Immobilization of endo-inulinase on poly-d-lysine coated CaCO3 micro-particles

Inulinase is widely applied to produce high fructose syrup (HFS) which is used in the food and pharmaceutical industries. Numbers of different techniques such as physical adsorption, entrapment, ion exchange and covalent bonding have been used to immobilize enzymes on solid supports. In this study, an efficient technique for inulinase immobilization was developed which consists of adsorbing a cationic polyelectrolyte poly-d-lysine (PDL) on negatively charged calcium carbonate micro-particles (CaCO3-MPLs) and immobilizing inulinase through electrostatic interactions between negatively charged amino acids of inulinase and positively charged [CaCO3/PDL-MPLs] complex. The most effective CaCO3-MPLs which had the smallest diameter were manufactured using ultrasonic spray pyrolysis; a new application of this method to manufacture the enzyme support. This method enabled us to reduce the particle size to 2.9 μm which showed the highest average protein loading capacity of 21.3 ± 0.1 mg or 32.0 ± 1.3 U enzyme per gram of CaCO3-MPLs. Two sets of binding sites are available on the [CaCO3/PDL-MPLs] for inulinase, both resulted in positive cooperation with Hill coefficients of 2.3 and 6.8 for the first and second sets of binding sites, respectively. For the immobilized enzyme, the optimum temperature of activity increased from 50 to 55 °C and immobilization resulted in higher thermal stability. Storage stability of immobilized inulinase increased more than two-fold as demonstrated by half-lives (t1/2) which increased from 65.2 to 148 days for the free and immobilized inulinases, respectively.