The pH stability of free and immobilized lipase were studied by incubating at 25°C in phosphate buffers of varying pH (3-10) for 1 h. The hydrolytic activity was deter­mined at optimum pH and temperature. After the incubation time, the immobilized beads were separated from the buffers, filtered, and air dried at room temperature, and its catalytic activity was measured as described in Sect. 12.5.2. Relative activi­ties were calculated as the ratio of activity of immobilized enzyme after incubation to the activity at optimum reaction pH.

Enzyme surface contains large number of acidic and basic groups. Depending on the pH of the medium, the charges on these groups may vary. This in turn alters the activity, structural stability, and solubility of the enzyme molecule. Also a change in pH can lead to the breakage of ionic bonds that hold the tertiary structure of the enzyme and also affect the charges on amino acids inside the active site so that the enzyme cannot form enzyme-substrate complex. In short, the effect of acid H+ ions and basic OH- ions on enzymatic activity is caused by a change in stereo configura­tion at or in the neighborhood of enzyme molecule’s active site (Seyhan et al. 2002). Relative activities of both free and immobilized lipase as a function of pH are depicted in Fig. 12.5b.

The thermal stability of free and immobilized lipase were tested by incubating at various temperatures in the range of 25-60°C for 1 h at pH 7. Lipase activity at optimum temperature was found out as in Sect. 12.5.2. Relative activity as a func­tion of temperature is shown in Fig. 12.5c. Optimum temperature for free lipase was found to be 30°C and that of immobilized lipase was found to be 40°C. It shows that immobilization resulted in higher activation energy for the enzyme. The elevated activation energy than the free enzyme implies an increased energy barrier, which

Fig. 12.5 (a) Leakage of protein from the beads as a function of incubation time in phosphate buffer of pH 7.0; (b) pH stability of free and immobilized lipase; (c) temperature stability of free and immobilized lipase at pH 7; (d) storage stability of free and immobilized lipase at 4°C in dif­ferent buffers; (e) reusability of immobilized lipase forp-NPP hydrolysis; (f) time course of olive oil hydrolysis by immobilized lipase with and without cross-linking

means that the immobilized enzyme needs an elevated temperature to attain its highest activity. It was also noted that the hybrid matrix used here could withstand a temperature up to 60°C, beyond which there was slow disintegration of the matrix. This implies that immobilized B. cepacia lipase can be applied at higher tempera­ture. The study demonstrates that the hybrid matrix used here shows good thermal resistance which comes as an important property in industrial applications.