Production, purification and characterization of a-galactosidase from aspergillus ficuum nrrl 3135
Extracellular a-galactosidase (E.C. 126.96.36.199), a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. Melibiose, galactose and to a lesser extent, raffinose induced the enzyme. Optimum culture conditions were: pH, 6.6; temperature, 28°C; initial glucose concentration, 15 g/liter; raffinose concentration at the time of induction, 6.8 g/Liter. The approximate molecular weight of the enzyme was determined to be 70-85 kDa by HPLC gel permeation chromatography. The purified enzyme was remarkably stable at 0°C. It had a broad temperature optimum and maximum catalytic activity was at 60°C, retained 33% of its activity after 10 minutes at 65°C, had a pH optimum of 6.0, and retained 62% Of its activity after 12 hours at pH of 2.3. The Km for pnitrophenyl- a-D-galactopyranoside, o-nitrophenyl-a-Dgalactopyranoside, and m-nitrophenyl-a-Dgalactopyranoside are about 1462, 839, and 718 μM, respectively. The enzyme was competitively inhibited by mercury (19.8 μM), silver (21.5 μM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM), and fructose (60.3 mM). It was inhibited noncompetitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).
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Wodzinski, Rudy J.
Master of Science (M.S.)
College of Arts and Sciences
Length of Campus-only Access
Masters Thesis (Open Access)
Arts and Sciences -- Dissertations, Academic; Dissertations, Academic -- Arts and Sciences
Zapater, Ismael G., "Production, purification and characterization of a-galactosidase from aspergillus ficuum nrrl 3135" (1989). Retrospective Theses and Dissertations. 4252.