Immobilization of Lipase From Candida Rugosa on Hydrophobic and Hydrophilic Supports

Abstract

The aim of this study is to find the optimum conditions for the immobilization of Candida rugosa lipase and also to select the most suitable support maximizing the immobilized enzyme yield under these optimum conditions.Prior to the immobilization studies, optimum working conditions of soluble Candida rugosa lipase were investigated. Candida rugosa lipase expressed maximum activity in pH 6.5 phosphate buffer with 1.0 M NaCl solution at 30 0C. Indeed, lipase was fully active between 4 and 37 0C. Under these conditions, activity of free lipase was 12.2 U/mg enzyme.Immobilization studies were carried out under constant temperature and stirring rate. Immobilization of lipase on hydrophobic support, octyl-sepharose, was very rapid and the equilibrium was reached in 10 minutes. Immobilized enzyme ratio was maximized by the use of pH 7.0 phosphate buffer with 1.0 M NaCl at 37 0C. Also, optimum solid-liquid ratio was found to be 0.1 when 1 ml support was added to 9 ml of enzyme solution. Effect of enzyme loading on lipase immobilization on octyl-sepharose was investigated at two different temperatures. At 20 0C and in the specified range of enzyme concentration (0.25-75 mg/ml), the experimental data fitted well to the linear isotherms with a C value of 138.2. Also, at 4 0C, results were similar and the experimental data could be expressed with the linear isotherm with a C value of 209.1. Under these conditions, activity of immobilized enzyme was 410 U/ml hydrated support.In the case of immobilization on hydrophilic support, chitosan, the reaction was much slower and the amount of the immobilized enzyme was maximized at the end of 24th hour. Optimum conditions of immobilization were determined as pH 6.5 and 20 0C. In this case, addition of salt decreased the immobilization and, therefore, low ionic strengths favored immobilization. Again, optimum solid-liquid ratio was found to be around 0.1 when the 1 ml support was added to 9 ml. of enzyme solution. The effect of enzyme loading was investigated at 20 0C and in the specified range of concentration, data fitted well to the Langmuir isotherm where the constants were calculated to be qm.200 U adsorbed enzyme/ml hydrated support and K. 72.5 U/ml solution.The storage stability of chitosan immobilized lipase was investigated at 4 0C under two different conditions. When immobilized lipase was kept dry for 25 days, 50 % of the initial activity remained. However, if the same lipase was kept in phosphate buffer of pH 6.5, activity loss was only 10 % after a period of 2 months. Complementary work was also carried out to find the differences in the behavior of free and chitosan immobilized lipase against different substrates. The results showed that the presence of 33 % olive oil and 45.5 % corn oil in substrate yielded the maximum activity for free and chitosan immobilized lipases, respectively.