INTEC   05402
INSTITUTO DE DESARROLLO TECNOLOGICO PARA LA INDUSTRIA QUIMICA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Neutrase immobilization into chitosan and green coconut activated by different methods
Autor/es:
ADRIANO, WELLINGTON; CHINELATE, GERLA; MAMMARELLA, ENRIQUE; GONÇALVES, LUCIANA
Lugar:
Nashville, TN, Estados Unidos
Reunión:
Otro; AIChE Annual Meeting; 2009
Institución organizadora:
American Institute Of Chemical Engineers
Resumen:
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.