ISSN print edition: 0366-6352
ISSN electronic edition: 1336-9075
Registr. No.: MK SR 9/7
Production of Geotrichum candidum polygalacturonases via solid state fermentation on grape pomace
Kateřina Illková, Zuzana Zemková, Dana Flodrová, Jakub Jäger, Dagmar Benkovská, Jiřina Omelková, Renáta Vadkertiová, Janette Bobáľová, and Eva Stratilová
Faculty of Chemistry, Brno University of Technology, Purkyňova 118, CZ-612 00 Brno, Czech Republic
Abstract: Geotrichum candidum CCY 16-1-29 (teleomorph Galactomyces geotrichum) is able to grow and produce polygalacturonase of remarkable activities on pectin or grape pomace as a sole carbon source.
The highest activities of extracellular enzymes were found on the third and the seventh day of cultivation. After extraction
and precipitation, polygalacturonases produced in these cultivation periods were characterized. Production of multiple forms
of polygalacturonase was observed in both cultivation periods. Two major forms, polygalacturonase with random action pattern
(endo-PGase, EC 188.8.131.52) and oligogalacturonate hydrolase (exoPGase, exopolygalacturonase preferring oligogalacturonides
as substrates), as well as numerous minor forms were detected by IEF-PAGE using the print technique detection. EndoPGase was
identified by mass spectrometry. The major forms have similar isoelectric points (below pH 6.0) and pH optima (4.6 and 4.8,
respectively). pH optimum of 4.6 was associated with exoPGase and that of 4.8 with endoPGase. Both enzymes were stable after
freeze-drying and storage at 4°C. EndoPGase had molecular mass of about 29 kDa (36 kDa by SDS-PAGE) as determined by gel filtration,
temperature optimum of about 45°C and it was stable only below 35°C. Molecular mass of exoPGase was about 50 kDa, its temperature
optimum was about 60°C, and it was stable to 60°C. Optimal substrate for exoPGase was a pentamer, for endoPGase it was a pectate.
Values of K
for optimal substrate reached the values of 11.4 × 10−5 M for for exoPGase and 6.6 × 10−5 M for endoPGase. Pectin methylesterase as another pectolytic enzyme was also identified by mass spectrometry.
Keywords: Geotrichum candidum – grape pomace – exopolygalacturonase – endopolygalacturonase – pectin methylesterase
Full paper is available at www.springerlink.com.
Chemical Papers 66 (9) 852–860 (2012)