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Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii |
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| Authors: | W Schnitzhofer H-J Weber M Vr&#x;ansk P Biely A Cavaco-Paulo GM Guebitz |
| Institution: | aDepartment of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria bDepartment of Organic Chemistry, Graz University of Technology, Stremayrgasse 16, 8010 Graz, Austria cInstitute of Chemistry, Slovak Acadamy of Sciences, 84238 Bratislava, Slovak Republic dDepartment of Textile Engineering, University of Minho, 4800 Guimaraes, Portugal |
| Abstract: | Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes. |
| Keywords: | Polygalacturonase FFE Purification Pectinase Plant pathogen fungus |
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