Immobilisation of fructosyltransferase from Aspergillus aculeatus on epoxy-activated Sepabeads EC for the synthesis of fructo-oligosaccharides

9 pages, 7 figures.-- Printed version published Aug 1, 2005.The transfructosylating activity present in two commercial pectinase preparations (Pectinex Ultra SP-L, from Aspergillus aculeatus, and Rapidase TF, from Aspergillus niger) was studied. Pectinex Ultra SP-L, which has a high transferase/hydrolase ratio, was covalently immobilised on a polymethacrylate-based polymer (Sepabeads® EC) activated with epoxy groups. The influence of pore volume and average pore size on biocatalyst performance was studied for two of these carriers (Sepabeads EC-EP3 and EC-EP5). Several parameters that affect immobilisation such as buffer concentration, pH and amount (mg) of protein added per gram of support (varied over the range 30:1 to 200:1) were analysed. We found that Pectinex Ultra SP-L can be efficiently immobilised on these supports without adding any external salt or buffer. Using Sepabeads EC-EP5 –whose pore volume (1.67 cm3/g) and pore size (800 nm) are higher than those corresponding to Sepabeads EC-EP3– the activity towards sucrose reached 25.9 U/g biocatalyst. The immobilised fructosyltransferase was applied to the batch synthesis of fructo-oligosaccharides (FOS) using 630 g/l sucrose to shift activity towards transfructosylation in detriment of hydrolysis. The FOS concentration reached a maximum value of 387 g/l after 36 h (240 g/l 1-kestose, 144 g/l nystose and 3 g/l 1(F)-fructofuranosyl-nystose), which corresponds to 61.5% (w/w) of the total carbohydrates in the mixture. The features of these immobilised biocatalysts are very attractive for their application in batch and fixed-bed bioreactors.We thank Drs. Moreno Daminati and Paolo Caimi (Resindion) for providing us Sepabeads EC polymers and for technical help. We are grateful to Ramiro Martínez (Novozymes A/S, Spain) and Gemma Hernández (DSM, Spain) for enzyme samples. This research was supported by the Spanish CICYT (Project BIO2004-03773-C04-01). We thank the Spanish AECI, Gobierno Vasco and Instituto Danone for research fellowships.Peer reviewe