Effective Purification Procedure of Aspergillus Oryzae ?-Amylase From Solid State Fermentation Cultures Including Concanavalin a-Sepharose

Abstract

Solid state fermentation cultures of Aspergillus oryzae NRRL 3485 on moistened wheat bran produced high levels of α‐amylase as judged by the specific activity of water extracts (500–700 units/mg protein) and by the enzyme concentration (around 0.15 g/L) in those extracts. The purification of α‐amylase by affinity chromatography on Concanavalin A‐Sepharose is described. A first DEAE‐Sepharose chromatography step (binding of the enzyme contained in the water extract and further elution with 0.2 M NaCl, pH 5) was necessary in order to remove contaminants that hinder the binding of the glycoprotein α‐amylase to the lectin. Elution was performed with 7.5 mM α‐D‐methylmannoside. The enzyme was obtained highly concentrated and purified with a specific activity of 2000 units/mg protein and a recovery of around 60%, free of α‐glucosidase, amyloglucosidase and color contaminants. The purity of the enzyme preparation was assessed through nondenaturing gel electrophoresis and sucrose gradient centrifugation. An improvement of the performance of the purification procedure is presented in which the maximum capacities of both the anion exchanger and the lectin matrices were exploited.

Thermal Stability of Dehydrated α-Amylase in Trehalose Matrices in Relation to its Phase Transitions

“…u.-Technol., 30, 513-518 (1997) 0023-6438/97/050513 + 06 $25.00/0/fs960210 ©1997 Academic Press Limited Millipore filter. α-Amylase was around 0.12-0.15 g/L corresponding to 25-30% of the total protein contained in the fermentation water extract (18). The effect of pH on the stability of α-amylase was first studied in order to select the adequate pH for performing the thermal inactivation studies.…”

Thermostability and Browning Development of Fungal α-amylase Freeze-dried in Carbohydrate and PVP Matrices

“…The liquid phase was recovered by filtering through a gauze and centrifuging for 15 min at 10,000 ϫ g. The resulting supernatant was filtered through a 0.22 µm Millipore filter (Massachusetts, USA). α-Amylase was around 0.12-0.15 g/L corresponding to 25-30% of the total protein contained in the fermentation water extract (10). pH was adjusted to 8 to prevent enzyme deactivation.…”

“…pH was adjusted to 8 to prevent enzyme deactivation. (10). Following freeze-drying, the samples were transferred into evacuated desiccators and kept over saturated salt solutions of RH 11% (LiCl), 22% (CHCOONa) and 44% (K 2 CO 3 ) for one week at 25 °C.…”