Enzyme activity studies

Lipase activity values which are derived from titrimetric assay procedure can be quite dependent upon the source and type of substrate, the preparation of the substrate emulsion, other compounds of the reaction mixture, the methodology and instrumentation utilized. Lipase acts as triglycerides present in the olive oil to release the free fatty acids and glycerol. The released free fatty acids are titrated against a standard alkali, as the µ moles of free fatty acids liberated per mg of protein per minute. The lipolytic activity was determined by modified method of Oi et.al.

Method of preparation of immobilized enzymes 23

Immobilization using sodium alginate with calcium chloride

The technique involved is ionotrophic gelation where the cross –linking of alginates with Ca [[[2+]]] is employed. A 2 % sodium alginate gel was prepared by dissolving 2gm of sodium alginate powder in 100 ml of de-ionized water. The various concentration of lipase sodium alginate mixtures were prepared by adding the different concentration of lipase such as 300mg, 600mg, 900mg, and 1200mg and were mixed thoroughly to get homogenous mixture. The alginate gel containing lipase was then taken in syringe (nozzle diameter-1mm) and then injected drop wise into 100ml of freshly prepared 5% Cacl₂ solution with general stirring. So uniform size alginate beads were formed and kept aside for 20 minutes. The beads were washed with double distilled water, dried at room temperature and stored in refrigerator.

Immobilisation using sodium alginate with barium chloride

The alginate beads were prepared by the above same method by using 5% Bacl₂ solution instead of 5% Cacl₂ solution.

Estimation of rate of immobilization

After the recovery of alginate beads, the 1 ml of the Cacl₂ / Bacl₂ filter was taken and assayed by 0.02M sodium hydroxide solution using phenolphthalein as an indicator. This was carried out to find out the unentrapped enzyme present in the Cacl₂ / Bacl₂ solution. After the alginate beads were washed with 20ml of double distilled water for 3 times and filtered.1 ml of the filtrate was assayed by using 0.02M sodium hydroxide using phenolphthalein as an indicator. The activity was determined by modified method of Oi et.al. 4

Assay of Immobilized Lipase

Effect of temperature on free and immobilized lipase

All the batches of the assay medium containing free lipase and immobilized lipase were kept at different temperature such as 10 ° C , 20 ° C, 25 ° C,30 ° C, 35 ° C, 37 ° C, 40 ° C, 45 ° C, 50 ° C, 60 ° C, 70 ° C, & 80 ° C for 30 minutes. The activity of all batches were determined. For free enzyme and calcium and barium alginate beads, the results were noted.

Effect of pH on free and immobilized lipase

All the batches of assay medium containing free lipase and immobilized lipase were maintained by different pH such as 5.0, 5.5, 6.0, 6.2, 6.4, 6.8, 7.0, 7.3, 7.4, 7.5, 7.8 and 8.0 for 30 minutes and then the activity was determined. For free enzyme, calcium and barium alginate beads, the results were noted. The optimum pH were determined and used the same pH for further investigation.

Kinetic studies of immobilized lipase

Method: Different concentrations of olive oil were used as a substrate in free lipase and immobilized lipase. The concentration of substrate such as 5%, 10%, 15%, 20%, 30%, 40%, 50% & 60% and their activity were determined. Many enzymes show typical hyperbolic curve reality to the reaction velocity to the substrate concentration with a gradual approach to saturation of the enzyme with substrate. There were two cordial points in such plots.

Km, half maximal velocity of the substrate concentration.

Vmax (or) the maximal velocity which the rate approaches at infinitely high substrate concentration.

The Michaelis - Menten equation is an algebraic expression of the hyperbolic shape of such curves, in which the important terms are substrate concentrations [s], initial velocity (V_o), Vmax and Km.

Transformation of the Michaelis – Menten equation is called as Line weaver Burk Equation. For enzyme obeying the Michaelis – Menten relationship, a plot at 1/V_o against 1/[s] yield a straight line. From the values at slope and intercept, the values of Km and Vmax are to be determined 5 . The kinetic data for calcium and barium alginate beads were calculated.

Particle size determination of alginate beads

The method employed is optical microscopy. The stage and eye piece micrometer were placed in their respective position and the later is standardized using the stage micrometer. Randomly, few particles were placed on a glass slide and measurement was made using eye piece micrometer for approximately 50 particles. The reading were tabulated the actual, the actual size was calculated and the average bead size was found. The volume and surface area of the alginate beads were calculated. The date for calcium and barium alginate beads was tabulated.

Leaching studied performance

Average weight of 1.5 gm of dried immobilized beads of alginate were taken and added in a beaker containing 100ml of the distilled water. 1ml of solution from the beaker was taken with interval of 1 hour up to 7 hours, and the amount of lipase leached were determined.

Physical properties

The dry calcium alginate beads and barium alginate beads with immobilized lipase are almost spherical in shape. The average diameter of calcium alginate bead is 0.897 nm and the average volume and surface area of the bead are 0.38 mm 3 and 2.53 mm 2 respectively. The average diameter of dried barium alginate bead is 0.694 nm and the average volume and surface area of the bead are 0.75 mm 3 and 1.512 mm 2 respectively.

Temperature studies

Activities of free lipase, immobilized calcium & barium alginate beads (0.6 gms of lipase) at different temperatures of operation have been studied. The experimental data have been tabulated in Table 1. The maximum activity of the free lipase, immobilized calcium & barium alginate beads are observed at 37 ° C, 37 ° C to 40 ° C & 30 ° C respectively. It is interesting to note that at high temperature, when activity of free enzyme decreases sharply, the activity of calcium alginate beads retained its activity at 40 ° C. But in the case of barium alginate bead can’t retained its activity at 37 ° C also.

Table 1: Estimation of optimum temperature

pH studies

A variation of activity of free lipase, immobilised calcium & barium alginate beads (0.6 gms of lipase) at different pH values has been tabulated in Table II. The optimum pH for free lipase, immobilized calcium & barium alginate beads are 7.8, 6.4 & 5.5 respectively.

Table 2: Estimation of optimum pH

Kinetic studies

Kinetic studies by Michaelis-Menten and Line- Weaver Burk method on immobilized lipase in calcium and barium alginate beads have been carried out and the data have been tabulated in Table 3 and experimental data have been plotted in figure 1&2 for determining Km and Vmax. It is clear that the rate of reaction for calcium and barium alginate beads are uniform after a certain substrate concentration which establishes the fact that the rate of forward diffusion to the active sites and the backward diffusion of product are constant and systematic the values of the Km and Vmax for calcium alginate beads are 0.289 and 0.18 and the values for the Km and Vmax for barium alginate beads are 0.661 and 0.515 respectively.

Table 3 : Kinetic studies of immobilized beads

Figure 1 : Michaelis-Menton Kinetics

Figure 2: Line Weaver Burk plot

Leaching studies

The calcium alginate beads with immobilized lipase can be used repeatedly for successive batches as the maximum amount of leaching is only 2.53U/mg/min. But in case of barium alginate beads,the maximum amount of leaching is 15.99U/mg/min. While comparing the barium alginate beads with the calcium alginate beads, the calcium alginate bead showed better results.

Acknowledgement

The authors wish to thank Mrs.Kavitha (chairperson) and Mrs. Anitha Prasad (member of management) of Gautham College of Pharmacy, Bangalore, India for providing facilities to carry out the research work.