Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose

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-amyl...

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Autores principales: Terebiznik, Mauricio R., Pilosof, Ana María Renata, Moreno de Colonna, Silvia
Publicado: 1996
Materias:
Aspergillus oryzae
Triticum aestivum
Acceso en línea:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01458884_v19_n5_p341_Terebiznik
http://hdl.handle.net/20.500.12110/paper_01458884_v19_n5_p341_Terebiznik
Aporte de:
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires
id paper:paper_01458884_v19_n5_p341_Terebiznik
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spelling paper:paper_01458884_v19_n5_p341_Terebiznik2023-06-08T15:12:29Z Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose Terebiznik, Mauricio R. Pilosof, Ana María Renata Moreno de Colonna, Silvia Aspergillus oryzae Triticum aestivum 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. Fil:Terebiznik, M.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Pilosof, A.M.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1996 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01458884_v19_n5_p341_Terebiznik http://hdl.handle.net/20.500.12110/paper_01458884_v19_n5_p341_Terebiznik
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-134
collection Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic Aspergillus oryzae
Triticum aestivum
spellingShingle Aspergillus oryzae
Triticum aestivum
Terebiznik, Mauricio R.
Pilosof, Ana María Renata
Moreno de Colonna, Silvia
Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
topic_facet Aspergillus oryzae
Triticum aestivum
description 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.
author Terebiznik, Mauricio R.
Pilosof, Ana María Renata
Moreno de Colonna, Silvia
author_facet Terebiznik, Mauricio R.
Pilosof, Ana María Renata
Moreno de Colonna, Silvia
author_sort Terebiznik, Mauricio R.
title Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
title_short Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
title_full Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
title_fullStr Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
title_full_unstemmed Effective purification procedure of Aspergillus oryzae α-amylase from solid state fermentation cultures including Concanavalin A-Sepharose
title_sort effective purification procedure of aspergillus oryzae α-amylase from solid state fermentation cultures including concanavalin a-sepharose
publishDate 1996
url https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01458884_v19_n5_p341_Terebiznik
http://hdl.handle.net/20.500.12110/paper_01458884_v19_n5_p341_Terebiznik
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