(485au) Neutrase Immobilization Into Chitosan and Green Coconut Activated by Different Methods

The specificity of enzymes and their ability to catalyze reactions at low temperatures and neutral pH make them attractive for applications in biochemical and industrial fields. However, they are expensive, have low operational stability and the recovery of the free enzyme from the medium is not economically viable, what has hindered their application in large scale processes. The enzyme immobilization on insoluble supports allows not only the reuse of the protein but also to modulate the catalyst properties. Besides, the use of an insoluble enzyme derivative enables the operation of continuous processes, fast ending of reactions, controlled product formation, easiness of reaction mixture removal and adaptability for several engineering purposes and making possible its use in a larger number of processes. Neutrase, a bacterial endoprotease produced by Bacillus subtilis presents considerable interest due to a wide variety of possible applications, for example, transformations of cheese whey proteins in high value food, as protein hydrolysates with low contents of phenylalanine that are an important issue for chemical and pharmaceutical industries. Thus, the aim of this work will be developing a neutrase immobilization protocol using chitosan and green coconut fiber, cheap and abundant materials in Brazil with very interesting properties as supports for enzyme immobilization, as well as, using different activation methods and comparing to soluble enzyme. Chitosan beads will be prepared by dissolving in a solution of acetic acid 5%. The obtained solution will be dropped into a gently stirred NaOH 0.1mol.L-1 solution. By contrast, green coconut fiber will be cut and sieved to obtain particles between 32 and 35 mesh being washed with distilled water and dried at 60 °C. Both supports will be activated with glutaraldehyde, glycidol and epichlorohydrin before enzyme immobilization. A solution of neutrase (5mg enzyme.g of support -1) in 0.2 mol.L-1 tris-maleic buffer at different pH value (5..3-8.5) will be added to the activated support and kept under mild stirring at 25°C for different process times. Soluble and immobilized neutrase activities will be assessed via spectrophotometer analysis at 700 nm according to the TCA- Lowry assay. One unit of activity is defined as the amount of enzyme that hydrolyzes casein to produce equivalent color to 1 µg of tyrosine per minute at pH 8.1 and 50 °C. The derivatives will be analyzed as immobilization yield, coupling yield and stabilization factor at 55°C and compared to soluble enzyme. According to literature, it is expected a high immobilization yield, an immobilized enzyme with a broader pH profile, indicating the effectiveness of support in providing resistance to wide variation in pH. Moreover, to enhance the optimum temperature and thermal stability of the immobilized enzyme compared to the soluble one.