conver-sion of esters. In order to reduce the cost of 2-phenethyl alcohol, a slight change of the increasing conversion seems not significant. A molar ratio of 3.0:1 was selected as the optimised condition in the following tests.
The effect of enzyme loading on the conversion of 2-phenethyl alcohol was studied in the range of 5–25% (w/w, based on the weight of all the reactants) and using the optimised molar ratio
(3.0:1) obtained from the previous result. A positive effect of enzyme loading (5–20%) on the conversion was demonstrated by the increased conversion rate of 2-phenethyl alcohol (p > 0.05) However, a further increased enzyme loading from 20% to 25% (w/w of all the reactants) caused a slight reduction of conversion.
To obtain a high conversion of 2-phenethyl alcohol, 20% (w/w of all the reactants) was selected as the optimum enzyme loading applied to the subsequent transesterification of butter oil with 2-phenethyl alcohol.
Proper agitation at an optimum speed plays an essential role in enhancing external mass transfer. To observe the effect of mass transfer limitations, experiments were carried out at various agitation speeds ranging from 50 to 250 rpm. It was found (Fig. 4b) that the conversion of 2-phenethyl alcohol significantly increased as the shaking speed increased from 50 to 100 rpm. However, only a slight increment was observed when the speed was further increased
to 250 rpm, which demonstrates that the mass transfer resistance had reached a minimum level.
With the increment of shaking speed, the decreased film thickness around the solid lipase
particles led to the decrease of the mass transfer resistance.
The optimised conversion of 2-phenethyl alcohol was at 100 rpm.
Fig. 4c shows a significant difference in the conversion of 2-phenethyl alcohol within the studied temperature range according to the ANOVA test result (p < 0.05).
The conversion rate increased gradually as the incubation temperature increased from 20 to 40 C. This is because the melting point of butter oil is around 35 C, which means the oil phase and the alcohol phase may not mix well below 35 C. By increasing the incubation temperature,
all the reactants will be mixed well and the mass transfer will be enhanced. A slight reduction of conversion was obtained when incubation temperature increased from 40 to 60 C. This indicated
that the enzyme may suffer from thermal denaturation due to the long reaction time at high temperatures.
It was suggested that almost all the non-thermophilic enzymes will be inactive when temperatures
rise above 45 C (Yong & Al-Duri, 1996).
In our experiment, the reaction may reach its fastest rate around 40 C, which could be one reason for the insignificant difference of the conversion rate observed from 40 to 60 C. Therefore, an optimal reaction temperature should consider both the enzyme stability and the reaction rate of transesterification. To protect the enzyme from thermal deactivation and to obtain high conversion the reaction temperature was kept at 40 C for the subsequent experiment.
Under the optimised conditions, the time-course reaction was conducted within a 20-h period.
Fig. 4d shows that the conversion of 2-phenethyl alcohol in the reaction solutions increased gradually with time from 4 to 8 h (p < 0.05). Only a slight increment of conversion was observed as the reaction time was further increased from 8 to 20 h.
In summary, under the optimised conditions of enzyme-catalysed reaction, the total yield of C4–18 2-phenethyl esters was about 817 mg per gram butter oil. The optimal reaction conditions
led to an 80.0% conversion of 2-phenethyl alcohol.