Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
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 |
_version_ | 1782201213641555968 |
---|---|
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. |
format | Text |
id | pubmed-3068306 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | SAGE-Hindawi Access to Research |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT galdinoalexsandrosobreira biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT silvarobertonascimento biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT lottermannmurieletaborda biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT alvaresalicecunhamorales biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT demoraeslidiamariapepe biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT torresfernandoararipegoncalves biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT defreitassoniamaria biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae AT ulhoaciranojose biochemicalandstructuralcharacterizationofamy1analphaamylasefromcryptococcusflavusexpressedinsaccharomycescerevisiae |