pNPP as a substrate. The experiments were carried out in triplicate. The error bar reflects the reproducibility for each data point. The enhancement of initial enzymic rates is a challenge particularly relevant to industry [10,12]. The transesterification activity of lipases has already been exploited in the synthesis and kinetic resolution of several compounds [10][11][12]. In order to illustrate this, we decided to look at a and b. The effect of ultrasonic pre-treatment (at various power ratings) on lipase stability in acetonitrile Figure 4 a and b. The effect of ultrasonic pre-treatment (at various power ratings) on lipase stability in acetonitrile. pH tuned lipase (1 mg) in acetonitrile (100 μl) was ultrasonicated at various power ratings (i.e. 110 W, 66 W and 44 W) continually. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be maintained at 40 ± 1°C throughout. The value on the x-axis denotes the total time during which the ultrasonicator was 'on'. Samples were extracted at various intervals (i.e. 1, 2, 3, and 4 h) and an esterase assay was performed using pNPP as a substrate. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be maintained at 40 ± 1°C throughout. The value on the x-axis denotes the total time during which the ultrasonicator was 'on'. Samples were extracted at various intervals (i.e. 1, 2, 3, and 4 h) and an esterase assay was performed using pNPP as a substrate. the production of biodiesel from Jatropha oil. Biodiesel is a diesel-equivalent processed fuel consisting of short chain alkyl (methyl or ethyl) esters of fatty acids, which can be used (alone, or blended with conventional diesel fuel) in unmodified diesel-engine vehicles [20]. It is a more environmentally-friendly fuel and its enzymatic preparation has attracted considerable attention [20][21][22]. The enzymatic route involves the lipase-catalysed tranesterification of plant oils with ethyl/methyl alcohol. The use of Jatropha oil as the starting material is favourable, given for instance its inedibility and easy cultivation, even on wasteland [14,15]. Both chemical [14] and enzymic [15] preparations have been described in the literature, and earlier studies have demonstrated the benefit of a solvent-free approach [22,23].
We decided to carry out pretreatment of lipase in Jatropha oil directly before initiating transesterification with the addition of ethyl alcohol. The Burkholderia cepacia lipase was chosen owing to its responding better to ultrasonic pre-irradiation. In addition, because outputs of 110 and 66 W were observed to yield better results when using organic solvents, these settings were again adopted (Figure 6). Pretreatment for 2 or 3 h at 110 W gave the optimum result, that is a 79% conversion over 24 h. The untreated enzyme (used as control) gave only 34% conversion over the same time period.
The effect of ultrasonic pre-irradiation (for various time intervals) on the transesterification activity of Burkholderia cepacia lipase with Jatropha oil Figure 6 The effect of ultrasonic pre-irradiation (for various time intervals) on the transesterification activity of Burkholderia cepacia lipase with Jatropha oil. In a control (without ultrasonicated lipase) a 34% conversion was obtained (data not shown). The experiments were carried out in triplicate. The error bar reflects the reproducibility for each data point.

Structural and morphological changes in the lipase preparation as a result of ultrasonication
In order to determine whether pretreatment affected the protein structure, the Burkholderia cepacia lipase was purified and the effects of pre-irradiation were investigated (Figure 7). Although no inactivation was observed, the pure protein was most affected, with maximum activity resulting after pre-irradiation for 1 h.
The far-UV CD spectra of the untreated and irradiated samples were identical, indicating that ultrasonic irradiation had not affected the lipase's secondary structure (figure 8a). However two differences could be seen in the 250-300 nm spectral region (Figure 8b). Signals in the near-UV CD spectral region are diagnostic of the microenvironments of aromatic residues phenylalanine, tyrosine and tryptophan. Signals in the 250-270 nm region are attributable to phenylalanine, with signals in the 270-290 nm region attributable to tyrosine, and those in the 280-300 region indicate the presence of tryptophan [24]. The CD spectra (Figure 8b) showed that ultrasonic preirradiation led to perturbation of the tyrosine and tryptophan environments. Ultrasonic pre-irradiation enhanced the negative band intensity, indicating slight perturbation of the tertiary structure [24].
The SEM of the untreated and irradiated lipase samples ( Figure 9) showed that definite morphological changes were brought about by ultrasonication. Initially, the preparation was more or less monolithic in nature, however, following pre-treatment, small spheres or, in the case of pure enzyme, a fine powder could be observed on the surface. This is thought to have increased the surface area of the catalyst. We can therefore conclude that activation of the lipase by ultrasonic irradiation originated both in morphological and structural changes at the molecular level.

Conclusion
Limited data on the effects of ultrasonication on enzyme activity are available in the literature. While in some cases, ultrasonication results in the loss of enzymic activity, in The effect of ultrasonic pre-irradiation on the stability of purified Burkholderia cepacia lipase in buffer Figure 7 The effect of ultrasonic pre-irradiation on the stability of purified Burkholderia cepacia lipase in buffer. Purified Burkholderia cepacia lipase (366 U) was dissolved in buffer and ultrasonicated at 110W. In this study, we carried out ultrasonication for 5 minutes, followed by a break of 10 minutes between each cycle. This ensured that the temperature of the sample could be maintained at 40 ± 1°C throughout. The value on the x-axis denotes the total time during which the ultrasonicator was 'on'. Samples were extracted at various intervals (i.e. 0.5, 1, 1.5 and 2 h) and an esterase assay was performed using pNPP as a substrate. The experiments were carried out in triplicate. The error bar reflects the reproducibility for each data point. others it leads to the enhancement of the reaction rate. In this study we demonstrated that ultrasonic pretreatment enhanced enzyme activity in water and organic media.

Experimental
Jatropha oil was obtained from Dr. Jayaveera, Jawaharlal Nehru Technological University Oil Technological Research Institute, Anatapur, India. Burkholderia cepacia (PS) and Pseudomonas fluorescens (AK) were kind gifts from Amano Enzyme Inc., Nagoya, Japan. p-Nitrophenylpalmitate (pNPP) and Sephadex G-75 were bought from Sigma Chemical Co., St Louis, USA. All solvents were of anhydrous grade and were obtained from J. T. Baker, USA. They were further dried by being gentle shaken with 3 Å molecular sieves (E. Merck, Mumbai, India).

Enzyme preparations
Lipase from Burkholderia cepacia (50 mg) was dissolved in 0.5 ml of 20 mM sodium phosphate buffer (pH 7.0). Pseudomonas fluorescens lipase (50 mg) was dissolved in 0.5 ml of 20 mM sodium phosphate buffer (pH 8.0). The samples were then lyophilized for 48 h. The resulting dried powders obtained were labelled as pH tuned enzymes [10].

Ultrasonic treatment
Ultrasonic treatment was performed using an Elma transsonic digital ultrasonic unit (model T 490 DH) from Elma & Co. KG Hans Schmidbauer Gmbh, Singen, Germany. A fixed frequency of 40 kHz and various power ratings specified in the figure legends were employed. The ultrasonicator bath was equipped with a temperature control.