Enzymatic Esterification of Oleic Acid with Lauryl Alcohol

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ABSTRACT In the present work, esterification of oleic acid with Lauryl alcohol, catalyzed by Rhizopus oryzae lipase (ROL) in a biphasic solvent system has been studied. Effect of pH, water concentration, solvent concentration, alcohol concentration and enzyme concentration; on activity of ROL have been studied. Activity of 2.103moles oleic acid consumed per mg enzyme per minute was obtained when 20mg enzyme dissolved in 1 ml buffer was used in each batch. At this enzyme concentration, 52.28 % esterification was achieved in one hour reaction time and 92.98% esterification has been obtained in 5 hours of reaction time. When 40mg enzyme dissolved in 1 ml buffer was used in each batch, 76.83% esterification was achieved in one hour reaction time and 94.38% esterification was obtained in 5 hours of reaction time. Key words: Oleic acid, lauryl alcohol, Rhizopus oryzae lipase, esterification, biphasic system.

INTRODUCTION Lipases, also known as triacylglycerol ester hydrolases, are enzymes that cleave ester bonds of triacylglycerols with the subsequent release of free fatty acids, diacylglycerols, monoacylglycerols and glycerol. Lipases are also able to catalyze the reverse reactions (esterification, interesterification and transesterification) provided that the aqueous medium is replaced by an organic or a biphasic aqueous/organic medium. Although ester synthesis can be chemically catalyzed by acids or bases, the use of enzyme technology offers environmental advantages and a reduction in energy consumption [1]. Furthermore, lipases show high selectivity including stereo-selectivity and give products of high purity and improved quality [2]. A range of fatty acid esters is now being produced commercially using immobilized lipase in non-aqueous solvents. For example, esters produced from long-chain fatty acids (1220 carbon atoms) as well as short-chain fatty acids (28 carbon atoms) are used increasingly in the food, detergent, plasticizer, lubricant, cosmetic and pharmaceutical industries [3]. The use of enzymes in biphasic systems instead of aqueous media offers several important advantages such as a reduction in enzyme substrate and/or product inhibition, the solubilisation of hydrophobic

compounds, the possibility of shifting thermodynamic equilibria towards the desired reaction [4]. R. miehei catalyzed esterifications of fatty acids with alcohols have been studied by several groups [5-8]; kinetic data in biphasic systems are scarce. Al-Zuhair et al. [9] have determined the kinetics of the esterification of butyric acid with methanol catalyzed by a free R. miehei lipase in a micro aqueous system containing enzyme in a suspension in hexane and a biphasic system (n-hexane and water). A kinetic model was determined for the micro aqueous phase but not for the biphasic system. Oliveira et al. [4] have studied the reaction of oleic acid with ethanol using a free R. miehei lipase and immobilized R. miehei lipase on accurel EP700 in a hexane and a phosphate buffer system. However, the rate expression for the free enzyme was based on the total liquid volume, without taking into account the phase ratios and the enzyme concentration. This limits the applicability of this model considerably. Baron et al. [10] reported the esterification of oleic acid with n-butanol using a crude lipolytic extract of Penicillum coryophilum in AOT/n-heptane reversed micelles. The present study has been aimed for the esterification of oleic acid with ROL enzyme in biphasic solvent system to determine the most suitable reaction conditions.

MATERIALS AND METHODS Materials Rhizopus oryzae lipase (ROL) was purchased from Sigma-Aldrich Co. USA. All the other chemicals such as Oleic acid, isooctane, lauryl alcohol, ethanol, methanol, potassium hydroxide and phenolphthalein indicator, were of AR grade and CDH make. Methods The experiments were conducted in a batch setup with magnetic stirring. For these experiments reaction mixture consisted of two phases: an organic phase (1.7g oleic acid and 1.67 g lauryl alcohol dissolved in 7 mL iso-octane) and an aqueous phase consisting of enzyme dissolved in 1mL phosphate buffer solution (pH 7). The reaction mixture was agitated at room temperature at a stirrer speed of 800 rpm. The reaction was stopped by adding 10 ml of hot (50C) methanol. Standard ASTM test method has been used for estimating the acid value (ASTM D 138698, 2004) of collected samples at different reaction time to determine moles of free fatty acids (FFAs) consumed. The activity of lipase has been expressed as moles of FFAs consumed per mg enzyme per minute of reaction time. RESULTS AND DISCUSSION Various reaction parameters such as pH, temperature, oleic acid to water, oleic acid to solvent, oleic acid to alcohol ratio and enzyme concentration affect the activity of lipase and percentage of esterification in biphasic system during esterification of oleic acid with

