1. INTRODUCTION:

The application of enzymes as biocatalysts in supercritical carbon dioxide has been described in 1980’s by Randolph et al (1985), Hammond et al (1985) and Nakamura et al (1986). Since then, various researchers reported the applicability of SC-CO₂as a solvent for enzyme catalysis (Kasche et al 1988, Dumont et al 1992, Chulalaksananukul et al 1993). The potential advantages of employing enzymes for esterification in the organic media are increased substrate solubility, shift of equilibrium towards product, enhanced specificity and thermostability. It has also been reported that the nature of the solvent could affect the enzyme activity and thus its stability (Kery et al 1990). Therefore, it is necessary to choose a suitable solvent, facilitating higher activity and stability of enzymes to get higher yields.

Further, supercritical carbon dioxide has been reported to exhibit properties similar to those of organic solvents and could serve as an alternative to classical organic solvents (Varma et al 2008). Enzymatic reactions in SC-CO₂ have been considered to be a viable alternative to classical organic solvents because of certain advantages like increased transport properties due to low viscosity and high density and ease of post reactional separations (Chulalaksananukul et al 1993).

The present work focuses on synthesis of ethyl butyrate ester from butyric acid and ethyl caprate using lipase from Candida rugosa in SC-CO2. The scope of the work includes

Assessment of stability of enzyme under supercritical carbon dioxide environment, Effect of pressurization and depressurization on stability of enzyme, Effect of incubation time, temperature, enzyme concentration and reaction pressure on the Alcoholysis reaction.

2. MATERIALS AND METHODS

2.1. Materials

2.1.1. Enzymes

Lipase from Candida rugosa [CRL] Type-VII was procured from Aldrich Chemicals [Milwaukee, WI, USA] .

2.1.2. Chemicals

Butyric acid and ethyl caprate were obtained from Aldrich chemicals [Milwaukee, WI, USA]. Ethyl stearate was from Sigma Chemicals Co. [St. Louis, MO, USA]. Tributyrin [Glycerol tributyrate] was from Merck [Darmstadt, Germany]. Methanol and n-octanol, were from Sd fine-Chem Ltd. [Mumbai, India]. Phenolphthalein and sodium hydroxide were from SISCO Research Laboratories Pvt. Ltd. [Mumbai, India]. All substrates were dried over molecular sieves before use. The solvents were distilled before use.

2.2. Methodology

2.2.1.Hydrolytic activity of lipase

Activity of lipase was measured as per the procedure recommended by Tietz and Fereick (1966). The hydrolytic activity value was calculated using following equation:

Enzyme activity

One unit of hydrolytic activity has been defined as one mmol of butyric acid released per minute per mg enzyme. The hydrolytic activity of C. rugosa was 32,000 U/g; All the experiments were repeated to check the consistency of data and the average of the results were reported.

2.2.2. Preparation of surfactant - coated lipase

Since surface coated lipase was found to be more effective than normal lipase (Nambula Annapurna devi 2006 ), it was prepared according to the methods given elsewhere (Kamiya et al 1995).

2.2.3. Esterification in Supercritical Carbon dioxide [SC-CO2]

Esterification reactions were carried out in a 100 ml high-pressure reactor [Berghoff autoclave, Germany] containing appropriate amount substrate and specified quantities of enzyme at different temperatures for 24 h of incubation time. After the temperature attained the set value, carbon dioxide was pumped into the reactor at different pressure levels through storage tank. Sampling was done through a micrometer valve in to 3 ml vial at atmospheric pressure. The reactor was provided with an inbuilt magnetic stirrer for continuous mixing of reactants. The product was estimated using GC.

2.2.4. Buffer saturation of solvent

Isooctane solvent was saturated with buffer [0.1 M phosphate buffer, pH 7.0] at a ratio of 10:1 [V/V] [solvent: buffer] (Prapulla 1995; Prapulla et al 1998). The buffer saturated organic solvent was used directly for the synthesis of esters.

3. ALCOHOLYSIS IN SUPERCRITICAL CARBON DIOXIDE [SC-CO2]

Fig.1 : Supercritical carbon dioxide experimental set-up

4. RESULTS AND DISCUSSIONS

4.1 Stability of Lipase enzyme in SC-CO2

In order to assess the stability of the enzyme in supercritical carbon dioxide, 100 mg of lipase from Candida rugosa was put into a high pressure reactor [100 ml]. Carbon dioxide was compressed by a piston pump up to the required pressure and the enzyme was incubated under these conditions for 24 h at 100 bar and 40ºC and 70ºC respectively. After treatment, the enzyme was measured for its residual hydrolytic activity. Table 1 shows the residual activity of the lipase enzyme that has been treated at two different temperatures.

Table 1: Residual hydrolytic activity of CRL treated at 100 bar pressure and at two different temperatures.

Incubation time (h)	Residual hydrolytic activity*
Incubation time (h)	40ºC	70ºC
1	100	95
5	98	78
10	98	56
24	98	50

* Value of untreated enzyme is set to 100 %.

There was a significant change in activity of lipase exposed to two distinct temperatures after high pressure treatment. The residual hydrolytic activity of lipase treated at 40ºC was lost marginally (2 %), while the lipase exposed to 70ºC high temperature lost more than 50 % of its hydrolytic activity, and the enzyme powder changed its color completely to caramel brown and then to firm pieces. On the other hand, the lipase preparation exposed to 40ºC did not change color and remained as white powder. These results were in accordance with the results reported, Bauer et al (2000).

