Immobilized Inulinase on Grafted Alginate Beads Prepared by the One-Step and the Two-Steps Methods Enas N. Danial, † Magdy M. M. Elnashar,* ,‡ and Ghada E. A. Awad † Chemistry of Natural and Microbial Products Department and Centre of Scientific ExcellencesPolymers DepartmentsAdVanced Materials & Nanotechnology Laboratory, National Research Center, El-Behooth St., Dokki, Cairo, Egypt Grafted alginate beads were prepared using the Encapsulator by two methods, the one-step and the two-step. The methods of grafting were characterized by thermal gravimetric analysis and infrared (IR). The glass transition (Tg) of both grafted gel beads showed gradual thermal improvement over the control gel. However, the one-step method showed higher Tg (231 °C) compared to the two-step method (220 °C). Both methods were also evaluated for immobilization of an important industrial enzyme, inulinase, to produce fructose, which is good for diet regimens and suitable for diabetics. The one-step method showed an enzyme loading capacity (ELC) of 530 U/g gel beads compared to 336 U/g gel beads for the two-step method. Accordingly, the one-step method has been chosen for further optimization. The ELC has been optimized to reach 1627 U/g gel using our locally prepared crude enzyme compared to 10.9 U/g by another author using purified inulinase. The immobilization process improved as did the enzyme’s thermal stability, from 50 to 60 °C, which is the most suitable temperature used in food industries to prevent microbial contamination. The enzyme’s thermal stability test at 60 °C and for an incubation time of 2 h, revealed a drastic decrease of the free enzyme activity to 21%, compared to 89% retention of activity for the immobilized enzyme. The immobilization process improved as well the enzyme’s shelf stability, where the free enzyme lost all of its activity at room temperature after 28 days, the immobilized enzyme retained over 77% of its initial activity. These results are encouraging to produce high fructose syrup on the industrial scale as the carrier is efficient and the method is simple and economic. 1. Introduction Inulinases are one of the most important enzymes used in industries. They are -fructan fructanohydrolases (EC 3.2.1.7), which hydrolyze inulin to produce fructose and fructo-oligosac- charides, both of which are important ingredients in the food and pharmaceutical industries. 1 Fructose is four times sweeter than glucose, easy to digest, and used as a sweetener in food industries as well as a remedy for people on diets and for diabetics. The best way to produce fructose in high yield is via enzymatic reaction (inulinase), where 95% pure fructose could be produced after one step of the enzymatic hydrolysis of inulin. Inulin is widely accumulated in the underground organs of chicory, dahlia, and Jerusalem artichoke. There are many techniques to immobilize enzymes, such as adsorption, covalent, encapsulation, entrapment, and cross- linking. 2–6 Each technique has it pros and cons; however, covalent technique has the advantage of keeping the enzymes well bound to the carrier, avoiding enzyme diffusion, 7 and this is why it is widely preferred on the industrial scale. Covalent immobilization of enzymes will enable the reuse of the immobilized enzymes tens of times and consequently reduce their cost as well as their products’ cost. Unfortunately, efficient commercial carriers suitable for immobilization of enzymes used in industries are few and relatively expensive. 8 Biopolymers, such alginates, carrageenans, and chitosans, are commercially available, have diverse features, and are available at a reasonable cost, which could make them good candidates for immobilizing enzymes. 9 However, they have some major drawbacks, such as low thermal stabilities and a lack of active functional groups to covalently immobilize enzymes. In our laboratory, we succeeded in the past few years in covalently immobilizing two industrial enzymes, penicillin G acyalse and -galactosidase, on carrageenan grafted with synthetic and natural polymers, respectively. 10–12 Recently, we succeeded in covalently im- mobilizing inulinase on alginate grafted with a synthetic polymer using the two-step method. 13 Alginate beads were prepared using the Encapsulator apparatus with a nozzle of 500 µm to produce gel beads of around 1000 µm. The alginate beads were hardened first with calcium alginate, then were soaked in polyelthylen- imine to incorporate amino groups, which were cross-linked to the amino groups of inulinase via glutaraldehyde that acted as well as a mediator to link the enzyme to the modified gel. The FTIR technique has proved the presence of the aldehydic group. The novel grafted alginate immobilized the inulinase covalently with an enzyme loading capacity of 461 U/g gel. The enzyme’s thermal stability has been improved from 50 °C for the free enzyme to 55 °C for the immobilized one, knowing that immo- bilization usually improves the enzyme’s thermal stability. 10,11 On the industrial scale, inulin hydrolysis is carried out at 60 °C in order to prevent microbial contamination and also because it permits the use of higher inulin substrate concentrations due to the increased solubility. Thus, a thermostable inulinolytic enzyme would be expected to play an important role in fructose- food industries. 1 Accordingly, in this work, we were aiming to further improve and optimize the enzyme loading capacity and the immobilized enzyme’s thermal stability. To achieve our goal, we prepared: (a) a new batch of crude inulinase from Penicillium funicu- losum with a specific activity of 381 U/mg, which is higher than that used in the previous publication (300 U/mg); 13 * To whom correspondence should be addressed. Fax: (202) 3370931. E-mail: magmel@gmail.com. † Chemistry of Natural and Microbial Products Department. ‡ Centre of Scientific ExcellencesPolymers Department. Ind. Eng. Chem. Res. 2010, 49, 3120–3125 3120 10.1021/ie100011z  2010 American Chemical Society Published on Web 03/08/2010