Lipases find application in many practical/ industrial processes (Bjorkling et al. 1991). They have been used to generate chiral entities from alcohols, carboxylic acid esters, cyanohydrins, chlorohydrins, diols, amines, diamines and amino alcohols (Jaeger et al. 1999), which are used as building blocks for a variety of pharmaceuticals and other fine chemicals. At present, the majority of the thermophilic lipases that have been purified and characterized are obtained from Bacillus sp (Kim et al. 1994). To use these enzymes for industrial purposes, the most desirable step is to improve the features of biological catalyst to suit the industrial demand.

In the present investigation attempts have been made to further improve the catalytic properties of the enzyme by the use of different immobilization techniques.

The culture was grown under optimal conditions for lipase production in media containing wheat bran and nutrient broth (1% w/v each), pH 8.0, seeded with 2% inoculum and incubated at 60ºC for 48 hrs. The cell free supernatant containing the lipase was used for further studies. Protein estimation was done by Lowry's method (Lowry et al. 1951).

The enzyme activity was determined according to the method of Sigurgisladottir et al.1993 with slight modification.

Purification of enzyme was first done by ammonium sulphate precipitation followed by hydrophobic interaction chromatography on Phenyl Sepharose (Sigma) column.

Immobilization of lipase was done on HP-20 beads (Diaion) and silica (Merck).

A comparative study was done on immobilized and aqueous enzyme for different parameters, such as effect of temperature, esterification studies, physicochemical properties, and thermo-inactivation.

The strain was identified as Bacillus sp.

Organic solvents benzene, hexane, acetone had stimulatory effect on the lipase activity while ethanol, methanol, propanol and dioxane inhibited the enzyme activity.

Good adsorption was seen in HP 20 (with and without cross linking) and silica, where attachment to lipase was up to the extent of 78, 79 and 86% respectively. The protein and lipase adsorption was fast and maximum binding was observed after 45 min for protein and 30 min for lipase (Figure 1a and Figure 1b).

Optimum pH for lipase activity was same in the case of aqueous and immobilized form. Optimum temperature of immobilized (CL) enzyme shifted from 60ºC to 65ºC.

Half-life of aqueous and immobilized lipase (HP-20) was calculated to be 85-90 min and 4 hrs respectively at 70ºC (Figure 2).

The effect of incubation time on esterification reaction indicates that ester synthesis was essentially complete by 9-10 hrs (Figure 3).

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