The Characteristics Of The Method Of Enzyme Immobilization
The experiment was carried out to meet the objective of studying the immobilization techniques and to study the characteristics of the enzyme immobilization by gel entrapment. An immobilized enzyme is an enzyme that is attached to an inert, insoluble material.
In general, method of enzyme immobilization coul be classified into three: cross-linking, entrapping and carrier-binding. As this experiment was highlighted about the entrapping method, the method is actually based on confining native enzyme in strictly defined space, limited by insoluble porous barrier. In other words, the enzyme is trapped in insoluble beads or microspheres.Basically, the preparation of alginate beads involves mixing of different concentration of sodium alginate and alpha-amylase solutions in buffer, followed by dripping of the solution into the solution of calcium chloride, CaCl2 to form a gel.
During the preparation of different concentration of sodium alginate, the colour variaty of those solutions were seen. The 3% sodium alginate has white translucent colour and the mixture is not as sticky as 6% and 9% sodium alginate. The colour of the higher concentration of sodium alginate were white and yellowish, where they were very sticky and quite hard to be stirred and put into the syringes. After approximately 10 drops of each of enzyme aginate solution were dripped into 0.2M CaCl2 solution and left aside for at least 0.5 hours, few beads were observed to float. Those which floated cannot be used for analysing and weighing as they were trapped with air bubbles and might not mixed homogenously while stirring. The other beads were seen clearly on the bottom of the beaker
The total weight of 3% alginate beads formed is 0.34 g. The values are increasing for the total weight of medium and large beads of 6% and 9% alginate, which are 0.65 g and 0.96 g, respectively. The shape and colour of beads formed for the smaller one were round-shape and translucent, while the tadpole-shape beads were formed for the medium and larger beads. The increased concentration of sodium alginate leads to the increased in weight of 10 beads formed.Galazzo and Bailey, 1990 once stated that the physical entrament process requires that the immobilized enxyme should be large enough to be kept inside the gel matrix, but the substrate and product should be small enough to pass through the pores of the gel.In this method, the amylase enzyme is expected to be immobilized due to the surface of beads as well as be contained inside the beads.
The purpose of using CaCl2 solution is to allow the beads to harden. Theoritically, when the beads are formed in CaCl2, calcium ions displace sodium ions and form a gel matrix. The amylase that would be immobilized on the bead surface is competing with the calcium ions to move toward the beads and this may deter some surface binding of amylase from occuring. From the experiment, the results were obtained as predicted and the study objectives were met.