lauryl alcohol. Except temperature, all other parameters as mentioned above, has been studied and the results are discussed below. Effect of pH The effect of pH was studied in the pH range of 5.5 to 8. The results are given in the Table-1. Maximum activity of enzyme was found to be 2.103moles oleic acid consumed per mg enzyme per minute at pH 7, corresponding to esterification of 52.28% in one hour time. The trend of variation of percentage of maximum enzyme activity and esterification with change in pH is shown in Fig.-1.

Table-1: Effect of pH on activity of Rhizopus oryzae lipase for esterification of oleic acid with lauryl alcohol
{Reaction Conditions: oleic acid 1.7 g, lauryl alcohol 1.67g, temperature 30C,agitation speed 800 rpm, reaction time 60 mins, ROL 20mg dissolved in 1 ml buffer, and iso-octane 7 ml}

pH

5.5 6.0 6.5 7.0 7.5 8.0

Total oleic acid consumed (moles) 2116.8 2250.3 2320.0 2524.7 2183.5 2081.2

Enzyme activity (moles oleic acid consumed/ mg enzyme.min) 1.764 1.875 1.933 2.103 1.819 1.734

% of maximum enzyme activity 83.88 89.17 91.92 100.00 86.52 82.46

percentage esterification 44.92 46.12 50.37 52.28 47.40 45.18

Fig.-1: Effect of pH on ROL activity and esterification Effect of solvent (iso-octane) concentration Organic phase of oleic acid and lauryl alcohol (6 and 9 mmol respectively) dissolved in iso-octane ranging from 4 ml to 16 ml and aqueous phase of 20mg ROL in 1 ml buffer (pH7), agitated on magnetic stirrer for 60 minutes was studied. The results of this study are shown in Table-2. Maximum activity of enzyme was found to be 2.0754moles oleic

acid consumed per mg enzyme per minute at 7 ml iso-octane, corresponding to esterification of 52.14% in one hour time. The trend of variation of percentage of maximum enzyme activity and esterification with change in solvent ratio is shown in Fig.-2.

Table-2: Effect of iso-octane on activity of Rhizopus oryzae lipase for esterification of oleic acid with lauryl alcohol
{Reaction Conditions: oleic acid 1.7 g, lauryl alcohol 1.67g, pH 7, temperature 30C, agitation speed 800 rpm, reaction time 60 mins, ROL 20mg dissolved in 1 ml buffer }

IsoTotal oleic octane acid (ml) consumed (moles) 4 2422.35 7 2490.58 10 2185.06 13 2081.17 16 2081.17

Enzyme activity (moles oleic acid consumed/ mg enzyme.min) 2.019 2.075 1.820 1.734 1.734

% of maximum enzyme activity 97.26 100.00 87.73 83.56 83.56

percentage esterification 50.71 52.14 45.74 43.57 43.51

Fig.-2: Effect of solvent concentration on ROL activity and esterification Effect of Lauryl Alcohol concentration The effect of lauryl alcohol concentration was studied at 1:1, 1:1.5, 1:2 (mol:mol) ratio with 40 mg enzyme. The results are given in the Table-3. Maximum activity of enzyme was found to be 3.058moles oleic acid consumed per mg enzyme per minute at 1:1.5 (mol:mol) ratio, corresponding to esterification of 76.83% in one hour time. The trend of variation of percentage of maximum enzyme activity and esterification with change in alcohol ratio is shown in Fig.- 3.

Table 3: Effect of alcohol on activity of Rhizopus oryzae lipase for esterification of oleic acid with lauryl alcohol
{Reaction Conditions: oleic acid 1.7 g, pH 7, temperature 30C, agitation speed 800 rpm, reaction time 60 mins, ROL 40mg dissolved in 1 ml buffer, and iso-octane 7 ml}

Lauryl alcohol (gm) 1.11 1.67 2.22

Total oleic acid consumed (moles) 3017.50 3670.00 3185.29

Enzyme activity (moles oleic acid consumed/ mg enzyme.min) 2.515 3.058 2.654

% of maximum enzyme activity 82.23 100.00 86.80

percentage esterification 66.66 76.83 73.07

Fig.-3: Effect of Lauryl alcohol concentration on ROL activity and esterification