4.2 Effect of Pressurization and depressurization on activity of enzyme

The enzyme preparation (100 mg) was incubated for 60 minutes in supercritical carbon dioxide at 100 bar and 40ºC. Then, the reactor was depressurized to atmospheric pressure (duration for complete depressurization is 10 minutes) and again pressurized to 150 bar pressure. This procedure was repeated 10 times. Afterwards, the enzyme preparation was assayed for its residual hydrolytic activity. Table 2 shows the enzyme activity after 10 pressurization and depressurization steps with SC-CO2.

Table 2: Effect of pressurization and depressurization on the activity of enzyme

No. Cycles	Residual hydrolytic activity (%)
1	100
2	100
3	98
4	98
5	98
6	98
7	98
8	97
9	97
10	97

This experiment reveals that there was no significant influence of pressurization and depressurization. The lipase remained active and showed more than 97 % hydrolytic activity during 10 cycles. This was much in agreement with the results reported in Željko Knez et al( 2009).

4.3 Effect of Incubation time

The results depicted in Fig. 2 shows the effect of incubation time on the synthesis of ethyl butyrate at 0.05 moles of substrate and 100 bar pressure in SC-CO2 at an enzyme concentration of 20 % w/w at 40°C. The percentage esterification gradually increased with incubation time and reached a saturation limit after 10 hrs. This accelerated effect is the result of activation of enzyme due to formation of water (Goldberg et al 1990). Optimum esterification obtained in SC-CO2 was 75 %. Hence, an incubation time of 10 h was fixed for the study of the interactive effects of other reaction parameters.

4.4 Effect of Temperature

The results depicted in Fig.3 show the effect of temperature on the synthesis of Ethyl butyrate by alcoholysis.

The percent esterification increased linearly with an increase in temperature, reached a maximum value of 92 % at 40°C and then decreased beyond this temperature. The decrease in esterification at higher temperatures can be attributed to enzyme inactivation. This inactivation was due to the water being released into the reaction medium due to higher esterification [%] ( Yuji shimada 2002). On the other hand, enzyme inactivation can also be due to increased solubility of butyric acid at higher temperatures and thus acidification of microaqueous phase of lipase (Manjoin et al 1991). Enzymatic hydrolysis of benzoyl-benzoin catalyzed by Candida cylindracea (CCL) lipase carried out in SCCO2 did not catalyze the reaction in an atmospherical condition (Nuray Celeb et al 2007) .

4.5 Effect of enzyme concentration

Fig. 4 shows the influence of enzyme concentration on alcoholysis reaction while keeping the pressure constant at 100 bar.

The percent esterification increased with increase in enzyme concentration. The maximum percentage esterification [92 %] was observed at higher enzyme concentration [25 % w/w]. This was attributed to the better contact between substrate and enzyme, thus leading to higher percentage esterification.

The extent of alcoholysis remained constant even at higher enzyme concentration of 30 %. The hydration of biocatalyst can be more at higher conversions not only because of formation of more water but also because the polarity of reaction medium gets reduced as the substrates [acid and alcohol] were converted to less polar ester product. This change in polarity causes water to partition more onto biocatalyst (Mensah et al 1998).

4.6 Effect of reaction pressure

Keeping the temperature constant at 40°C, the effect of reaction pressure was studied (Fig. 5).

The maximum percentage esterification was obtained at 150 bar pressure [96 %]. However, the reaction pressure has no significant influence on enzyme stability. As the pressure increased from 50 to 200 bar, esterification increased up to 150 bar, and on further increase in the pressure, a decreased esterification was noticed. The decreased esterification at higher pressures can be due to loss in activity of enzyme or the result of increased solvent-solute interactions. It was known that the increase in reaction pressure resulted in increased solvent-solute interactions (Vermue et al 1992) which affects the partitioning of substrate between the solvent and enzyme. Decrease in esterification at lower pressure [50 bar] can be due to the adsorption of the formed ester to the enzyme, causing enzyme inhibition (Steytler et al 1991). However, the increase in reaction pressure leads to decrease in the substrate concentration at active site of the enzyme [i.e, resorption of substrate], resulting in lower esterification (Vermue et al 1992).

5. CONCLUSIONS:

Alcoholysis of ethyl caprate with butyric acid was carried out in supercritical carbon dioxide using SCL from Candida rugosa. The lipase was very stable at low temperature [40ºC] and loss in activity was aggravated when high temperatures [70ºC] were employed under supercritical conditions. The reaction temperature was found to have a significant effect on percent esterification. At higher temperatures, percentage esterification decreased. This could be due to partial thermal denaturation. However, reaction pressure has got marginal impact on the extent of alcoholysis and enzyme stability. No significant loss was noticed during pressurization and depressurization conditions even after 10 cycles. The maximum alcoholysis of 96 % was obtained under the following reaction conditions:

Substrate concentration 0.05 moles [1:1 ratio of ester and acid], An enzyme concentration of 25 % w/w, at 150 bar reaction pressure and at 40ºC temperature and 10 h of incubation period.

Moreover, the synthesized ester will be free from solvent [CO2 can be removed at atmospheric pressure] and can thus find use in food applications.

6. ACKNOWLEDGEMENTS:

The authors are thankful to CFTRI-Mysore for providing the supercritical extraction equipment for experimentation. The authors are also thankful to the Principal Dr. KV.L. Raju and the management of MVGR College of Engineering, Vizianagaram for their constant support and encouragement.

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Received on 14.07.2011 Accepted on 10.10.2011