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EFFECTIVE PURIFICATION PROCEDURE OF ASPERGILLUS ORYZAE α‐AMYLASE FROM SOLID STATE FERMENTATION CULTURES INCLUDING CONCANAVALIN A‐SEPHAROSE
Author(s) -
TEREBIZNIK MAURICIO R.,
PILOSOF ANA M.R.,
MORENO SILVIA
Publication year - 1995
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1995.tb00539.x
Subject(s) - aspergillus oryzae , chromatography , concanavalin a , solid state fermentation , chemistry , affinity chromatography , sepharose , elution , amylase , fermentation , enzyme , specific activity , bran , lectin , enzyme assay , biochemistry , organic chemistry , in vitro , raw material
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 m M 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.