Effect of enzyme concentration The effect of enzyme concentration on activity of ROL was studied by taking 20mg, 40mg & 60mg of enzyme with 1.67g oleic acid as substrate. The activity of enzyme and corresponding esterification in 1 hour reaction time is shown in Table-4. From the table it can be seen that maximum acivity was obtained to be 2.103 (moles oleic acid consumed/ mg enzyme.min) with 20mg enzyme concentration. The percentage esterification in 1 hour with 20mg has been obtained to be 52.28%, which has increased to 76.83% of esterification in case of 40mg enzyme used. However, when 60mg enzyme was used, the percentage esterification in 1 hour was 77.53% indicating no appreciable

change in esterification. Thus there was no significant increase in percent esterification with increase in enzyme concentration beyond 40mg enzyme with 1.67g oleic acid.

Table-4: Effect of Rhizopus oryzae lipase concentration on esterification of oleic acid with lauryl alcohol
{Reaction Conditions: oleic acid 1.7 g,lauryl alcohol 1.67g, pH 7, temperature 30C, agitation speed 800 rpm, reaction time 60 mins, enzyme dissolved in 1 ml buffer, and iso-octane 7 ml}

Enzyme amount (mg) 20 40 60

Total oleic acid consumed (moles) 2524.70 3670.00 3703.52

Enzyme activity (moles oleic acid consumed/ mg enzyme.min) 2.103 1.529 1.028

% of maximum enzyme activity 100.00 72.70 67.27

Percentage esterification

52.28 76.83 77.53

It is expected that percentage esterification would increase with increase in time of esterification. Therefore, the reaction was carried out for 24 hours with 20 and 40mg enzyme and the extent of esterification was measured at different time intervals. Data are given in Table-5 and the trend of increase in esterification with time of reaction is shown in Fig.-4. From Table- 5, it can be seen that with 20mg enzyme in 3 hours, 89.47% esterification has been obtained. Beyond that the rate of esterification has become very slow and in 7 hours 93.68% esterification has been achieved and it appears that esterification has reached the equilibrium stage. In case of 40mg enzyme used, in 3 hours, 92.98% esterification has been obtained and in 5 hours 94.38% esterification has been obtained. Further this reaction was continued upto 8 hours and no increase in esterification was achieved beyond 5 hours which again shows that equilibrium for esterification has reached around 94.38%.

Table-5 :Effect of Rhizopus oryzae lipase concentration on esterification of oleic acid with lauryl alcohol
{Reaction Conditions: oleic acid 1.7 g, lauryl alcohol 1.67g, pH 7, temperature 30C, agitation speed 800 rpm, reaction time 24 hours, enzyme dissolved in 1 ml buffer, and iso-octane 7 ml}

Time Oleic (min) acid initially present (moles)

Oleic acid consumed (moles) at 20 mg enzyme in reaction mixture

Oleic % % acid Esterification Esterification consumed at 20 mg at 40 mg (moles) enzyme in enzyme in at 40 mg reaction reaction enzyme mixture mixture in reaction mixture

10 20 30 45 60 90 120 180 240 300 360 420 1440

4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47 4776.47

920.67 1088.23 1423.52 2060.58 2530 2731.17 3904.7 4273.5 4374.1 4441.1 4474.7 4474.7 4474.7

1021.21 1390.0 2127.80 2965.95 3670.0 3804.2 4340.64 4441.17 4474.70 4508.23 4508.23 4508.23 4508.23

19.27 22.78 29.81 43.14 52.96 57.17 81.74 89.47 91.58 92.98 93.68 93.68 93.68

21.38 29.10 44.54 62.09 76.83 79.64 90.87 92.98 93.68 94.38 94.38 94.38 94.38

Fig.-4: Percentage esterification with time CONCLUSIONS The most suitable reaction conditions for the esterification of oleic acid with lauryl alcohol in biphasic solvent system have been found to be, pH: 7.0, temperature: 30 o C, agitation speed 800 rpm, enzyme dissolved in 1 ml buffer, and iso-octane 7 ml, Oleic acid : lauryl alcohol ratio 1:1.5 (mol:mol). The equilibrium level for esterification has been obtained around 94.4%. ACKNOWLEDGEMENTS The author acknowledges the financial support provided by CSIR, New Delhi, as JRF to the first author for this research work. REFERENCES

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