Lipase activity of both soluble and immobilized enzyme was measured by using 0.1% w/v of p-nitrophenyl palmitate (p-NPP) in ethanol (95%) as substrate by mod­ifying the method of Hung et al. (2003). The reaction mixture consists of 100 pl substrate, 1 ml 0.05 M phosphate buffer of pH 7, and 50 pl of free lipase solution (stock 1 mg/ml) or 1 g immobilized lipase beads. The mixture was kept at 30°C for 5 min followed by termination of the reaction by addition of 2 ml of 0.5 N Na2CO3

Sodium alginate+

Dropped into 0.1M CaCl2 solution

alginate

=» carrageenan

Burkholderia cepacia lipase

Fig. 12.2 Immobilization of Burkholderia cepacia lipase by cross-linking with glutaraldehyde followed by entrapment in hybrid matrix of alginate and carrageenan solution. After this, the reaction mixture was centrifuged at 10,000 rpm for 10 min. Absorbance of the end product p-nitrophenol, which is released by lipase hydrolysis of p-NPP, was measured at 410 nm in a spectrophotometer (Spectronic 4001, USA) against an enzyme-free blank solution. A molar extinction coefficient of 15,000/M/ cm for p-nitrophenol was used (Okahata et al. 1995) . The amount of enzyme required to hydrolyze 1 |rmol/min of p-NPP under the similar assay conditions was defined as one unit of lipase activity (U).

The efficiency of lipase entrapment was evaluated in terms of activity yield as follows:

Activity yield (%) = Specific activity of immobilized lipase x100 Specific activity of free lipase