Immobilization of pancreatic porcine lipase in calcium and barium alginate comparative studies of their kinetic properties and screening the polymer for suitability
V Prasanth, G Parthasarathy, A Puratchikody, A Balaraman, B Vinod, N Chandrasheker, S Mathew
barium alginate, immobilization, km, sodium alginate, vmax
V Prasanth, G Parthasarathy, A Puratchikody, A Balaraman, B Vinod, N Chandrasheker, S Mathew. Immobilization of pancreatic porcine lipase in calcium and barium alginate comparative studies of their kinetic properties and screening the polymer for suitability. The Internet Journal of Pharmacology. 2008 Volume 6 Number 2.
Immobilization of enzymes often incurs an additional expense and is only undertaken if there is a sound economic (or) process advantage in the use of immobilized enzyme rather than free enzyme. The most benefit derived from immobilization is the easy separation of enzymes from the product of the catalysed reaction.This prevents the enzymes from the contaminating the product, minimizing downstream processing costs and possible effluent handling problems. In this study ionotropic gelation method for immobilization of lipase using calcium and barium alginate was evaluated. This was simple and inexpensive to perform and also, gives a product with good retention of activity and high operation stability. The Km and Vmax values for calcium alginate beads are more than the barium alginate beads, from Line Weaver Burk Plot, which is the reciprocal of Michaeleis Menton curve. Lipases are one among the most widely used biocatalyst in number of industries. They have a wide range of applications in biochemical as well as pharmaceutical industries. They are used for its enantioselective applications. Due to number of reasons like low stability (Thermal as well as Chemical) and high extraction costs, it is difficult with pure enzymes in industries. Immobilization promises re-use and more stability of enzyme preparation. Immobilization of pancreatic porcine lipase in an inert carrier of calcium alginate and determined its activity in comparison with immobilized enzyme in barium alginate alginate matrix. The immobilized enzyme can use several times while free enzyme can’t use more than one. Lipase is one of the pharmaceutically important enzymes. Lipase has applications as industrial catalysts for the resolution of racemic alcohols in the preparation of some prostaglandins, steroids and carboxylic nucleoside analogues 1 . The matrix chosen is alginate and lipase has been immobilized. To ascertain the desired properties and for their applications, a comparative evaluation of immobilised lipase relative to free enzyme in terms of activity ,stability (pH, temperature profile studies),rate of reaction (Michaelis Menton kinetics) have been performed. Rate measurements have been made over a range of substrate concentration. In the present study we have investigated comparative kinetic properties of pancreatic porcine lipase in calcium and barium alginate.
Materials And Methods
Pancreatic porcine lipase, Sodium chloride, Sodium alginate, Calcium chloride, 0.2M Disodium hydrogen phosphate, 0.2M Sodium dihydrogen phosphate, Sodium taurocholate were from Nice research laboratories Pvt Ltd, and Gum Arabic and reagent grade water from local market.
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.
Activity studies of immobilized enzyme
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
Gum Arabic – Olive oil emulsion
Reagent grade water
3 M sodium chloride
0.075M Calcium Chloride
0.027M Sodium Taurochloate
Amount of beads – 100mg
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
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 (Vo), 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/Vo 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.
Results And Discussion
Pancreatic porcine lipase, the experimental enzyme has been immobilized by entrapping within calcium alginate and barium alginate. The rate of immobilization was quite satisfactory. The optimum temperature for free enzyme is 37°C, and calcium alginate beads are 37- 40 ° C and for barium alginate bead is 30 ° C. In temperature studies the calcium alginate beads can withstand more temperature than barium alginate beads. While comparing to kinetic studies calcium alginate beads showed less Km value compare to barium alginate beads. Less Km value indicates more affinity of enzyme towards the substrate concentration and vice versa.
Km value indicating affinity of enzyme towards enzyme concentration and Vmax indicating maximum velocity. From the above studies we concluded that calcium alginate beads are showing less Km compare to barium alginate beads and moreover that calcium alginate can withstand at high temperature also. Depending upon these different parameters were discussed below and concluded.
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.
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.
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.
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.
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.
Immobilization of pancreatic porcine lipase were carried out successfully using calcium alginate and barium alginate by cross linking method and the polymer nature is found to influence the entrapment efficiency and release characteristics of the enzyme from the beads. This studies indicates that barium alginate beads cannot withstand high temperature, leaching studies shows poor results and kinetic studies shows also not up to the mark while compare to calcium alginate beads. It is concluded that calcium alginate is suitable for entrapment of pancreatic porcine lipase. This promises re-use of pancreatic porcine lipase.
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.