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Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae

An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4)(+) and inhibited by Cu(+2) and Hg(+2). Signifi...

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Autores principales: Galdino, Alexsandro Sobreira, Silva, Roberto Nascimento, Lottermann, Muriele Taborda, Álvares, Alice Cunha Morales, de Moraes, Lídia Maria Pepe, Torres, Fernando Araripe Gonçalves, de Freitas, Sonia Maria, Ulhoa, Cirano José
Formato: Texto
Lenguaje:English
Publicado: SAGE-Hindawi Access to Research 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068306/
https://www.ncbi.nlm.nih.gov/pubmed/21490699
http://dx.doi.org/10.4061/2011/157294
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author Galdino, Alexsandro Sobreira
Silva, Roberto Nascimento
Lottermann, Muriele Taborda
Álvares, Alice Cunha Morales
de Moraes, Lídia Maria Pepe
Torres, Fernando Araripe Gonçalves
de Freitas, Sonia Maria
Ulhoa, Cirano José
author_facet Galdino, Alexsandro Sobreira
Silva, Roberto Nascimento
Lottermann, Muriele Taborda
Álvares, Alice Cunha Morales
de Moraes, Lídia Maria Pepe
Torres, Fernando Araripe Gonçalves
de Freitas, Sonia Maria
Ulhoa, Cirano José
author_sort Galdino, Alexsandro Sobreira
collection PubMed
description An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4)(+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1(w), points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels.
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spelling pubmed-30683062011-04-13 Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae Galdino, Alexsandro Sobreira Silva, Roberto Nascimento Lottermann, Muriele Taborda Álvares, Alice Cunha Morales de Moraes, Lídia Maria Pepe Torres, Fernando Araripe Gonçalves de Freitas, Sonia Maria Ulhoa, Cirano José Enzyme Res Research Article An extracellular alpha-amylase (Amy1) whose gene from Cryptococcus flavus was previously expressed in Saccharomyces cerevisiae was purified to homogeneity (67 kDa) by ion-exchange and molecular exclusion chromatography. The enzyme was activated by NH(4)(+) and inhibited by Cu(+2) and Hg(+2). Significant biochemical and structural discrepancies between wild-type and recombinant α-amylase with respect to K(m) values, enzyme specificity, and secondary structure content were found. Far-UV CD spectra analysis at pH 7.0 revealed the high thermal stability of both proteins and the difference in folding pattern of Amy1 compared with wild-type amylase from C. flavus, which reflected in decrease (10-fold) of enzymatic activity of recombinant protein. Despite the differences, the highest activity of Amy1 towards soluble starch, amylopectin, and amylase, in contrast with the lowest activity of Amy1(w), points to this protein as being of paramount biotechnological importance with many applications ranging from food industry to the production of biofuels. SAGE-Hindawi Access to Research 2011-03-30 /pmc/articles/PMC3068306/ /pubmed/21490699 http://dx.doi.org/10.4061/2011/157294 Text en Copyright © 2011 Alexsandro Sobreira Galdino et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Galdino, Alexsandro Sobreira
Silva, Roberto Nascimento
Lottermann, Muriele Taborda
Álvares, Alice Cunha Morales
de Moraes, Lídia Maria Pepe
Torres, Fernando Araripe Gonçalves
de Freitas, Sonia Maria
Ulhoa, Cirano José
Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title_full Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title_fullStr Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title_full_unstemmed Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title_short Biochemical and Structural Characterization of Amy1: An Alpha-Amylase from Cryptococcus flavus Expressed in Saccharomyces cerevisiae
title_sort biochemical and structural characterization of amy1: an alpha-amylase from cryptococcus flavus expressed in saccharomyces cerevisiae
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068306/
https://www.ncbi.nlm.nih.gov/pubmed/21490699
http://dx.doi.org/10.4061/2011/157294
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