XXXV Reunião Anual da SBBqResumoID:9207


Production, purification and characterization of intracellular invertase of Aspergillus terreus.


Almeida, M.N.; Fontes,G.C.; Coitinho, J.B; Barbosa, D.S.; Rezende, S.T.; Guimarães, V.M.

Instituto de Biotecnologia Aplicado à Agropecuária-BIOAGRO, Departamento de Bioquímica e Biologia Molecular- UFV- Viçosa- MG

Invertases (β-D-fructofuranoside fructo hydrolase, E.C, 3.2.1.26) are enzymes which catalyze the hydrolysis of sucrose into glucose and fructose. It is a great interest enzyme due to its applications, and among those, the production of inverted sugar thoroughly used on food industry. These enzymes are also used on pharmacist industry for the production of syrup. In this work, intracellular invertase of Aspergillus terreus was produced, purified and characterized. Substantial intracellular invertase activity was produced during fungal growth on sucrose at 30°C in mineral liquid medium for 48 hours under agitation. It was not detected invertase activity in the supernatant. The mycelium was macerated with liquid nitrogen, suspended in lyse solution and centrifuged (15,300 g) for 30 min at 4°C. The supernatant was used as a crude enzyme preparation. This preparation was concentrated by ultrafiltration through a 10 kDa polyethersulfone membrane filter and this solution was applied to an ion exchange column. The fraction that showed the highest invertase activity was submitted to a hydrophobic interaction chromatography. The preparation obtained in this process was analyzed by SDS PAGE that showed a highly purified protein. The maximum activity of invertase against sucrose was in pH 4.7 and temperature of 60°C. The values of KM and Vmax for the hydrolysis of sucrose were 6.8 mM and 0.43 mM min-1 respectively and for the hydrolysis of raffinose were 13.25 mM and 5.6 mM min-1 respectively. It was also evaluated the ion effect. The activity of invertase was smaller when pre incubated in the presence of SDS and Cu(SO)4. Contrarily, MnCl2 increased this activity. The activation energy to the sucrose hydrolysis was 29.84 J mol-1 and to raffinose was 33.11J mol -1.

Supported by FAPEMIG and CNPq