Cargando…

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially...

Descripción completa

Detalles Bibliográficos
Autores principales: Álvarez-Cao, María-Efigenia, Cerdán, María-Esperanza, González-Siso, María-Isabel, Becerra, Manuel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786279/
https://www.ncbi.nlm.nih.gov/pubmed/31601209
http://dx.doi.org/10.1186/s12934-019-1222-x
_version_ 1783458045770072064
author Álvarez-Cao, María-Efigenia
Cerdán, María-Esperanza
González-Siso, María-Isabel
Becerra, Manuel
author_facet Álvarez-Cao, María-Efigenia
Cerdán, María-Esperanza
González-Siso, María-Isabel
Becerra, Manuel
author_sort Álvarez-Cao, María-Efigenia
collection PubMed
description BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.
format Online
Article
Text
id pubmed-6786279
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-67862792019-10-17 Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides Álvarez-Cao, María-Efigenia Cerdán, María-Esperanza González-Siso, María-Isabel Becerra, Manuel Microb Cell Fact Research BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry. BioMed Central 2019-10-10 /pmc/articles/PMC6786279/ /pubmed/31601209 http://dx.doi.org/10.1186/s12934-019-1222-x Text en © The Author(s) 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Álvarez-Cao, María-Efigenia
Cerdán, María-Esperanza
González-Siso, María-Isabel
Becerra, Manuel
Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title_full Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title_fullStr Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title_full_unstemmed Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title_short Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
title_sort optimization of saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786279/
https://www.ncbi.nlm.nih.gov/pubmed/31601209
http://dx.doi.org/10.1186/s12934-019-1222-x
work_keys_str_mv AT alvarezcaomariaefigenia optimizationofsaccharomycescerevisiaeagalactosidaseproductionandapplicationinthedegradationofraffinosefamilyoligosaccharides
AT cerdanmariaesperanza optimizationofsaccharomycescerevisiaeagalactosidaseproductionandapplicationinthedegradationofraffinosefamilyoligosaccharides
AT gonzalezsisomariaisabel optimizationofsaccharomycescerevisiaeagalactosidaseproductionandapplicationinthedegradationofraffinosefamilyoligosaccharides
AT becerramanuel optimizationofsaccharomycescerevisiaeagalactosidaseproductionandapplicationinthedegradationofraffinosefamilyoligosaccharides