CONICET | Buscador de Institutos y Recursos Humanos

Lignocellulosic biomass such as agricultural and forestry residues , municipal solids waste and dedicated crops provide a low cost feedstock for biological production of fuels and chemicals , which offer economics, environmental and strategic advantages. These materials generally contains up to 75% of cellulose and hemicellulose, which cannot be easily converted to simple monomeric sugars due to their recalcitrant nature. Although the cost of producing sugars from these materials and converting them into ethanol, for instance, has been reduced in the last years, additional cost reductions are desirable to achieve competitiveness vis a vis the existing conventional fuels. Enzymatic hydrolysis of such celulosic material by cellulases enzymes is the most promising approach to get high product yields vital to economic success. However, the high cost of cellulases enzymes production hinders the application of these enzymes to bioethanol production. The enzyme immobilization allows the easy catalyst recovery and its subsequent reuse, reducing the processes costs and facilitating the implementation of the enzymatic process in industrial scale. Besides, immobilization of the enzyme may avoid autolysis and increase the enzyme stability, allowing continuous operation of the reactor for long periods. Therefore, cellulases stabilization and immobilization may help the achievement of the searched competitiveness of the use of cellulosic biomass as raw material to produce fuels. The aim of the present work is to study the immobilization of Cellulclast (Novo) into chitosan, sodium alginate, gelatin and different hybrid supports produced by the replacement of chitosan by sodium alginate or gelatin. Chitosan, 1.0, 2,5 and 5.0% and the alginate and gelatin hybrid was prepared by dissolving the polymer(s) in a 5% acetic acid solution, which is sprayed in a 0.1M ammonium hydroxide solution, pH 8.0, cured for two hours. The formed beads were activated with 2,5% glutaraldehyde, pH 7.0, for 90 minutes, at 25ºC. Immobilization of the enzyme in chitosan and chitosan hybrids was performed in 0.1 M of phosphate buffer, pH 7,0, at 27 0°C (2,5%chitosan/2.5%alginate was also immobilized at 4°C) , for 16 hours, offering 10mg of enzyme/g support. Enzyme immobilization into 2.5 % alginate was performed by dissolving enzyme and polymer in water and spraying the resulting solution in a 0.2 M calcium chloride solution. Immobilization into 2.5% gelatin was performed by dissolving enzyme and polymer in water and spraying the resulting solution in cold water. The enzyme inside the gel (calcium alginate and gelatin) was crosslinked by contacting the beads with a 0,5% glutaraldehyde solution, pH 7.0, for 90 minutes, at 25ºC and 4°C. Enzyme immobilization was followed measuring protein contents (Bradford method) and enzymatic activity (FPU) in the supernatant. The enzyme hydrolytic activity is determined using of Wathman paper filter n°1 with citrate buffer 50 mM (pH 4.8) 50°C for 1 h. Reducing sugars generated are quantified by DNS (acid 3,5-dinitrosalicilic) method It was evaluated immobilization yield, recovery activity, thermal stability (at 65°C) and the influence of temperature and pH in the enzyme activity. Derivates Cellulclast 2,5%alginate / 2.5%chitosan, obtained at 4°C e 27°C, showed to be 2.1 and 2.8 more stable than soluble enzyme, with immobilization yield of 55% e recovery activity of 40%. Both derivatives, Cellulclast immobilized into 1% chitosan and into 2,5%chitosan/2,5%alginate, showed the temperature of maximum activity (51°C) 5 grades higher than the determined for the soluble enzyme. The pH for maximum activity changed from 4.2 for soluble enzyme to the range between 2.5 and 3 for the immobilized enzyme.

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