title: Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method.
abstract: Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123-128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 μg of polygalacturonic acid, and 45 μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAM-CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina.
authors.forename: Gastón E
authors.initials: GE
authors.lastname: Ortiz
authors.affiliation: Downstream Processing Laboratory, Jacobs University Bremen, D-28759 Bremen, Germany.
authors.forename: María E
authors.initials: ME
authors.lastname: Guitart
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAM-CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina.
authors.forename: Edgardo
authors.initials: E
authors.lastname: Albertó
authors.affiliation: Downstream Processing Laboratory, Jacobs University Bremen, D-28759 Bremen, Germany.
authors.forename: Héctor M
authors.initials: HM
authors.lastname: Fernández Lahore
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico Chascomús (IIB-INTECH, UNSAM-CONICET), Universidad de San Martín, San Martín, Buenos Aires 1650, Argentina. Electronic address: mblasco@bioimanay.com.
authors.forename: Martin
authors.initials: M
authors.lastname: Blasco
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D011096:Polygalacturonase / Q000378:metabolism*; D012430:Ruthenium Red / Q000737:chemistry*; D014783:Viscosity
chemical_list: D012430:Ruthenium Red; D011096:Polygalacturonase
keywords: Assay; Endo-polygalacturonases; High throughput; Microplate; Pectinases; Ruthenium red