Candida
rugosa lipase was immobilized on Celite by acetone precipitation and adsorption. The
immobilized enzyme was used for catalyzing olive oil hydrolysis in an aqueous medium without
any added emulsifiers. Adsorption on Celite gave better results in terms of effectiveness factor,
reuse, and stability compared to acetone precipitation. Adsorbed lipase exhibited good hydrolytic
activity but was never more efficient than soluble enzyme. The effectiveness factor of lipase adsorbed
on Celite was found to be 0.90. The fatty acid formation was linearly increased (P < 0.05) up to an
enzyme concentration of 0.5 mg. The progress curve of the olive oil hydrolysis gave a typical
hyperbolic form. After immobilization, the optimal reaction pH was shifted from 7 to 6.5 and the
optimal reaction temperature from 40 to 45 °C. Although the lipase immobilized on Celite exhibited
higher stability, the rapid loss of enzyme activity appeared to be the main problem during repetitive
use. The parameters, specific production rate, and maximum product value were estimated for
each experimental data set by applying the Logistic-2 model.
Keywords: Candida
rugosa
lipase; adsorption; immobilization; olive oil; hydrolysis
Polyphenol oxidase (PPO) obtained from wheat bran catalyzed the oxidation of 4-methyl catechol. Phenolic compounds found naturally in crude extract played role as an endogeneous substrate and activity of crude extract needed correction. Activity versus enzyme concentration gave a linear plot at high substrate concentration whereas a nonlinear plot was obtained at low substrate concentration which proved the presence of endogeneous substrate. Adsorption on celite and extraction with polyvinylpyrrolidone (PVPP) caused the removal of phenols. Adsorption of PPO on celite yielded a 4-fold increase in specific activity whereas extraction with PVPP yielded a 2.5-fold increase in specific activity compared to the crude extract. The kinetics of PPO catalyzed oxidation obeyed Michaelis-Menten model; Km and Vmax values were found as 218 mM and 99 microM/min, respectively. The enzyme was inhibited by ethyl alcohol, dithiothreitol (DTT) and isoproterenol and exhibited heat stability up to a temperature of 90 degrees C. The optimum pH of the enzyme was found to be 5.0.
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